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An inhalational antiviral strategy for the potential use of nitric oxide during influenza infection McMullin, Bevin Brent 2012

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An Inhalational Antiviral Strategy for the Potential Use of Nitric Oxide during Influenza Infection by  Bevin Brent McMullin RT, Cariboo College, 1992 MAppSc, Charles Sturt University, 2004  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in  The Faculty of Graduate Studies (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (VANCOUVER)  September 2012  ©Bevin Brent McMullin, 2012  Abstract  Since the discovery in the late 1980’s that the endothelium relaxing factor is nitric oxide (NO) there has been intensive scientific pursuit to understand the many roles of NO in biological systems. NO is a messenger molecule with both paracrine and autocrine functions. NO is produced by phagocytes as part of the immune system as a non-specific antimicrobial which may be effective against Influenza. Influenza is a virus that infects millions of people each year resulting in hundreds of thousands of deaths. While vaccination and antivirals have helped reduce the death toll, their effectiveness is limited because of the rapidly evolving nature of the influenza virus and the development of resistance. The recent 2009 influenza pandemic has highlighted the need for new and novel antivirals. We hypothesize that the direct exposure of influenza viruses to gaseous nitric oxide (gNO) will have an antiviral effect. We also show that it is feasible and safe to deliver inhaled gNO to humans at antiviral concentrations using an intermittent high dose regimen.  ii  Preface In Chapter 1, an independent search of the literature was done in collaboration with Tom Oliver, manager of Clinical Epidemiology and Biostatistics, McMaster University. I then reviewed and synthesized the papers for inclusion in Chapter 1 and throughout the remaining chapters. A version of Chapter 2 has been submitted for publication. I was responsible for the in-vitro testing and proof of concept. Gilly Regev-Shoshani was responsible for the pH and nitrite portion of the study. Gilly Regev-Shoshani, Chris Miller and I were responsible for manuscript preparation. A version of Chapter 4 has been published as: Gaseous Nitric Oxide Bactericidal Activity Retained During Intermittent High-Dose Short Duration Exposure, Miller C, McMullin B, Ghaffari A, Stenzler A, Pick N, Road J, Av-Gay Y, Nitric Oxide 20 (2009) 16-23. Copyrighted material used by permission from Elsevier. The initial concept was developed and patented by Chris Miller, Alex Stenzler and myself. I was responsible for all NO exposure and testing of bacteria, virus and cell lines. Evaluation of cells was done by Abdi Ghaffari and Neora Pick. Manuscript preparation was done by Chris Miller and myself. Funding for the project was provided by Pulmonox Medical Inc. and Cardinal Health Care. A version of Chapter 5 has been published as: A Phase I Clinical Study of Inhaled Nitric Oxide in Healthy Adults, Chris Miller, Minna Miller, Bevin McMullin, Gilly Regev, Lena Serghides, Kevin Kain, Jeremy Road, Yossef Av-Gay. Journal of Cystic Fibrosis, available online 18 April 2012. The study had UBC CREB Approval #H09-00872 and Health Canada approval TPD CTA #129958. Under the supervision of Chris Miller, I took part in the study design, review and implementation, including space allocation, patient recruitment, evaluation, set-up, monitoring of iii  treatments and lung function testing. I was responsible for data collection, collation and review. Gilly Regev-Shoshani and Lena Serghides performed nitrite, cytokine/chemokines and angiopoietin assays and provided interpretation of results. Chris Miller and I were responsible for manuscript preparation.  iv  Table of Contents Abstract ....................................................................................................................................... ii Preface........................................................................................................................................ iii Table of Contents ........................................................................................................................ v List of Tables ............................................................................................................................. ix List of Figures ............................................................................................................................. x Acknowledgements .................................................................................................................... xi Dedication ................................................................................................................................ xiii Chapter I: Introduction to Nitric Oxide and Influenza............................................................... 1 1.1 Nitric Oxide .......................................................................................................................... 1 1.1.1 Nitric Oxide Biochemistry ............................................................................................. 1 1.1.2 Nitric Oxide in the Immune System .............................................................................. 3 1.1.3 Nitric Oxide Delivery .................................................................................................... 4 1.1.4 Gaseous Nitric Oxide as an Antibacterial ...................................................................... 5 1.1.5 Inhalation of Nitric Oxide .............................................................................................. 6 1.2 Nitric Oxide as an Antiviral .................................................................................................. 7 1.2.1 Literature Search ............................................................................................................ 8 1.2.2 Baltimore Classification............................................................................................... 11 1.2.3 Viruses Susceptible to the NO Donor, SNAP .............................................................. 14 1.3 Influenza ............................................................................................................................. 15 1.3.1 Nitric Oxide and Influenza........................................................................................... 16 1.4 Hypothesis........................................................................................................................... 18 Chapter 2: Gaseous Nitric Oxide and Influenza: An in-vitro model ........................................ 20 2.1 Introduction ......................................................................................................................... 20 2.2 Materials and Methods ........................................................................................................ 20 2.2.1 Viruses and Cell Lines ................................................................................................. 20 2.2.2 Gaseous Nitric Oxide Delivery .................................................................................... 21 2.2.3 Post Infection Effect of Gaseous Nitric Oxide ............................................................ 22 2.2.4 Virucidal (Cell-free) Effect of Gaseous Nitric Oxide .................................................. 22 2.2.5 Nitrites and pH Content ............................................................................................... 23 v  2.2.6 Effect of Nitrites, pH and Combination on H1N1 Virion (Cell-free) .......................... 23 2.2.7 Nitric Oxide Effect on Dried H1N1 ............................................................................. 23 2.2.8 Statistical Analysis ....................................................................................................... 24 2.3 Results ................................................................................................................................. 24 2.3.1 Post Infection Effect of Gaseous Nitric Oxide in Cells ............................................... 24 2.3.2 Virucidal Effect of Gaseous Nitric Oxide on Cell-free Virions .................................. 27 2.3.3 Nitrite Concentrations, pH and Their Effect on Cell-free H1N1 Virions .................... 30 2.4 Summary ............................................................................................................................. 32 Chapter 3: A Mechanism by which Nitric Oxide Inhibits the Infectivity of Influenza ............ 34 3.1 Introduction ......................................................................................................................... 34 3.2 Materials and Methods ........................................................................................................ 34 3.2.1 Influenza Viruses ......................................................................................................... 34 3.2.2 Gaseous Nitric Oxide Delivery .................................................................................... 35 3.2.3 Hemagglutination Inhibition Assay ............................................................................. 36 3.2.4 Nitrosylation Assay...................................................................................................... 36 3.2.5 Nitric Oxide Effect on Dried H1N1 ............................................................................. 37 3.2.6 Hemagglutinin.............................................................................................................. 37 3.3 Results ................................................................................................................................. 37 3.3.1 Hemagglutination Inhibition Assay ............................................................................. 37 3.3.2 Nitrosylation Assay...................................................................................................... 39 3.4 Summary ............................................................................................................................. 41 Chapter 4: The Safe Delivery of Nitric Oxide: An Intermittent Dosing Strategy .................... 42 4.1 Background ......................................................................................................................... 42 4.2 Materials and Methods ........................................................................................................ 43 4.2.1 In-vitro Nitric Oxide Exposure Device ........................................................................ 43 4.2.2 In-vitro Nitric Oxide Delivery Regimen ...................................................................... 44 4.2.3 Lung Epithelial Cell Culture ........................................................................................ 45 4.2.4 Lung Epithelial Cell Proliferation Assay ..................................................................... 46 4.2.5 Lung Epithelial Cell Migration Assay ......................................................................... 47 4.2.6 Clonogenic Cell Survival Assay .................................................................................. 47 4.2.7 Statistical Analysis ....................................................................................................... 48 vi  4.3 Results ................................................................................................................................. 48 4.3.1 Antimicrobial Effect of Intermittent gNO In-vitro ...................................................... 48 4.3.2 Proliferation, Migration and Mutagenesis of Pulmonary Cells in the Presence of ...... 49 Exogenous gNO .................................................................................................................... 49 4.4 Summary ............................................................................................................................. 51 Chapter 5: A Phase I Safety Trial of Nitric Oxide in Healthy Adult Humans ......................... 52 5.1 Background ......................................................................................................................... 52 5.2 Methods and Materials ........................................................................................................ 53 5.2.1 Intermittent High Dose Inhaled Nitric Oxide Delivery ............................................... 53 5.2.2 Measurement of Changes in Pulmonary Function ....................................................... 54 5.2.3 Oxygenation and Vital Sign Measurements ................................................................ 54 5.2.4 Blood Chemistry and Hematology Measurements ...................................................... 55 5.2.5 Analysis of Nitric Oxide Metabolism .......................................................................... 55 5.2.6 Systemic Inflammation and Endothelial Activation Assays ........................................ 56 5.3 Statistical Analysis .............................................................................................................. 56 5.4 Results ................................................................................................................................. 57 5.4.1 Safe Delivery of 160 ppm Nitric Oxide Gas: Inhaled Nitric Oxide, Nitrogen Dioxide and Oxygen Measurements. .................................................................................................. 57 5.4.2 Vital Signs and Clinical Safety During and After gNO Delivery: Blood Pressure, Oxygenation, Methemoglobin and Nitrite/Nitrate Levels. ................................................... 58 5.4.3 Normal Lung Function and Anti-Inflammatory Response During and After gNO Delivery................................................................................................................................. 61 5.4.4 Inflammatory Cytokines and Endothelial Activation Factors ..................................... 61 5.5 Summary ............................................................................................................................. 62 Chapter 6: Discussion, Summary, Future Directions and Conclusion...................................... 63 6.1 Discussion ........................................................................................................................... 63 6.1.1 gNO Can Act as an Antiviral Against Influenza ......................................................... 63 6.1.2 gNO Acts as an Antiviral Through Nitrosylation ........................................................ 65 6.1.3 Intermittent Delivery of 160 ppm gNO can be a Viable Treatment Regime ............... 68 6.1.4 The Safe Treatment of Normal Healthy Adult Humans .............................................. 74 6.2 Summary ............................................................................................................................. 79 6.3 Future Directions ................................................................................................................ 79 vii  6.4 Conclusion .......................................................................................................................... 83 Works cited ............................................................................................................................... 84 Appendices ................................................................................................................................ 95 Appendix 1: Supporting Graphs for Chapter 4 ......................................................................... 95 Appendix 2: The Safety Trial ................................................................................................. 102  viii  List of Tables Table 1.1 Literature Search Strategies …………………………………………….…….…........10 Table 1.2 Viruses Susceptible to the NO Donor SNAP……….……...………………………….14 Table 2.1 Amount of Nitrites Found in Treated and Control Wells After Exposing to NO or Air……………………………………..…………………………………………………..….….31 Table A2.1 Synopsis of the Safety Trial Protocol……………….………….……..………...…102  ix  List of Figures Figure 2.1 Influenza H1N1 Infected MDCK Cells Exposed to 160 ppm gNO……….…………25 Figure 2.2 Influenza H3N2 Infected MDCK Cells Exposed to 160 ppm gNO………….………26 Figure 2.3 Influenza B infected MDCK Cells Exposed to 160 ppm gNO…………..……….….27 Figure 2.4 H1N1 Virions Exposed to 80 ppm or 160 ppm gNO……………………….…....…..28 Figure 2.5 H3N2 Virions Exposed to 80 ppm or 160 ppm gNO…………………….…….….....29 Figure 2.6 Influenza B Virions Exposed to 80 ppm or 160 ppm gNO….…………….……...….30 Figure 2.7 Virucidal Effect of Nitrites and Low pH on H1N1………………………….…...…..32 Figure 3.1 Hemagglutination Inhibition Assay……………………………..…….……..……….39 Figure 3.2 The Nitrosylation of Influenza Hemagglutinin……………………………….……...41 Figure 4.1 The Effect of Intermittent 160ppm gNO on Influenza…………………………....….49 Figure 5.1 Summary of all Treatments of Methemoglobin Kinetics, Over One, 4 hour Cycle………………………………………………………...……………….…………….…….59 Figure 5.2 Cumulative Summary of Methemoglobin Kinetics………………………………..…60 Figure A1.1 Comparison of Bacterial Survival Curves between Intermittent Short Duration and Continuous Exposure to gNO……………………………………………………………..….….95 Figure A1.2 Effect of gNO on Proliferation of Epithelial Cells…………………………..……..98 Figure A1.3 Effect of 160ppm gNO on A549 Cells Migration………………………....…….....99 Figure A1.4 Lung Epithelial Cell Clonogenic Survival Assay…………………………………100 Figure A1.5 Cytotoxic Effects of Nitric Oxide…………………………………………………101  x  Acknowledgements  I wish to gratefully acknowledge the members of my supervisory committee. I thank Dr. Jeremy Road for his encouraging me to pursue graduate studies and for his tireless support and dedicated advocacy and supervision. I am eternally grateful to Dr. Chris Miller who has mentored me for many years and who has allowed me to be a part of the journey that is nitric oxide. I thank Dr. Martin Petric for patiently opening the world of virology for me to explore. Lastly, I am indebted to Dr. Yossef Av-Gay who opened his lab to me and has always welcomed my pursuit of understanding and patiently shepherded me along my path. Thank you. I thank Dr. Jim Hudson for opening up his lab to me when I was first embarking into the world of virology and for Dr. Selvarani Vimalanathan for teaching me the technical aspects of virology and her enduring friendship. I wish to thank Xingji Zheng and Dennis Wong for their friendship, support and willingness to help whenever I was lost. I thank Valerie Poirier for her support. I thank Adam Crowe for his assistance with the SNOB experiments. I wish to thank Joe Miller and Gal Av-Gay for technical assistance. I am grateful, and thank Mary Ko for her efficiency in running the lab and her unconditional support in the Av-Gay lab. I thank Minna Miller and Dr. Gilly Regev-Shoshani for all of their guidance, support and encouragement.  xi  I thank Dr. Abdi Ghaffari for his willingness to help support all of our nitric oxide questions and collaborations. I wish to thank Experimental Medicine for all their support. I thank Dr. Norman Wong who was the first to encourage me to pursue advanced education. I thank Dr. Vincent Duronio for his tireless support. I thank Patrick Carew and Cornelia Reichelsdorfer for helping me successfully navigate graduate school. I am grateful to Bruce Murray and Alex Stenzler who have, from afar, always supported and encouraged my endeavours. I am grateful to Dr. Barb Conway for guidance in manuscript preparation. Research in the Yossef Av-Gay laboratory was funded by Pulmonox Medical Inc. We thank the Lotte & John Hecht Memorial Foundation for their support of Drs. Av-Gay and Miller. A portion of the viral research was funded by a grant from United States Department of Defence, Defence Advanced Research Projects Agency (DARPA). I received a fellowship from the Canadian Lung Association. And finally, I wish to acknowledge my parents Brent and Barbara who made all this possible and who have supported me for all these many years. I am grateful for the love and support of my children, Hayden, Keenan, Montana, Malachi, Takoda and Talon. Most of all I wish to thank Dolly, my best friend, my eternal companion and my wife whose love and patience have kept me going and who will always be my partner.  xii  Dedication  To my loving and supportive family  xiii  Chapter I: Introduction to Nitric Oxide and Influenza 1.1 Nitric Oxide Nitric oxide (NO) is a small molecule produced by the innate immune response in organs and cells exposed to bacterial and viral infections. These include the nasopharyngeal airway, lungs, circulating neutrophils and macrophages. It has been suggested that prolonged exposure to NO is microbicidal and its antimicrobial characteristics are in addition to its role as a key biological messenger. NO is a highly reactive free radical synthesized from L-arginine by a family of enzymes called nitric oxide synthases (NOS). NO is a broad non-specific antimicrobial, effective against a wide range of bacteria, parasites, fungi and viruses. 