WO2017130066A1

WO2017130066A1 - Methods of treatment of respiratory tract infections and cystic fibrosis

Info

Publication number: WO2017130066A1
Application number: PCT/IB2017/000118
Authority: WO
Inventors: David Greenberg; Asher TAL; Yossef Av-Gay
Original assignee: Advanced Inhalation Therapies (Ait) Ltd.
Priority date: 2016-01-27
Filing date: 2017-01-27
Publication date: 2017-08-03

Abstract

The present invention provides a method for treating cystic fibrosis in a human subject in need thereof, wherein the method comprises repeatedly administering to the human subject a gas mixture comprising nitric oxide at a concentration from about 144 to about 176 ppm for a first period of time, followed by a gas mixture containing no nitric oxide for a second period of time, wherein the administration is repeated for a time sufficient to: a) reduce the level of at least one inflammatory biomarker in the human subject when compared to the level of the inflammatory biomarker prior to the administration; b) reduce the microbial density by 1 to 2 log units as measured by colony forming units in the human subject when compared to the microbial density prior to the administration; or c) a combination thereof.

Description

METHODS OF TREATMENT OF RESPIRATORY TRACT INFECTIONS

AND CYSTIC FIBROSIS

RELATED APPLICATIONS

[1] This application claims the priority of U.S. Patent Application No. 62/

287,654, filed January 27, 2016; entitled "TREATMENT OF RESPIRATORY TRACT INFECTIONS AND CYSTIC FIBROSIS," which is incorporated herein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[2] The present invention, in some embodiments thereof, relates to therapy, and more particularly, but not exclusively, to methods and devices for treating respiratory tract infections or cystic fibrosis in human subjects.

BACKGROUND OF THE INVENTION

[3] Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. The classical signs of acute inflammation are pain, heat, fever, redness, swelling, and loss of function.

[4] Respiratory tract infections (RTIs) are a major cause of hospitalization, economic burden, mortality, and morbidity worldwide. In the United States (US) alone, RTIs result in over 1.5 million hospitalizations annually. Bronchiolitis is defined as an infection of the small airways. It is also the most common manifestation of acute lower respiratory infection (ALRI) in early infancy, and is the leading cause of global child mortality.

[5] Cystic fibrosis (CF) is an inherited monogenic disorder that presents as a multisystem disease that causes severe lung damage and nutritional deficiencies. CF affects cells that produce mucus, sweat, and digestive juices. The defective gene causes these secretions to become thick and sticky and affect the ability of organs such as the lungs and pancreas to function efficiently. Human subjects diagnosed with, or suffering from CF are highly prone to environmental opportunistic bacterial infections leading to prolonged and chronic lung infections. This results in reduction in the life expectancy of human subjects diagnosed with or suffering from CF due to excessive lung tissue destruction.

SUMMARY OF THE INVENTION

[6] In one embodiment, the present invention provides a method for treating cystic fibrosis in a human subject in need thereof, wherein the method comprises repeatedly administering to the human subject a gas mixture comprising nitric oxide at a concentration from about 144 to about 176 ppm for a first period of time, followed by a gas mixture containing no nitric oxide for a second period of time, wherein the administration is repeated for a time sufficient to: a) reduce the level of at least one inflammatory biomarker in the human subject when compared to the level of the inflammatory biomarker prior to the administration; b) reduce the microbial density by 1 to 2 log units as measured by colony forming units in the human subject when compared to the microbial density prior to the administration; or c) a combination thereof.

[7] In one embodiment, the human subject suffers from a microbial infection associated with cystic fibrosis.

[8] In one embodiment, the microbial infection is caused by a pathogenic microorganism.

[9] In one embodiment, the pathogenic microorganism is selected from the group consisting of Pseudomonas alcaligenes, non-mucoid and mucoid Pseudomonas aeruginosa, Aspergillus fumigates, Staphylococcus aureus, Haemophilus influenza, Burkholderia cepacia complex, Klebsiella pneumonia, Escherichia coli, methicillin- resistant Staphylococcus aureus (MRSA), methicillin- sensitive Staphylococcus aureus (MSSA), Stenotrophomonas maltophilia, Achromobacter spp., Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, non-tuberculous mycobacteria (NTM) species, non-mucoid Pseudomonas aeruginosa, Mycobacterium abscessus complex (MABSC), mucoid Pseudomonas aeruginosa, and Acinetobacter baumannii.

[10] In one embodiment, the first time period is 30 minutes and the second time period is from about 3 to about 5 hours.

[11] In one embodiment, the administration is repeated 6 times per day.

[12] In one embodiment, the nitric oxide is repeatedly administered for a period of time from about one day to three weeks. [13] In one embodiment, the nitric oxide is repeatedly administered for 5 days.

[14] In one embodiment, the intermittent inhalation comprises: (1) repetitive administration of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, continuously for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

[15] In one embodiment, the intermittent inhalation comprises: (1) repetitive administration of 5 cycles per day of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

[16] In one embodiment, the at least one inflammatory biomarker is selected from the group consisting of C-reactive protein (CRP), TNFa, TNF RII, IL- 1β, IL-lra/IL-lF3, IL-2, IL-4, IL-5, IL-6, IL-8, CXCL8/IL-8, IL-10, IL-12 p70, IL- 17A, GM-CSF, ICAM-1, IFN-gamma, MMP-8, MMP-9, VEGF and IL-12p70, neutrophils, lymphocytes and eosinophils count, neutrophil elastase activity, alpha- 1- antitrypsin (AAT), haptoglobin, transferrin, an immunoglobulin, granzyme B (GzmB), eosinophil cationic protein (ECP), eotaxin, tryptase, chemokine C-C motif ligand 18 (CCL18/PARC), RANTES (CCL5), surfactant protein D (SP-D), lipopolysaccharide (LPS)-binding protein and soluble cluster of differentiation 14 (sCD14).

[17] In one embodiment, the at least one inflammatory biomarker is C- reactive protein (CRP). [18] In one embodiment, the method further comprises monitoring at least one on-site oximetric parameter in the subject, the on-site parameter being selected from the group consisting of: oxyhemoglobin saturation (SpC^); methemoglobin (SpMet); perfusion index (PI); respiration rate (RRa); oxyhemoglobin saturation (SpCh); total hemoglobin (SpHb); carboxyhemoglobin (SpCO); methemoglobin (SpMet); oxygen content (SpOC); and pleth variability index (PVI).

[19] In one embodiment, the method further comprises monitoring at least one additional on-site spirometric parameter in the subject, the at least one additional on-site parameter being selected from the group consisting of: forced expiratory volume (FEV1); maximum mid-expiratory flow (MMEF); diffusing capacity of the lung for carbon monoxide (DLCO); forced vital capacity (FVC); total lung capacity (TLC); and residual volume (RV).

[20] In one embodiment, the method further comprises monitoring at least one on-site parameter in the gas mixture inhaled by the subject, the on-site parameter being selected from the group consisting of: end tidal CO₂ (ETCO₂); nitrogen dioxide (NO₂), nitric oxide (NO); serum nitrite/nitrate; and fraction of inspired oxygen (F1O₂).

[21] In one embodiment, the method further comprises monitoring at least one off-site bodily fluid parameter in the subject, the parameter being selected from the group consisting of: a bacterial and/or fungal load; urine nitrite; blood methemoglobin; blood pH; a coagulation factor; blood hemoglobin; hematocrit ratio; red blood cell count; white blood cell count; platelet count; vascular endothelial activation factor; renal function; an electrolyte; a pregnancy hormone; serum creatinine; and liver function.

[22] According to further aspects of the present invention, there are provided devices and systems for effecting the intermittent inhalation described herein in any of the methods described herein, as is detailed hereinafter.

[23] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[24] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[25] Figure 1 shows the Forced Expiratory Volume in 1 second (FEVi) observed in one patient, prior to treatment according to some embodiments of the present invention.

[26] Figure 2 shows the change in Forced Expiratory Volume in 1 second

(FEVi) relative to baseline values, observed in one patient treated according to some embodiments of the present invention.

[27] Figure 3 shows the change in Forced Expiratory Volume in 1 second

(FEVi) as a percentage of predicted value, observed in one patient treated according to some embodiments of the present invention.

[28] Figure 4 shows the Lung Clearance Index (LCI) observed in one patient treated according to some embodiments of the present invention.

[29] Figure 5 shows the mean perfusion index (PI) observed in one patient treated according to some embodiments of the present invention.

[30] Figure 6 shows the C-reactive protein (CRP) observed in one patient treated according to some embodiments of the present invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[31] The present invention, in some embodiments thereof, relates to therapy, and more particularly, but not exclusively, to methods and devices for treating respiratory tract infections or cystic fibrosis in human subjects.

[32] The principles and operation of the present invention may be better understood with reference to the figures and accompanying descriptions. [33] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[34] Cystic fibrosis (CF) is a genetic disorder in which mutations in the epithelial chloride channel, CF transmembrane conductance regulator (CFTR), impairs various mechanism of innate immunity. Chronic lung infections caused by pathogenic microorganisms are the leading cause of morbidity and mortality in human subjects diagnosed with, or suffering from CF. Early antibiotic eradication treatment of human subjects diagnosed with, or suffering from CF for the most prevalent bacterial pathogen, Pseudomonas aeruginosa, has considerably increased the life expectancy in CF, however still the vast majority of adult human subjects diagnosed with, or suffering from CF suffer from chronic lung infections which are difficult to treat due to biofilm formation and the development of antibiotic resistant strains of the virulent. Other species found in CF airways include antibiotic resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA), members of the Burkholderia cepacia complex, Haemophilus influenzae, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, non-tuberculous mycobacteria (NTM) species and various strict anaerobic bacteria. In addition, poor clearance of mucus from the bronchi causes general breathing difficulties in human subjects diagnosed with, or suffering from CF.

