Respiratory diseases, such as asthma and chronic obstructive pulmonary disease (COPD), are pathological conditions affecting the airways of the respiratory system. Currently more than 90 million people are suffering from respiratory diseases, and COPD is predicted to become the third leading cause of death in the world by 2030. Inhalation devices are commonly used in the treatment of respiratory diseases, where an aerosolised medication is delivered to the lungs of the patient via inhalation. The inspiratory flow rate is one of the main factors affecting the drug deposition in the lung, but is currently not controlled in most inhalation systems. The purpose of this master thesis is to design, manufacture and characterize a passive flow regulator device for portable inhalation systems. The designed prototype utilises the principle of a Venturi nozzle and membrane deflection to create a variable flow constriction, which acts as a negative feedback loop for the flow rate regulation. The flow regulator is based on a previously working device used for controlling exhalation flow rate in the range of 3 L/min. Experimental results are evaluated and compared to an analytical solution of the classical Venturi design. Additionally, membrane deflection is measured to analyse the physical behaviour of the membrane within the device. The flow regulating device is scaled up for inhalation flow rate ranges (>30 L/min) and a flow rate sensor is constructed for the measurements. The passive control of flow rates using a deflecting membrane is deemed promising. However, further improvements of the scaled up model used for inhalation are necessary.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

This work focuses on the development of a spray dried adenoviral vector for its application as a thermally stable and inhalable vaccine against tuberculosis. / As the global public health community continues to strive for more equitable vaccine access, thermal instability of liquid vaccines continues to be a significant challenge due to strict cold-chain temperature requirements. Dry powder vaccines offer a favourable alternative, with the ability to retain vaccine efficacy at ambient temperature conditions. In the form of dry powder, vaccines against respiratory diseases can also be administered via inhalation for targeted delivery to the lung tissue. A processing technique known as spray drying is particularly promising for the development of thermally stable and inhalable dry powder vaccines, offering a method of continuous and scalable production. Spray drying is widely used in the pharmaceutical industry and can effectively encapsulate and immobilize labile biologics, like adenoviral vectors, within a glassy carbohydrate matrix to help retain biologic function. However, pulmonary delivery of a thermally stable, viral vectored dry powder vaccine has yet to be demonstrated. This thesis focuses on improving the formulation of a carbohydrate excipient blend of mannitol and dextran encapsulating a human serotype 5 adenovirus (AdHu5), with the goal of producing an inhalable vaccine with sufficient viral potency for in vivo murine testing. First, the impact of cryoprotective agents used for frozen storage of the stock adenovirus was investigated with respect to viral activity retention, thermal stability and inhalation properties of the dry powder after spray drying. Trehalose was considered a preferred cryoprotective agent, compared to glycerol traditionally used for adenoviral cryo-storage, allowing for the preparation of a high potency viral dry powder with 1.5 log loss of viral titre after processing and thermal aging. Further investigation of the dextran mass ratio and dextran molecular weight used within the excipient blend revealed that incorporating mannitol in a 1:3 ratio with 500 kDa dextran can further improve viral activity to achieve 0.8 log loss of viral titre after aging. Through controlled drying dynamics, this formulation led to improved activity retention and thermal stability, in addition to desirable aerosolization properties for pulmonary delivery. Using this optimized formulation, custom-made intratracheal dosator devices were evaluated for pulmonary powder delivery in mice. The method of powder loading in the device was found to be a significant factor of device performance in vivo when determining if the critical powder mass dosage could be delivered. Successful intratracheal delivery of the AdHu5-vectored dry powder was achieved with a pipette-tip loading dosator and led to a strong bioactive response. Overall, this work indicates the feasibility of murine pulmonary delivery and immunological testing of a thermally stable, adenoviral-vectored vaccine in dry powder form. / Thesis / Master of Applied Science (MASc) / Most vaccines currently available on the market must be stored and transported at temperatures ranging from 2-8 ⁰C to properly maintain their function, with some vaccine requiring temperatures as low as -80 ⁰C. The equipment required to maintain such temperatures is costly and is a significant limitation for developing nations trying to secure vaccine access. As an alternative to traditional liquid vaccine formulations, dry powder vaccines offer stability at room temperature without the need for expensive equipment and can also be administered through inhalation. Using a processing method called spray drying, an active vaccine component can be encapsulated in a carefully selected sugar formulation which forms a protective coating as the particles dry to provide stabilization. Since the efficacy of such dry powder vaccines must be first evaluated with mouse models, the focus of this work was to improve an existing blend of sugars to produce a dry vaccine powder that contains high enough dosage for mouse testing. Processing losses from spray drying were minimized through careful selection of vaccine cryoprotective agents, in addition to optimizing the blend ratio and molecular weight of sugars used for encapsulation. Successful delivery of the optimized powder to the lungs of mice was also accomplished after analyzing the suitability of a variety of custom-made handheld devices. This work shows that inhalable dry powder vaccine delivery is a promising solution to help improve temperature stability and achieve more equitable access to vaccines globally.