1.1.1 Nitric Oxide Biochemistry  As outlined by Stamler, NO is a short-lived chemical transmitter that diffuses freely across membranes. The molecule possesses a small dipole moment because of the similar electronegativities of oxygen and nitrogen, making it essentially hydrophobic. Its reactivity is due to the unpaired electron in the outer valence orbital of its oxygen constituent. NO is almost unreactive as a free radical compared to other oxygen radicals. Indeed, NO decays within seconds after its synthesis, if left unbound in solution, because it reacts with either molecular oxygen or superoxide (Stamler 1992). NO has been implicated, in-vivo, in a number of diverse physiological processes. The biochemical pathways in these processes share two common features: the enzymatic synthesis of NO from L-arginine and the formation of an iron-nitrosyl complex in a target (heme) protein to evoke the functional response. The broader chemistry of NO, however, involves an array of  interrelated redox forms: nitrosonium cation (NO+), nitric oxide (NO•), and nitroxyl anion (NO-) (Stamler 1992, Fang 2004). NO interacts strongly with molecular oxygen to form dinitrotrioxide (N2O3), or with superoxide (O2-) to form peroxynitrite (ONOO-). The reaction with superoxide can be diminished by superoxide dismutase (SOD), which removes O2- from ONOO to form hydrogen peroxide (H2O2). NO also binds to sulfhydryl groups (SH) and unsaturated fatty acids. NO can be “stored” by covalent interaction with glutathione to form S-nitrosoglutathion. Superoxide dismutase thereby prevents the loss of NO to peroxynitrite, instead forming hydrogen peroxide and increasing the cyclase stimulatory capacity of the cell (Stamler 1992). NO can potentially be regenerated, endogenously, from ONOO- in two steps: first, cytochrome C oxidase reduces peroxynitrite to nitrite (NO2-), followed by the enzyme nitrate reductase reducing nitrite to NO (Degroote 1999). Nitrate reductase exists in two isoforms: a mitochondrial type and an endoplasmic reticulum resident protein. Both receive the electrons needed for nitrite reduction to NO from either NADH or NADPH, and interact with flavoproteins (FAD prosthetic groups) and cytochromes. NO is produced commercially by oxidating ammonia at 750°C to 900°C using platinum as a catalyst. In the lab, NO is generated by reducing nitric acid with copper or by reducing nitrous acid with sodium nitrite or potassium nitrite. For the studies that follow we used commercially prepared NO. Gaseous NO concentration can be determined using a simple chemiluminescent reaction where NO is combined with ozone to produce NO2, which is detected by gas chromatography-mass spectrometry  (GC-MS),  or  by  Fourier  transform  infrared  spectroscopy  (FTIR).  Chemiluminescence is the most commonly used method of determining NO concentration in the  2  laboratory setting. Clinically, electrochemical analysis is the most common method of analyzing both NO and nitrogen dioxide (NO2). 1.1.2 Nitric Oxide in the Immune System  While this dissertation does not deal specifically with the role of the NOS pathway within the host, it is nonetheless important to understand how the mammalian host protects itself by increasing the expression of the enzymatic pathways of NOS to produce NO. NO provides a broad spectrum, front line antimicrobial. The NOS systems reviewed and summarized by Bogdan are: First, in addition to macrophages, a large number of other immune-system cells produce and respond to NO. Second, and contrary to previous views, all known isoforms of NO synthase— neuronal NOS (nNOS, or NOS1), iNOS and endothelial NOS (eNOS, or NOS3)—operate in the immune system. (The nNOS and eNOS isoforms are also known collectively as constitutive... Third, the activity of NO is not restricted to the site of its production. As an uncharged gas, NO• radicals are highly diffusible. Low-molecular weight S-nitrosothiols (such as Snitrosoglutathione), S-nitrosylated proteins, and nitrosyl-metal complexes can function as longdistance NO vehicles, which liberate NO either spontaneously or after cleavage by ectoenzymes found on cells such as T and B lymphocytes. Fourth, in contrast to cytokines, the interaction of NO is not restricted to a single defined receptor; rather, it can react with other inorganic molecules (such as oxygen, superoxide or transition metals), structures in DNA (pyrimidine bases), prosthetic groups (such as heme) or proteins(leading to S-nitrosylation of thiol groups, nitration of tyrosine residues or disruption of metal–sulphide clusters such as zinc-finger domains or iron–sulphide complexes). Considering that many of the targets of NO are themselves regulatory molecules (for example, transcription factors and components of various signalling cascades), it is evident that NO frequently exerts heterogeneous and diverse phenotypic effects. It is now clear that iNOS is detrimental in some infectious disease processes and that it helps to counteract excessive immune reactions, protects to some degree against autoimmunity and functions as an intra- and intercellular signalling molecule shaping the immune response. In addition, nNOS and eNOS are now known to participate in important immunological processes such as apoptosis, cell adhesion, autoimmunity and perhaps antimicrobial defence. The demonstration of iNOS expression by macrophages and other cell types in tissues from patients with a wide variety of infectious, autoimmune and degenerative diseases has disproved the claim that iNOS does not occur in the human immune system. Because the regulation, expression and function of the NOS isoforms are so complex, NO-based therapies against infectious, autoimmune or malignant diseases are not easy to design. This should not, however, 3  discourage immunologists from future research on NO, especially considering that they have been confronted with similar problems in the field of cytokines for years (Bogdan 2001). As Bogdan outlines, the regulation of NOS can be difficult to control and therapies can be difficult to design. For this reason, we have chosen to use gaseous NO from an exogenous source that has been commercially prepared to a known concentration (fully described in Chapter 2). 1.1.3 Nitric Oxide Delivery  The experimental methodologies used to study NO were identified in a search of the literature summarized by Reiss (1998). The methodologies were organized into three main categories: 1. Manipulation of the iNOS pathways. 2. NO-derived from a donor molecule. 3. Genetically modified animals (i.e., knock-out mice). We propose a fourth methodology, direct delivery of gaseous NO (gNO). We evaluated all four delivery methodologies for their potential for translation to a therapeutic intervention. The first method of manipulating the enzymatic pathway to target a specific site of infection is difficult to achieve. NO is ubiquitous throughout the body and plays a wide range of functions depending on the location and concentration of the NO generated (Fang 2004). Additionally NO is a significant part of the inflammatory cascade as an effector and messenger molecule (Bogdan 2001). Therefore, the precise delivery of NO generated through enzymatic manipulation has the potential for unwanted consequences because it is difficult to control and it is difficult to titrate for a therapeutic application.  4  The second method of using NO donors such as sodium nitroprusside (SNP), S-nitroso-Lacetylpenicillamine (SNAP), or 3-morpholino-sydononimine (SIN-1) allows for NO to be delivered to the site of an infection. However, the precise control and release of the NO donated to the site of an infection can be difficult to achieve, especially since the release of NO by these donors often requires a highly acidic environment for NO release. Also, few donors are capable of sustaining the kind of controlled NO release required to be effective. The third method, knocking out iNOS genes in transgenic mice is not practical as a treatment in humans because our understanding of the human immune system is incomplete and there is no practical way to alter human genes (as described in the above section on NO and the immune system). We propose gNO as an alternate method of assessing antimicrobial properties with the potential to be readily translated into a therapy. Therapeutic gNO is applicable to areas where NO can be delivered topically, for example, the skin, or to the upper and lower respiratory tract where gNO can be inhaled (Ghaffari 2006). While the topical delivery of gNO is readily achievable, the delivery of gNO to the respiratory tract can cause some unwanted side effects by reacting with oxygen to form radicals (e.g., NO2), and crossing into blood and binding with hemoglobin forming methemoglobin. 1.1.4 Gaseous Nitric Oxide as an Antibacterial  Recently, our group and others have reported that continuous in-vitro exposure to exogenous gNO is efficacious as a non-specific antimicrobial agent against a broad range of microorganisms including Gram positive, Gram negative and multi-drug resistant strains of bacteria, yeast, and mycobacteria (McMullin 2005, Stenzler 2004, Ghaffari 2005). Long et al  5  have shown clinically that gNO doses lower than 80 parts per million (ppm) are not bactericidal (Long 2005), while we have shown that 160 ppm is an effective bactericidal concentration of gNO (Miller 2009, McMullin 2005). These studies demonstrated that, for bacteria, 160 ppm was the minimum inhibitory concentration (MIC) during five hours of continuous exposure. Additionally, exogenous NO may potentially reduce inflammatory sequelae associated with the high levels of endogenous NO that are part of the inflammatory cascade (Ghaffari 2006, Rezakhanloua 2011). We have also shown that multiple eight-hour daily exposures to 160–200 ppm gNO is neither toxic nor mutagenic to human cell lines used to model dermal wounds (Ghaffari 2006). These observations led to the successful treatment of a critically colonized, nonhealing, lower leg ulcer in a human subject using gNO (Miller 2004). We hypothesize that gNO may also be effective as an inhaled antimicrobial treatment against pulmonary pathogens. The main consideration when using inhaled gNO is the formation of methemoglobin, because NO has a high affinity for hemoglobin. The formation of methemoglobin reduces the oxygen carrying capacity of hemoglobin. Another concern is that NO may be harmful to cells in its capacity as a nitrosative agent. 1.1.5 Inhalation of Nitric Oxide  NO gas is classified as a drug and is currently administered by inhalation as a selective pulmonary vasodilator to infants on mechanical ventilation. NO is approved for use in neonates with hypoxic respiratory failure associated with pulmonary hypertension (FDA 1999). The most commonly used dose is 20 ppm and a dosing range of 1- 80 ppm is approved for treating pulmonary hypertension. Consequently, significant knowledge regarding the safe delivery of gNO as a selective, short acting pulmonary vasodilator has been established over the past decade  6  (FDA Guidance 2000). Due to potential complications, particularly in the lungs, the use of gNO as a microbicidal agent has raised safety concerns. (Hurford 2005). Some preliminary data on NO safety is available from animal studies. Hergott reported the effects of intermittent delivery of 160 ppm inhaled NO for thirty minutes every 3.5 hours for a 24 hour period in a rat model of Pseudomonas aeruginosa pneumonia. This therapeutic regimen successfully reduced pulmonary bioburden and leukocyte infiltration without a significant change in myeloperoxidase, plasma nitrates and methemoglobin (Hergott 2006). In a second animal trial, 160 ppm gNO was administered to cattle for approximately 30 minutes to treat Bovine Respiratory Disease Complex (BRDc). BRDc is a condition associated with five pathogenic viruses often seen when cattle are transported and housed in close proximity to each other. The treatment was well tolerated physiologically and metabolically by the cattle (Schaefer 2006). Additionally, these results showed that gNO given prophylactically or upon initial detection of infection significantly decreased the clinical symptoms of the disease and significantly lowered the incidence of recurring BRDc or a secondary bacterial pneumonia when compared to the control cattle. To realize our goal of a human therapy, the safe delivery of gNO will need to be assessed.  1.2 Nitric Oxide as an Antiviral Viruses can be grouped into seven families based on the Baltimore classification system, which groups viruses based on their method of replication and type of genomic structure (Baltimore 1971). Viruses from all seven classes have the ability to infect humans and cause disease. Over the last century, some common viral infections such as measles, mumps, rubella and polio have been controlled with the use of vaccines. Some infections such as influenza are partially  7  controlled through seasonal vaccination but the threat of a pandemic persists because the influenza virus evolves rapidly (Palese 2007). Viruses that cause the common cold, HIV, hepatitis and many others that cause diseases have no vaccine, and once infection occurs treatment can be difficult. 1.2.1 Literature Search  Methodology - A systematic review of the literature was conducted following standard systematic review methodological approaches (Higgins 2009). Given the anticipated lack of literature on the topic, the systematic review was intended to act as an evidentiary foundation on which to base novel exploratory analyses of the role of NO in influenza viral infectivity. A consultant with methodological expertise was recruited to assist in the design and implementation of the literature search. Articles were selected for inclusion by one reviewer with content expertise in viral infectivity. Strategy - The literature search strategy was designed to address questions related to the role of NO as an antiviral, specifically as an antiviral for human influenza virus. The literature was searched using MEDLINE (OVID: 1996 through August 2009), EMBASE OVID: (1996 through August 2009), the Cochrane Library (OVID; Issue 3, 2009), the Canadian Medical Association InfoBase, the National Guideline Clearinghouse, and the NHS evidence database. Reference lists of related papers and recent review articles were also scanned for additional citations. The literature search of the electronic databases combined medical subject headings (MeSH), heading terms and text search terms to identify the body of published evidence on NO as an antiviral agent (Table 1.1). 8  Studies were to report data on outcomes informing: 1) whether NO was a more effective antiviral therapy compared to standard antiviral therapy with M2 channel blockers or neuraminidase inhibitors; 2) whether NO was effective in inhibiting human influenza virus replication and infectivity; and 3) whether NO delivered in supra-physiologic concentrations was more effective than using NO donors or manipulating the enzymatic pathways to either increase or block NO production and, if so, the mechanism of action. Outcomes of interest included the inhibition of the replication of viral RNA synthesis, inhibition of infectivity, and viral inactivation.  9  Table 1.1 Literature Search Strategies Search Terms 1  nitric oxide/ or nitric oxide.ti. or Nitric Oxide Donors/ or S-nitroso-n-acetylpenicillamine/ or S-nitroso-n-acetylpenicillamine.mp. or s nitroso n acetylpenicillamine.mp.  2  AMANTADINE/ or amantadine.mp. or RIMANTADINE/ or rimantadine.mp. or neuraminidase inhibitor$.mp.  3  influenza, human/ or (influenza and human).ti. or human influenza.ti. or influenza.ti. or flu.ti. or influenza a virus/ or influenza a virus.mp. or influenza b virus/ or influenza b virus.mp. or h1n1.mp. Medline  Embase  1  (56288)  (69811)  2  (2035)  (6238)  3  (38350)  (19722)  4  2 or 3 (18868)  2 or 3 (24251)  5  1 and 4 (30)  1 and 4 (83)  6  from 5 keep 1-30 (30)  from 5 keep 1-83 (83)  CMA Infobase  NGC  Human Influenza (19)  1. Keyword: human influenza  1  2. Treatment/Intervention: antiviral (26)  1  NHS Evidence Database  Cochrane  “Human Influenza” (13)  1. Influenza (29)  Study Selection Criteria - Articles were selected for inclusion in the systematic review of the evidence if they reported data on the role of NO as an antiviral. Given the nature of the topic, it was recognized that the strength and volume of the evidence from the published literature would likely be modest. For that reason, no study limitations were placed on the search of the literature. 10  In descending order of preference, the minimum levels of evidence needed to inform the clinical questions were considered to be clinical practice guidelines, systematic reviews, meta-analyses, randomized controlled trials, non-randomized comparative studies, prospective single-cohort studies, retrospective single-cohort studies, and case studies. In the event that sufficient published data were not available, unpublished data collected through direct contact with experts in the field was considered eligible for review. Articles were excluded from the systematic review of the evidence if they were reported in a language other than English. Synthesizing the Evidence - Given the paucity of evidence, data was not pooled from literature identified by the search. Each paper was evaluated individually for the antiviral potential of NO, and for which of the three experimental methodologies, described above, generated the antiviral result. 1.2.2 Baltimore Classification  A detailed review of the literature revealed that under experimental conditions, NO has antiviral potential against at least one virus within each of the Baltimore Classification System categories. Below is a brief summary of the current literature for each class as it relates to the method of NO delivery and the antiviral potential. Group I viruses are double-stranded DNA and are usually associated with replicating cells since they require the cell’s polymerase for replication. Interferon gamma stimulation in-vitro has led to the production of NO which has been shown to inhibit the growth of Herpes Simplex Virus (HSV-1) (Karupiah 1993, Komatsu 1996). In an in-vivo model, inhibition of NOS2 has been shown to improve HSV-1-induced pneumonia in mice (Adler 1997). Epstein-Barr Virus 11  (Mannick 1994), Vaccinia Virus (Karupiah 1993, Harris 1995, Melkova 1995), Ectromelia Virus (Karupiah 1993) and Marek’s Disease Virus (Djeraba 2000) have been shown to be inhibited by enzymatic manipulation of NOS in-vitro. Adenovirus has been inhibited in-vitro with the use of a NO donor (Cao 2003). In vivo enzymatic inhibition of NO led to an increase in Ectromelia Virus in mice (Karupiah 1993). Group II viruses are single-stranded DNA viruses. Porcine Parvovirus has been inhibited invitro by both NO donors and enzymatic manipulation of NOS (Wei 2009). Group III viruses are double-stranded RNA viruses. NO donors inhibited both Reovirus (Reoviridae) (Pertile 1996) and Infectious Bursal Disease Virus (Birnaviridae) (Poonia 2005). These groups of viruses represent two of the major families making up class III. Group IV viruses are positive sense, single-stranded RNA viruses. Many viruses in this class have been found to be susceptible to NO. In-vitro studies with NO donors have shown effects on Poliovirus (Lopez Guerrero 1998, Komatsu 1996), Coxsackie Virus (Zaragoza 1997, Zell 2004), Rhinovirus (Sanders 1998), Mouse Hepatitis Virus (Lane 1997), Sindbis Virus (Tucker 1997), SARS (Coronavirus) (Kayaerts 2004, Akerstrom 2005 and reviewed by Cinatl 2005,), and Dengue (Charnsilpa 2005, Takhampunya 2006). In-vivo results that showed inhibition by NO include: Theiler’s Murine Encephalomyelitis Virus (Oleszak 1997), Coxsackie (Lowenstein 1996), Japanese Encephalitis Virus (JEV) (Lin 1997, Saxena 2000), Mouse Hepatitis Virus (MHV) (Lin 1997) and Sindbis Virus (Tucker 1997).  12  Group V viruses are negative sense, single-stranded RNA viruses. Many viruses in this class have been found to be inhibited by NO. In-vitro studies with NO donors have demonstrated effects on Influenza (Rimmelzwaan 1999), Bunyaviridae (Klingstrom 2005, Simon 2006) and Rhabdovirus (Bi 1995). In-vitro antiviral effects with enzymatic increase of NOS were noted in Influenza (Imanishi 2004, Komatsu 1996), Respiratory Syncytial Virus (RSV) (Hobson 2008), and Rhabdovirus (Komatsu 1996). In-vivo results show NO inhibits of RSV (Stark 2005) and Rhabdovirus (Bi 1995). Group VI viruses are a family of viruses that use reverse transcriptase to convert positive sense ssRNA into DNA. In-vitro and in-vivo models of NO delivery by both donors and NOS increased inhibition of Friend Murine Leukemia Virus (Akarid 1993). Human Immunodeficiency Virus (HIV) gp120 has been shown in-vitro to be inhibited by NO ( Koka 1995, Kong 1996). The pros and cons of NO use in HIV infection were reviewed by Torre (2002), and helped to illustrate the potential challenges of using NO against viruses. Group VII viruses are double-stranded DNA viruses that use reverse transcriptase for replication. NO inhibited Hepatitis B Virus replication in the livers of transgenic mice (Guidotti 2000). As we see from the above list, NO affects a wide range of viruses, covering all the classifications. Within the viral life cycle, there are multiple targets for NO to bind to and, thereby, disrupt the viral life cycle.  