[35] In addition, human subjects diagnosed with, or suffering from CF are highly susceptible to infections with non-tuberculous mycobacteria. Mycobacterium abscessus complex (MABSC) is a complex of three closely related "species" of rapidly growing mycobacterium. Significant differences exist between M. abscessus isolates; Abscessus ssp abscessus (sensu stricto) is the most virulent compared to M. abscessus ssp bolletii Mycobacterium massiliense. With current antibiotic options, M. abscessus is a chronic incurable infection for most CF patients (negative culture>12m on therapy). MABSC is one of the most antibiotic-resistant RGM: very few drugs are potentially active, and of these, only few can be administered by the oral route. [36] Bronchiolitis is defined as an infection of the small airways. It is also the most common manifestation of acute lower respiratory infection (ALRI) in early infancy, and is the leading cause of global child mortality. Viral bronchiolitis is currently the most common reason for pediatric hospital admission in the US, accounting for almost 20% of all-cause infant hospitalizations. Viral etiology is the main cause, and among the respiratory viruses, respiratory syncytial virus (RSV) is believed to be the most important viral pathogen causing ALRI in young children. The disease is common mainly in the first year of life. The clinical signs and symptoms are consistent with hypoxia, difficulty breathing, coryza, poor feeding, cough, wheeze and crepitations on auscultation, and in some cases respiratory failure.

[37] In some aspects of the present invention, intermittent dosing and delivery by inhalation of nitric oxide, cycling between high concentrations of nitric oxide for a relatively short period of time and longer periods of no or low concentration of nitric oxide has been shown to overcome the problems of nitric oxide toxicity in humans of all ages. In some embodiments, it has been shown that the high concentration of nitric oxide, delivered according to an intermittent regimen, is effective in overwhelming the nitric oxide defense mechanisms of pathogens, and hence that at such a high concentration, nitric oxide exhibits a pronounced antimicrobial effect.

[38] In one embodiment, the present invention provides a method that administers nitric oxide to a human subject, wherein the administration is short durations of high concentrations of nitric oxide, that improves lung function and reduce microbial infections and inflammatory symptoms in human subjects diagnosed with, or suffering from CF, while not causing lung injury or other signs of adverse effects. In one embodiment, forced expiratory volume in 1 sec (FEVi), LCI, mean perfusion index, and C-reactive protein (CRP) levels were improved. It was thus demonstrated that intermittent inhalation of 160 ppm nitric oxide or more is safe and well tolerated in human subjects diagnosed with, or suffering from CF and is beneficial in terms of alleviation of CF symptoms.

[39] Herein-throughout, whenever the term "nitric oxide" is used in the context of inhalation, it is to be understood that nitric oxide is inhaled in the gaseous state. Cystic Fibrosis:

[40] According to some embodiments of the present invention, there is provided a method of treating cystic fibrosis in a human subject (e.g., a human subject afflicted with cystic fibrosis, a human subject diagnosed with cystic fibrosis, or a human subject suffering form cystic fibrosis). Diagnosis of cystic fibrosis can be effected by methods known in the art, including the methods described in the Examples section that follows.

[41] The method according to some embodiments comprises subjecting the human subject to intermittent inhalation of a gaseous mixture that comprises nitric oxide, as described in any one of the embodiments pertaining to intermittent inhalation, and any combination thereof.

[42] According to embodiments of the present invention, the method of treating a human subject suffering from CF encompasses any beneficial therapeutic effect exhibited in a human subject diagnosed with, or suffering from CF, including, for example, amelioration of a symptom of CF (e.g., improvement of a pulmonary function), amelioration of a medical condition associated with CF (e.g., reduction of a microbial infection associated with CF, reduction of the load of a pathogenic microorganism which is associated with CF, reduction of inflammation), amelioration of an adverse effect caused by another treatment of CF, reduction of mortality in human subjects diagnosed with, or suffering from CF and general improvement of the medical and mental condition of a human subject diagnosed with, or suffering from, CF.

[43] In some embodiments, a method of treating CF as described herein is regarded as a method of treating a CF patient (e.g., a subject afflicted by cystic fibrosis, a subject diagnosed by cystic fibrosis), and encompasses a method of ameliorating a symptom of CF (e.g., improvement of a pulmonary function), ameliorating a medical condition associated with CF (e.g., treatment of a microbial infection associated with CF, reduction of the load of a pathogenic microorganism which is associated with CF, reduction of inflammation), ameliorating an adverse effect caused by another treatment of CF, prolonging the life time of a human subject diagnosed with, or suffering from CF, and/or generally improving a medical and/or mental condition of a human subject diagnosed with, or suffering from CF. [44] In terms of following the efficacy of the treatment of CF in a human subject diagnosed with, or suffering from CF, it is generally accepted that pulmonary function is one of the most simple and direct marker for alleviating the symptoms of CF, and hence that improvement of a pulmonary function is a human subject represents a beneficial treatment of a human subject diagnosed with, or suffering from CF.

[45] One of the primary complications of CF is the accumulation of airway phlegm, which contains predominantly bacteria, inflammatory cells, polymeric DNA, and F-actin. The bacterial colonizations and infections are most often caused by Staphylococcus aureus, Pseudomonas aeruginosa, and Haemophilus influenzae. Escherichia coli and Klebsiella pneumoniae present as chronic colonization develop in the airways. Burkholderia cepacia has been isolated in older human subjects and is associated with a rapid decline in pulmonary function progressing to death.

[46] Without being bound by any particular theory, it is assumed that nitric oxide, delivered in an exogenous gaseous form, easily enters the pulmonary system and acts by pulmonary vasodilatation, reducing bacterial load, reducing inflammation, and alleviating other clinical symptoms.

[47] Nasal nitric oxide concentration has been found to be significantly lower in human subjects diagnosed with, or suffering from CF than in controls, and this reduced nitric oxide may play a role in bronchial obstruction and reduced defense to bacterial infections observed in human subjects diagnosed with, or suffering from CF.

[48] In some embodiments, the method as described herein, in any one of the embodiments thereof, and in any combination thereof, is effected by improving one or more physiological parameters in a human subject diagnosed with, or suffering from CF which worsen by a medical condition associated with CF. An improvement of any of these parameters is indicative of the beneficial effect of the treatment by intermittent inhalation of nitric oxide, according to any one of the embodiments described herein.

[49] According to some embodiments of the present invention, the method is effected by improving at least one pulmonary function (spirometric parameter), such as, but not limited to, Forced Expiratory Volume in 1 second (FEVi), Forced Vital Capacity (FVC), FEVi/FVC ratio or FEVi% and Forced Expiratory Flow (FEF). [50] The spirometric parameter Forced Vital Capacity (FVC) is the volume of air measured in liters, which can forcibly be blown out after full inspiration, and constitutes the most basic maneuver in spirometry tests.

[51] The spirometric parameter Forced Expiratory Volume in the 1st second

(FEV1) is the volume of air that can forcibly be blown out in one second, after full inspiration. Average values for FEVi depend mainly on sex and age, whereas values falling between 80 % and 120 % of the average value are considered normal. Predicted normal values for FEVI can be calculated on-site and depend on age, sex, height, weight and ethnicity as well as the research study that they are based on.

[52] The spirometric parameter FEVi/FVC ratio (FEVi%) is the ratio of

FEVi to FVC, which should be approximately 75-80 %. The predicted FEVi% is defined as FEVi% of the patient divided by the average FEVi% in the population appropriate for that patient.

[53] The spirometric parameter Forced Expiratory Flow (FEF) is the flow

(or speed) of air coming out of the lung during the middle portion of a forced expiration. It can be given at discrete times, generally defined by what fraction remains of the forced vital capacity (FVC), namely 25 % of FVC (FEF25), 50 % of FVC (FEF5₀) or 75 % of FVC (FEF₇₅). It can also be given as a mean of the flow during an interval, also generally delimited by when specific fractions remain of FVC, usually 25-75 % (FEF25-75). Measured values ranging from 50-60 % up to 130 % of the average are considered normal, while predicted normal values for FEF can be calculated on-site and depend on age, sex, height, weight and ethnicity as well as the research study that they are based on. Recent research suggests that FEF₂5-75% or FEF₂5-5o% may be a more sensitive parameter than FEVi in the detection of obstructive small airway disease. However, in the absence of concomitant changes in the standard markers, discrepancies in mid-range expiratory flow may not be specific enough to be useful, and current practice guidelines recommend continuing to use FEVi, VC, and FEVi/VC as indicators of obstructive disease.