Inhaled corticosteroids (ICS) are used to address inflammatory illnesses including asthma and COPD, with delivery commonly achieved using pressurised metered dose inhalers (pMDI). Hydrofluoroalkanes (HFAs) have been introduced as an alternative propellant to chlorofluorocarbons (CFCs) to reduce their environmental impact. However, the thermodynamic properties of HFAs are poorly understood and are different to those of CFCs. It is essential, therefore, to characterise the drugs and excipients used in HFA inhalers in order to obtain a comprehensive understanding of the device performance and the therapeutic efficacy. This work has developed different analytical methods to study the complexation between ICS and CD which are added to enhance the solubility of inhaled drugs in pMDI propellant systems providing rational control of suspension vs. solution formulations and hence their dose uniformity and stability. The Nuclear Magnetic Resonance (NMR) method developed has shown weaker complexation between budesonide and the derivatised CDs DIMEB and TRIMEB in organic solvents compared to D2O with the strength of the complex formed being ranked as D2O > MeOD > CDCl3 > CD3CN. The derivatisation of the CD also shows a marked difference in complexation with budesonide with the strength of the association being ranked as DIMEB > βCD > TRIMEB. Studies of various ICS compounds with TRIMEB in the fluorinated propellant HPFP showed the association to be greatest in budesonide, followed by beclomethasone dipropionate, momestasone furoate and fluticasone propionate. Surface-enhanced Raman scattering (SERS) has been used for the detection of corticosteroids in water using thiol functionalised βCD as a complementary study to NMR. This has been utilised to evaluate the host-guest complexes formed and provides further insight into the complexation of the compounds by their inclusion into the CD cavity. The structural data obtained using the NMR and SERS approaches developed have provided a fundamental insight into the physical chemistry of these interactions at a molecular level.

No description available.

In asthma therapy, the use of theophylline to prevent bronchial spasm and glucocorticoids to decrease inflammation is widely indicated. Apart from the acute asthma attack oral theophylline is treated for chronic therapy in order to minimize inflammation and to enhance the efficiency of corticosteroids and recover steroids’ anti-inflammatory actions in COPD treatment. The preferred application route for respiratory disease treatment is by inhalation, such as dry powder inhalers (DPI) being the delivery systems of first choice. As shown recently, there is an advantageous effect if the drugs are given simultaneously which is caused by a synergistic effect at the same target cell in the lung epithelia. Therefore, it seems rational to combine both substances in one particle. This type of particle has the advantage over a combination product containing both drugs in a physical mixture which occurs rather randomly deposition leading to API segregation and non-dose-uniformity. Dry powder inhalers (DPIs) is a type of therapeutic pharmaceutical formulations usually present in the solid form. Due to the nature of the solid-state, an understanding of chemical and physical properties must be established for acquiring optimum performance of the active pharmaceutical ingredients (APIs). In recent year, generation of DPIs is a destructive procedure to meet the micron size. Such processes are inefficient and difficult to control. Moreover, according to current researches on combination APIs formulation, this type of DPIs performed a greater variability in does delivery of each active, leading to poor bioavailability and limit clinical efficient. This result suggest that combination formulations require advanced quality and functionality of particles with suitable physicochemical properties. Hence, in order to production of binary and combination DPIs products, the aim of this study was to develop the spray drying and ultrasonic process for engineering of combination drug particles that will be delivered more efficiently and independently of dose variations to the lung. Microparticles were produced by spray drying or/and ultrasonic technique. The processing parameters and addition of excipients (polymers) were optimized using a full factorial design such that microparticles were produced in a narrow size range suitable for inhalation. Employing excipients resulted in high saturation environment leading to minimized sphere particles when compared to conventional solvent. Solid state characterization of microparticles using powder x-ray diffraction and differential scanning calorimetry indicated that the particles contained crystalline but no cocrystal. The combination particles comparable to or better than micronized drug when formulated as a powder blended with lactose. It was concluded that the use of HPMC enhanced crystallinity suitable for inhalation; and combination particles improved uniform distribution on the stage of NGI.