13  1.2.3 Viruses Susceptible to the NO Donor, SNAP  Current testing methodologies summarized by Reiss state: In-vitro, for most (but not all) viruses studied, prior activation of the cell to have enzyme activity of NO before infection is associated with inhibition of viral replication. This has been accomplished by providing NO donors, by co-culture with activated macrophages as a source of diffusing NO, by directly activating NOS in cells with cytokines or through other cell surface receptors (Reiss1998). The use of donors, in particular SNAP, closely approximates the use of gNO in experimental testing. As outlined above NO has an antiviral effect. The NO donor SNAP has antiviral effects on a variety of viruses in in-vitro testing models (Table 1.2). NO donor molecules usually require a reduction in pH in order to release NO. The duration and concentration of the NO release can be difficult to control and quantify. Our methodology of using gNO eliminates the pitfalls of donor molecules by using a precise concentration of NO for a precise time. Table 1.2 Viruses Susceptible to the NO Donor SNAP Baltimore Classification I I II IV IV IV V V  Family Name  Virus  Marek’s virus Adenoviridae Adenovirus Parvoviridae Parvovirus Picornaviridae Coxsackie Coronaviridae SARS Flaviviridae Dengue Bunyaviridae Hantavirus Orthomyxoviridae Influenza Herpesviridae  Naked enveloped Disease enveloped naked naked naked enveloped enveloped enveloped enveloped  or Genome Architecture dsDNA dsDNA ssDNA (+) ssRNA (+) ssRNA (+) ssRNA (-) ssRNA (-) ssRNA  14  1.3 Influenza For centuries, influenza has affected human health both seasonally and through recurring pandemics. Significant disease reduction has been achieved through vaccination efforts. However, circulation of seasonal Influenza A and B viruses continue to cause morbidity and mortality, particularly in patients with pre-existing pulmonary conditions. Seasonal influenza is responsible for over 36,000 deaths and 200,000 hospitalizations at a cost of $10 billion each year in the United States (Thompson 2003, Tosh 2010). Two subtypes of Influenza A virus, H3N2 and H1N1, circulate within the human population. In contrast to Influenza A, which has a broad host range, Influenza B has a limited host range of only humans and seals, and it mutates at a much slower rate than Influenza A. Nevertheless, Influenza B viruses cause significant disease and are the predominant circulating strain of influenza virus in approximately one in every three cases (CDC Influenza update May22-September 3, 2011). A pandemic can occur when human viruses acquire mutations directly or by re-assortment with other mammalian viruses that adapt them for transmission and replication in human hosts. In 2009, the world experienced a global, Phase 6 pandemic caused by a novel swine origin Influenza A/California/04/2009(H1N1) virus. During previous pandemics, influenza viruses needed more than six months to spread, allowing sufficient time to develop new vaccines. The new H1N1 virus spread worldwide in less than six weeks (WHO 2009). Had the virulence been higher, the mortality prior to release of the vaccine would have been catastrophic. This rapid spread of viral infection is of grave concern as there may not be time for an effective vaccine to be produced or there may not be sufficient quantities of vaccine available. Viral infection control methods are mainly dependent on antiviral agents to interrupt transmission and limit outbreaks.  15  Two classes of antiviral medications are currently used to treat and prevent influenza infections: the adamantanes and neuraminidase inhibitors. The adamantine derivatives, amantadine and rimantadine, act on the M2 protein of Influenza A. They are not effective on Influenza B and the development of wide spread amantadine resistance in H3N2 (99%) and H1N1 (10%) strains during the 2008-2009 season has limited their utility (Tosh 2010). Fortunately, the recent pandemic Influenza A/California/04/2009 virus and some other Influenza A and B viruses are still susceptible to neuraminidase inhibitor drugs, Zanamivir (inhaled) and Oseltamivir (oral) (Tosh 2010, Moscana 2005). Zanamavir (Relenza) and Oseltamivir (Tamiflu) are licensed worldwide for the treatment and prevention of influenza. Oseltamivir-resistant viruses have recently increased in circulation, especially among H1N1 viruses (Dharan 2009). New antivirals have been developed in recent years, but given the rapidly evolving nature of antiviral resistance, other options warrant exploration. 1.3.1 Nitric Oxide and Influenza  In an influenza model using NOS2 deficient mice (NOS2  -/-  ), it was demonstrated that in the  absence of NO production, the interferon response of the immune system was sufficient to clear the infection. In the same experiment using a wild type mouse, infection with influenza virus resulted in excessive NO production that led to a fatal pneumonitis (Karupiah 1998). The interferon class of cytokines can inhibit viral replication and signal other molecules of the immune system. One of the roles of NO in the immune system is to enhance the inflammatory response. The inflammatory process was well reviewed by Zaki et al who stated that “excessive production of NO and O2 in the respiratory tract during influenza and other respiratory virus infection plays a profound role in airway inflammation and pathogenesis of pneumonia” (Zaki 2007). This finding can be contrasted with Rimmelzwaan who showed that NO donors in an in16  vitro model inhibited the replication of the influenza virus during the early stages of infection by inhibiting viral RNA synthesis (Rimmelzwaan 1999). Reiss stated that “in most cases an in-vitro anti-viral inhibition translated to comparable in-vivo results” (Reiss 1998). It is important at this point to review the previous discussion of NO. The above results point out that there may be a difference in comparing NO efficacy between the endogenous NOS pathways and the exogenous delivery of NO via donors and gNO. Rimmelzwaan demonstrated that the direct application of NO inhibits influenza, whereas Akaike et al. (and others) have shown that the enzymatic pathway and the associated profound inflammatory response can be detrimental to the host. There appears to be a fine balance between beneficial and detrimental effects of NO that is essential for the successful resolution of influenza. (Reviewed in Pamer 2009). Observations in our laboratory have shown that the direct application of an exogenous gas supply of NO can inhibit the infection of influenza in-vitro in MDCK cells. Li, in a recent report, demonstrated “that Influenza A virus infection activates IL-32 and iNOS expression by a heretofore unrecognized complex mechanism, in which the two pro-inflammatory factors regulate each other through positive and negative feedback regulatory loops” (Li 2009). An area of future interest would be to explore regulatory feedback mechanisms of inflammation and the interaction of exogenously applied gNO. We speculate that the application of exogenous gNO may have a feedback mechanism that dampens the proinflammatory effects of NO in addition to its anti-viral potential. Interestingly, NO overproduction has been noted during infection by HIV-1, an enveloped RNA virus with similarities to the Influenza Virus (Koka 1995, Kong 1996). Torre et al reported that in HIV-1 infection, endogenous NO had both positive and deleterious effects (Torre 2002). The  17  challenge is to direct NO at the right concentration and for the right duration to maximize its antiviral potential. It is this antiviral potential we seek to understand with Influenza.  1.4 Hypothesis Over the past decade our research group has pioneered the direct use of exogenous gNO as a broad spectrum antimicrobial (Miller 2009, Ghaffari 2006 and McMullin 2005). We have developed a testing methodology that allows the precise delivery of a known concentration of gNO at physiologic temperature and humidity. The delivery of gNO is readily translated from research models to therapy. We have successfully eradicated bacteria (including drug resistant strains), mycobacteria, fungi and parasites in in-vitro testing in our lab (Miller 2004, McMullin2005 and Ghaffari 2007). NO has successfully treated wounds colonized with bacteria (reviewed in Jones 2010). It is our intention to undertake an exploration of the antiviral potential of gNO against Human Influenza Virus and to determine an effective dosing regimen for the safe delivery of gNO to humans. We hypothesize that the direct application of gNO inhibits the influenza virus and can be safely inhaled by humans. We propose the following specific objectives: 1. Determine the effect of the direct application of gNO on the influenza virus in an in-vitro testing methodology. 2. Determine a mechanism of action by which gNO may inhibit the influenza virus. 3. Identify a viable treatment regime that would allow antimicrobial concentrations of gNO to be safely administered to humans. 4. Validate the safety of the potential treatment regime in normal healthy adult humans.  18  The successful completion of these objectives will contribute to scientific knowledge and will pioneer exploration of the antimicrobial potential of gNO.  19  Chapter 2: Gaseous Nitric Oxide and Influenza: An in-vitro model 2.1 Introduction This chapter shows the in-vitro antiviral effect of gNO on Influenza at two stages: the cell-free virions and post-infection of the host cell. To our knowledge, there are no reports on the evaluation of the highest approved dose (80 ppm) or the antibacterial dose of 160 ppm gNO to identify the antiviral potential of gNO on influenza viruses. Therefore we chose to use these two doses of gNO with two different testing methodologies. The first method was to infect MadinDarby Canine Kidney Epithelial (MDCK) cells with influenza and then expose to gNO. The second method was to expose influenza virions to gNO prior to infecting MDCK cells. The British Columbia Center for Disease Control (BCCDC) supplied the strains of influenza used in this study. We chose to use an H3N2 strain to represent seasonal influenza, an H1N1strain to represent a recent pandemic-like influenza, and Influenza B. These three strains of influenza are representative of current strains that may be circulating in the human population. Laboratory observation has shown that as the concentration of gNO increases, the pH decreases and nitrite concentration increases in the testing media (unreported internal communication). To ensure that gNO is responsible for the antiviral effect we observed, and not reduced pH or increased nitrite concentration, we undertook an exploration of the effect of gNO on influenza.  2.2 Materials and Methods 2.2.1 Viruses and Cell Lines  MDCK cells (ATCC CCL-34) were obtained from the American Type Culture Collection and maintained in Dulbecco Minimal Essential Medium (DMEM) supplemented with 5% Fetal 20  Bovine Serum (FBS), and incubated at 37°C in a humidified atmosphere containing 5% CO2, without antibiotics or antimycotic agents. MDCK cells were grown as monolayers in 75-cm2 cell culture flasks. Passages between 3 and 15 were used for these experiments. All viral strains were obtained from the laboratory stock of the BCCDC. Stocks of Influenza A viruses, A/Denver/1/1957 (H1N1), A/Victoria/3/75(H3N2) and Influenza B Virus, B/Hong Kong/5/72 were grown in MDCK cells for 48 hours, with medium containing 2 µg/mL modified trypsin (treated with TPCK) without serum. All stock viruses were prepared as clarified cell-free supernatants. Virus concentrations for stocks were determined by standard plaque assay on MDCK cells (Hayden1980). Virus titres for these stocks were 3×107 plaque forming units (pfu)/mL (H3N2), 6×106 pfu/mL (H1N1) and 1×105 pfu/mL (Influenza B). 2.2.2 Gaseous Nitric Oxide Delivery  The design and validity of the continuous horizontal-flow gNO delivery device used in this study has been described in detail elsewhere (Ghaffari 2005). In brief, the device consisted of two cylindrical Plexiglas® exposure chambers with separate gas entry ports and a common exit port. These chambers were surrounded by an airtight Plexiglas® jacket to create a thermally isolated environment. This jacket enclosed an electrical heater unit controlled by an internal thermostat (Invensys Appliances Control, Carol Stream, Illinois, USA), that provided stable temperatures inside the chamber. Independent lines from each of the two exposure chambers delivered samples of the gas mixtures to a NO/NO2/O2 electrochemical analyzer (AeroNOx, Pulmonox Medical Inc, Tofield, AB, Canada) to detect the exact composition of the gases in the mixture. Gases were supplied from pressurized cylinders at a constant pressure of 50 pounds per square inch. These included 10,000 ppm NO diluted in nitrogen (N2) (Airgas, Chicago, USA), and  21  medical air (Praxair, Mississauga, ON, Canada). These gases were then mixed together at predetermined concentrations using a dilution manifold and a digital mass flow meter (TSI Inc., Shoreview, MN, USA). A gas mixture of 80 or 160 ppm was delivered to the exposure chamber at a rate of 10.0 L/min at 70-90% relative humidity at a set temperature of approximately 37°C, through two independent humidifiers (MR850, Fisher & Paykel Healthcare, CA, USA). The control chamber contained only medical air at a flow rate of 10 L/min. 2.2.3 Post Infection Effect of Gaseous Nitric Oxide  Confluent monolayers of MDCK cells grown in six-well plates were washed once with phosphate buffered saline (PBS), then infected with influenza virions at a concentration of 200 pfu/well. The plates were continuously shaken for 45-60 minutes at 37°C to allow for virus adsorption. The inocula were removed and replaced with 1 mL of saline (containing 0.5% FBS) per well. Infected plates were treated with either 160 ppm gNO (treatment) or medical air (control) for 1, 2 and 2.5 hours and after each time point, saline was removed and replaced with 2 mL/well of overlay medium consisting of 2X DMEM supplemented with 0.5% agarose and 2 µg/mL TPCK- trypsin. After two days of incubation at 37°C, the infected cells were fixed with 3% buffered formalin, then stained with 0.1% crystal violet and the number of plaques was counted. 2.2.4 Virucidal (Cell-free) Effect of Gaseous Nitric Oxide  Each virus (1000 pfu/mL) was treated with 80 or 160 ppm gNO in saline containing 0.5% FBS, for 10-180 minutes. At the end of each time point, virus infectivity was measured using the plaque reduction assay. Confluent MDCK cells were grown in six-well culture plates (Cotar Inc, NY, USA) and infected with virus from both the control and the gNO treated samples to give 22  around 100–250 plaques per well (for optimal visualization). The plates were incubated in 5% CO2 at 37°C for one hour. Following one hour, for viral absorption, the viral inocula were removed and cells were then cultured for two days with 2 mL/well overlay medium consisting of 2X DMEM supplemented with 0.5% agarose and 2 µg/mL TPCK-trypsin. After incubating for two days at 37°C, the infected cells were fixed with 3% buffered formalin, stained with 0.1% crystal violet and the number of plaques counted. 2.2.5 Nitrites and pH Content  NO has a short half-life in vivo of a few seconds. Therefore, we measured NO indirectly by determining the levels of more stable NO metabolites such as nitrites and nitrates. Nitrite concentration at the end of each treatment was measured using Griess reagent (Green 1982). A sample (100 L saline) was taken from each treatment and control plate (one well) and tested for the nitrite concentration and pH. 2.2.6 Effect of Nitrites, pH and Combination on H1N1 Virion (Cell-free)  Since nitrites and a reduced pH were found in the treated samples, the individual effect of each was tested. H1N1 (10 μL or 100,000 pfu/mL) was placed into each of the following conditions: 1) 100 μL of saline, 2) saline with 10 mM nitrites, 3) saline with citric acid (pH 3.5) and 4) a combination of citric acid and 10 mM nitrites (pH 3.5). After one minute, virus infectivity was measured using the plaque reduction assay, as explained above. 2.2.7 Nitric Oxide Effect on Dried H1N1 Dried virions were tested to control for the change in pH noted when exposing virions suspended in saline or media. Aliquots of 20L H1N1 containing approximately 10,000 pfu, diluted in  23  saline (containing 0.5% FBS), were spotted onto a sterile glass slide (25x15mm) and allowed to dry inside a biosafety cabinet for about 20 minutes. Glass slides were treated with a flow of 10 L/m of 160 ppm gNO for 60 and 120 minutes in the exposure chamber described above. Controls were treated with air. Samples were reconstituted in 1mL PBS, and virus infectivity was measured using a plaque reduction assay, as outlined above. 2.2.8 Statistical Analysis The results were analyzed using the unpaired Student’s t-test for comparison between any two groups. Group means were statistically tested by least squares means (two-tailed t-test). Data analysis and graphical presentation were completed using a commercial statistics package (Graphpad-Prism V 3.0, GraphPad Software Inc., USA). Unless otherwise specified, p < 0.05 indicated statistical significance. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  2.3 Results 2.3.1 Post Infection Effect of Gaseous Nitric Oxide in Cells  MDCK cells were infected with influenza virions for one hour and treated with either 160 ppm gNO or air (control) for 1, 2 and 2.5 hours. Rimmelzwaan demonstrated that NO donors inhibited viral replication. This first experiment was done to assess whether gNO would inhibit viral replication after the cells had been infected (Rimmelzwaan 1999). The viability of MDCK cells was confirmed by direct microscopic visualisation. The six-well plates were stained to visualize plaque formation, then counted and reported as control versus  24  treatment. H1N1 was found to be the most sensitive to gNO, resulting in about a 30% reduction of plaques formed after 2 and 2.5 hours of treatment (Figure 2.1).  110 100  PFUs  90 80 70 60 50 0  30  60  90  120  150  180  Time (min) Figure 2.1 Influenza H1N1-Infected MDCK Cells Exposed to 160 ppm gNO. MDCK cells were exposed to influenza for one hour to allow for infection. The cells were then treated with air (control- solid line) or 160 ppm gNO (Treatment- dotted line). Results were reported as the mean ± standard error of the mean from at least three independent measurements. No change from control was observed after two hours treatment post-infection with H3N2. A 25% inhibition was achieved after 2.5 hours (Figure 2.2).  25  175  PFUs  150 125 100 75 0  30  60  90  120  150  180  Time (min)  Figure 2.2 Influenza H3N2-Infected MDCK Cells Exposed to 160 ppm gNO. MDCK cells were exposed to influenza for one hour to allow for infection. The cells were then treated with air (control- solid line) or 160 ppm gNO (Treatment- dotted line). Results were reported as the mean ± standard error of the mean from at least three independent measurements.  Treating Influenza B-infected cells for up to 2.5 hours with 160 ppm gNO did not show any effect on the virus when compared to control (Figure 2.3).  26  120 110  PFUs  100 90 80 70 60 0  50  100  150  200  Time (min) Figure 2.3 Influenza B-infected MDCK Cells Exposed to 160 ppm gNO. MDCK cells were exposed to influenza for one hour to allow for infection. The cells were then treated with air (control- solid line) or 160 ppm gNO (Treatment- dotted line). Results were reported as the mean ± standard error of the mean from at least three independent measurements.  2.3.2 Virucidal Effect of Gaseous Nitric Oxide on Cell-free Virions  All three viruses were suspended in saline and exposed to either 80 or 160 ppm of gNO for varying lengths of time. Controls were treated with air for the same period of time. Virus infectivity was measured using a plaque assay with MDCK cells. gNO was shown to have a time and dose dependent effect on all three viruses. Exposing H1N1 to a continuous dose of 80 ppm gNO resulted in a 20% reduction in the ability to infect after one hour, a 50% reduction after two hours, and complete inactivation after three hours. Exposing H1N1 to 160 ppm resulted in viral inactivation of about 65% at 30 minutes and complete inactivation after one hour (Figure 2.4).  27  1500  PFUs  1000  500  0 0  50  100  150  200  Time (min)  Figure 2.4 H1N1 Virions Exposed to 80 ppm or 160 ppm gNO. The influenza virions were treated with 80 ppm gNO (solid line, control-square, treatment-open squares) or 160 ppm gNO (dotted line, control-triangle, treatment-open triangle). Infectivity was measured using a standard plaque assay. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  Repeating the same experiment with H3N2 produced similar results, compared to H1N1, with slightly higher susceptibility to gNO. Using 80 ppm, the treatment caused a small reduction in viral load after one hour and complete inhibition after two hours. When using 160 ppm on H3N2, complete inhibition was reached after 30 minutes of treatment (Figure 2.5).  