[54] It is noted that in some embodiments, other spirometric parameters, as these are defined and described herein below, may be used to follow the progression and efficacy of CF treatment by intermittent inhalation of 160 ppm nitric oxide, and/or to follow safety parameters of the treatment. [55] According to some embodiments, FEVi is monitored as an on-site parameter, as defined hereinafter, which is indicative of the beneficial effect of the intermittent inhalation of nitric oxide, as provided herewith. In general, an increase in the FEVi level is regarded as a desired effect in human subjects diagnosed with, or suffering from CF, wherein an increase of at least 3 percent in the FEVi baseline level of the patient (before commencing the treatment) is regarded as a notable improvement. In some embodiments, the method is effected such that FEVi level is increased by at least 3, 5, 10, 15 or 20 percent during and/or after the intermittent inhalation (e.g., during and/or after the entire time period intermittent inhalation of nitric oxide is effected) of nitric oxide, as described herein.

[56] According to some embodiments of the present invention, the CF is associated with a microbial infection, that is, the human subject diagnosed with, or suffering from CF treated by a method as described herein suffers from a microbial infection. According to some embodiments of the present invention, the microbial infection is caused by one or more pathogenic microorganisms which can be for example, a Gram-negative bacterium, a Gram-positive bacterium, a virus and a viable virion, fungi and parasites.

[57] According to some embodiments, the method of treating CF comprises treating a microbial infection associated with CF (a microbial infection that typically develops in a human subject diagnosed with, or suffering from CF), and/or reducing a load of a pathogenic microorganism that causes a microbial infection associated with CF (also referred to as a pathogenic microorganism associated with CF).

[58] CF is typically associated with respiratory microbial infections caused by certain pathogens (pathogenic microorganisms associated with CF). These include, for example, Pseudomonas alcaligenes, non-mucoid and mucoid Pseudomonas aeruginosa, Aspergillus fumigates, Staphylococcus aureus, Haemophilus influenza, Burkholderia cepacia complex, Klebsiella pneumonia, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), methicillin- sensitive Staphylococcus aureus (MSSA), Stenotrophomonas maltophilia, Achromobacter spp., Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, Mycobacterium abscessus complex (MABSC), non- tuberculous mycobacteria (NTM) species, and Acinetobacter baumannii. [59] Such microbial infections can be regarded as a secondary condition to

CF, or as an opportunistic infection in human subjects diagnosed with, or suffering from CF.

[60] According to some embodiments, the pathogenic microorganism which is associated with CF is selected from the group consisting of Pseudomonas alcaligenes, methicillin-sensitive Staphylococcus aureus (MSSA), Achromobacter spp., Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, A. fumigates, Mycobacterium abscessus complex (MABSC), non-mucoid P. aeruginosa and mucoid Pseudomonas aeruginosa. According to some of these embodiments, the method as described herein comprises treating a microbial infection associated with CF and/or reducing the load of the pathogenic microorganism that causes the microbial infection (pathogenic microorganism associated with CF).

[61] As demonstrated in the Examples section that follows below, the method presented herein has been demonstrated to reduce the load of several strains of pathogens known to cause debilitating and even fatal infections in human subjects diagnosed with, or suffering from CF.

[62] According to embodiments of the present invention, the method is effected so as to reduce the load of the pathogenic microorganism in the subject by at least one log unit during the intermittent inhalation treatment.

[63] The term "log unit" as used herein to describe a change in the load of a pathogenic microorganism, also known as "log reduction" or "log increase", is a mathematical term used to show the relative number of live microbes eliminated from a system by carrying out the method of intermittent inhalation of nitric oxide, as presented herein. For example, a 5 log units reduction means lowering the number of microorganisms by 100,000-fold, that is, if a sample has 100,000 pathogenic microbes on it, a 5-log reduction would reduce the number of microorganisms to one. Hence, a 1 log unit reduction means the number of pathogenic microbes is 10 times smaller, a 2 log reduction means the number of pathogens is 100 times smaller, a 3 log reduction means the number of pathogens is 1000 times smaller, a 4 log reduction means the number of pathogens is 10,000 times smaller and so forth.

[64] As known in the art, CF is typically associated with a state of inflammation in at least one bodily site, e.g. the lungs, or an acute, chronic, local or systemic inflammation, cause by one or more medical conditions, including but not limited to pathogenic infections. Inflammation in human subjects diagnosed with, or suffering from CF can also be regarded as a secondary condition to CF (a medical condition associated with CF). According to some embodiments of the present invention, the method is effected by reducing the level of inflammation associated with CF.

[65] Reduction in inflammation associated with CF is typically regarded as a beneficial effect of the treatment of CF. Similarly, a reduction of a level of an inflammatory biomarker associated with CF can be regarded as an indication of efficacy of the method of treating a human subject diagnosed with, or suffering from CF as presented herein. For an exemplary provision of methods and discussion regarding the relationship of systemic inflammation to prior hospitalization in adult human subjects diagnosed with cystic fibrosis, see Ngan, D. A. et al, BMC Pulmonary Medicine, 2012, 12(3).

[66] In the context of some embodiments of the present invention, inflammatory or inflammation biomarkers associated with CF include, without limitation, serum/blood levels of C-reactive protein (CRP), cytokines such as interleukins IL-6 and IL-Ιβ, alpha- 1 -antitrypsin (AAT), haptoglobin, transferrin, various immunoglobulins, granzyme B (GzmB), chemokine C-C motif ligand 18 (CCL18/PARC), surfactant protein D (SP-D), lipopolysaccharide (LPS)-binding protein, and soluble cluster of differentiation 14 (sCD14).

[67] The term "cytokine", as used in the context of embodiments of the present invention, includes chemokines, interferons, interleukins, lymphokines and tumor necrosis factor.

[68] Following is a brief description of four non-limiting exemplary inflammatory biomarkers associated with CF.

[69] Tumor Necrosis Factor alpha (TNFa) signals to the body to bring the neutrophil white blood cells to the site of infection or injury. TNFa is known as a cytokine, or a cell-signaling protein. TNFa acts like a "first responder" at an accident by signaling to the body where the most damage is so that the immune system can respond effectively, which is to send neutrophils.

[70] Nuclear Factor kappa B (NFkB) is a transcription factor protein complex that acts as a switch for certain genes. When NFkB is allowed to enter the nucleus, which it does through the aid of TNFa, it turns on the genes which allow cells to proliferate, mature, and avoid destruction through apoptosis (programmed cell death). This allows white blood cells to replicate and effect their activity in cleaning up the infected or injured area. NFkB is similar to the priority setting on a communications line by opening all channels available for the quickest response.

[71] Interleukin-6 (IL-6) is a cytokine that dictates the neutrophils to destroy themselves and draws monocytes, another type of white blood cell, to the infected or injured area instead. The monocytes create macrophages which clean up the debris and pathogens through phagocytosis, the process by which macrophages degrade dead cells and other particles whole.

[72] C-Reactive Protein (CRP) is a "pattern recognition receptor" protein, which means it marks recognized debris for removal, that is produced by the liver in response to IL-6 levels and binds to the surface of dead and dying cells, and also to certain forms of bacteria. CRP acts as a form of signal for the macrophages to ingest something through phagocytosis, and thus helps in the ultimate clearing of debris during inflammation.

[73] According to some embodiments, monitoring the level of an inflammatory biomarker associated with CF is useful in determining the course and effect of the treatment of inflammation associated with CF. In some embodiments, the level of a biomarker associated with CF in the serum extracted from the subject, based on a baseline of the serum level in the subject before commencement of the treatment, is reduced by at least 3, 5, 10, 15, 20, 30, 35, 40, 50 or at least 60 percent during the treatment.

[74] In some embodiments, the biomarker associated with CF is CRP, and the serum level of CRP is reduced during the intermittent inhalation treatment by at least 3, 5, 10, 15, 20, 30, 35, 40, 50 or at least 60 percent, compared to the baseline level in the subject before commencement of the treatment.

[75] In some embodiments, the biomarker associated with CF is a cytokine, such as, but not limited to, TNFa, IL-Ιβ, IL-6, IL-8, IL-10 and/or IL-12p70, and the serum level of the cytokine(s) is reduced by at least 3, 5, 10, 15, 20, 30, 35, 40, 50 or at least 60 percent , compared to the baseline level in the subject before commencement of the treatment. In some embodiments, the cytokines used as inflammatory biomarkers in the method presented herein are IL-6 and IL-Ιβ. [76] According to some embodiments of the present invention, there is provided a method of reducing a load of a pathogenic microorganism in a human subject by subjecting the human subject to intermittent inhalation of a gas mixture comprising nitric oxide at a concentration of at least 160 ppm.

[77] According to some embodiments of this aspect, the human subject is a human subject diagnosed with, or suffering from CF, as described herein.

[78] In some embodiments, the pathogenic microorganism causes a microbial infection associated with CF, as described herein. According to some embodiments, the pathogenic microorganism is selected from the group consisting of Pseudomonas alcaligenes, non-mucoid and mucoid Pseudomonas aeruginosa, Aspergillus fumigates, Staphylococcus aureus, Haemophilus influenza, Burkholderia cepacia complex, Klebsiella pneumonia, Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), methicillin- sensitive Staphylococcus aureus (MSSA), Stenotrophomonas maltophilia, Achromobacter spp., Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, Mycobacterium abscessus complex (MABSC), and non-tuberculous mycobacteria (NTM) species, and Acinetobacter baumannii.

[79] According to some embodiments, the pathogenic microorganism is selected from the group consisting of Pseudomonas alcaligenes, methicillin-sensitive Staphylococcus aureus (MSSA), Achromobacter spp. , Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, Aspergillus fumigates, non- mucoid Pseudomonas aeruginosa and mucoid Pseudomonas aeruginosa.