Die Aspergillose ist eine Erkrankung des Respirationstraktes bei Vögeln, vor allem in Gefangenschaft gehaltenen tropischen Papageien, Greifvögeln und Pinguinen. Itraconazol ist hierbei ein häufig eingesetztes Therapeutikum und wird in der Vogelmedizin per os verabreicht (JONES und OROSZ 2000; KELLER 2011). Es zeigten sich gute Wirksamkeiten und Resistenzlagen. Bei über 90 % der Aspergillus fumigatus Isolate aus Wild- und Hausvögeln konnte eine MHK von 0,5 µg/ml eruiert werden (BEERNAERT et al. 2009). Allerdings kam es nach oraler Gabe von Itraconazol zu einem verzögerten Wirkungseintritt sowie, vor allem bei Graupapageien, zu Nebenwirkungen (KRAUTWALD-JUNGHANNS 2011b; PSCHERER 1995). Um ausreichende Wirkspiegel in Lunge und Luftsäcken zu erreichen, ist die Verabreichung einer hohen oralen Dosis notwendig. Dies führt wiederum zu hohen systemischen Konzentrationen und somit zu hohen potenziellen Nebenwirkungen (LUMEIJ et al. 1995). Ein Ziel in der Verbesserung der Therapiemöglichkeiten der Aspergillose ist es daher, einen hohen lokalen Wirkspiegel mit gleichzeitig geringen systemischen Konzentrationen zu erreichen. Ein Ansatz besteht in der inhalativen Verabreichung von z. B. Nanosuspensionen, da hier das Medikament direkt in Lunge/Luftsäcke appliziert werden kann. Ziel dieser Arbeit war die Sammlung von ersten Daten über den Einsatz einer neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem an Tauben (Columba livia f. domestica). Hierzu wurden in einer ersten Studie pharmakokinetische Spiegel in Blut sowie Lungengewebe nach einmaliger Gabe zweier Konzentrationsstufen (1 % und 10 %) erfasst. In der zweiten Studie wurde die Verträglichkeit sowie die Pharmakokinetik nach 14 tägiger Inhalation der Suspension in den Dosierungsstufen 1 %, 4 % und 10 % evaluiert. In der ersten Studie wurden die Tauben in zwei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten einmalig über 30 min die Itraconazol-Nanosuspension per inhalationem in den Dosierungsstufen 1 % und 10 % bzw. isotonische Natriumchlorid-Lösung. Keines der Tiere zeigte Nebenwirkungen, die in Zusammenhang mit der Medikation gestellt werden konnten. Zur Erstellung eines pharmakokinetischen Profils wurden Blutproben nach 1 h, 4 h, 24 h, 48 h, 72 h und 96 h sowie Lungengewebe von je vier Tauben nach der Euthanasie entnommen und darin die Itraconazol sowie die OH Itraconazol Konzentrationen bestimmt. Alle vorhandenen Werte zu einem gegebenen Zeitpunkt wurden gemittelt, woraus sich ein zusammengesetztes Profil ergab. Nach singulärer Verabreichung zeigten sich geringe systemische Spiegel mit einer Peak-Konzentration von 0,01 µg/ml Itraconazol 4 h post inhalationem bzw. 0,04 µg/ml OH-Itraconazol 24 h post inhalationem bei der 1%igen Nanosuspension und 0,065 µg/ml Itraconazol 24 h nach der Inhalation bzw. 0,365 µg/ml OH Itraconazol 24 h nach der Inhalation bei der 10%igen Dosierungsgruppe. In den Lungen konnten weitaus höhere Spiegel mit 9,1 µg/g Itraconazol und 0,223 µg/g OH Itraconazol 1 h post inhalationem bei der 1%igen Dosierungsgruppe bzw. 91,13 µg/g Itraconazol 1 h nach der Inhalation und 1,081 µg/g OH Itraconazol 4 h nach der Inhalation bei der 10%igen Dosierungsgruppe detektiert werden. Bei der zweiten Studie wurden die Tiere in drei Hauptgruppen sowie eine Placebogruppe randomisiert aufgeteilt und erhielten 14 Tage lang über je 30 min die Itraconazol-Nanosuspension per inhalationem in den Konzentrationen 1 %, 4 % und 10 % bzw. istotonische Natriumchlorid Lösung. Zur Abklärung der Verträglichkeit wurden die Tiere täglich adspektorisch, sowie alle sieben Tage ausführlich klinisch untersucht. Weiterhin wurden vor und nach der Inhalationsperiode detaillierte Blutuntersuchungen durchgeführt. Bei keiner der Untersuchungen konnten Unverträglichkeitsreaktionen auf das zu testende Medikament festgestellt werden. Auch nach 14 tägiger Gabe konnten im Plasma nur geringe Itraconazol-/OH Itraconazol-Konzentrationen gemessen werden. Aufgrund der teilweise sehr hohen Lungenspiegel (17,14 µg/g bei der 4%igen und 185 µg/g bei der 10%igen Nanosuspension je 24 h post inhalationem) und hohen Eliminationshalbwertszeiten (von über 40 h) sind jedoch hohe und langanhaltende Wirkspiegel am Infektionsort gegeben. Abschließend kann gesagt werden, dass die erlangten Daten über den Einsatz der neuartigen Itraconazol-Nanosuspension zur Verabreichung per inhalationem bei Tauben keine klinischen Nebenwirkungen erkennen ließen und sich ein effektives pharmakokinetisches Profil zeigte. Mit den hohen lokalen Lungenspiegeln nebst geringen systemischen Konzentrationen und somit geringen zu erwartende Nebenwirkungen erscheint das Medikament durchaus zum Einsatz gegen die Aspergillose bei Vögeln geeignet zu sein.