28  1200  PFU/mL  1000 800 600 400 200 0 0  10  20  30  40  50  60  120  180  Time (min) Figure 2.5 H3N2 Virions Exposed to 80 ppm or 160 ppm gNO. The influenza virions were treated with 80 ppm gNO (solid line, control-square, treatment-open squares) or 160 ppm gNO (dotted line, control-triangle, treatment-open triangle). Infectivity was measured using a standard plaque assay. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  Influenza B showed a similar pattern to Influenza A, although it was less susceptible to gNO, with a 50% reduction after one hour and 100% inhibition after two hours. When the lower gNO concentration of 80 ppm was used, a similar pattern to H1N1 was seen (Figure 2.6).  29  PFU/ml  1500  1000  500  0 0  30  60  90  120  150  180  Time (min) Figure 2.6 Influenza B Virions Exposed to 80 ppm or 160 ppm gNO. The influenza virions were treated with 80 ppm gNO (solid line, control-square, treatment-open squares) or 160 ppm gNO (dotted line, control-triangle, treatment-open triangle). Infectivity was measured using a standard plaque assay. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  2.3.3 Nitrite Concentrations, pH and Their Effect on Cell-free H1N1 Virions  A solution exposed to gNO produces nitrites and lowers the pH (Table 1). This directly correlates with time of exposure and concentration of gNO. The pH was reduced to a pH between 3.5-4.6 after of three hours of treatment with 80 ppm gNO or two hours with 160 ppm. Exposure time and increased gNO concentration resulted in lower pH and higher nitrates in the solution. A correlation was found between the level of nitrite found in the exposed solution and the percentage of virus inhibition. In general, the higher the nitrite concentration and the lower the pH value, the higher the percentage viral inhibition.  30  Table 2.1 Amount of Nitrite Found in Treated and Control Wells After Exposing to NO or Air. Saline (100 µL) was exposed to 80 ppm or 160 ppm gNO. The pH was measured with a standard pH meter. Nitrite concentration was measured using Griess reagent. gNO  Time  ppm  Min.  80 80 80 160 160 160  60 120 180 30 60 120  H1N1Treated Nitrite(µM)/ pH  H1N1Control Nitrite(µM)/ pH  H3N2Treated Nitrite(µM)/ pH  H3N2Control Nitrite(µM)/ pH  Inf. BTreated Nitrite(µM)/ pH  Inf. BControl Nitrite(µM)/ pH  156/5.8 226/5.2 270/4.6 171/5.6 215/5.1 350/4.1  23/6.6 18/6.6 32/6.5 13/6.7 19/6.7 25/6.7  174/6.8 317/4.2 375/4.2 198/4.8 280/4.1 380/3.7  21/6.8 32/6.6 31/6.7 15/6.8 24/6.7 22/6.7  193/5.5 250/4.6 350/4.1 133/6.1 196/5.1 340/4.4  5/6.6 7/6.6 5/6.6 8/6.6 7/6.9 7.5/6.8  To determine whether the pH or the nitrites alone were responsible for the inhibition, H1N1 infectivity was tested under a variety of conditions, including saline containing 10 mM sodium nitrite (pH 6.5), saline with reduced pH (3.5), and a combination of 10 mM sodium nitrite and low pH (3.5). The combination of sodium nitrite and acid is known to produce NO (Weller 2005). As can be seen in Figure 2.7, sodium nitrite had no significant effect on the survival of H1N1. Acidified saline with a reduced pH caused 40% reduction in H1N1 pfu/mL while the 5 mM NO producing solution resulted in 94% inhibition of H1N1. Complete inhibition was attained in one minute, using a 10 mM NO-producing solution at pH 3.7. The short time for complete inhibition is due to the higher concentrations used in the acidified nitrite solution compared to the relatively lower concentration in the gas treated solutions. This experiment suggested that the NO gas itself was the factor inhibiting viral infectivity, and not some other chemical combination or pH. We performed an additional experiment to substantiate this supposition.  31  PFUs/ml  150  100  50  0 Control  Nitrites  pH  Acidified nitrites  Figure 2.7 Virucidal Effect of Nitrite and Low pH on H1N1. H1N1 (10 μL) was placed into 100 μL of control (saline) and treatment (nitrite, low pH or a combination of both) vials. After one minute, virus infectivity was measured using a plaque reduction assay. Results were reported as the means ± standard error of the mean from at least three independent measurements.  To eliminate changes in the surrounding solution (pH) as a factor in the virucidal effect of gNO on the virions, we tested the effect of gNO on dried virus (H1N1). A known concentration of virus from stock was diluted in saline and dried on a glass slide. The dried virus was then placed in the same exposure chamber used for all the other testing and exposed to gNO (160 ppm) or air (control) for 60 and 120 minutes at a constant flow rate of 10 L/min. Results show that viral infectivity was reduced by 85% (152 ± 26 pfu/mL for treatment compared to 980 ± 56 pfu/mL for control) after two hours of exposure.  2.4 Summary We conclude that gNO has an antiviral effect on a representative cross section of Influenza A H3N2 and H1N1 subtypes and Influenza B. This effect is dose dependent and begins to occur at the highest dosage approved for use of inhaled NO for full term infants (80 ppm). At a dose of  32  160 ppm a significant inhibitory effect is exerted on the early infection of Influenza A. Influenza B was not similarly affected during infection but its virions were susceptible in a cell free environment. We also show that gNO has an inhibitory effect on the virions independent of the effect caused by a reduced pH. Future research should focus on expanding these experimental observations to elucidate the mechanism of its antiviral action.  33  Chapter 3: A Mechanism by which Nitric Oxide Inhibits the Infectivity of Influenza 3.1 Introduction In the previous chapter we demonstrated that gNO is an antiviral against influenza. The next step is to identify the mechanism by which gNO inhibits influenza. From the literature review outlined in Chapter 1 we note that, aside from the report by Rimmelzwaan, there was no other report of NO inhibiting influenza. One report speculated that NO may be able to nitrosylate several highly conserved cysteines on the influenza hemagglutinin as a potential mechanism (Colasanti 1999, Fisher 1998). We propose to identify a mechanism of action by which gNO inhibits the infectivity of influenza by using two methods that focus on the hemagglutinin. The first method uses the hemagglutination inhibition assay to assess if hemagglutinin is a target. The second method is a simplified, one step version of the biotin switch method using an S-nitrosylation binding agent (SNOB) to identify nitrosylation.  3.2 Materials and Methods 3.2.1 Influenza Viruses  All viral strains were obtained from the laboratory stock of the BCCDC. Stocks of Influenza A viruses, A/Denver/1/1957 (H1N1), A/Victoria/3/75(H3N2) and Influenza B Virus, B/Hong Kong/5/72 were grown in MDCK for 48 hours, with 2 µg/mL of medium containing TPCK treated trypsin (Sigma) without serum. All stock viruses were prepared as clarified cell-free supernatants. Virus contents for stocks were determined by standard plaque assay on MDCK 34  cells (Hayden 1980). Virus titres for these stocks were 3×107 pfu/mL (H3N2), 6×106 pfu/mL (H1N1) and 1×105 (Influenza B) pfu/mL. 3.2.2 Gaseous Nitric Oxide Delivery  The design and validity of the continuous horizontal-flow gNO delivery device used in this study has been described in detail elsewhere (Ghaffari 2005). In brief, the device consisted of two cylindrical Plexiglas® exposure chambers with separate gas entry ports and a common exit port. These chambers were surrounded by an airtight Plexiglas® jacket to create a thermally isolated environment. This jacket enclosed an electrical heater unit controlled by an internal thermostat (Invensys Appliances Control, Carol Stream, Illinois, USA), that provided stable temperatures inside the chamber. Independent lines from each of the two exposure chambers delivered samples of the gas mixtures to a NO/NO2/O2 electrochemical analyzer (AeroNOx, Pulmonox Medical Inc., Tofield, AB, Canada) to detect the exact composition of the mixed gases. Gases were supplied from pressurized cylinders at a constant pressure of 50 psi. These included 10,000 parts per million (ppm) NO diluted in N2 (Airgas, Chicago, USA), and medical air (Praxair, Mississauga, ON, Canada). These gases were mixed together at pre-determined concentrations using a dilution manifold and a digital mass flow meter (TSI Inc., Shoreview, MN, USA). A range of 40 – 20,000 ppm gNO was delivered to the exposure chamber at a rate of 10.0 L/min at 70-90% relative humidity at a set temperature of 37°C, through two independent humidifiers (MR850, Fisher & Paykel Healthcare, CA, USA). Control chamber contained only medical air at a flow rate of 10 L/min.  35  3.2.3 Hemagglutination Inhibition Assay Influenza A/Victoria/3/75(H3N2) virions were exposed to gNO as described above. Serial 1:1 dilutions were performed in 96 well plates with U-shaped bottoms (Corning, USA). A 50µL volume of virions exposed to gNO or air were added to 50µL of PBS and serially diluted, then 50 µL of 0.7% guinea pig red blood cells (RBCs) were added to each well and mixed. After 30 minutes at room temperature, each tray was visually examined for agglutination. If agglutination occurred the RBCs would be suspended in a matrix. If no agglutination occurred the RBC’s would settle to the bottom of the well and form a doughnut shape. 3.2.4 Nitrosylation Assay The SNOB reagent was used to detect cysteine residues that were S-nitrosylated by NO. The SNOB assay is a single-step assay to detect S-nitrosylated proteins with SNOB for detection via a Western Blot technique (Tocris Bioscience, Missouri, USA). Using virions exposed to gNO, as described previously, stock SNOB reagent solution (20X) was diluted into a lysate sample to give a working solution of 1X, which was incubated at 37°C for 30 minutes in reduced light conditions. Proteins were separated by sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) and transferred from the gel onto a nitrocellulose membrane. The transferred proteins were then visualised by staining with ponceau red. Excess stain was removed by washing with 1% acetic acid, and the membrane was blocked with 5% (w/v) bovine serum albumin (BSA) overnight at 4°C. To visualize SNOB, streptavidin‐conjugated to HRP (Invitrogen, California, USA) was added to the membrane for 30 minutes and rinsed, leaving only bound HRP-SNOB. A chemiluminescent substrate, Super Signal West Pico (Thermo Scientific, Illinois, USA), was used to visualize the labelled proteins by exposure to x-ray film.  36  3.2.5 Nitric Oxide Effect on Dried H1N1  To control for a reduced pH when exposing virions suspended in liquid media to gNO, we tested dried virions. Aliquots of H1N1 (20 L containing approximately 10,000 pfu) diluted in saline (containing 0.5% FBS), were spotted onto a sterile glass slide (25x15mm) and dried in a biosafety cabinet for 20 minutes. Glass slides were treated with a flow of 10 L/min of 160 ppm gNO for 60 and 120 minutes in the exposure chamber described above. Controls were treated with air. Samples were reconstituted in 20 L PBS, and S-nitrosylation was assessed using the nitrosylation assay. 3.2.6 Hemagglutinin  Two commercially available hemagglutinins (HA) were purchased to determine if gNO was Snitrosylating hemagglutinin: Influenza A H1N1 (A/California/7/2009 HA) protein and H3N2 (A/Brisbane/10/2007 ) HA protein (Sino Biological, China). Each HA was reconstituted as per the manufacturer’s specifications. H1N1, HA, and H3N2 HA were exposed to either gNO or air as per described above and in parallel with the exposure of the corresponding virions. Following exposure, the HAs were examined using the nitrosylation assay.  3.3 Results 3.3.1 Hemagglutination Inhibition Assay  As outlined in Chapter 2, gNO inhibits the infectivity of influenza. In the hemagglutination inhibition assay, Influenza H3N2 exposed to gNO at 160 ppm for three hours only had a modest effect when compared to the control. This experiment was done at a flow rate of 2 L/min NO which controls for effect on the pH as compared to the experiments in Chapter 2 that were 37  conducted at a flow rate of 10 L/min. The different flow rates were used in order to control for pH. At the lower flow rate of 2 L/min the reduction in pH is more gradual and remains above pH 6.5 for the duration of the exposure. This method is used when a particular experiment, such as the hemagglutination assay, may be sensitive to reduced pH. However, at 2 L/min the formation of NO2 is more rapid, leading to concentrations that did not impact the experiment but nevertheless would not be clinically acceptable (discussed further in Chapter 5). To overcome the excessive production of NO2, the flow rate can be increased to 10 L/min, however, the increased flow rate also increases the rate at which pH decreases. As noted in Chapter 2, there was some effect inhibiting infectivity when the pH was reduced. We, therefore, theorized that a reduced pH may play a role in inhibiting agglutination. To further explore this relationship, we tested a range of pH from 6.5 - 3.5. When the pH was lower than 5 and combined with gNO the effect became more pronounced, compared to just a lower pH (Figure 3.1). A low pH alone had only a modest effect on the virions, and agglutination was not fully inhibited. As the concentration of gNO increased, the rate of agglutination increased.  38  Serial dilutions 1/2  1/128  1/64 1/32 1/16 C-6.5 Tx-6.5 C-5.5 Tx-5.5 C-4.5 Tx-4.5 C-3.5 Tx-3.5  pH  Figure 3.1 Hemagglutination Inhibition Assay. Three hours of continuous 160 ppm NO exposure to Influenza A/Victoria H3N2. Influenza A H3N2 virions were exposed to 160 ppm gNO at a flow rate of 2 L/min, with a pH ranging from 6.5 to 3.5. Control (C) exposed to air. Treatment (Tx) exposed to 160 ppm. 3.3.2 Nitrosylation Assay Using Influenza A/Denver/1/1957 (H1N1) suspended in PBS as described in Chapter 2, we observed nitrosylation with the SNOB assay (Tocris, USA) via Western Blot analysis, following SDS-page gel electrophoresis to separate the viral proteins. Nitrosylation was visible via an allor-none reaction of the SNOB binding to the site(s) of nitrosylation compared to the control sample. The control sample was exposed to air and the treated sample was exposed to 160 ppm gNO for two hours at 10 L/min. To control for the pH changes that occur when exposing PBS to gNO (as described fully in Chapter 2), we dried the H1N1 virions onto glass slides and exposed them to either 160 ppm gNO or air. Therefore the effect of a reduced pH on the virion was eliminated from this experiment. These exposures where done in parallel with the above experiment (see Section 2.2.4) at 160 ppm gNO or air delivered at 2l/min. The dried virions were reconstituted with PBS and then SDS-PAGE gel-electrophoresis to separate the proteins followed by Western Blot to 39  show nitrosylation. Dried virions demonstrated the same pattern of nitrosylation as the virions suspended in PBS. Note that in Chapter 2, the use of dried virions, controlled for pH, and demonstrated that a low pH was not solely responsible for inhibition. This is different from the hemagglutination inhibition assay in which a low pH is integral to showing an effect of gNO and is of interest because it demonstrates that inhibition can occur at a relatively normal pH. Total proteins from the virion were separated on a SDS-PAGE gel and transferred to the nitrocellulose paper for Western Blot. The size of the band corresponded to its reported size of 59kDa for hemagglutinin. Using the same H1N1 and Influenza B strains as in Chapter 2, we demonstrated nitrosylation of the protein in the band similar to the known size of hemagglutinin. No nitrosylation was observed in either of the commercially purified hemagglutinins, H1N1-HA or H3N2-HA, when exposed to gNO (Figure 3.2). The commercially produced HAs were both in their native (HA0, uncleaved) state. Even when inducing conformational change with trypsin, reduced pH or both, no evidence of nitrosylation was seen on the Western Blot.  40  A 90kDa 80kDa  50kDa  Lane  1  2  3  4  5  6  7  8  9  10  B 90kDa 80kDa  50kDa  Figure 3.2 The Nitrosylation of Influenza Virion. A- shows the proteins transferred from an SDS-PAGE gel to nitrocellulose for Western Blot. Proteins were visualized by staining with ponceau red dye. Excess dye was washed away with 1% acetic acid before blocking with 5% bovine serum albumin overnight and then continuing with standard Western Blot. B- results of the Western Blot showing nitrosylation of a protein that migrates like hemagglutinin on the influenza samples treated with 160 ppm.(Lane: 1. Positive control BSA, 2. Control H1N1, 3. Treated H1N1-1h, 4. Treated -2h, 5. Treated dried H1N1 -2h, 6. Control H1-HA, 7. Treated H1HA, 8. Control H3-HA, 9. Treated H3-HA, 10. Pre-stained protein marker.)  3.4 Summary We demonstrated one potential mechanism by which gNO inhibits the infectivity of the influenza virus is hemagglutinin inhibition likely due to the S-nitrosylation of the hemagglutinin. This provides a novel antiviral with the potential for limiting infectivity and ameliorating the burden of influenza on our society. However, before the potential of gNO as an antiviral can be achieved, further work must be done to understand the mechanism of action and the role of pH. The specific identification of hemagglutinin as a mechanism of action will be discussed in the Future Directions section of Chapter 6. Additionally, there are several issues regarding the safety of inhaling gNO that must be overcome if gNO is to be considered a viable clinical therapy. 41  Chapter 4: The Safe Delivery of Nitric Oxide: An Intermittent Dosing Strategy 4.1 Background As described in the preceding chapters, we identified 160 ppm gNO as an antiviral concentration in-vitro, and proposed the nitrosylation of hemagglutinin as a mechanism of action. The next step in translating our findings to a viable clinical therapy was to determine an effective delivery method that would be safe for humans to inhale. In order to determine the efficacy of the dosing regimen, we included three strains of bacteria that cause respiratory infections, in addition to influenza. While the bactericidal effect was not one of the specific aims of this dissertation, the results presented hereafter are significant, and illustrate the unique potential of gNO as a broad, non-specific antimicrobial.1 Prior to testing this hypothesis in an animal model, a number of challenges need to be addressed. The main challenge being that the administration of inhaled continuous gNO at 160 ppm for five hours (h) would unequivocally lead to methemoglobinemia and potential hypoxemia. To circumvent this problem, we explored an inhaled gNO delivery regimen using a high-dose of gNO with a limited duration of time (160 ppm gNO for 30 min) with sufficient time in between treatments (3.5 h) to avoid significant methemoglobinemia; thereby reducing the potential for host cell toxicity. The purpose of this in-vitro study was to explore the possibility that this dosing regimen could retain the desired antimicrobial effect as seen in continuous gNO delivery.  1  A version of Chapter 4 has been published as Gaseous Nitric Oxide Bactericidal Activity Retained During Intermittent High-Dose Short Duration Exposure, Miller C, McMullin B, Ghaffari A, Stenzler A, Pick N, Road J, Av-Gay Y, Nitric Oxide 20 (2009) 16-23.  42  Additionally, we examined the ability of host cells to tolerate high-dose gNO exposure. For this experiment we used human lung epithelial carcinoma cells.  4.2 Materials and Methods 4.2.1 In-vitro Nitric Oxide Exposure Device  The design and validity of the continuous horizontal-flow gNO delivery device used in this study has been described in detail elsewhere (Ghaffari 2005). In brief, the device consisted of two cylindrical Plexiglas© exposure chambers with separate gas entry ports and a common exit port. These chambers were surrounded by an airtight Plexiglas© jacket to create a thermally isolated environment. This jacket enclosed an electrical heater unit controlled by an internal thermostat (Invensys Appliances Control, Carol Stream, Illinois, USA), that provided stable temperatures inside the chamber. Independent lines from each of the two exposure chambers provided samples of the gas mixtures to a NO/NO2/O2 electrochemical analyzer (AeroNOx, Pulmonox Medical Inc., Tofield, AB, Canada) to detect the exact composition of the gases in the mixture. Gases were supplied from pressurized cylinders at a constant pressure of 50 psi. These included 800 ppm medical-grade NO diluted in N2 (ViaNOx-H, Viasys Healthcare), medical air, O2 and CO2 (Praxair, Mississauga, ON, Canada). These gases were then mixed together at pre-determined concentrations using a dilution manifold and a digital mass flow meter (TSI Inc., Shoreview, MN, USA). The gas mixture was delivered to the exposure chamber at a flow rate of 2.0 L/min at 90% relative humidity through two independent humidifiers (MR850, Fisher & Paykel Healthcare, CA, USA).  