[80] As discussed hereinabove, in some embodiments, the load of the pathogenic microorganism is reduced by the presently claimed method by at least 1 log units during the intermittent inhalation.

[81] According to some embodiments of the present invention, there is provided a method of reducing a level of an inflammatory biomarker associated with CF in a human subject by subjecting the human subject to a treatment by intermittent inhalation of a gas mixture comprising nitric oxide at a concentration of at least 160 ppm.

[82] According to some embodiments, the inflammatory biomarker associated with CF, and/or a change in its normal physiological level, is associated with cystic fibrosis and/or with complications and other medical conditions associated with CF. Reducing a level of an inflammatory biomarker associated with CF in a human subject diagnosed with, or suffering from CF is indicative of treating inflammation (as a secondary medical condition) in a human subject diagnosed with, or suffering from CF.

[83] According to some embodiments, the inflammatory biomarker, which is targeted for reduction by the presently claimed method is selected from the group consisting of C-reactive protein (CRP), a cytokine, alpha- 1 -antitrypsin (AAT), haptoglobin, transferrin, an immunoglobulin, granzyme B (GzmB), chemokine C-C motif ligand 18 (CCL18/PARC), surfactant protein D (SP-D), lipopoly saccharide (LPS)-binding protein and soluble cluster of differentiation 14 (sCD14).

[84] In some embodiments of this aspect of the present invention, the inflammatory biomarker associated with CF is C-reactive protein (CRP). A rate of reduction as a result of the intermittent inhalation is at least 3, 5, 10, 15, 20, 30, 35, 40, 50 or at least 60 percent , compared to a baseline level of the biomarker in the patient.

[85] In some embodiments of this aspect of the present invention, the inflammatory biomarker associated with CF is a cytokine is selected from the group consisting of TNFa, IL-Ιβ, IL-6, IL-8, IL-10 and IL-12p70. In some embodiments, the inflammatory biomarkers are IL-6 and IL-Ιβ. A rate of reduction in the level of a cytokine as a result of the treatment is at least 3, 5, 10, 15, 20, 30, 35, 40, 50 or at least 60 percent , compared to a baseline level of the biomarker in the patient.

[86] According to some embodiments of this aspect, the human subject is a cystic fibrosis patient, as described herein.

Intermittent Inhalation:

[87] As presented hereinabove, any of the methods provided herewith comprise subjecting the human subject to intermittent inhalation of a gas mixture comprising nitric oxide at a concentration of at least 160 ppm.

[88] The term "intermittent" is used herein and in the art as an antonym of

"continuous", and means starting and ceasing an action and/or performing an action in intervals.

[89] By "intermittent inhalation" it is meant that a human subject breathes a mixture of gases that contains an indicated concentration of nitric oxide intermittently; hence while the volume of the inhaled mixture of gases may not change significantly during the intermittent inhalation, the chemical composition of the mixture changes according to a predetermined regimen, as described herein below. The human subject therefore inhales a gas mixture comprising nitric oxide at a concentration of at least 160 ppm for predetermined periods of time, and between these periods of time the human subject inhales a gaseous mixture that is essentially devoid of nitric oxide (e.g., ambient air or another nitric oxide-free mixture).

[90] Herein and throughout, "a nitric oxide-containing gaseous mixture" or

"a gas mixture comprising nitric oxide" is used to describe a gaseous mixture that contains at least 160 ppm nitric oxide. The nitric oxide-containing mixture can comprise 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm and even higher concentrations of nitric oxide. Other gaseous mixtures mentioned herein include less than 160 ppm nitric oxide or are being essentially devoid of nitric oxide, as defined herein.

[91] By "essentially devoid of nitric oxide" it is meant no more than 50 ppm, no more than 40 ppm, no more than 30 ppm, no more than 20 ppm, no more than 10 ppm, no more than 5 ppm, no more than 1 ppm, no more than lOOppb, and no more than 10 ppb including a nitric oxide concentration below measurable limits.

[92] According to some embodiments of the present invention, the intermittent inhalation includes one or more cycles, each cycle comprising continuous inhalation of a gaseous mixture containing nitric oxide at the specified high concentration (e.g., at least 160 ppm) for a first time period, followed by inhalation of a gaseous mixture essentially devoid of nitric oxide for a second time period. According to some embodiments of the present invention, during the second period of time the subject may inhale ambient air or a controlled mixture of gases, which is essentially devoid of nitric oxide, as defined herein.

[93] In some embodiments, the first time period spans from 10 minutes to

45 minutes, or from 20 to 45 minutes, or from 20 to 40 minutes, and according to some embodiments, spans about 30 minutes.

[94] According to some embodiments of the present invention, the second time period ranges from 3 hours to 5 hours, or from 3 to 4 hours, and according to some embodiments the second time period spans about 3.5 hours. [95] According to some embodiments of the present invention, this inhalation regimen is repeated 1-6 times over 24 hours, depending on the duration of the first and second time periods.

[96] In some embodiments, a cycle of intermittent delivery of nitric oxide, e.g., 160 ppm for 30 minutes followed by 3.5 hours of breathing no nitric oxide, is repeated from 1 to 6 times a day. According to some embodiments, the cycles are repeated 5 times a day. Alternatively the cycles are repeated 3 times a day.

[97] According to some embodiments of the present invention, the regimen of 1-5 cycles per day is carried out for 1 to 21 days, or from 2 to 14 days, or from 3 to 10 days. According to some embodiments of the present invention, the intermittent inhalation is effected during a time period of 2 weeks. However, longer time periods of intermittent nitric oxide administration as described herein, are also contemplated.

[98] For example, according to some embodiments, the intermittent inhalation comprises: (1) repetitive administration of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, continuously for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

[99] According to some embodiments, the intermittent inhalation comprises: (1) repetitive administration of 5 cycles per day of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

Safety:

[100] As discussed hereinabove, intermittent inhalation of 160 ppm of nitric oxide has been shown to be safe in human subjects of all ages. Safety has been demonstrated by monitoring one or more physiological parameters in the human and while minding that no substantial adverse change is effected in the monitored parameters, as a safety measure of the method presented herein. According to any one of the embodiments of the present invention, the intermittent inhalation is effected while monitoring one or more physiological parameters in the human subject.

[101] In some embodiments, the methods disclosed herein are effected while monitoring various parameters relevant for maintaining the desired dosage and regimen, relevant to the safety of the procedure and relevant for efficacy of the treatment.

[102] According to any one of the embodiments of the present invention, the method is effected while monitoring one or more physiological parameters in the human and while minding that no substantial adverse change is effected in the monitored safety parameters, as a safety measure of the method presented herein.

[103] In some embodiments, the method is carried out while maintaining safety measured which include non-invasive monitoring of bodily fluid chemistry, such as perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (Sp0₂/Sa0₂/DO), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), as these physiological parameters are known in the art. Typically, these on-site physiological parameters are monitored by pulse oximetry.

[104] Other parameters, also monitored as a safety measure on the presently disclosed method, according to some embodiments thereof, are off-site physiological parameters which are typically determined by collecting bodily samples using noninvasive (e.g., urine, feces or sputum samples) and invasive (e.g., blood or biopsy) method.

[105] For example, off-site physiological parameters which are typically measured by invasive methods may include serum nitrite/nitrate (NCV/NCV), blood methemoglobin, a complete blood cells count (CBC), blood chemistry /biochemistry (electrolytes, renal and liver function tests etc.) and coagulation tests.

[106] Off-site physiological parameters which are typically measured by non-invasive methods may include urine nitrite/nitrate (N0₂7N03^_), pregnancy tests in urine, and bacterial and fungal load in sputum, urine or feces. [107] In some embodiments, the method is carried out while maintaining safety measures which include controlling the mixture of inhaled gases and monitoring the exhaled gases, which is effected by standard means for monitoring and controlling, on-site, the contents and/or flow of the mixture to which the subject is subjected to, or that which is delivered through a delivery interface, and/or while monitoring on-site exhaled gases and controlling the intake by feedback in real-time. In some embodiments, the method is effected while monitoring the concentration of nitric oxide, 0₂, CO2 and NO2 in the gaseous mixture to which the human is exposed to or exhales.

[108] In some embodiments, the concentration of nitric oxide in the nitric oxide-containing gaseous mixture is controlled so as not to deviate from a predetermined concentration by more than 10 %. For example, the method is carried out while the concentration of nitric oxide, set to 160 ppm, does not exceed substantially the margins of 144 ppm to 176 ppm.

[109] Similarly, the NO2 content in a nitric oxide-containing gaseous mixture is controlled such that the concentration of NO2 is maintained lower than 5 ppm.

[110] Further, oxygen level in the nitric oxide-containing gaseous mixture is controlled such that the concentration of O2 in the mixture ranges from about 20 % to about 25 %.

[I l l] Alternatively or in addition, the oxygen level in the nitric oxide- containing gaseous mixture is controlled such that the fraction of inspired oxygen (Fi0₂) ranges from about 20 % to about 100 %.

[112] The phrase "fraction of inspired oxygen" or "Fi0₂", as used herein, refers to the fraction or percentage of oxygen in a given gas sample. For example, ambient air at sea level includes 20.9 % oxygen, which is equivalent to F1O2 of 0.21. Oxygen-enriched air has a higher F1O2 than 0.21, up to 1.00, which means 100 % oxygen. In the context of embodiments of the present invention, F1O2 is kept under 1 (less than 100 % oxygen).