一氧化二氮，俗稱笑氣，是現代臨床麻醉最為常用的一種麻醉劑。然而，關於一氧化二氮效能及安全性的研究至今仍存在爭議。近來，一個稱為ENIGMA的臨床研究項目對2,050個施行大手術的病人接受麻醉情況及術後併發癥進行了研究。研究發現手術中施行一氧化二氮麻醉的病人術後傷口感染率較之對照組上升了35%。另一方面，對423個ENIGMA病人的長期隨訪研究發現，在術中接受一氧化二氮麻醉的病人中慢性術後痛的發病率相對對照組降低了56%。 對於這些臨床發現的分子機制，目前知之甚少。 因此我們進行了一系列實驗來研究一氧化二氮導致術後感染以及預防慢性痛的分子機制。 / 一氧化二氮對基因穩定性的影響 / 在對93個接受直腸結腸大型外科手術的病人進行的隨機對照試驗中，我們比較了接受一氧化二氮麻醉及其對照組病人的外周白細胞脫氧核糖核酸（DNA）損傷情況和術後傷口感染率。通過單細胞凝膠電泳（彗星實驗），我們發現術中一氧化二氮麻醉顯著增加了手術24小時后病人的DNA損傷情況 （p < 0.001）。且這種變化是劑量依賴的，r = 0.33; p = 0.03。並且，在DNA損傷程度及術後傷口感染率間存在顯著相關。術後DNA損傷程度每增加十個單位，傷口感染率則隨之增加17%。 / 一氧化二氮對DNA損傷應答及修復的影響 / 利用大鼠模型，我們對一氧化二氮導致基因不穩定的機制進行了研究。Sprague Dawley大鼠暴露於一氧化二氮中2小時後，我們對其DNA損傷應答及修復基因的轉錄情況進行了檢測。脂多糖（LPS）注射大鼠模擬了圍手術期的炎癥反應。我們發現LPS刺激的白細胞經一氧化二氮處理后，其編碼DNA連接酶IV的LIG4基因的轉錄量顯著降低（p < 0.05）。LIG4基因的下調導致了一氧化二氮麻醉後圍手術期的免疫抑制效應。 / NMDA受體抑制在一氧化二氮預防性鎮痛中的作用 / 在大鼠慢性神經痛模型中，我們檢測了一氧化二氮鎮痛作用的機制。我們發現一氧化二氮處理組的機械痛覺過敏相較對照組顯著降低（p = 0.001）。一氧化二氮處理后，神經痛大鼠脊髓背角中的c-Fos表達量也顯著降低，這表明了一氧化二氮對神經元活性的影響。該影響可能是NMDA受體抑制的結果。另外，我們還觀察到一氧化二氮的鎮痛特徵與NMDA受體非競爭性拮抗劑MK-801的鎮痛效果相似。 / 基因表達改變在一氧化二氮預防性鎮痛中的作用 / 一氧化二氮鎮痛效果的遲發性和延續性提示了除受體拮抗之外的其他作用機制的存在。我們發現在大鼠坐骨神經壓迫損傷模型中，一氧化二氮處理顯著降低了同側脊髓背角組織中LIG4基因的轉錄及表達（p = 0.006）。同時一氧化二氮處理降低了星形膠質細胞在同側脊髓背角中的活化。我們的研究表示一氧化二氮影響DNA修復， 抑制脊髓背角基因的表達，從而起到預防性鎮痛的作用。 / 總而言之，一氧化二氮通過抑制鉀硫氨酸合成酶，削弱DNA修復和基因組穩定性，從而成為導致術后傷口感染的危險因素。另一方面，這一機制也阻止了脊髓背角中異常突觸的建立，從而預防了神經損傷導致的慢性神經痛的建立。另外，一氧化二氮的鎮痛機制也和NMDA受體抑制作用有關。 / Nitrous oxide is a commonly administered anesthetic and analgesic agent in contemporary clinical anesthesia. However, the efficacy and safety of nitrous oxide delivery remains a subject of debate. The recent Evaluation of Nitrous oxide In a Gas Mixture for Anaesthesia (ENIGMA) Trial found that nitrous oxide administration, in 2,050 patients undergoing major surgery, increased the incidence of wound infection by 35%. On the other hand, in a long term follow-up study of 423 ENIGMA patients in Hong Kong, the risk of chronic postsurgical pain was reduced by 56% in patients who received nitrous oxide in the index surgery. Little is known about the mechanisms associated with these clinical observations; we therefore conducted a series of experiments to determine the molecular changes after nitrous oxide administration leading to postoperative wound infection and preventive analgesia. / Genomic Instability after Nitrous Oxide Administration / In a randomized controlled trial of 93 patients undergoing major colorectal surgery, we compared the changes of deoxyribonucleic acid (DNA) damage in circulating leukocytes and rates of wound infection in patients who were exposed to nitrous oxide or not. Using single cell gel electrophoresis (CometAssay), we found that intraoperative nitrous oxide administration produced significant DNA damage, 24 hours after surgery, compared with controls, p < 0.001. The changes were dose-dependent, r = 0.33; p = 0.03. In addition, there was a significant correlation between DNA damage and postoperative wound infection. For every 10 units increase in the percentage of DNA in tail after surgery compared with baseline, there was 17% increase in the risk of wound infection. / DNA Damage Response and Repair / In a rat model, we explored the mechanism of genomic instability after nitrous oxide administration. In Sprague Dawley rats exposed to nitrous oxide anesthesia for 2 hours, we tested the transcription of an array of DNA damage response and repair genes. Lipopolysaccharide (LPS) was added to mimic postoperative inflammation. In the mRNA that were extracted and analyzed by real-time polymerase chain reaction (RT-PCR), we found the transcription of gene encoding for DNA Ligase IV (LIG4 gene) was significantly reduced after nitrous oxide administration in LPS-stimulated leukocytes (p < 0.05). The down regulation of LIG4 gene contributed to perioperative immunosuppression following nitrous oxide exposure. / Role of N-methyl-D-aspartate receptor (NMDAR) Blockade for Preventive