43  4.2.2 In-vitro Nitric Oxide Delivery Regimen Bacterial cultures were obtained from the American Type Culture Collection (ATCC), Influenza A/Victoria/3/75(H3N2) was provided by The BCCDC and clinical samples were provided courtesy of the Cystic Fibrosis Clinic at British Columbia Children’s Hospital. The bacteria used for this study were Staphylococcus aureus (a clinical multi-drug resistant strain), Escherichia coli (a strain isolated from the lungs of a nosocomial pneumonia patient) and Pseudomonas aeruginosa (a lethal, antibiotic-resistant strain, isolated from a deceased cystic fibrosis patient). The organisms were grown according to the standard operating procedures of the certified main hospital laboratory. From these cultures, a 0.5 McFarland standard with 108 colony forming units per milliliter (cfu/mL) was prepared and further diluted 1:1000 with sterile saline to105 cfu/mL in a volume of 20 mL. The concentration of 105 cfu/mL, or 105 pfu/mL for virions, represents an accepted threshold for determining infection (Bowler 2003). We observed in previous in-vitro NO studies that the constituents of bacterial support media contain molecules to which NO readily binds, reducing the available NO to target the bacteria (Keynes 2003). We, therefore, suspended the bacteria in 0.9% saline, rather than a nutrient broth media. Saline maintains the bacteria and virions in stasis. Aliquots were then pipetted, in triplicate, into six-well, flat bottomed, cell culture dishes with lids (Corning 3516, Corning, NY). When the temperature and gNO concentration were at steady state, the diluted cultures were incubated in the control and treatment arms of the chamber. To simulate a minimal and safe gNO pulmonary exposure with an acceptable antimicrobial effect, an intermittent exposure protocol was designed. The pathogens in the treatment arm of the chamber were exposed to 160 ppm gNO for 30 minutes, followed by exposure to medical air for 3.5 hour, or they were exposed to gNO continuously. The intermittent gNO exposure cycle was repeated for a total of six complete cycles in a 24hour  44  period for treating bacteria. Intermittent gNO administration for influenza was three cycles in a 12hour period. Pathogens in the control arm were exposed to continuous medical air only. To measure the effect of gNO on bacterial viability, cultures were sampled at 0, 4, 6, 8, 12, 16 and, if needed, 24 hour. The samples were then spread on Columbia agar containing 5% sheep’s blood (PML Microbiologicals, Willsonville, OR) and incubated at 37ºC for 24 hours. The virions were sampled at 0, 4, 8, and 12 hours, followed by a standard plaque assay to assess viability. A technologist blinded to the exposure performed the colony or plaque counts. The total ppm-hours for the intermittent dosing regimen and continuous exposure regimens were calculated as follows: Total ppm-hours = [gNO ppm concentration x Duration of exposure] x number of exposures.  The intermittent dosing regimen for six 30-minute exposure cycles would be calculated as follows: Total ppm-hours = 160 ppm x 0.5 hour x 6 treatment cycles = 480 ppm-hours For a continuous exposure of four hours the total ppm-hours would be: Total-ppm-hours= 160 ppm x 4.0 hours = 640 ppm-hours 4.2.3 Lung Epithelial Cell Culture  A549 cells (human lung epithelial carcinoma) were obtained from the ATCC. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 100 units/mL penicillin, 100 units/mL streptomycin sulfate, 2mM L-glutamine and 1% essential  45  amino acids, and grown in a humidified 37ºC, 5% CO2 incubator. For growth and analysis of THP-1 cell cytotoxicity, the monocytic cell line THP-1 (ATCC 202, Rockville, MA) was cultured in RPMI 1640 (Hyclone, Logan, UT), supplemented with 5% FBS, (Hyclone, Logan, UT), and 2 mM L-glutamine (StemCell Technologies, Vancouver, BC). Cells that were three passages or younger were used for all experiments. The cells were seeded in 96-well microtiter dishes at a density of 3x105 cells per well and incubated at 37ºC, 5% CO2 for 24 hours, either continuously in the presence of 200 ppm gNO, or intermittently (with the same regimen as described previously) in the presence of 160 ppm gNO. The negative control consisted of THP-1 cells incubated in the presence of medical air. As a positive control for cytotoxicity, THP-1 cells were treated for six hour with 5% H2O2, and stained with propidium iodide (PI), confirming the correlation between PI staining and THP-1 cell death during this assay. Cytotoxicity analysis was performed by flow cytometry as described elsewhere (Pick 2004). 4.2.4 Lung Epithelial Cell Proliferation Assay A colorimetric methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cytotoxic effects of gNO on lung epithelial cell proliferation (Roehm 1991, Rasouli-Nia 2004). A549 cells were seeded at a density of 5 x104 per well in a 12-well plate (Costar). Cells were treated with 160 ppm gNO for 30 minutes followed by 3.5 hours of medical air. This intermittent treatment was repeated for up to 72 hours. The control group was continuously exposed to medical air (commercially prepared sterile air supplied in compressed gas cylinders) for 72 hours. After each exposure period, the MTT assay was used to determine the number of viable cells. MTT was dissolved in PBS at a concentration of 2 mg/mL and 200 µL of MTT was added to each well. After five hours of incubation at 37ºC, MTT end products were solubilised by replacement of the culture medium with dimethyl sulfoxide (DMSO), and the optical density at 540 nm was 46  recorded. The MTT analysis is dependent on the reduction of the tetrazolium salt and MTT by the mitochondrial dehydrogenase of viable cells to form a blue formazan product. 4.2.5 Lung Epithelial Cell Migration Assay To analyze the effect of 160 ppm gNO on cell migration and attachment, A549 cells were seeded at a density of 1 x 106 cells per 60 mm culture dish and incubated at 37ºC in DMEM containing 10% FBS to form a confluent monolayer. Monolayers were scratched with a plastic pipette tip to mimic a wound with a uniform width. Suspended cells were washed away and the media was replaced by DMEM containing 2% FBS. These cultures were then exposed continuously to 160 ppm gNO or medical air for up to 72 hours. Serial light micrographs of the wound areas were taken daily for the next three days. Individual frames were stored, calibrated, and measured using an image analyzer (Quartz PCI, Pleasanton, CA, USA) to determine the distance migrated across the wound. Migration distance was expressed as a percentage of the wound width on day 0. 4.2.6 Clonogenic Cell Survival Assay Exponentially growing A549 cells were seeded in 60 mm culture dishes at a density of 200–600 cells per dish. The dishes were incubated overnight at 37ºC to allow the cells to attach. The cultures were then subjected to different doses of γ-radiation (60Co gammacell; Atomic Energy of Canada Limited, Mississauga, ON, Canada) and 160 ppm of gNO, continuously, for 24 hours at 37ºC, or air alone as a control. Following each treatment, the culture medium was replaced with fresh DMEM supplemented with 2% FBS, and incubated at 37ºC for 10–14 days. Cell colonies were fixed in 70% ethanol, stained with 10% methylene blue, and counted. The surviving colonies were plotted as a function of treatment received.  47  4.2.7 Statistical Analysis The results were analyzed using the unpaired Student’s t-test for comparison between any two groups. Group means were statistically tested by least squares means (two-tailed t-test). Data analysis and graphical presentation were done using a commercial statistics package (GraphpadPrism V 3.0, GraphPad Software Inc., USA). Unless otherwise specified, p < 0.05 indicated statistical significance. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  4.3 Results 4.3.1 Antimicrobial Effect of Intermittent gNO In-vitro Previous studies demonstrated that continuous delivery of 160 ppm gNO resulted in a 100% lethal dose (LD100) representing a 5 log10 reduction in cfu/mL at 5 hours (SD = 1) (McMullin 2005). Conversely, the proposed intermittent delivery regimen in this study showed that the average LD100 was 16 hours (SD = 0.0) for a 5 log10 reduction in cfu/mL. The resulting survival curves for each organism, comparing the continuous versus the proposed intermittent regimen are shown in Appendix 1, Fig. 1. This intermittent delivery strategy resulted in a 100% bactericidal effect (5 log10 reduction in concentration) against S. aureus, P. aeruginosa, and E. coli. These bacteria are associated with human pulmonary pathogenesis. The strategy resulted in a 75% reduction of the influenza virions after only three cycles. These experiments demonstrated that four cycles of 160 ppm gNO for 30 minutes followed by air exposure for 3.5 hours resulted in complete death for the bacteria tested (Appendix 1, Fig. A1.1), and severely inhibited influenza (Figure 4.1). NO2 levels were measured during the studies and were analyzed at a concentration of 15–18 ppm at flows of 2 L/ min.  48  200  PFU's/ml  150 100 50 0 0  2  4  6  8  10  Time(hours)  Figure 4.1 The Effect of Intermittent 160 ppm gNO on Influenza. Influenza A/Victoria H3N2. Influenza virions were exposed to either air (control) or 160 ppm gNO for 30 minutes, then air for 3.5 hours (treatment). The result was measured using a standard plaque assay. Squares represent control conditions; triangles represent treatment conditions; circles represent intermittent gNO exposure cycles (not to scale). Results were reported as the mean ± standard error of the mean from at least three independent measurements.  4.3.2 Proliferation, Migration and Mutagenesis of Pulmonary Cells in the Presence of Exogenous gNO The effect of gNO on pulmonary epithelial cell proliferation was measured using the MTT assay. Proliferation data were expressed as absorbance at 540 nm representing formazan production in metabolically active and viable cells (Appendix 1, Fig. A1.2). Cytotoxic and cytostatic effects were not observed in epithelial cells exposed to the intermittent exposure regimen of 160 ppm gNO. In fact, cells demonstrated a slightly higher growth rate in the presence of gNO after 72 hours of exposure. Interestingly, this dose of gNO exhibited significant antimicrobial properties in previously reported in-vitro studies (McMullin 2005, Ghaffari 2007). The result of a migration assay revealed that epithelial cells did not lose their ability to migrate in the presence of gNO  49  (Appendix 1, Fig. A1.3A). Cell migration was monitored and photographed for three days post exposure. The distance migrated by epithelial cells was measured and expressed as a relative percentage of the original injury size. Cells in both treated and control groups were able to repopulate 70% of a 300 µm gap by day three (Appendix 1, Fig. A1.3B). Sensitivity of A549 lung epithelial cells to 160 ppm gNO was determined using the clonogenic survival assay. Various doses of gamma (γ) radiation, a genotoxic agent, were used as positive controls. As shown in Appendix 1 Fig. A1.4, epithelial cells were not sensitive to 160 ppm intermittent gNO exposure compared to gamma radiation. A significant difference was noted between the control and the gNO treated cells, with the gNO treated cells improving. Of interest, it is purported that NO orchestrates wound healing and influences cell behaviour (Witte 2002). It is possible that NO may act as an effecter molecule in epithelial cell proliferation. To determine if gNO exposure was harmful to human immune cells important for lung defence, the survival of THP-1 monocytes and macrophages was measured after continuous exposure to 200 ppm gNO or medical air as a negative control. The dose of 200 ppm was used to test for a dose 25% higher than what was used in the exposure regimen. A continuous exposure to gNO was used to simulate long term multiple exposures of up to 10-fold greater duration threshold exposure than what was used in the exposure regimen. After 24 hours of exposure, almost 75% of undifferentiated THP-1 monocytes survived, whether they were treated with gNO or air (Appendix 1, Fig. A1.5A). In addition, there was no significant difference in survival between gNO exposure and air only. When THP-1 cells were first differentiated into macrophages before exposure to gNO or air, they had a slightly lower survival rate compared to monocytic cells, at approximately 55% for treatment with air and 60% for treatment with gNO (Appendix 1, Fig. A1.5B). In this case, survival of gNO-treated 50  macrophages was increased, compared to the macrophages exposed to air only. Since the undifferentiated THP-1 monocytes were not sensitive to treatment with the continuous, long term, high end doses of gNO, these same cell lines were further tested for differences in survival when treated with an intermittent regimen of 160 ppm for 30 minutes and 3.5 hours of 20 ppm gNO (repeated for five cycles) or 20 ppm continuously for 24 hours. A control of 20 ppm gNO was used since this is the most commonly prescribed dose of gNO used to treat full term infants with persistent pulmonary hypertension (FDA 1999). The histograms of the two PI-stained populations are shown in Appendix 1, Fig. A1.5C. Except for a small population of PI stained cells in the 160/20 ppm gNO treatment group, no significant population shifts were observed between the cells exposed to 160/20 ppm and the cells exposed to 20 ppm gNO. These observations were consistent with the previous results, indicating that high-dose gNO delivered in an intermittent regimen for a prolonged period of time to representative pulmonary host cells does not induce the same cytotoxicity seen for bacteria at similar doses and durations.  4.4 Summary In this study we showed that the antimicrobial properties of continuous gNO were retained when an intermittent dosing strategy was employed. We demonstrated that the intermittent dosing strategy of 160 ppm gNO for 30 minutes followed by a 3.5 hours recovery time was effective at reducing the pathogen load and was generally well tolerated by host cells. In the bacterial models we tested, a 100% or 5 log10 reduction was observed within three cycles of intermittent gNO exposure. Intermittent gNO produced a 75% reduction in the influenza model. We speculate that high-dose (160 ppm) intermittent delivery of gNO may be useful as a first line inhaled antimicrobial treatment to augment the innate defence system. The next step is to assess whether this dosing strategy can be safely inhaled. 51  Chapter 5: A Phase I Safety Trial of Nitric Oxide in Healthy Adult Humans 5.1 Background In Chapter 4 we demonstrated that gNO is an effective anti-viral in-vitro at a dose of 160 ppm. Eradication of the bacteria tested and inhibition of influenza was maintained with intermittent dosing using 160 ppm gNO. The next step was to undertake safety trials in healthy humans. Patient safety is a concern when using gNO as a microbicidal agent in clinical applications (Hurford 2005). Accordingly, we sought to determine whether the treatment strategy outlined in Chapter 4 would lead to unacceptably high methemoglobin levels, acute pulmonary injury or impairment of pulmonary function in healthy volunteers. Based on published methemoglobin kinetics and the dose required for antimicrobial effect (Miller 2007), we developed a treatment model using high dose gNO (160 ppm) for a short duration (30 minutes) to limit methemoglobinemia and reduce the risk of other toxic side effects (Borgese 1987, Young 1994, Young 1996). We demonstrated support for this dosing regimen in Chapter 4 through in-vitro studies showing that this strategy provides an effective antimicrobial effect and appears to be non-toxic to pulmonary cells (Ghaffari 2007, Miller 2009). In this chapter we report a prospective, phase one, open label safety study in ten healthy adults, acting as their own controls. Each subject inhaled 160 ppm gNO for 30 minutes, five times a day, for five consecutive days. Safe delivery of gNO was assessed and defined as the ability to deliver a stable concentration of NO without excessive NO2 levels while maintaining acceptable arterial hemoglobin oxygen saturation (SpO2). Subject safety was determined by the absence of acute pulmonary injury, pulmonary function impairment or presentation of other adverse effects. The subjects were assessed by monitoring vital signs, methemoglobin levels, lung function, blood  52  chemistry  (including  nitrite/nitrates),  hematology,  prothrombin  time,  inflammatory  cytokine/chemokines levels and endothelial activation (angiopoietin ratio). These parameters were compared to baseline measurements (prior to initiation of gNO) and to various time points during gNO administration, and at three, seven and 21 days post gNO administration.  5.2 Methods and Materials The protocol used was approved by the University of British Columbia Clinical Research Ethics Board, Vancouver Coastal Health Research Institute and the Therapeutic Products Directorate of Health Canada (TPD, CTA Number 129958). After obtaining informed consent, healthy adult volunteer subjects were screened by physical exam, pulmonary function tests, blood work and a medical history to exclude any pre-existing condition. Ten subjects passed the screening and were enrolled into the study. Treatment was initiated within five days of enrolment. Subjects were housed in a hospital ward and administered inhaled treatments of 160 ppm gNO for 30 minutes every four hours, five times a day, for five consecutive days. During the treatment period (Day1-5) several parameters were monitored (see below). Subjects returned for follow-up evaluations three, seven and 21 days after the final gNO exposure on day five. 5.2.1 Intermittent High Dose Inhaled Nitric Oxide Delivery  To obtain approval for this clinical trial, all components of the gNO delivery system required approval from the Therapeutic Product Directorate of Health Canada. Subjects were administered gNO as close to the mouth as possible through a modified disposable mouthpiece to minimize NO2 production. Inspiration was spontaneously initiated by the subject from a conventional intermittent positive pressure breathing respirator (Mark-7, Carefusion, USA) in fixed flow mode, delivering 48 L/min. Gas flow was verified with a calibrated mass flow meter 53  (TSI, USA). An NO (INOmax, Ikaria, USA) concentration of 800 ppm at 12 L/min was titrated into the distal delivery port of the mouthpiece connected to the respirator during the inspiratory phase only. The Mark 7 respirator was supplied by an air/oxygen blender (Bird Sentry, Carefusion, USA) set to deliver 26% oxygen. The target gas mixture was 160 ppm gNO with a NO2 concentration of less than 5 ppm and an O2 level between 21 and 25%. Inspiratory NO, NO2 and O2 levels were continuously monitored by sampling from the mouthpiece sample port (located about 6 mm from the subject’s mouth) with an AeroNOx (Pulmonox, AB, Canada) NO, NO2 and O2 electrochemical analyzer. Delivery safety was determined by the number of occasions that NO2 exceeded 5 ppm, NO exceeded 10% variation or O2 dropped below 20% during gNO treatments. 5.2.2 Measurement of Changes in Pulmonary Function  At time of screening, and on days two, eight, 12 and 26, subjects were administered full pulmonary function tests (PFT), including lung diffusing capacity (DLCO), by a trained technician using a calibrated pulmonary function system (Jaeger MasterScreen, VIASYS Healthcare, USA). Bedside spirometry (Microloop by Micro Medical, England) was performed on days one, three and four. The effect of gNO on lung function and DLCO was determined by changes between baseline, treatment days and follow up days.  5.2.3 Oxygenation and Vital Sign Measurements Full physicals were performed by a pulmonary physician on initial screening, and on days eight, 12 and 26. Abbreviated physical examinations were performed by a registered nurse prior to each daily initiation of treatment on days 1-5. Oxygenation was measured with a pulse oximeter 54  (Rad 57, Masimo Corporation, USA) according to manufacturer’s guidelines to measure functional oxygen saturation of arterial hemoglobin (SpO2) and heart rate. These parameters were measured continuously during every gNO treatment and for 3.5 hours after the first treatment of each day. Cardiovascular status was determined by monitoring heart rate, blood pressure, respiratory rate and temperature. Values were recorded prior to the start of each treatment, following a five minute rest. Vital signs (except temperature) were recorded 15 minutes after the start of the treatment and at the end of gNO treatment. After the first treatment of the day was completed, vital signs were recorded every 30 minutes until the start of the second treatment of the day. 5.2.4 Blood Chemistry and Hematology Measurements  Hematological assessment included a complete blood count and differential (hemoglobin, hematocrit, red blood cell count, white blood cell count, white blood cell differential, and platelet count). The blood chemistry profile included serum creatinine, and liver function tests: aspartate aminotransferase (AST) serum glutamic oxaloacetic transaminase (SGOT), alkaline phosphatase, and gamma-glutamyl transferase (GGT). The effect of gNO on coagulation was determined by assessing prothrombin time (PT) and its derived measures of prothrombin ratio (PR) and international normalized ratio (INR). 