[113] According to some embodiments, fraction of inspired oxygen (F1O2) in the nitric oxide-containing gaseous mixture is 0.2. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.25. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.3. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.35. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.4. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.45. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.5. In an altemate embodiment, the F1O2 in the nitric oxide- containing gaseous mixture is 0.55. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.6. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.65. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.7. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.75. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.8. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.85. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.9. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.95.

[114] In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.25 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.3 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.35 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.4 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.45 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.5 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.55 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.6 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.65 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.7 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.75 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.8 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.85 to 0.95. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.9 to 0.95. [115] In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.9. In an altemate embodiment, the F1O2 in the nitric oxide- containing gaseous mixture is 0.2 to 0.85. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.8. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.75. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.7. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.65. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.6. In an altemate embodiment, the F1O2 in the nitric oxide- containing gaseous mixture is 0.2 to 0.55. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.5. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.45. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.4. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.35. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.2 to 0.3. In an altemate embodiment, the F1O2 in the nitric oxide- containing gaseous mixture is 0.2 to 0.25.

[116] In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.25 to 0.9. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.3 to 0.85. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.35 to 0.8. In an altemate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.4 to 0.75. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.45 to 0.7. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.5 to 0.65. In an alternate embodiment, the F1O2 in the nitric oxide-containing gaseous mixture is 0.55 to 0.6.

[117] In some embodiments, the nitric oxide-containing gaseous mixture is formed by combining a stock supply of nitric oxide with air, which dilutes the stock supply of nitric oxide to the desired concentration. In some embodiments, the stock supply of nitric oxide is combined with air and oxygen to keep the Fi0₂ above 0.20. The ratio of nitric oxide, air and/or oxygen can be varied to achieve the desired nitric oxide concentration and F1O2. _[us_] The phrase "end tidal CO2" or "ETCO2", as used herein, refers to the partial pressure or maximal concentration of carbon dioxide (CO2) at the end of an exhaled breath, which is expressed as a percentage of CO2 or the pressure unit mmHg. Normal values for humans range from 5 % to 6 % CO2, which is equivalent to 35-45 mmHg. Since CO2 diffuses out of the lungs into the exhaled air, ETCO2 values reflect cardiac output (CO) and pulmonary blood flow as the gas is transported by the venous system to the right side of the heart and then pumped to the lungs by the right ventricles. A device called capnometer measures the partial pressure or maximal concentration of CO2 at the end of exhalation. In the context of embodiments of the present invention, a capnometer is used and ETCO2 levels are monitored so as to afford a warning feedback when ETCO2 is more than 60 mmHg.

[119] Levels of respiratory NO, NO2 and O2 concentration levels (both inhaled and exhaled; inspiratory and expiratory gases) are typically monitored continuously by sampling from a mouthpiece sample port located in an inhalation mask NO, NO2 and O2 equipped with an electrochemical analyzer. In the context of embodiments of the present invention, safety considerations requires the absolute minimization of the number of occasions in which NO2 levels exceed 5 ppm, nitric oxide concentration variations exceeding 10 %, and F1O2/O2 levels drop below 20 % during nitric oxide administration.

[120] It is noted that a sharp elevation of inflammatory biomarkers may be associated with a phenomenon called "cytokine storm", which has been observed in subjects undergoing nitric oxide inhalation treatment. Hence, monitoring inflammatory biomarkers while performing the method as described herein has an additional role in safety considerations pertaining to the method, according to embodiments of the present invention, wherein no significant increase in inflammatory markers is an indication of safety.

[121] In some embodiments, monitoring the one or more physiological parameters is effected by noninvasive measures and/or mild invasive measures.

[122] In some embodiments, monitoring the physiological parameter(s) in the subject is effected by on-site measurement and analysis techniques based on samples collected sporadically, continuously or periodically from the subject on-site in real-time at the subject's bed-side, and/or off-site measurement and analysis techniques based on samples collected sporadically or periodically from the subject which are sent for processing in a off-site which provides the results and analysis at a later point in time.

[123] In the context of some embodiments of the present invention, the phrase "on-site measurement and analysis techniques" or "on-site techniques", refers to monitoring techniques that inform the practitioner of a given physiological parameter of the subject in real-time, without the need to send the sample or raw data to an off-site facility for analysis. On-site techniques are often noninvasive, however, some rely on sampling from an invasive medical device such as a respiratory tubus, a drainer tube, an intravenous catheter or a subcutaneous port or any other implantable probe. Thus, the phrase "on-site parameters", as used herein, refers to physiological parameters which are obtainable by online techniques.

[124] Other than the trivial advantage of real-time on-site determination of physiological parameters, expressed mostly in the ability of a practitioner to respond immediately and manually to any critical change thereof, the data resulting from realtime online determination of physiological parameters can be fed into the machinery and be used for real-time feedback controlling of the machinery. In the context of embodiments of the present invention, the term "real-time" also relates to systems that update information and respond thereto substantially at the same rate they receive the information. Such real-time feedback can be used to adhere to the treatment regimen and/or act immediately and automatically in response to any critical deviations from acceptable parameters as a safety measure.

[125] Hence, according to embodiments of the present invention, the term

"on-site parameter" refers to physiological and/or mechanical and/or chemical datum which is obtainable and can be put to use or consideration at or near the subject's site (e.g., bed-side) in a relatively short period of time, namely that the time period spanning the steps of sampling, testing, processing and displaying/using the datum is relatively short. An "on-site parameter" can be obtainable, for example, in less than 30 minutes, less than 10 minutes, less than 5 minutes, less than 1 minute, less than 0.5 minutes, less than 20 seconds, less than 10 seconds, less than 5 seconds, or less than 1 second from sampling to use. For example, the time period required to obtain on-site parameters by a technique known as pulse oximetry is almost instantaneous; once the device is in place and set up, data concerning, e.g., oxygen saturation in the periphery of a human subject, are available in less than 1 second from sampling to use. [126] In the context of some embodiments of the present invention, the phrase "off-site measurement and analysis techniques" or "off-site techniques", refers to techniques that provide information regarding a given physiological parameter of the subject after sending a sample or raw data to an offline, and typically off-site facility, and receiving the analysis offline, sometimes hours or days after the sample had been obtained. Off-site techniques are oftentimes based on samples collected by mild invasive techniques, such as blood extraction for monitoring inflammatory cytokine plasma level, and invasive techniques, such as biopsy, catheters or drainer tubus, however, some off-site techniques rely on noninvasive sampling such as urine and stool chemistry offline and off-site analyses. The phrase "off-site parameters", as used herein, refers to physiological parameters which are obtainable by off-site laboratory techniques.

[127] Hence, according to embodiments of the present invention, the term

"off-site parameter" refers to physiological and/or mechanical and/or chemical datum which is obtain and can be put to use or consideration in a relatively long period of time, namely that the time period spanning the steps of sampling, testing, processing and display ing/using the datum is long compared to on-site parameters. Thus, an "off-site parameter" is obtainable in more than 1 day, more than 12 hours, more than 1 hour, more than 30 minutes, more than 10 minutes, or more than 5 minutes from sampling to use.

[128] An "off-site parameter" is typically obtainable upon subjecting a sample to chemical, biological, mechanical or other procedures, which are typically performed in a laboratory and hence are not performed "on-site", namely by or near the subject's site.

[129] Noninvasive measures for monitoring various physiological parameters include, without limitation, sputum, urine and feces sampling, pulse oximetry, nonintubated respiratory analysis and/or capnometry. Invasive measures for monitoring various physiological parameters include, without limitation, blood extraction, continuous blood gas and metabolite analysis, and in some embodiments intubated respiratory analysis and transcutaneous monitoring measures. Intense invasive measures include biopsy and other surgical procedures.

[130] The term "pulse oximetry" refers to a noninvasive and on-site technology that measures respiration-related physiological parameters by following light absorption characteristics of hemoglobin through the skin (finger, ear lobe etc.), and on the spectroscopic differences observed in oxygenated and deoxygenated species of hemoglobin, as well as hemoglobin species bound to other molecules, such as carbon monoxide (CO), and methemoglobin wherein the iron in the heme group is in the Fe ⁺ (ferric) state. Physiological parameters that can be determined by pulse oximetry include, for example, SpC>2, SpMet and SpCO.

[131] The phrase "nonintubated respiratory analysis", as used herein, refers to a group of noninvasive and on-site technologies, such as spirometry and capnography, which provide measurements of the physiological pulmonary mechanics and respiratory gaseous chemistry by sampling the inhaled/exhaled airflow or by directing subject's breath to a detector, all without entering the subject's respiratory tract or other orifices nor penetrating the skin at any stage.

[132] The term "spirometry" as used herein, refers to the battery of measurements of respiration-related parameters and pulmonary functions by means of a noninvasive and on-site spirometer. Following are exemplary spirometry parameters which may be used in the context of some embodiments of the present invention:

[133] The spirometric parameter Tidal volume (TV) is the amount of air inhaled and exhaled normally at rest, wherein normal values are based on person's ideal body weight.

[134] The spirometric parameter Total Lung Capacity (TLC) is the maximum volume of air present in the lungs.