Analgesia with Nitrous Oxide / Using a rat model of chronic neuropathic pain, we tested the mechanisms underlying nitrous oxide analgesia. In Sprague Dawley rats undergoing unilateral constrictive injury to the sciatic nerve, we found that mechanical hyperalgesia was significantly reduced with nitrous oxide compared with controls (p = 0.01). In addition, c-Fos expression was decreased in spinal dorsal horn suggesting that neuron excitability was reduced after nitrous oxide administration which could be caused by blockade of the NMDA receptor. Interestingly, the characteristics of analgesia were similar to that provided by MK-801, a noncompetitive antagonist of NMDA receptors. / Transcriptional Changes for Nitrous Oxide Analgesia / The onset of nitrous oxide analgesia was delayed and outlasted actual receptor antagonism. We therefore explored mechanisms, other than NMDA receptor blockade, for nitrous oxide analgesia. Specifically, in rats undergoing sciatic nerve constrictive injury, we found that transcription of LIG4 gene was down-regulated in the ipsilateral spinal dorsal horn after nitrous oxide administration (p = 0.006). There was a decrease in DNA ligase IV expression and a reduction in the activation of astrocytes. Our data suggested that regulation of DNA repair and suppression spinal dorsal horn gene transcription is one of the alternative mechanisms for nitrous oxide analgesia. / In summary, nitrous oxide administration is a risk factor for postoperative wound infection. This is related to irreversible inhibition of the enzyme methionine synthase, impaired DNA repair and genomic instability. The same mechanism however prevented aberrations of synaptic regeneration in the spinal dorsal horn and could therefore prevent the development of chronic neuropathic pain after direct nerve injury. Nitrous oxide analgesia was also related to NMDA receptor blockade. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Yan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 126-157). / Abstract also in Chinese. / Declaration of origination --- p.I / Abstract: --- p.II / Acknowledgements --- p.VIII / Table of Contents --- p.X / List of Tables --- p.XV / List of Figures --- p.XVI / List of Abbreviations --- p.XVIII / Chapter Part I: --- Literature Review --- p.1 / Chapter Chapter 1 --- A review of nitrous oxide: Historical, clinical and mechanistic Perspectives --- p.2 / Chapter 1.1 --- History of Nitrous Oxide --- p.2 / Chapter 1.2 --- Clinical Pharmacology of Nitrous Oxide --- p.4 / Chapter 1.3 --- Evaluation of Nitrous oxide In the Gas Mixture for Anesthesia (ENIGMA) Trial --- p.9 / Chapter 1.4 --- Efficacy and Toxicity of Nitrous Oxide: Biochemical and Molecular Mechanisms --- p.14 / Chapter 1.4.1 --- Immunosuppression Following Nitrous Oxide Administration --- p.14 / Chapter 1.4.2 --- Analgesia with Nitrous Oxide - Molecular Mechanisms --- p.17 / Chapter 1.4.2.1 --- Direct molecular target --- p.17 / Chapter 1.4.2.2 --- Interaction with γ aminobutyric acid type A (GABAA) receptors --- p.19 / Chapter 1.4.2.3 --- Regulation of opioid system --- p.20 / Chapter 1.4.2.4 --- Regulation of noradrenergic neurons --- p.21 / Chapter 1.4.2.5 --- N-methyl-d-aspartate (NMDA) receptor inhibition --- p.22 / Chapter 1.4.2.6 --- Long-term Preventive Analgesia with Nitrous Oxide --- p.23 / Chapter 1.4.3 --- Summary --- p.24 / Chapter Part II: --- Experiments --- p.26 / Chapter Chapter 2 --- Study Hypothesis and Objectives --- p.27 / Chapter 2.1 --- Genomic Instability after Nitrous Oxide Administration --- p.27 / Chapter 2.2 --- DNA Damage Response and Repair --- p.28 / Chapter 2.3 --- NMDA Receptor Blockade for Preventive Analgesia with Nitrous Oxide --- p.28 / Chapter 2.4 --- Transcriptional Changes for Nitrous Oxide Analgesia --- p.28 / Chapter Chapter 3 --- Genomic Instability After Nitrous