5.2.5 Analysis of Nitric Oxide Metabolism A commercially available noninvasive pulse oximeter (Rad 57, Masimo Corporation, USA) was used to measure methemoglobin saturation (SpMet). These parameters were measured continuously during every gNO treatment and for 3.5 hours after the first treatment of the day.  55  Daily serum samples were collected and frozen at -80°C. The serum nitrite/nitrite levels were measured using the Griess reagent method (Green 1982). 5.2.6 Systemic Inflammation and Endothelial Activation Assays  Heparinized plasma was collected and frozen at -80°C, at baseline, and on days one, two, four and five of gNO treatment, and on follow-up days three, seven and 21. Plasma cytokine levels were assessed using the human inflammation cytokine bead array kit (BD Bioscience, Canada). Plasma levels of angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) were determined by ELISA (R&D Systems, USA).  5.3 Statistical Analysis Descriptive characteristics of the subjects prior to, during, and at the end of the study were tabulated and expressed as mean ± standard deviation. Differences in continuous variables (methemoglobin, serum nitrate and SpO2 levels) over the course of the study were analyzed utilizing repeated measures analysis of variance. Categorical events (number of subjects with a particular adverse event) were determined by constructing 95% confidence limits for the incidence rate. Differences between important continuous variables at two specific times were evaluated with the paired t-test. Important categorical events such as changes in clinical pulmonary function and lung diffusion changes, changes in serum inflammatory markers, hematology, clinical chemistry and incidence of adverse events were analyzed by constructing 95% confidence limits for the incidence rates. The data was analyzed using the unpaired MannWhitney test for comparison between any two groups and ANOVA for repeated measures of variance. Baseline comparisons were analyzed by repeated measures ANOVA with Bonferroni post-test for parametric data or Friedman test with Dunn’s post-test for non-parametric data. 56  Data was analyzed and presented graphically using a commercial statistics package (GraphPadPrism V 3.0, GraphPad Software Inc., USA). Unless otherwise specified, p < 0.05 indicated statistical significance. Results were represented by mean ± standard deviation from at least three independent measurements.  5.4 Results 5.4.1 Safe Delivery of 160 ppm Nitric Oxide Gas: Inhaled Nitric Oxide, Nitrogen Dioxide and Oxygen Measurements.  A total of 250, thirty minute gNO treatments at a target concentration of 160 ppm were administered to ten subjects (five male, five female) during the study period. The age of the subjects ranged from 20 to 62 years old. As defined by the study parameters, the treatments were well tolerated and no significant adverse events were observed. There were three minor adverse events reported. One subject reported bruising of the arm from multiple attempts to draw blood. (Bleeding that could be caused by a decrease in platelet cGMP levels was not observed or reported by the subjects or coagulation assays.) Two separate subjects reported a numbing sensation of the tongue during gNO treatment. We speculated that the subjects were pressing the mouthpiece too tightly between the tongue and roof of their mouths. This was resolved by instructing the subjects to relax and reposition the mouthpiece. Inspired NO levels were monitored continuously and recorded at the start, at 15 minutes and at the end of gNO treatment. A total of 750 measurements of NO were recorded during the study. The average inspired NO was 163.3ppm (SD=4.0). The highest NO concentration recorded was 177 ppm.  57  NO2 levels were also monitored continuously and recorded at the start, at 15 minutes and at the end of gNO treatment. No subject experienced NO2 > 5 ppm. The highest NO2 recorded (n=750) during the treatments was 2.8 ppm with a mean of 2.32 and a 95% confidence level of 2.17-2.47 ppm. This was consistent with the performance specifications provided by the analyzer manufacturer of 1.56 ppm (SD=0.3). The fractional inspired oxygen percentage was analyzed continuously during gNO treatments. The goal was to adjust the blender to ensure greater than 20.8% or ambient levels of oxygen without resulting in adverse NO2 levels (greater than 5 ppm). Of the 300 recorded values, the average oxygen percentage was 22.0% (SD=0.22). 5.4.2 Vital Signs and Clinical Safety During and After gNO Delivery: Blood Pressure, Oxygenation, Methemoglobin and Nitrite/Nitrate Levels.  During and after gNO exposure, all vital signs remained within normal limits for age and with respect to baseline values. Specifically, there was no drop in blood pressure (potentially due to the vasodilator effect of gNO) during or after gNO treatments. There were 300 valid SpO 2 values at 15 and 30 minutes during the administration of gNO. There were no sudden incidences of hypoxemia (< 85% SpO2) during gNO administration, or after gNO termination. The lowest observed SpO2 was 93%. SpO2 (%) levels decreased slightly over time, between the pretreatment and post-treatment but were not statistically or clinically significant. ANOVA analysis determined that this decrease was not associated with the five repeated exposures to gNO over the course of the same day. Inhaled NO is reduced in the systemic blood stream by hemoglobin, which results in an increase in methemoglobin levels. To ensure that inhaling gNO did not compromise oxygen carrying  58  capacity, methemoglobin percent levels (SpMet) were kept below 5%. SpMet was monitored continuously and recorded every 15 minutes during gNO delivery and at every 30 minutes after the first treatment of the day. During subsequent daily treatments of gNO the SpMet was recorded prior to and immediately after treatment. Follow up measurements were recorded three, seven and 21 days after the final gNO exposure on day five. Of the 930 recorded SpMet measurements all remained below 5%. The initial baseline SpMet was 0.16% (SD=0.10). The highest SpMet was observed at the end of the 30-minute treatment and was 2.5% with an average increase of 0.9% (SD=0.08). SpMet increased slightly between pre-treatment and post treatment (p<0.001). ANOVA analysis determined that this increase was not associated with repeated treatments on the same day. Results comparing pre- and post-treatment SpMet for all ten subjects for every gNO treatment were plotted and are presented in Figure 5.1.  %MetHgb  1.5  1.0  0.5  0.0 0  60  120  180  240  Time (min)  Figure 5.1 Summary of all Treatments of Methemoglobin Kinetics, Over One, Four-hour cycle. Each subject breathed 160 ppm gNO for 30 minutes followed by 210 minutes recovery, breathing room air. The graph of methemoglobin is the composite of all (n=250) gNO treatments. Results were reported as the means ± standard error of the mean from at least three independent measurements.  59  As predicted, methemoglobin concentration rose by approximately 1% and returned to baseline after 3.5 hours prior to next gNO treatment. As can be seen, the maximum (SpMet) occurred at thirty minutes and returned rapidly to baseline between treatments. There was no cumulative effect on SpMet after five daily treatments for five consecutive days (Figure 5.2). Follow-up SpMet measurements on three, seven and 21 days after the final gNO exposure on day five did not demonstrate any residual increase in SpMet.  %Met Hgb  1.5  1.0  0.5  0.0  1  2  3  4  5  8  12  26  Time (days)  Figure 5.2 Cumulative Summary of Methemoglobin Kinetics. The methemoglobin kinetics of all (250) treatments cumulated over the entire duration of the study. Each treatment (30 min) for each subject for the entire duration of the study is shown, as well as all the methemoglobin measurements from day 0 to 26. Results were reported as the mean ± standard error of the mean from at least three independent measurements.  Methemoglobin is reduced by an enzymatic reductase resulting in an increase in blood nitrite/nitrate levels. There was no significant difference in serum nitrite/nitrate levels from baseline through the trial. It should be noted that one subject had significantly higher peak nitrite and nitrate values (p<0.001) as well as a slightly different baseline value (p=0.038) compared to the other subjects.  60  There were no statistically or clinically significant changes in blood coagulation parameters, clinical chemistry or hematological parameters from baseline to completion of day five. Although there was a decrease in eosinophils from baseline (0.15 giga/L; SD=0.12;p=0.104) to 0.19 giga/L (SD=0.19), it was not significant (p=0.104). There was a 1% increase in neutrophils from a baseline of 0 to 0.01 giga/L (0.03). At the completion of treatment, these were not statistically or clinically significant (p=0.169).  5.4.3 Normal Lung Function and Anti-Inflammatory Response During and After gNO Delivery  Results of the pulmonary function tests revealed no abnormalities for any subject during gNO treatments or three weeks after exposure. Specifically, airflow, as measured by Forced Expiratory Volume in one second (FEV1) and maximum mid-expiratory flow (MMEF), was not different from baseline during the course of the study. Other lung function measurements like DLCO, Forced Vital Capacity (FVC), Total Lung Capacity (TLC) and Residual Volume (RV) did not change from baseline measurement through day 28. 5.4.4 Inflammatory Cytokines and Endothelial Activation Factors  To assess whether gNO treatment resulted in inflammation or endothelial activation we quantified cytokine and Ang-1 and Ang-2 levels in peripheral plasma at baseline, on days one, two, four, and five of gNO treatment, and on post treatment days three, seven and 21. Preliminary results showed cytokine levels of TNF, IL-6, IL-8, IL-10, IL-1b and IL-12p70 were unaffected by inhalation of gNO compared to baseline. Comparisons between baseline cytokine levels and levels at each of the sampling time points for all 10 participants resulted in no 61  significant differences (compared by repeated measures ANOVA with Bonferroni post-test for parametric data, or Friedman test with Dunn’s post-test for non-parametric data). Additionally, preliminary results also showed that the vascular endothelium activation factors Ang-1, Ang-2 and the Ang-2/Ang-1 ratio were not affected in this study.  5.5 Summary The results presented show the promise that a high dose of inhaled gNO delivered intermittently can be safely administered within clinical guidelines for NO. NO2 levels were kept within an acceptable range without compromising the subjects’ vital signs. The treatments were well tolerated by all subjects and no severe adverse events were noted during the study. We also found that intermittent (every four hours) inhalation of 160 ppm gNO could be delivered five times a day for five consecutive days without an adverse increase in methemoglobin. We have provided evidence that the resulting metabolic burden was within normal parameters for serum nitrite/nitrate concentrations. Furthermore, we have shown that this treatment regimen does not appear to be pro-inflammatory or alter pulmonary function in healthy adults. These results suggest that gNO could be explored further to evaluate efficacy in otherwise stable subjects with mild pulmonary infections. Should these studies prove to be safe and effective, we speculate that high antimicrobial doses of intermittent gNO administration may be useful as a first line inhaled antimicrobial treatment to augment the innate defence system or, possibly, as an adjunct to antibiotics.  62  Chapter 6: Discussion, Summary, Future Directions and Conclusion 6.1 Discussion 6.1.1 gNO Can Act as an Antiviral Against Influenza  The results presented here demonstrate that gNO profoundly affects the ability of Influenza A and B virions to infect and replicate in MDCK cells. To our knowledge, this is the first study to report this. Interestingly, it was shown that virions suspended in normal saline and exposed to gNO lose their ability to infect MDCK cells. Conversely, when MDCK cells were first infected with Influenza A, then exposed to gNO, the virucidal effect of gNO was modest. The antiviral effect of gNO on Influenza A during infection was consistent with the effect shown by Rimmelzwaan using the organic donor SNAP on Influenza A viruses (Rimmelzwaan 1999). Rimmelzwaan demonstrated that NO released from SNAP inhibited Influenza A and B virus at an early stage of viral replication by inhibiting viral RNA synthesis. Other studies using different viruses have shown that NO donor compounds have antiviral effects within infected host cells. Studies using Coxsackie virus demonstrated that NO interrupted the viral life cycle via NOmediated S-nitrosylation of the cysteine residue inhibiting protease activity in the 3C protease (Saura 1999, Zell 2004). Harris et al. demonstrated that several processes in the late stages of viral replication, including viral DNA replication, viral protein synthesis and virion maturation, were greatly inhibited by IFN--induced NO in Vaccinia Virus (Harris 1995). Other in-vitro studies utilizing chemical donors of NO have shown inhibition of viral replication in DNA and RNA viruses. Reports have suggested that NO inhibits viral proteins, RNA synthesis and viral replication by modifying molecules such as reductases and proteases required for replication (Tosh 2010, Croen 1993, Zell 2004, Zaragoza 1997). Direct virucidal activity on cell-free 63  virions, as we observed, has not previously been reported. There are multiple targets on the virion surface to which NO could bind and disrupt the infection process. However, little is known about the antiviral mechanism by which NO acts on virions before they bind to cellular receptors and begin replication. In addition to its direct antimicrobial activity, NO also functions as a reactive nitrogen intermediate and can react with oxygen intermediates, such as hydrogen peroxide (H2O2) and superoxide (O2−) to form a variety of antimicrobial molecular species (Radi 1991). Colasanti et al. theorized that NO may be able to affect hemagglutinin (HA), a surface protein, by nitrosylation of the cysteine moieties within its structure (Colasanti 1999). This structural change could alter HA stoichiometry during its interaction with the sialic acid receptor or could prevent fusion of the virion with the epithelial cell membrane (Fischer 1998). Results from our studies support the theory that NO inactivates the virion, thereby preventing infection of the host cell. Influenza A virions, regardless of strain, were slightly more susceptible than Influenza B in this study. Since we only tested one strain of Influenza B, future work is required to identify why there are susceptibility differences between Influenza A and B. Broadening the testing to multiple strains will help determine whether the difference in susceptibility is maintained throughout all Influenza B viruses or if it is restricted to specific strains. We are currently screening a wide variety of viruses against gNO to assess which are more or less susceptible. This may shed some light on the mechanism of action causing the antiviral activity of NO and identify future avenues of investigation. We discovered a correlation between the lengths of time saline is exposed to gNO and antiviral effectiveness. Regardless of the concentration of gNO (80 or 160 ppm), the antiviral effect coincided with a specific range of nitrite concentration and acidic pH. We theorize that gNO diffuses into the saline and reacts with water to produce nitrous acid (HNO2), resulting in a drop 64  in pH and accumulation of nitrite (NO2-) ions. The resulting pH, along with the nitrite concentration, was within the same range found to be antifungal and antibacterial in other studies using a combination of acid and nitrites (acidified nitrites) (Weller 2001, Anyim 2005 and Hardwick 2001). As far as we know, there have been no reports on the antiviral effects of acidified nitrites. Interestingly, we found that neither acid nor nitrite on their own are antiviral, however, when combined they are an effective antiviral combination. This combination of acid and nitrites has been previously shown to produce NO (Weller 2001). In our study, the low pH alone produced some antiviral effect (40% inhibition) but a much greater effect (100% inhibition) was found in combination with nitrites. This suggests that NO had a direct antiviral effect that was not wholly attributable to the low pH or nitrite level. To support this idea, we showed that virions dried on a glass slide, then exposed to dry gNO at 10 L/min had reduced infectivity. Since the virus was not suspended in liquid, we eliminated the effect of the low pH environment on the virions. The gas treatment, in this case, reduced infectivity (by 85%), thus, we speculate that there are targets on the virion that may bind to NO and prevent infection of cells. 6.1.2 gNO Acts as an Antiviral Through Nitrosylation  To elucidate a mechanism of action, we first chose to use the hemagglutination inhibition assay. We demonstrated that gNO inhibits the ability of influenza hemagglutinin to agglutinate guinea pig red blood cells. The results were not as dramatic as we had anticipated unless the pH was reduced. Perhaps a reduced pH is beneficial because, in a low pH environment, the nitrites in solution will react with the acid to produce more NO. We reduced the pH via one of two methods: by adding hydrochloride acid or by increasing the concentration of gNO, which converts to nitrous acid. We noted that the inhibition was dose and pH dependent. Of note, pH 65  alone had a modest effect on hemagglutination, similar to the effect outlined in Chapter 2. However, when a reduced pH was combined with gNO the effect was more profound. This indicated that the inhibition of influenza infectivity was due at least in part to gNO reacting with the influenza surface protein, hemagglutinin. Hemagglutinin (HA) is a membrane glycoprotein from the influenza virus that comprises over 80% of the envelope proteins present in the virus particle. In natural infection, inactive HAο matures into HA1 and HA2 outside the cell via the action of one or more trypsin-like, argininespecific endoproteases secreted by the bronchial epithelial cells. Binding of HA to sialic acidcontaining receptors on the surface of its target cell attaches the virus particle to the cell and forms an endosome. The low pH encountered in endosomes induces an irreversible conformational change in HA2, releasing the hydrophobic “fusion peptide”. The virus then penetrates the cell and releases its contents, including the RNA genome, into the cytoplasm, mediated by fusion of the endocytosed virus particle’s own membrane and the endosomal membrane. Hemagglutinin plays a major role in the determination of host range restriction and virulence (Günther-Ausborn 2000, Hoffman 1997, Marjuki 2006, Sinobiological.com).The potential of NO to inhibit hemagglutinin was first postulated by Colasanti, based on the findings of Fischer, in a review paper on the S-nitrosylation of viral proteins. Colasanti states: “Although further studies are needed to clarify the effect of NO on the Influenza A virus life cycle, viralencoded hemagglutinin may represent a target for this diatomic mediator. In fact, mutation of Cys551, Cys559, and Cys562 residues close to the C terminus of the hemagglutinin-2 subunit does not allow palmitylation and reduces the ability to induce syncytia.” (Colasanti 1999, Fischer 1998) Akerstrom reported that NO inhibited the fusion step by reducing palmitoylation of the  66  SARS-CoV S protein (Akerstrom 2009). To date we have been unable to locate any published report to either confirm or refute the ability of NO to nitrosylate hemagglutinin. The results from the hemagglutination assays were sufficient to give us the confidence to proceed to determine if NO nitrosylated the influenza hemagglutinin. For this study, rather than using the traditional biotin switch method (Jaffrey 2001), we used a newly available, one-step Snitrosylation binding (SNOB) reagent. SNOB reagents detect cysteine residues that have been Snitrosylated by NO. SNOB can be used as part of a single-step assay to label S-nitrosylated proteins with biotin for detection by Western Blot or proteomic analysis. When the whole influenza virion was exposed to 160 ppm gNO, as described in Chapter 2, nitrosylation was detected using the SNOB reagent and visualized via Western Blot. Conversely, we demonstrated that no nitrosylation was found on either of the commercially provided HAs. These HAs are secreted as recombinant hemagglutinin of Influenza A virus (A/Brisbane/10/2007 (H3N2)) which comprises 526 amino acids and Influenza A virus H1N1 (A/California/07/2009 ) which comprises 521 amino acids. Both HAs have a predicted molecular mass of 59 kDa (SinoBiological.com). These two HAs are expressed with a C-terminal polyhistidine (H3N2), or fused to a polyhistidine at the C-terminus (H1N1), thereby lacking the cysteines that would be the target of nitrosylation. Several reports have shown that the cysteines located in the cytoplasmic tail are essential for pore formation and, thereby, infectivity (Wagner 2005, Fischer 1998, Naeve 1990). Of note, in order to further assess the two commercial HAs, we exposed them to a reduced pH and to trypsin to see if inducing a conformational change would open sites on the HA susceptible to nitrosylation, but no effect was identified. We demonstrated that gNO inhibits the infectivity of influenza through nitrosylation of the virions. This reaction is probably mediated by nitrosylation of the cysteines in the cytoplasmic tail of hemagglutinin. While we are 67  confident that nitrosylation of hemagglutinin occurs, there is the potential of other targets, in particular the M2 ion channel (Gaston, 1999). We demonstrated, for the first time, that gNO could inhibit the infectivity of the influenza virus through nitrosylation of the virion. This suggests a mechanism whereby gNO, a novel antiviral, may have the potential for limiting infectivity. Before the potential of gNO as an antiviral can be achieved, several challenges regarding its safety must be overcome. As described previously, the highest dose of NO approved for inhalation is 80 ppm. While we had some success with this dose against influenza we demonstrated that a dose of 160 ppm gNO is more efficacious. However, a dose of 160 ppm gNO cannot be inhaled continuously, because it will bind with hemoglobin to form methemoglobin. As methemoglobin accumulates, it will reduce the availability of oxygen to the host. Also the inhaled levels of NO2 need to be kept below accepted levels. Therefore, an alternate strategy needs to be developed. 