[135] The spirometric parameter Vital Capacity (VC) is the maximum amount of air that can expel from the lungs after maximal inhalation, and is equal to the sum of inspiratory reserve volume, tidal volume, and expiratory reserve volume.

[136] The spirometric parameter Slow Vital Capacity (SVC) is the amount of air that is inhaled as deeply as possible and then exhaled completely, which measures how deeply a person can breathe.

[137] The spirometric parameter Forced Vital Capacity (FVC) is the volume of air measured in liters, which can forcibly be blown out after full inspiration, and constitutes the most basic maneuver in spirometry tests.

[138] The spirometric parameter Forced Expiratory Volume in the 1st second

(FEVi) is the volume of air that can forcibly be blown out in one second, after full inspiration. Average values for FEVi depend mainly on sex and age, whereas values falling between 80 % and 120 % of the average value are considered normal. Predicted normal values for FEVi can be calculated on-site and depend on age, sex, height, weight and ethnicity as well as the research study that they are based on.

[139] The spirometric parameter FEVi/FVC ratio (FEVi%) is the ratio of

FEVi to FVC, which in adults should be approximately 75-80 %. The predicted FEV1% is defined as FEVi% of the patient divided by the average FEVi% in the appropriate population for that person.

[140] The spirometric parameter Forced Expiratory Flow (FEF) is the flow

(or speed) of air coming out of the lung during the middle portion of a forced expiration. It can be given at discrete times, generally defined by what fraction remains of the forced vital capacity (FVC), namely 25 % of FVC (FEF25), 50 % of FVC (FEF5₀) or 75 % of FVC (FEF₇₅). It can also be given as a mean of the flow during an interval, also generally delimited by when specific fractions remain of FVC, usually 25-75 % (FEF25-75%). Measured values ranging from 50-60 % up to 130 % of the average are considered normal, while predicted normal values for FEF can be calculated on-site and depend on age, sex, height, weight and ethnicity as well as the research study that they are based on. Recent research suggests that FEF₂5-75% or FEF₂5-5o% may be a more sensitive parameter than FEVi in the detection of obstructive small airway disease. However, in the absence of concomitant changes in the standard markers, discrepancies in mid-range expiratory flow may not be specific enough to be useful, and current practice guidelines recommend continuing to use FEVi, VC, and FEVi/VC as indicators of obstructive disease.

[141] The spirometric parameter Negative Inspiratory Force (NIF) is the greatest force that the chest muscles can exert to take in a breath, wherein values indicate the state of the breathing muscles.

[142] The spirometric parameter MMEF or MEF refers to maximal (mid- expiratory flow and is the peak of expiratory flow as taken from the flow-volume curve and measured in liters per second. MMEF is related to peak expiratory flow (PEF), which is generally measured by a peak flow meter and given in liters per minute. [143] The spirometric parameter Peak Expiratory Flow (PEF) refers to the maximal flow (or speed) achieved during the maximally forced expiration initiated at full inspiration, measured in liters per minute.

[144] The spirometric parameter diffusing capacity of carbon monoxide

(D_LCO) refers to the carbon monoxide uptake from a single inspiration in a standard time (usually 10 sec). On-site calculators are available to correct D_LCO for hemoglobin levels, anemia, pulmonary hemorrhage and altitude and/or atmospheric pressure where the measurement was taken.

[145] The spirometric parameter Maximum Voluntary Ventilation (MVV) is a measure of the maximum amount of air that can be inhaled and exhaled within one minute. Typically this parameter is determined over a 15 second time period before being extrapolated to a value for one minute expressed as liters/minute. Average values for males and females are 140-180 and 80-120 liters per minute respectively.

[146] The spirometric parameter static lung compliance (Cst) refers to the change in lung volume for any given applied pressure. Static lung compliance is perhaps the most sensitive parameter for the detection of abnormal pulmonary mechanics. Cst is considered normal if it is 60 % to 140 % of the average value of a commensurable population.

[147] The spirometric parameter Forced Expiratory Time (FET) measures the length of the expiration in seconds.

[148] The spirometric parameter Slow Vital Capacity (SVC) is the maximum volume of air that can be exhaled slowly after slow maximum inhalation.

[149] Static intrinsic positive end-expiratory pressure (static PEEPi) is measured as a plateau airway opening pressure during airway occlusion.

[150] The spirometric parameter Maximum Inspiratory Pressure (MIP) is the value representing the highest level of negative pressure a person can generate on their own during an inhalation, which is expresented by centimeters of water pressure

(cmH₂0) and measured with a manometer and serves as n indicator of diaphragm strength and an independent diagnostic parameter.

[151] The term "capnography" refers to a technology for monitoring the concentration or partial pressure of carbon dioxide (CO₂) in the respiratory gases. End-tidal CO₂, or ETCO₂, is the parameter that can be determined by capnography. [152] Gas detection technology is integrated into many medical and other industrial devices and allows the quantitative determination of the chemical composition of a gaseous sample which flows or otherwise captured therein. In the context of embodiments of the present invention, such chemical determination of gases is part of the on-site, noninvasive battery of tests, controlled and monitored activity of the methods presented herein. Gas detectors, as well as gas mixers and regulators, are used to determine and control parameters such as fraction of inspired oxygen level (F1O2) and the concentration of nitric oxide in the inhaled gas mixture.

[153] According to some embodiments of the present invention, the measurement of vital signs, such as heart rate, blood pressure, respiratory rate and a body temperature, is regarded as part of a battery of on-site and noninvasive measurements.

[154] The phrase "integrated pulmonary index", or IPI, refers to a patient's pulmonary index which uses information on inhaled/exhaled gases from capnography and on gases dissolved in the blood from pulse oximetry to provide a single value that describes the patient's respiratory status. IPI, which is obtained by on-site and noninvasive techniques, integrates four major physiological parameters provided by a patient monitor (end-tidal CO2 and respiratory rate as measured by capnography, and pulse rate and blood oxygenation SpC>2 as measured by pulse oximetry), using this information along with an algorithm to produce the IPI score. IPI provides a simple indication in real time (on-site) of the patient's overall ventilatory status as an integer (score) ranging from 1 to 10. IPI score does not replace current patient respiratory parameters, but used to assess the patient's respiratory status quickly so as to determine the need for additional clinical assessment or intervention.

[155] According to some of the embodiments described herein, the monitored physiological or chemical parameters include one or more of the following parameters:

Perfusion Index (PI);

Respiration Rate (RRa);

Oxyhemoglobin Saturation (Sp0₂);

Total Hemoglobin (SpHb);

Carboxyhemoglobin (SpCO);

Methemoglobin (SpMet); Oxygen Content (SpOC); and

Pleth Variability Index (PVI),

and/or at least one off-site parameter selected from the group consisting of: serum nitrite/nitrate (N0₂7N0₃ ^");

serum or urine nitrite/nitrate (NCV/NCV) and

blood methemoglobin.

[156] According to some of the embodiments described herein, the monitored physiological or chemical parameters include one or more of the following parameters:

Perfusion Index (PI);

Respiration Rate (RRa);

Oxyhemoglobin Saturation (Sp0₂);

Total Hemoglobin (SpHb);

Carboxyhemoglobin (SpCO);

Methemoglobin (SpMet);

Oxygen Content (SpOC); and

Pleth Variability Index (PVI),

and/or at least one off-site parameter selected from the group consisting of: serum nitrite/nitrate (N0₂7N0₃ ^"); and

skin salinity.

[157] According to some of the embodiments described herein, the method is conducted while monitoring at least one of the following on-site parameters in the gas mixture inhaled by the human subject:

End Tidal C0₂ (ETC0₂);

Nitrogen dioxide (NO₂),

Nitric oxide (NO); and

Fraction of inspired oxygen (F1O₂).

[158] According to some of the embodiments described herein, the monitored physiological or chemical parameters further include one or more of the following parameters:

a urine level of nitrogen dioxide (urine nitrite level) (an off-line parameter); a vital sign selected from the group consisting of a heart rate, a blood pressure, a respiratory rate and a body temperature (an on-line parameter); a hematological marker (an off-line parameter), such as, but not limited to, a hemoglobin level, a hematocrit ratio, a red blood cell count, a white blood cell count, a white blood cell differential and a platelet count;

a coagulation parameter (an off-line parameter) such as, but not limited to, a prothrombin time (PT), a prothrombin ratio (PR) and an international normalized ratio (INR);

a serum creatinine level (an off-line parameter);

a liver function marker (an off-line parameter) selected from the group consisting of a aspartate aminotransferase (AST) level, a serum glutamic oxaloacetic transaminase (SGOT) level, an alkaline phosphatase level, and a gamma-glutamyl transferase (GGT) level;

a vascular endothelial activation factor (an off-line parameter) selected from the group consisting of Ang-1, Ang-2 and Ang-2/Ang-l ratio.

[159] It is noted that a sharp elevation of inflammatory biomarkers may be associated with a phenomenon called "cytokine storm", which has been observed in subjects undergoing nitric oxide inhalation treatment. Hence, monitoring inflammatory biomarkers while performing the method as described herein has an additional role in safety considerations pertaining to the method, according to embodiments of the present invention, wherein no significant increase in inflammatory markers is an indication of safety.