Oxide Administration: A Randomized Controlled Trial --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Methods and Materials --- p.36 / Chapter 3.2.1 --- Study Participants --- p.36 / Chapter 3.2.2 --- Study Procedures --- p.36 / Chapter 3.2.3 --- Randomization --- p.37 / Chapter 3.2.4 --- Anesthetic Care --- p.37 / Chapter 3.2.5 --- Postoperative Care --- p.38 / Chapter 3.2.6 --- Measurement of Genomic Instability --- p.44 / Chapter 3.2.7 --- Statistical Analysis --- p.47 / Chapter 3.2.8 --- Sample Size Calculation --- p.47 / Chapter 3.3 --- Results --- p.48 / Chapter 3.4 --- Discussion --- p.61 / Chapter 3.4.1 --- Principal Findings --- p.61 / Chapter 3.4.2 --- Comparison to Other Studies --- p.61 / Chapter 3.4.3 --- Strengths and Limitations --- p.63 / Chapter 3.4.4 --- Implications --- p.64 / Chapter 3.4.5 --- Conclusions --- p.64 / Chapter Chapter 4 --- DNA Damage Response and Repair After Nitrous Oxide Administration / Chapter 4.1 --- Introduction --- p.65 / Chapter 4.1.1 --- DNA Damage Response and Repair Pathways --- p.65 / Chapter 4.1.2 --- Nitrous oxide and DNA Damage Response and Repair --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Animals --- p.70 / Chapter 4.2.2 --- Nitrous Oxide Administration --- p.70 / Chapter 4.2.3 --- Lipopolysaccharide-Induced Infection Model --- p.72 / Chapter 4.2.4 --- Sample Collection and Preparation --- p.72 / Chapter 4.2.5 --- Single Cell Gel Electrophoresis (CometAssay) --- p.72 / Chapter 4.2.6 --- RNA Extraction for Gene Transcription Study --- p.72 / Chapter 4.2.7 --- Reverse Transcription Polymerase Chain Reaction (RT PCR) --- p.73 / Chapter 4.2.8 --- Quantitative RT PCR --- p.74 / Chapter 4.2.9 --- Statistical Analysis --- p.76 / Chapter 4.3 --- Results --- p.76 / Chapter 4.3.1. --- Genome Instability in Rat Leukocytes after Nitrous Oxide Administration --- p.76 / Chapter 4.3.2. --- Effect of Nitrous Oxide on the Transcription of DNA Damage Response Genes --- p.78 / Chapter 4.3.3. --- Effects of Nitrous Oxide on DNA Damage Response Genes in Animals Treated with Lipopolysaccharide --- p.80 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Principal Findings --- p.84 / Chapter 4.4.2 --- Implications --- p.84 / Chapter 4.4.3 --- Limitation of our Study --- p.85 / Chapter 4.4.4 --- Conclusions --- p.87 / Chapter Chapter 5 --- Preventive Analgesia with Nitrous Oxide: Role of NMDA Receptor Blockade / Chapter 5.1. --- Introduction --- p.88 / Chapter 5.1.1. --- The ENIGMA Trial: Long Term Follow-up --- p.89 / Chapter 5.1.2. --- Nitrous oxide prevents chronic postsurgical pain: putative mechanisms --- p.89 / Chapter 5.1.3. --- Hypothesis --- p.90 / Chapter 5.2. --- Materials and methods --- p.91 / Chapter 5.2.1. --- Animals --- p.91 / Chapter 5.2.2. --- Chronic constriction injury (CCI) to induce neuropathic pain --- p.91 / Chapter 5.2.3. --- Behavioral test --- p.93 / Chapter 5.2.4. --- Nitrous oxide administration --- p.93 / Chapter 5.2.5. --- Dizocilpine (MK-801) pretreatment --- p.94 / Chapter 5.2.6. --- Tissue Collection, Preparation and Western Blot --- p.94 / Chapter 5.2.7. --- Statistical Analysis --- p.96 / Chapter 5.3. --- Results --- p.96 / Chapter 5.3.1. --- Preventive Analgesia with Nitrous oxide --- p.96 / Chapter 5.3.2. --- Nitrous oxide analgesia via NMDA receptors block --- p.99 / Chapter 5.3.3. --- Preventive analgesia with NMDA receptors Blockade --- p.101 / Chapter 5.4. --- Discussion --- p.103 / Chapter 5.4.1 --- Principal Findings --- p.103 / Chapter 5.4.2 --- Our Findings compared with Other Studies --- p.103 / Chapter 5.4.3 --- Limitations of the Study --- p.104 / Chapter 5.4.4 --- Conclusions --- p.105 / Chapter Chapter 6 --- Transcriptional Changes for Nitrous Oxide Analgesia --- p.106 / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.1.1 --- Neuro-immune Interactions in the Development of