6.1.3 Intermittent Delivery of 160 ppm gNO can be a Viable Treatment Regime  We reported in Chapter 4 that the bactericidal effect of 160 ppm gNO was preserved during intermittent delivery, compared to continuous delivery of the same concentration. In the influenza model, greater than 75% reduction in the number of pfu was achieved after three treatments of 30 minutes. While this result did not match the 100% reduction in pfu observed during continuous exposure, it should be noted that the intermittent total gNO delivered was 240 ppm-hour compared to 320-480 ppm-hour for continuous exposure. If we continued the intermittent treatment for one more cycle, we could predict a further reduction to match the continuous result. Unfortunately, our testing methodology for influenza limited our exposures to a maxiumum time of nine hours. Beyond this time, the stability of the virions became  68  unpredictable. We are currently revising our methodology to allow us to expand our exposure times and extend our results. Overall, these are highly relevant findings if gNO is to be considered for inhalational use as an antimicrobial agent to treat pulmonary infections such as bacterial pneumonia or influenza. NO has a high affinity for metal ions and reacts readily with hemoglobin to produce methemoglobin. At high levels, methemoglobin can interfere with oxygen transport. Due to the large gas to blood surface area in the lungs, rapid conversion of hemoglobin to methemoglobin by continuous exposure to gNO causes hypoxemia. This may be especially pertinent as the target populations for gNO antimicrobial therapy are likely already to have pulmonary complications. Rather than using continuous delivery of gNO, with its higher potential for inducing methemoglobinemia, an alternative high-dose (160 ppm), short duration (30 min) strategy was hypothesized for a pulmonary application model of gNO. This study was designed to evaluate whether an intermittent high-dose gNO delivery regimen could preserve the antimicrobial effect of continuous gNO delivery while avoiding elevated methemoglobin levels. We showed that when gNO was administered to three bacterial strains and Influenza A, all of which were associated with pulmonary disease, at 160 ppm for 30 minutes, repeated every four hours, the same potent antimicrobial activity was observed as when 160 ppm gNO was delivered continuously. Despite taking about 10 hours longer to achieve the same antimicrobial effect as continuous delivery of gNO, this intermittent regimen may allow for a safer approach with reduced side effects. However, this gNO delivery regimen may not be as effective in an in-vivo setting where bacteria might replicate to unacceptable levels between gNO exposures. We speculate that continuous exposure to gNO is not needed to kill pathogens and an intermittent exposure is effective because of the affinity of NO to bind target bacteria and viruses. 69  NO, in gaseous form, passes unhindered through cell membranes, targeting a wide range of macromolecules. In mycobacteria, after thiol detoxification sites are overwhelmed, free NO in the cytosol binds with iron complexes such as aconitase, interrupting cellular respiration (Miller 2004). NO can further react with reactive oxygen species, resulting in the production of peroxynitrite, which is known to destroy DNA (Fang 2004). It appears that this regimen places a significant burden on bacteria, not allowing the bacteria time to sufficiently replenish or increase thiol production. However, future work in an in-vivo biological model is required to verify whether this effect is retained. The rationale for the 30 minutes of 160 ppm gNO therapy was based on published methemoglobin kinetic studies (Young 1994 and 1996, Borgese 1987). The predicted half-life of methemoglobin in humans is approximately one hour. The anticipated rise in methemoglobin during the 30 minute, 160 ppm gNO treatment was calculated to be approximately 1%. The 3.5 hour interim period would allow the methemoglobin concentration to return to baseline, at which time another 30 minutes gNO treatment of 160 ppm could be given. The total 24 hour NO metabolic burden for this six-cycle regimen was calculated to be 480 ppm-hour. In reality, in more than 10 years of successfully treating term infants with inhaled NO, methemoglobinemia levels have not been shown to be a reported risk factor, even in rare cases where methemoglobin reductase was impaired. 80 ppm NO for 24 continuous hours, as used in early neonatal and adult clinical trials, resulted in 1920 ppm-hour (FDA 1999). However, in clinical practice, the usual accepted dose is 20 ppm, for a total exposure to the lung, over 24 hours, of 480 ppm-hour. Thus, the metabolic burden for the intermittent antimicrobial regimen of six cycles of 160 ppm gNO for 30 min, is the same (480 ppm-hour) as that currently being used in neonatal practice. We hypothesized that this treatment combination could be a safe therapeutic regimen from the 70  standpoint of methemoglobinemia and NO metabolic burden, but this needed to be confirmed in an animal model. A series of in-vitro mechanistic studies demonstrated the effectiveness of gNO as a bactericide. This antimicrobial activity was dependent on 160 ppm gNO overwhelming bacterial thiol-based detoxification mechanisms and maintaining pressure on the thiol pathways so that bacteria could not replenish this defence reservoir (Miller 2004, Meister 1988). Eukaryotic cells (human) have much higher thiol levels and can cope with high levels of NO better than prokaryotes (Meister 1988). Thus, host cells should tolerate NO stress more effectively than bacteria. The data reported here provides evidence that for up to 72 hours, proliferation of the sensitive A549 pulmonary epithelial carcinoma cell line exposed to 160 ppm gNO was equivalent or better than exposure to medical air. A549 cells exposed to gNO also exhibited normal function and survival as demonstrated in a healing process model. In this study, A549 cells were used as a surrogate for primary (normal) lung epithelial cells. Previous studies have also used these cell lines to predict the behaviour of primary cells, since cell lines are already established by others and much easier to grow in culture (Rasouli-Nia 2004). A549 cells, along with gNO-treated THP-1 monocytes (undifferentiated) and macrophages, were shown to survive as well as, or better than control cells exposed to medical air. This data supports our hypothesis that the host will tolerate a high-dose, short duration gNO treatment strategy. In earlier work exploring the mechanisms of the antimicrobial action of NO, it was found that 40 ppm and 80 ppm gNO was not an effective dose for killing bacteria. Other in-vitro and in-vivo work reported in the literature concluded that doses below 90 ppm would not be clinically efficacious (Jean 2002, Webert 2000). This was also confirmed clinically by Long et al., who showed that inhaled 80 ppm gNO was ineffective at reducing the bacterial load in patients with 71  TB (Long 2005). We speculate that had they used a higher dose, their results may have shown a significant reduction in bacterial load. Administration of NO donors, such as polyethyleneimine cellulose NONOate polymer, SNAP, sodium nitroprusside (SNP), and molsidomine (Nethoxycarbomyl- 3-morpholinyl-sidnonimine), to deliver NO may be effective as antimicrobial agents (DeGroote 1999, Loskove1995, Slivka 1994, Maragos 1991). Acidified nitrite derivatives have been shown to kill mucoid P. aeruginosa and decrease bacterial load in cystic fibrosis patients (Yoon 2006). The challenge with donors and nitrite derivatives is achieving a consistent and effective antimicrobial delivery. Nitrites require a specific pH level to release NO, and donors also require acidification or another catalyst to release NO. This further exacerbates the potential toxicity of these NO carrier compounds (Nauer 1998). gNO is not compounded like other drugs, it is carried within inert nitrogen as a 0.0160% active ingredient. As such, toxicity is derived directly from the molecule. Aerosol delivery of a compound to the lung may be problematic with regard to particle size and deposition to target sites. An advantage of gNO delivery to the lung is readily achievable, uniform diffusion and dispersion within the lungs. NO2 formation during gNO therapy is a concern, and may be exacerbated at high levels of NO delivery. Occupational safety and health standards limit the NO2 exposure of workers to 5 ppm or less (CDC 1998). Adverse effects of high NO2 have been reported throughout the literature and are potentially hazardous when NO is inhaled (Hurford 2005). NO is an approved drug for term infants, is inhaled continuously and may be inhaled for weeks (FDA 1999). The acceptable NO2 levels for inhaled NO delivery have been established at 2 ppm (Guidance for Industry 2000). Previous in-vitro work has shown that high levels of NO2 are not bactericidal and, further, high levels of NO2 (18 ppm) did not have deleterious toxic effects on a wide range of host cells 72  (Ghaffari 2006 and 2007). Nevertheless, 18 ppm NO2 is unacceptable from a clinical perspective and the attitude adopted should be to keep the inhaled NO2 level as low as possible during gNO therapy. NO is a free radical and formation of NO2 is concentration and time dependent. NO2 formation is further accelerated as the fractional inspired oxygen concentration increases, such as in the case of the compromised pulmonary patient. This is a significant challenge should gNO therapy be considered for further study as an inhaled antimicrobial agent. To date, there are no approved delivery devices capable of delivering 160 ppm gNO, let alone having the ability to keep the inhaled NO2 below 2 ppm. However, we were able to construct an unapproved prototype system that, in preliminary studies, proved effective. We hypothesized that increasing the total flow of gas from the 2 L/min used in the exposure chamber to that required for ventilation will reduce the dwell time and result in a significant reduction to 2–3 ppm NO2 formation. Prior to this regimen being extrapolated to human testing, it would be prudent to ensure that inhaled NO2 levels are kept below 2 ppm. These results suggest the use of high-dose, intermittent gNO as an inhaled antimicrobial agent has promise. We have calculated that the resulting metabolic burden would be within normal safety levels for methemoglobin. Further, we have shown this treatment regimen to be non-toxic to representative cells residing in the host airway. These results suggest that gNO should be explored for use against pathogens associated with pulmonary infections. However, clinical devices need to be engineered to deliver high-dose gNO to animal models while keeping the concomitant NO2 levels below 2 ppm. If this challenge can be overcome, animal models could be examined to evaluate and support these findings to further examine histopathological and toxicological sequelae. In this study we demonstrated that the intermittent dosing strategy of 160 ppm gNO for 30 minutes followed by a 3.5 hour recovery time is effective at reducing the pathogen load and is 73  generally well tolerated by host cells. We speculated that high-dose (160 ppm), intermittent dosing of gNO may be useful as a first line inhaled antimicrobial treatment to augment the innate defense system. The next step was to assess whether this dosing strategy could be safely delivered to the respiratory tract in a way that could be tolerated by the host. 6.1.4 The Safe Treatment of Normal Healthy Adult Humans The results provided in our treatment of 10 healthy adults show that 160 ppm gNO can be safely administered. The concentration of 160 ppm gNO was previously identified as an effective antimicrobial dose against a variety of microorganisms as described previously in in-vitro and invivo animal models (McMullin 2005; Ghaffari 2007; Miller 2009). We successfully demonstrated that 160 ppm gNO can be safely delivered five times a day for five consecutive days. This strategy of intermittent dosing maintained the delivered NO and O2 levels, and the resulting NO2, methemoglobin concentration, nitrite/nitrate measurements and vital signs were well within acceptable clinical parameters for the duration of the study. To the best of our knowledge, this is the first time that an antimicrobial dose of NO delivered to healthy volunteers has been reported, compared to the delivery of up to 80 ppm which is the approved dose for inhalational use in full term infants. The 100% increase in dose, albeit intermittent, might have caused problems. However, our findings were surprisingly similar and even slightly better than using continuous delivery of inhaled 80 ppm, with regard to concomitant NO2 and methemoglobin concentration. Methemoglobin levels reported in the supporting documentation for prescribing gNO show that methemoglobin levels rose to at least 5% with 35% of the subjects exceeding 7% during delivery of 80 ppm (INOmax package insert). They reported the mean peak concentration of NO2 for delivery of 80 ppm was 2.6 ppm. The ability to deliver 160  74  ppm gNO without increasing risk, compared to continuous 80 ppm gNO, is likely due to the intermittent dosing strategy utilized in this study. Our main concern prior to the study was the potential increase in methemoglobin levels. Several studies have evaluated the formation of methemoglobin during inhalation of gNO (Borgese 1987, Young 1994). Healthy volunteers have previously breathed gNO continuously at 32, 64, or 128 ppm for three hours, and 512 ppm for 55 min before methemoglobin reached 5% (Young 1996). The rise in methemoglobin, as well as the decay following cessation of inhaling the gNO in that study, followed a first order pharmacokinetics model. The clearance time-constants were between 39 and 91 minutes. The predicted half-life of methemoglobin in humans is approximately one hour. We hypothesized that administering high dose gNO for 30 minutes followed by a 3.5 hour break from treatment would allow methemoglobin levels to return to baseline between treatments (Miller 2009). The anticipated rise in methemoglobin during the 30 minutes, 160 ppm gNO treatment was calculated to be 1%. The actual average rise in methemoglobin percent for the 10 individuals for a single 30 minute gNO treatment was consistent with these kinetic estimates at 0.9% (SD=0.08), considering the ±1% absolute accuracy of the pulse oximeter. This study also confirmed that the predicted 3.5 hour interim period allowed the methemoglobin concentration to return to baseline, at which time another 30 minute gNO treatment of 160 ppm could be administered. This regimen was repeated for four more cycles; then repeated for five days without a significant clinical increase in baseline methemoglobin concentration. Thus, we have shown that the intermittent dosing strategy administering 160 ppm gNO appears to be safe from the standpoint of methemoglobin production and metabolic burden. Furthermore, the methods and results of this study seem to address the concerns about methemoglobinemia, asphyxia and 75  death associated with high concentrations of inhaled NO, using this intermittent dosing strategy (Hurford 2005). The limitations of this and other studies with regard to gNO delivery are that the NO and NO 2 levels are only known at the entry point into the subjects’ respiratory tract and the actual resulting levels of oxides of nitrogen in the lung are unknown. To date, studies indicate that acute pulmonary injury, pulmonary edema, hemorrhage, changes in surface tension of surfactant, reduced alveolar numbers and airway responsiveness may be caused by high airway levels of NO, NO2 and other oxides of nitrogen (Hurford 2005). It has been reported that human cells have significantly elevated thiol levels and can cope with high concentrations of NO better than prokaryotes (Meister 1988). We have reported that the same concentration level of gNO that is cidal to microbes may be tolerated by the host cells (Miller 2007). This is not surprising since the body’s innate defence mechanism relies on NO released from macrophage and neutrophils to act as one of its key non-specific antimicrobial agents (Fang 2004; MacMicking 1997). Despite this resilience to nitrosative stress, it may well be prudent in future studies to screen subjects for thiol and methemoglobin reductase deficiencies. Based on the available literature, it was predicted that high nitrogen oxide derivatives or acidic changes in lung fluid might manifest effects on pulmonary function tests performed on individuals with healthy lungs. This study demonstrates that 160 ppm gNO delivered as outlined, appears to have minimal impact on lung function. Specifically, acute inflammation in the airways as measured by flow rates was not detected. It is possible that potential deleterious airway reactivity could be masked by the ameliorative smooth muscle relaxation that can be exerted by NO (Dupuy 1992). However, this seems unlikely as flow rates were maintained in the measurement periods well past the point when NO would still have been present. Perhaps in 76  unhealthy lungs, such as during a pulmonary infection, there may be more airway reactivity. Conversely, any potential bronchodilatory activity of gNO may prove to be an additional benefit of utilizing gNO as an antimicrobial agent to treat pulmonary infections associated with asthma and cystic fibrosis. The results of lung parenchymal injury as measured by lung volumes and DLCO after gNO treatments did not reveal any changes. Additional indicators such as plasma inflammatory cytokines, eosinphils and neutrophils showed no change. The earliest host responses to viral infection and lung injury involve cytokine release. Specifically, tumor necrosis factor and interferon gamma have been shown to be active in many cells lines and to lead to the generation and release of inflammatory mediators (O’shea 1997, Levy 1997, Staeheli 1990). Studies in adults suggest that prevention of lung injury decreases the amount of circulating cytokines. Ranieri (1999), showed that a “lung-protective strategy” of respiratory support reduced cytokine levels in both serum and bronchoalveolar lavage fluid of adult patients with adult respiratory distress syndrome (ARDS). Pulmonary cytokine levels also appear to be elevated in some infants on assisted ventilation (Kotecha 1996; Jobe 1998; Bagchi 1994). Pro-inflammatory mediators may be elevated because of fetal exposure to maternal inflammatory mediators, postnatal infection, or due to ventilator-induced injury of preterm lungs (Zimmermann 1995). In addition, the vascular endothelial activation factors Ang-1, -2 and the Ang-2/Ang-1 ratio were unaffected by gNO treatments in our study. Elevated levels of plasma Ang-2 and an Ang-2/Ang-1 ratio favouring Ang-2 have been reported in patients with vascular endothelium activation and may have a prognostic role for mortality and clinical morbidity over the duration of critical illness (Conroy 2010; Conroy 2009; Lovegrove 2009; Yeo 2008). Recently it has been shown that plasma levels of Ang-1 and –2 correlated with severity of illness, morbidity (multiple organ 77  dysfunction), cardiovascular status, and 28-day mortality in patients with severe sepsis. (Ricciuto 2011). Three days and 28 days post-treatment were deemed sufficient time intervals to detect changes in lung diffusion, pulmonary function mechanics and inflammatory markers. These measurements were all unchanged compared to baseline values. We cannot exclude the possibility that some longer term change may occur in lung function, but the absence of any sign of an inflammatory reaction in the post treatment period makes this unlikely. Serum inflammatory markers may not have been sensitive enough to measure acute or even chronic changes in the lungs. Ideally, inflammatory markers from bronchoalveolar lavage would have been sampled, but in this study design it was not deemed ethical to perform this measurement on the healthy volunteers enrolled. It is important to deliver higher doses of gNO than the currently approved dose of 80 ppm since this dose is not likely to be an effective antimicrobial dose. In earlier in-vitro work exploring the mechanisms of the antimicrobial action of NO, it was found that 40 and 80 ppm gNO were not effective bactericidal doses. Other in-vitro and in-vivo work reported in the literature has concluded that doses below 90 ppm would not be clinically efficacious (Weibert 2000; Jean 2002). This has also been confirmed clinically by Long, who showed that continuous inhaled 80 ppm gNO for 72 hours was ineffective at decontaminating the airway in patients with Mycobacterium tuberculosis (Long 2005). We speculate, based on subsequent in-vitro results, that had they used a higher dose, their results may have shown a significant reduction in bacterial load. This study was not designed to address whether this gNO delivery regimen and dose would have an antimicrobial effect. It is possible that concentrations greater than 160 ppm gNO may be needed to achieve sufficiently high concentrations of NO in atelectatic or consolidated areas of the lung. Conversely, NO is a potent nitrosylating agent and could act as a mucolytic by 78  oxidizing disulfide bonds. This may improve both bronchial toilet and ventilation perfusion matching.  