Selected safety and efficacy parameters and criteria for monitoring safety:

[160] According to some embodiments of the present invention, the method as disclosed herein is such that no substantial change is observed in at least one of the monitored physiological parameters or a level of biomarkers pertaining to the safety and efficacy of the treatment presented hereinabove.

[161] In the context of the present embodiments, a change in a parameter or a level of a biomarker is considered substantial when a value of an observation (measurement, test result, reading, calculated result and the likes) or a group of observations falls notably away from a normal level, for example falls about twice the upper limit of a normal level.

[162] A "normal" level of a parameter or a level of a biomarker is referred to herein as baseline values or simply "baseline". In the context of the present embodiments, the term "baseline" is defined as a range of values which have been determined statistically from a large number of observations and/or measurements which have been collected over years of medical practice with respect to the general human population, a specific sub-set thereof (cohort) or in some cases with respect to a specific person. A baseline is a parameter/biomarker-specific value which is generally and medically accepted in the art as normal for a subject under certain physical conditions. These baseline or "normal" values, and means of determining these normal values, are known in the art. Alternatively, a baseline value may be determined from or in a specific subject before effecting the method described herein using well known and accepted methods, procedures and technical means. A baseline is therefore associated with a range of tolerated values, or tolerance, which have been determined in conjunction with the measurement of a parameter/biomarker. In other words, a baseline is a range of acceptable values which limit the range of observations which are considered as "normal". The width of the baseline, or the difference between the upper and lower limits thereof are referred to as the "baseline range", the difference from the center of the range is referred to herein as the "acceptable deviation unit" or ADU. For example, a baseline of 4-to-8 has a baseline range of 4 and an acceptable deviation unit of 2.

[163] In the context of the present embodiments, a significant change in an observation pertaining to a given parameter/biomarker is one that falls more than 2 acceptable deviation unit (2 ADU) from a predetermined acceptable baseline. For example, an observation of 10, pertaining to a baseline of 4-to-8 (characterized by a baseline range of 4, and an acceptable deviation unit of 2), falls one acceptable deviation unit, or 1 AUD from baseline. Alternatively, a change is regarded substantial when it is more than 1.5 ADU, more than 1 ADU or more than 0.5 ADU.

[164] In the context of the present embodiments, a "statistically significant observation" or a "statistically significant deviation from a baseline" is such that it is unlikely to have occurred as a result of a random factor, error or chance.

[165] It is noted that in some parameters/biomarkers or groups of parameters/biomarkers, the significance of a change thereof may be context- dependent, biological system-dependent, medical case-dependent, human subject- dependent, and even measuring machinery-dependent, namely a particular parameter/biomarker may require or dictate stricter or looser criteria to determine if a reading thereof should be regarded as significant. It is noted herein that in specific cases some parameters/biomarkers may not be measurable due to patient condition, age or other reasons. In such cases the method is effected while monitoring the other parameters/bi omarkers .

[166] A deviation from a baseline is therefore defined as a statistically significant change in the value of the parameter/biomarker as measured during and/or following a full term or a part term of administration the regimen described herein, compared to the corresponding baseline of the parameter/biomarker. It is noted herein that observations of some parameters/biomarkers may fluctuate for several reasons, and a determination of a significant change therein should take such events into consideration and correct the appropriate baseline accordingly.

[167] Monitoring methemoglobin and serum nitrite levels has been accepted in the art as a required for monitoring the safety of nitric oxide inhalation in a subject. Yet, to date, no clear indication that methemoglobin and serum nitrite levels remain substantially unchanged upon nitric oxide inhalation by a human subject.

[168] According to some embodiments of the present invention, the method comprises monitoring and/or improving at least one of the parameters/biomarkers described hereinabove.

[169] According to some embodiments, the monitored parameter is methemoglobin level.

[170] As methemoglobin levels can be measured using noninvasive measures, the parameter of percent saturation at the periphery of methemoglobin (SpMet) is used to monitor the stability, safety and effectiveness of the method presented herein. Hence, according to some embodiments of the present invention, the followed parameter is SpMet and during and following the administration, the SpMet level does not exceed 5 %, and preferably does not exceed 1 %. As demonstrated in the Examples section that follows, a SpMet level of subjects undergoing the method described herein does not exceed 1 %.

[171] According to some embodiments, the monitored parameter is serum nitrate/nitrite level.

[172] High nitrite and nitrate levels in a subject's serum are associated with nitric oxide toxicity and therefore serum nitrite/nitrate levels are used to detect adverse effects of the method presented herein. According to some embodiments of the present invention, the tested parameter is serum nitrite/nitrate, which is monitored during and following the treatment and the acceptable level of serum nitrite is less than 2.5 micromole/liter and serum nitrate is less than 25 micromole/liter.

[173] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site parameters which include perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (Sp0₂/Sa0₂/DO), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), and/or monitoring at least one or all off-site parameters which include serum nitrite/nitrate level.

[174] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site parameters in the gas mixture inhaled by the subject, which include end tidal C0₂ (ETCO₂), nitrogen dioxide (NO₂), nitric oxide (NO) and fraction of inspired oxygen (F1O₂).

[175] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site and/or off-site safety parameters pertaining to nitric oxide inhalation, e.g., methemoglobin formation, and while monitoring at least one, at least two, or all on-site and/or off-site efficacy parameters.

[176] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site and/or off-site safety parameters pertaining to nitric oxide inhalation, e.g., methemoglobin formation, and while monitoring at least one, at least two, or all on-site and/or off-site efficacy parameters pertaining to CF symptoms, which include, pulmonary functions and/or inflammatory biomarkers.

[177] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site and/or off-site safety parameters pertaining to nitric oxide inhalation, e.g., methemoglobin formation, and while monitoring at least one, at least two, or all on-site and/or off-site efficacy parameters pertaining to bronchiolitis symptoms, which include, pulmonary functions and/or inflammatory biomarkers.

[178] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site pulmonary function parameters (spirometric parameters), such as forced expiratory volume (FEVi), maximum mid-expiratory flow (MMEF), diffusing capacity of the lung for carbon monoxide (D_LCO), forced vital capacity (FVC), total lung capacity (TLC) and residual volume (RV).

[179] For example, the method according to some embodiments is effected while monitoring SpMet as an on-site parameter. Alternatively, the method is effected while monitoring SpMet and ETCO2 as on-site parameters. Alternatively, the method is effected while monitoring SpMet, ETCO2 and SpC as on-site parameters.

[180] Alternatively, the method according to some embodiments is effected while monitoring SpMet as one on-site parameter, and one off-site parameter, such as plasma or urine levels of Ν0₂7Ν(ν. Alternatively, the method is effected while monitoring SpMet and SpC>2 as on-site parameters, and serum nitrite/nitrate level as one off-site parameter. Alternatively, the method is effected while monitoring SpMet as one on-site parameter, and inflammatory biomarkers in the plasma (for efficacy) and serum nitrite/nitrate level as off-site parameters. Alternatively, the method is effected while monitoring SpC>2 as one on-site parameter, and bacterial load and serum nitrite/nitrate level as off-site parameters. Alternatively, the method is effected while monitoring SpC>2 as one on-site parameter, and inflammatory biomarkers in the plasma and pulmonary function parameters such as FEVi.

[181] Further alternatively, the method is effected while monitoring SpMet,

FEVi and SpC>2 as on-site parameters, and inflammatory biomarkers in the plasma and serum nitrite/nitrate level as off-site parameters.

[182] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site parameters which include SpMet, SpC>2 and FEVi, and/or monitoring at least one or all off-site parameters which include serum nitrite/nitrate level and inflammatory biomarkers in the plasma, and further monitoring one or more and in any combination of:

a urine NO2 level (an off-site parameter);

a vital sign (an on-site parameter);

a pulmonary function (an on-site parameter);

a hematological marker (an off-site parameter);

a coagulation parameter (an off-site parameter);

a serum creatinine level (an off-site parameter); a renal function marker (an off-site parameter);

a liver function marker (an off-site parameter);

a vascular endothelial activation factor (an off-site parameter).

[183] According to some of the embodiments described herein, the method is effected while monitoring at least one, at least two, or all on-site chemical parameters in the inhaled gas mixture, such as F1O2 and NO2.

[184] It is noted herein that for any of the abovementioned embodiments, that the method is effected while no substantial change is observed in any one or more than one or all of the monitored parameters described herein.

[185] According to some embodiments of the present invention, the method is effected while monitoring urine nitrite levels, such that the urine nitrite level is substantially unchanged during and subsequent to carrying out the method as presented herein. It is noted herein that urine nitrite levels may fluctuate for several known reasons, and a determination of a significant change therein should take such events into consideration and correct the appropriate baseline accordingly.

[186] According to some embodiments of the present invention, hematological markers, such as the hemoglobin level, the hematocrit ratio, the red blood cell count, the white blood cell count, the white blood cell differential and the platelet count, are substantially unchanged during and subsequent to carrying out the method as presented herein.

[187] According to some embodiments of the present invention, vascular endothelial activation factors, such as Ang-1, Ang-2 and Ang-2/Ang-l ratio, as well as the serum creatinine level and various liver function markers, such as the aspartate aminotransferase (AST) level, the serum glutamic oxaloacetic transaminase (SGOT) level, the alkaline phosphatase level, and the gamma-glutamyl transferase (GGT) level, are substantially unchanged during and subsequent to carrying out the method as presented herein.