Chronic Neuropathic Pain --- p.106 / Chapter 6.1.2 --- Nitrous Oxide Interferes Astrocytes and Glial Responses --- p.107 / Chapter 6.2 --- Materials and methods --- p.109 / Chapter 6.2.1 --- Chronic Constriction Injury Pain Model and Nitrous Oxide Administration --- p.109 / Chapter 6.2.2 --- Lumbar Dorsal Horn Tissue Collection, Preparation and Immunoflurescence --- p.109 / Chapter 6.2.3 --- RNA Extraction and Quantitative RT PCR for Gene Transcription Study --- p.110 / Chapter 6.2.4 --- Protein Extraction and Western Blot for protein Expression Study --- p.110 / Chapter 6.2.5 --- Statistical Analysis --- p.111 / Chapter 6.3 --- Results --- p.112 / Chapter 6.3.1 --- Time Course of LIG4 Gene Transcription after Constrictive Nerve Injury --- p.112 / Chapter 6.3.2 --- Nitrous oxide reduced DNA ligase IV expression in spinal dorsal horn --- p.114 / Chapter 6.3.3 --- Nitrous oxide Reduced Astrocytes Activation in Spinal Dorsal Horn --- p.116 / Chapter 6.4 --- Discussions --- p.119 / Chapter 6.4.1 --- Principal Findings --- p.119 / Chapter 6.4.2 --- Our Findings in Relation to Other Studies --- p.119 / Chapter 6.4.3 --- Limitations of Our Study --- p.120 / Chapter 6.4.4 --- Conclusions --- p.120 / Chapter Part III: --- Conclusions --- p.122 / Chapter Chapter 7 --- Conclusions and Future Perspectives --- p.123 / Chapter 7.1 --- Conclusions --- p.123 / Chapter 7.2 --- Future Perspectives --- p.124 / Chapter Part IV --- References --- p.126

La délivrance de médicaments lors de la mise en œuvre de thérapies inhalées est dépendante de nombreux facteurs. Parmi ceux-ci la morphologie des voies aériennes supérieures(VAS) et en particulier celle de la région glottique est déterminante dans les mécanismes de dépôt de particules par impaction inertielle. Dans le cadre de ce travail, il est examiné d’une part la dynamique glottique durant différentes modalités de respiration et d’autre part déterminé numériquement l’effet de ces mouvements et des conditions de respiration associées sur le dépôt des aérosols dans les VAS.Une étude clinique a été menée dans un premier temps sur un panel de 20 sujets sains (10 hommes et 10 femmes) au sein du service ORL de l’hôpital de la Timone à Marseille pour déterminer le mouvement glottique durant différentes tâches de ventilation allant d’une respiration normale avec moins de 20 cycles/min à une ventilation accélérée jusqu’à 90 cycles/min. L’acquisition des mouvements glottiques a été réalisée par imagerie numérique durant un examen de laryngoscopie avec mesure simultanée des débits associés aux différentes tâches de respiration.Les mesures expérimentales montrent que la géométrie glottique varie pendant la respiration en fonction de la tâche de respiration, du volume courant et de la fréquence respiratoire. Une étude statistique a permis d’isoler deux comportements types l’un où l’aire glottique demeure sensiblement constante durant la respiration et l’autre ou une variation de cette même aire est observée. Sur ce dernier groupe d’individus la variation d’aire maximale observée sur les hommes est de 26%, l’ouverture maximale étant atteinte durant la phase d’inspiration et l’aire minimale durant l’expiration.Ces résultats, ainsi que des données anatomiques, ont permis de construire un modèle géométrique idéalisé des VAS. Ce modèle reproduit fidèlement les principales singularités des voies extrathoraciques en apportant un grand soin à la description de la région glottique. Le transport et le dépôt d’aérosols dans ce modèle a été étudié en ayant recours à des simulations numériques3D de l’écoulement cyclique. Une étude paramétrique a permis d’évaluer l’influence sur le dépôt de l’écoulement cyclique, de la nature du gaz porteur (Air vs mélange d’hélium-oxygène), de la prise en compte de la dynamique glottique et de la taille des particules.Les résultats mettent