6.2 Summary We undertook an examination of the direct application of gNO to inhibit the influenza virus and the possibility of safely delivering gNO, in antimicrobial concentrations, to healthy humans. To accomplish this we undertook four specific objectives: 1. Determine the effect of the direct application of gNO on the influenza virus with an in-vitro testing methodology. 2. Determine a mechanism of action by which gNO inhibits the influenza virus. 3. Identify a viable treatment regime that would allow antimicrobial concentrations of gNO to be safely administered to humans. 4. Validate the safety of the potential treatment regime in normal healthy adult humans. We have successfully accomplished all of our stated objectives and we have demonstrated the antimicrobial potential of gNO.  6.3 Future Directions We feel that we have now pioneered a whole new field of the research into virology, the direct application of exogenous gNO. In Chapter 2 we demonstrated that gNO was capable of inhibiting influenza in an in-vitro testing methodology. We established and validated a testing methodology that would allow us to screen a variety of viruses. As described in Chapter 1, at least one virus from each of the Baltimore classes was shown to be inhibited by NO. Future research should focus on screening a wide 79  variety of viruses to determine which types are susceptible to gNO and determine at which point of their viral life cycle gNO is able to act. In this context, it would be of interest to see which viruses are susceptible to gNO as free virions before they infect the cells. This will allow us to further direct research towards viruses that may be susceptible to gNO. Additionally, it would be of interest to compare enveloped viruses and non-enveloped viruses to assess if treating with gNO before infection affects one more than the other. In Chapter 3 it was demonstrated that gNO nitrosylates the influenza virus (most likely the surface protein hemagglutinin), and we were able to detect the nitrosylation with SNOB. The use of SNOB broadens the prospects of identifying viral targets of nitrosylation. While we have identified that gNO most likely nitrosylates hemagglutinin, further studies should be done to understand how nitrosylation inhibits the influenza life cycle. While it has been shown that altering the cysteines in the cytoplasmic tail prevents pore formation, thereby disrupting the insertion of genetic material from the virus into the host (Naeve 1990, Fischer 1998), future studies should specifically outline how the nitrosylation of hemagglutinin inhibits the infectivity of influenza. Identification of targets can be accomplished by isolating the specific nitrosylated proteins using gel-electrophoresis and sequencing the specific protein band, followed by mass spectrophotometry to positively identify the protein. Another method that may also elicit the mechanism would be to use antibodies specific to the HA in order to positively identify the HA as a target of nitrosylation. Additionally, we would like to explore the potential of gNO to modify the ion channel proteins Influenza A/M2 and Influenza B/BM2. The M2 proteins form a proton-selective ion channel which is essential for virus uncoating (Gaston 1999, Mould 2003). The Influenza A/M2 protein is the target for the amantadine class of antiviral drugs. The amanatidines have no effect on Influenza B/BM2. 80  Perhaps the difference in susceptibility to gNO seen in Chapter 2 between Influenza A and Influenza B may be due to in the difference in strains of Influenza B. We only used one sub-type of Influenza B virus, specifically Influenza B/Hong Kong/5/72. Other sub-types of Influenza B should be tested to determine if the differences between Influenza A and B are consistent throughout or if they are limited to Influnenza B/Hong Kong/5/72. Expanding the number and variety of strains of influenza tested will help elucidate the mechanisms by which gNO inhibits infectivity. In Chapters 4 and 5, we provided evidence of a safe, intermittent dosing strategy for delivering effective antimicrobial doses (160 ppm) gNO to a variety of bacteria and influenza viruses. Then we demonstrated that this antimicrobial gNO delivery regimen was tolerated without adverse side effects when delivered to healthy adults. This data provides a strong rationale to justify further in-vivo studies. These studies could include animal models of respiratory infection as well as studies on stable non-healthy human subjects with mild respiratory infections. Of particular interest is the potential for gNO within the context of a pandemic caused by a respiratory pathogen. With the broad spectrum nature of gNO it may prove beneficial. For example, gNO has been shown to have antimicrobial properties against not only viruses, but also bacteria, fungi, and parasites, hence it is referred to as broad spectrum. In this context, gNO can be administered at any time during the infection cycle, even before the invading organism has been identified. Another condition that may benefit from gNO is cystic fibrosis, where bacteria grow out of control, forming a biofilm, because of impaired mucociliary clearance in the lung. This can lead to pneumonia, and also predispose the subject to viral infections or vice versa with a viral infection allowing a secondary bacterial infection. gNO can be useful as an antimicrobial in controlling the pathogens causing both primary and secondary infections. gNO may also have 81  mucolytic properties, due to its ability to disrupt sulphur bonds, thus warranting further investigation. Since the turn of the 21stcentury, the world has experienced two pandemics of viral origin; the 2004 SARS and 2009 H1N1. As we have shown, gNO is very effective at eradicating influenza before it infects a cell and retains effectiveness at reducing the number of infectious particles after the cell has been infected. With its broad, non-specific antimicrobial properties, gNO (160 ppm) may prove effective as a prophylactic treatment during a pandemic. Prophylactic treatment may prove to be useful for several reasons: first, gNO is very effective at preventing infectivity of host cells and, second, gNO is a non-specific broad spectrum antimicrobial, which allows therapy to be initiated before the infecting organism has been identified. Initiating therapy at the earliest possible time, even before the organism has been identified, may help prevent infection. Preventing infection can halt the spread of the organism, thereby limiting the progression of the infection and transmission to others. With continuous exposure of gNO, we were limited to only three hours continuously. High-dose gNO can be delivered intermittently over many days, which may prove to have therapeutic effects, which may also prove beneficial in a pandemic. Our results show the promise of gNO against influenza, and, when combined with Akerstrom’s (2009) results showing NO working against SARS, highlight the potential of gNO to disrupt a pandemic. Pandemics are not only a concern to humans, but also to animals. In 2004, an outbreak of highly pathogenic influenza (HPAI) H7N3 infected approximately 1.3 million domestic poultry in British Columbia. In an attempt to control the spread, depopulation of the infected birds was undertaken, and heightened surveillance of the workers involved. This intensive screening  82  identified two workers who became infected with the H7N3 virus. Fortunately, the virulence of this virus in humans was low and both workers recovered (Tweed 2004). We were provided access to the clinical isolates of the HPAI- H7N3, thanks to Dr. Martin Petric, clinical virologist at the BCCDC (recently retired). I exposed the clinical isolates to gNO, in the Level-3 biosafety lab at the BCCDC, using a modified methodology similar to that described in Chapter 2. I was able to demonstrate the complete inhibition of the infectivity of HPAI- H7N3 (results are not included in this work). This result is consistent with the result reported above and highlights that gNO may also prove beneficial not only for human infection but also for other species, such as domesticated poultry. Future work in this area is of great interest to our group.  6.4 Conclusion For the first time, gNO, delivered exogenously, was shown to inhibit the infectivity of the influenza virus. This may be achieved through nitrosylation of hemagglutinin by gNO. We developed and validated an intermittent dosing strategy that allowed for the delivery of an antimicrobial dose of 160 ppm gNO that eradicated bacteria and inhibited influenza. 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Zimmerman JJ: Bronchoalveolar inflammatory pathophysiology of bronchopulmonary dysplasia. Clin.Perinatol. 1995. 22: 429-456.  94  Appendices Appendix 1: Supporting Graphs for Chapter 4  A 400  300  1.0×10 03  200  100  1.0×10 00 0  5  10  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 20  15  Time (Hours)  B 400  300  1.0×10 03  200  100  1.0×10 00  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 0  1  2  3  4  5  6  7  Time (Hours)  Figure A1.1 Comparison of Bacterial Survival Curves Between Intermittent Short Duration and Continuous Exposure to gNO. Panel AB: represent the results of S. Aureus cultures; CD: P. aeruginosa cultures (cystic fibrosis isolate); EF: E. Coli cultures. Bacteria were either exposed to repeated cycles of 160 ppm gNO for 30 minutes followed by medical air for 3.5 hours (Panel A, C, E dashed line with triangles) or continuous 160 ppm gNO (Panel B,D, F dashed line with triangles). Treatment regimens were compared to medical air continuously as a control (squares), for up to 24 hours. Solid line at graph bottom represents gNO dose regimen. Results were reported as the means ± standard error of the mean from at least three independent measurements.  95  C 400  300  1.0×10 03  200  100  1.0×10 00 0  5  10  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 20  15  Time (Hours)  D 400  300  1.0×10 03  200  100  1.0×10 00  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 0  1  2  3  4  5  6  7  Time (Hours)  Figure A1.1 Comparison of Bacterial Survival Curves Between Intermittent Short Duration and Continuous Exposure to gNO. Panel AB: represent the results of S. Aureus cultures; CD: P. aeruginosa cultures (cystic fibrosis isolate); EF: E. Coli cultures. Bacteria were either exposed to repeated cycles of 160 ppm gNO for 30 minutes followed by medical air for 3.5 hours (Panel A, C, E dashed line with triangles) or continuous 160 ppm gNO (Panel B,D, F dashed line with triangles). Treatment regimens were compared to medical air continuously as a control (squares), for up to 24 hours. Solid line at graph bottom represents gNO dose regimen. Results were reported as the means ± standard error of the mean from at least three independent measurements.  96  E 400  300  1.0×10 03  200  100  1.0×10 00 0  5  10  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 20  15  Time  F 400  300  1.0×10 03  200  100  1.0×10 00  gNO Dosage (ppm)  Survival (cfu/mL)  1.0×10 06  0 0  1  2  3  4  5  6  7  Time  Figure A1.1 Comparison of Bacterial Survival Curves Between Intermittent Short Duration and Continuous Exposure to gNO. Panel AB: represent the results of S. Aureus cultures; CD: P. aeruginosa cultures (cystic fibrosis isolate); EF: E. Coli cultures. Bacteria were either exposed to repeated cycles of 160 ppm gNO for 30 minutes followed by medical air for 3.5 hours (Panel A, C, E dashed line with triangles) or continuous 160 ppm gNO (Panel B,D, F dashed line with triangles). Treatment regimens were compared to medical air continuously as a control (squares), for up to 24 hours. Solid line at graph bottom represents gNO dose regimen. Results were reported as the means ± standard error of the mean from at least three independent measurements.  97  4  *  Cell Proliferati on (OD540)  3 Air  2  gNO  1  0 24 h  48 h  72 h  Figure A1.2 Effect of gNO on Proliferation of Epithelial Cells. Following intermittent exposure to 160 ppm gNO for up to 72 hours, an MTT proliferation assay was carried out to determine the number of viable cells in culture. Data represents the amount of formazan production by metabolically active cells at OD540. The asterisk (* p < 0.01) denotes a significant difference in proliferation between gNO and control group at 72 hours.  98  % migration  B  8 0 6 0 4 0 2 0 0  * Air gNO  Day 1  Day 2  Day 3  Figure A1.3 Effect of 160ppm gNO on A549 Cells Migration. (A) Microscopic photographs illustrating epithelial cell migration behavior under exposure of gNO or medical air. (B) Quantitative comparison of percent migration relative to baseline (day 0) in both groups. The asterisk (*p < 0.01) denotes significant difference between migration rate of gNO and control group in day 1. Scale bar represents 100 µm.  99  *  70  Colony count  60 50 40 30  20 10 0 Air  8  4  2  0  gNO  γ radiation (Gy) Figure A1.4 Lung Epithelial Cell Clonogenic Survival Assay. A549 cells were exposed to air or to 160 ppm gNO for 24 hours. Cells were exposed to 0, 2, 4, or 8 gray of gamma radiation, Cell colonies were counted on day 10 post-treatment. Each bar represents the mean values from a minimum of three independent determinations ± standard deviation. The asterisk denotes a significant difference (p < 0.01) between gNO and gamma radiated group colony counts.  100  A 100  B  Survival (%)  Survival (%)  75 50 25 0 Air  gNO  75 50 25 0 Air  gNO  Figure A1.5 Cytotoxic Effects of Nitric Oxide. A. In-vitro studies of the effect of 200 ppm gNO (24 hours) on human macrophage survival. B. In-vitro studies of the effect of 200 ppm gNO (24 hours) on human monocytes survival. C. FACS histograms of monocytes survival. Toxicity was measured as death rate by flow cytometry with PI (Propidium Iodide). Black outline represents THP-1 monocytes exposed to intermittent 160/20 ppm gNO. Solid represents THP-1 monocytes exposed to continuous (24 hours) 20 ppm gNO.  101  Appendix 2: The Safety Trial  Table A2.1 Synopsis of the Safety Trial Protocol TITLE  A Phase I Open label Safety Study of Inhaled Gaseous Nitric Oxide (gNO) for Administration to Healthy Adults for Potential Use as a Frontline Treatment During a Pandemics  FUNDING  Lotte & John Hecht Memorial Foundation  INVESTIGATORS  Dr. Yossef Av-Gay Ph.D, Dr. Chris Miller, Ph.D, RT, Dr. Jeremy Road MD.  INDICATION  Respiratory Tract Infection  POPULATION  Healthy adult volunteers without pulmonary disease  PRIMARY OBJECTIVES          SECONDARY OBJECTIVES        STUDY DESIGN  Determine the safety of inhaled gNO for healthy subjects Determine the safety of a gNO delivery device in healthy subjects Determine the effect of inhaled gNO on Pulmonary Function Test (PFT) including Lung Diffusion (DL) % of predicted Determine the effect of inhaled gNO on FEV1 % Determine the Met-Hemoglobin percentage (MetHb) associated with inhaled gNO Determine the effect of inhaled gNO on lung inflammatory serum markers Determine adverse events associated with inhaled gNO Number of subjects with MetHb level >5% at any time point Number of subjects with study drug related bleeding at any time point Number of subjects experiencing sudden hypoxemia (SaO2%< 90%) MetHb levels increasing to > 5% over time Incidence of adverse events  Prospective, open label controlled single-Center study.  102  NUMBER OF SITES  Vancouver Coastal Health Region, Vancouver, Canada  ENROLLMENT GOALS  Up to 20 subjects will be recruited. Ten healthy subjects enrolled.  DURATION OF SUBJECT PARTICIPATION  Total time on study/visits:  5 weeks; 9 visits  Screening:  Day -7 Hx, PFT/DL, MetHb% & Bloodwork  Assessment:  Day 1 (Baseline): FEV1% ,MetHb %  Treatment:  Day 1-5: 160 ppm gNO Tx, Q4h x 5  Assessment:  Day 1-5: FEV1% & MetHb & Bloodwork Day 2,5: PFT/DL  Follow-up:  Day 8, 12, 26: PFT/DL,MetHb% & Bloodwork  TRIAL DRUG  Phramaceutical Grade Gaseous NO (delivered with air and oxygen as a carrier) administered by inhalation  ADMINISTRATION  ViaNOx-H Pharmaceutical Grade Gaseous NO (gNO) will be administered using a ViaSys II system  DOSE     Subjects will inhale 160 ppm on gNO Estimate of maximum total exposure to gNO (2,000 ppm hours)  E DOSING SCHEDULE    Inhalations are administered for 30 minutes for a maximum of five times for five days. Inhalations are performed every four hours (± 30 minutes) with a minimum of three hours between the end of one treatment and the beginning of the next treatment.  Concomitant medications    Allowed: Except as noted below all usual medications are allowed. Prohibited: All adjunctive therapies and medications are allowed     Primary endpoint     No unanticipated adverse events Mean change in FEV1 , PFT/DL, inflammatory cytokines and MetHb % predicted (absolute) from baseline 103  Secondary endpoints        SAFETY MONITORING    SUBJECT SELECTION    CRITERIA  Number of subjects with MetHb level >5% at any time point Number of subjects with study drug related bleeding at any time point Number of subjects experiencing sudden hypoxemia (SaO2%< 90%) MetHb levels increasing to > 5% over time Incidence of adverse events Oversight for this trial will be by an independent respirologist experienced in treating pulmonary disease and working with nitric oxide. The respirologist can recommend early termination of the trial for reasons of subject safety. Safety data will be monitored on a continual basis throughout the study.  Inclusion Criteria: 1. Written informed consent and HIPAA authorization 2. Male or female ≥19 years of age. 3. Female not pregnant at time of study i. FEV1 ≥ 75 % of predicted ii. Oxygen saturation on room air >90% at screening iii. Non-smoker for at least 6 months prior to screening and agrees not to smoke during the study iv.  Exclusion Criteria: v. Use of an investigational drug within 30 days of screening vi. Has symptoms or diagnosis of a pulmonary disease vii. History of frequent epistaxis (>1 episode/month) viii. Significant hemoptysis within 30 days (≥ 5 mL of blood in one coughing episode or > 30 mL of blood in a 24 hour period) ix. Methemoglobin >3% at screening x. Changes to antibiotics (e.g. azithromycin) from 7 days prior to screening through last treatment day . (Subjects may be taking antibiotics during this time period, but they cannot start, stop or change doses during this time period) 4. Organ transplant recipient. 5. For female subjects: pregnant or unwilling to practice birth control during participation in the study. 6. Presence of a condition or abnormality that in the opinion of the Investigator would compromise the safety of the patient or the quality of the data  104  STATISTICS  This pilot clinical evaluation is intended to demonstrate safety and proof of principle, and the sample size is not necessarily powered or nor intended to establish statistically significant differences. Treatment outcomes on a sample size of 10 subjects will be sufficient to confirm safety and determine initiation of a submission for a follow-on study. The safety of the treatment based on indicators will be helpful in establishing safety requirements for future efficacy trials. Nevertheless, descriptive characteristics of the subjects prior to, during, and at the end of the study will be tabulated. Differences between important continuous variables at two specific times will be evaluated with the paired t-test. Important categorical events such as reduction in viral/bacterial load, clinical FEV1% and PFT/DL improvement, changes in serum inflammatory markers and incidence of adverse events will be evaluated by constructing 95% confidence limits in their incidence.  105  

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