principalement en évidence une nette diminution de la part extrathoracique du travail respiratoire lors de l’emploi du mélange He-O2 et un écoulement de type jet en aval de la glotte durant l’inspiration associé à une recirculation sous le plancher glottique. Le mécanisme de dépôt principal étant l’impaction inertielle (pour les tailles des particules 1 - 10μm)le zone principale de dépôt est situé dans l’oropahrynx, quelles que soient les conditions pendant inspiration. La fraction de dépôt augmente rapidement avec le diamètre des particules atteignant près de 80% pour les particules de 10μm et diminuer deux fois pour He-O2 en comparaison avec air.Finalement, la dynamique du dépôt ne varie pas de façon significative entre le modèle où la glotte est considérée comme statique et celle où elle est mobile. Donc, dans les conditions de respiration normal le mouvement de la glotte peut être négligé. / During inhaled therapies several factors limits the amount of drug delivered to the lungs. E.g. the upper airways morphology and in particular the glottis, defined by the vocal-fold aperture, causes upper airways to narrow in a minimal cross section, which is determinant on aerosol depo­ sition by inertial impaction. This thesis aims to (i) investigate evolution of the glottal area during breathing, and (ii) predict the effects of a dynamic glottis and realistic airflow conditions on the aerosol deposition in upper airways using three-dimensional simulations.First, a clinical study was conducted on 20 healthy volunteers (10 males and 10 females) to explore the glottal motion during several specifie slow (below 20 cycles/min) and rapid breathing tasks (up to 90 cycles/min). The breathing was investigated simultaneously for the glottal area variations using laryngoscopie video recordings and for airflow rate using oral flowmeter.The experimental measurements showed that the glottal geometry observed during a breathing cycle can be extremely variable depending on the respiratory phase, tidal volume, and breathing frequency. Testing the dynamic behaviour of the glottis during breathing, two groups of subjects were identified: one with relatively constant glottal area and other with significant variations. In average, the variations for the latter group of subjects was observed for males at 26% comparing maximale and minimal glottal opening during inspiration and expiration respectively.The results of the clinical study together with anatomical morphological data served to create a madel with idealised geometry of upper airways. This madel represents the major geometrical characteristics of upper airways with special interest in the glottal region. Transport and deposition of aerosols was studied using 3D numerical cyclic simulations and parametrical analysis allowed to evaluate the influence of the cyclic flow, glottal dynamics, type of carrier gas (air or helium-oxygen mixture) and particle size on the deposition of aerosols in the upper airways.The numerical simulations demonstrated significant decrease of respiration work with He-02 and jet-like flow with recirculation zone in the oro-pharynx and downstream the glottal plane. The principal deposition mechanism is inertial impaction (for the particle diameters 1 - lOiJ.m) with most significant deposition region in the oro-pharynx. Important parameters for deposition are the particle size and the nature of carrier gas. For He-02 the deposition reaches two times smaller values than for air and the fraction of deposited particles increases significantly with diameter, reaching 80% of deposited efficiency for 10 iJ.m particles. Finally, the CFD results demonstrated negligible differences in aerosol transport and deposition between different glottal characteristics. Therefore, in normal breathing conditions the glottal motion can be neglected