[188] Oxygenation of the subject can be assessed by measuring the subject's saturation of peripheral oxygen (SpC^). This parameter is an estimation of the oxygen saturation level, and it is typically measured using noninvasive measures, such as a pulse oximeter device. Hence, according to some embodiments of the present invention, the followed parameter during and following the administration is SpC>2, and the level of SpC>2 is higher than about 89 %. [189] According to some embodiments of the present invention, various vital signs, such as the heart rate, the blood pressure, the respiratory rate and the body temperature; and various coagulation parameters, such as the prothrombin time (PT), the prothrombin ratio (PR) and the international normalized ratio (INR), are substantially unchanged during and subsequent to carrying out the method as presented herein. It is noted that these parameters are regarded as an indication that the general health of the subject is not deteriorating as a result of the medical condition and/or the treatment.

[190] According to some embodiments, the aforementioned general health indicators show an improvement during and subsequent to carrying out the method as presented herein, indicating that the treatment is beneficial to the subject.

[191] Thus, according to some embodiments of the present invention, the method as disclosed herein is effected such that general health indicators as described herein are at least remained unchanged or are improved.

Modes of administration and inhalation devices:

[192] The human subject can be subjected to the inhalation by active or passive means.

[193] By "active means" it is meant that the gaseous mixture is administered or delivered to the respiratory tract of the human subject. This can effected, for example, by means of an inhalation device having a delivery interface adapted for human respiratory organs. For example, the delivery interface can be placed intermittently on the human subject's respiratory organs, whereby when it is removed, the subject breaths ambient air or any other gaseous mixture that is devoid of nitric oxide, as defined herein.

[194] By "passive means" it is meant that the human subject inhales a gaseous mixture containing the indicated dose of nitric oxide without devices for delivering the gaseous mixture to the respiratory tract.

[195] For example, the subject can be subjected to 160 ppm or more nitric oxide in an intermittent regimen by entering and exiting an atmospherically controlled enclosure filled with the nitric oxide-containing mixture of gases discussed herein, or by filling and evacuating an atmospherically controlled enclosure which is in contact with a subject's respiratory tract. [196] According to some embodiments of the present invention, in any of the methods of treatment presented herein, the nitric oxide administration can be effected by an inhalation device which includes, without limitation, a stationary inhalation device, a portable inhaler, a metered-dose inhaler and an intubated inhaler.

[197] An inhaler, according to some embodiments of the present invention, can generate spirometry data and adjust the treatment accordingly over time as provided, for example, in U.S. Patent No. 5,724,986 and WO 2005/046426. The inhaler can modulate the subject's inhalation waveform to target specific lung sites. According to some embodiments of the present invention, a portable inhaler can deliver both rescue and maintenance doses of nitric oxide at subject's selection or automatically according to a specified regimen.

[198] According to some embodiments of the present invention, an exemplary inhalation device may include a delivery interface adaptable for inhalation by a human subject.

[199] According to some embodiments of the present invention, the delivery interface includes a mask or a mouthpiece for delivery of the mixture of gases containing nitric oxide to a respiratory organ of the subject.

[200] According to some embodiments of the present invention, the inhalation device further includes a nitric oxide analyzer positioned in proximity to the delivery interface for measuring the concentration of nitric oxide, oxygen and nitrogen dioxide flowing to the delivery interface, wherein the analyzer is in communication with the controller.

[201] According to some embodiments of the present invention, subjecting the subject to the method described herein is carried out by use of an inhalation device which can be any device which can deliver the mixture of gases containing nitric oxide to a respiratory organ of the subject. An inhalation device, according to some embodiments of the present invention, includes, without limitation, a stationary inhalation device comprising tanks, gauges, tubing, a mask, controllers, values and the likes; a portable inhaler (inclusive of the aforementioned components), a metered- dose inhaler, a an atmospherically controlled enclosure, a respiration machine/system and an intubated inhalation/respiration machine/system. An atmospherically controlled enclosure includes, without limitation, a head enclosure (bubble), a full body enclosure or a room, wherein the atmosphere filling the enclosure can be controlled by flow, by a continuous or intermittent content exchange or any other form of controlling the gaseous mixture content thereof.

[202] According to some embodiments of the invention, the intermittent inhalation is effected by intermittently subjecting the human subject to a gaseous mixture (the inhalant) by breathing cycle-coordinated pulse delivery, which contains nitric oxide at the indicated concentration (a nitric oxide-containing gaseous mixture). This mode of inhalation is referred to herein as intermittent breathing cycle- coordinated pulse delivery inhalation.

[203] According to an alternative aspect of some embodiments of the present invention, there is provided a method of treating an inflammatory disease or disorder in a human subject, which includes subjecting the human subject to intermittent inhalation of an inhalant, whereas the intermittent inhalation includes at least one cycle of a breathing cycle-coordinated pulse delivery inhalation of the inhalant for a first time period, followed by inhalation of essentially no nitric oxide for a second time period, wherein the breathing cycle-coordinated pulse delivery inhalation is configured to deliver about 80 ppm-hour of nitric oxide during at least one cycle.

[204] In the context of embodiments of the present invention, the term "nitric oxide-load" ("NO-load") refers to a certain cumulative amount of nitric oxide to which a subject, or a pathogen, is exposed to during inhalation treatment (e.g., the presently claimed treatment), which is estimated in terms of ppm-hour, namely the average concentration of nitric oxide in the inhalant multiplied by the overall time of exposure. The nitric oxide-load can be estimated per cycle of the treatment (NO-load per cycle), or per a time unit, such as a day (daily NO-load).

[205] According to some embodiments of the present invention, the intermittent delivery of nitric oxide to the subject is conducted such that the subject inhales nitric oxide at an nitric oxide-load that ranges from 600 ppm-hour to 2000 ppm-hour daily, wherein the intermittent delivery is effected such that the daily nitric oxide-load is inhaled in more than one session of uninterrupted administration.

[206] According to some embodiments of the present invention, the intermittent delivery is effected such that the daily nitric oxide-load is inhaled in one or more sessions of intermittent breathing cycle-coordinated pulse delivery inhalation, while the nitric oxide-load per cycle of each cycle is at least about 80 ppm-hour. Such nitric oxide-load per cycle can be obtained, for example, by configuring the pulse(s) to deliver, during one cycle, an inhalant having 160 ppm of nitric oxide for 30 minutes (the first time period). It is noted that other concentrations and other first time periods, which afford a nitric oxide-load of at least 80 ppm-hour per cycle, are also contemplated and encompassed by embodiment of the present invention.

[207] By "intermittent breathing cycle-coordinated pulse delivery inhalation" it is meant that the subject is subjected to a gaseous mixture that contains the indicated concentration of nitric oxide intermittently, and thus inhales such a nitric oxide-containing gaseous mixture by breathing cycle-coordinated pulse delivery two or more times with intervals between each inhalation. The subject therefore inhales the nitric oxide-containing gaseous mixture, then stops inhaling a nitric oxide- containing gaseous mixture by breathing cycle-coordinated pulse delivery and inhales instead a gaseous mixture that does not contain the indicated concentration of nitric oxide (e.g., air), then inhales again the nitric oxide-containing gaseous mixture by breathing cycle-coordinated pulse delivery, and so on and so forth.

[208] In some embodiments of this aspect of the present invention, "a nitric oxide-containing gaseous mixture" is used to describe a gaseous mixture that contains at least 160 ppm nitric oxide. The nitric oxide-containing mixture can comprise 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm and even higher concentrations of nitric oxide. Other gaseous mixtures mentioned herein include less than 160 ppm nitric oxide or are being essentially devoid of nitric oxide, as defined herein.

[209] In some embodiments "a nitric oxide-containing gaseous mixture" describes a gaseous mixture that delivers nitric oxide at 80 ppm-hour.

[210] By "essentially devoid of nitric oxide" it is meant no more than 50 ppm, no more than 40 ppm, no more than 30 ppm, no more than 20 ppm, no more than 10 ppm, no more than 5 ppm, no more than 1 ppm, no more than lOOppb, and no more than 10 ppb including a nitric oxide concentration below measurable limits.

[211] According to some embodiments of the present invention, the intermittent breathing cycle-coordinated pulse delivery inhalation includes one or more cycles, each cycle comprising breathing cycle-coordinated pulse delivery inhalation of a gaseous mixture containing nitric oxide at the specified concentration (e.g., at least 160 ppm) for a first time period, which is also referred to herein as the nitric oxide-load per cycle, followed by inhalation of a gaseous mixture containing no nitric oxide for a second time period. According to some embodiments of the present invention, during the second period of time the subject may inhale ambient air or a controlled mixture of gases which is essentially devoid of nitric oxide, as defined herein.

[212] In some embodiments, the first time period spans from 10 to 45 minutes, or from 20 to 45 minutes, or from 20 to 40 minutes, and according to some embodiments, spans about 30 minutes.

[213] According to some embodiments of the present invention, the second time period ranges from 3 to 5 hours, or from 3 to 4 hours, and according to some embodiments the second time period spans about 3.5 hours.

[214] According to some embodiments of the present invention, this inhalation regimen is repeated 1-6 times over 24 hours, depending on the duration of the first and second time periods.

[215] In some embodiments, a cycle of intermittent breathing cycle- coordinated pulse delivery of nitric oxide, e.g., 160 ppm for 30 minutes followed by 3.5 hours of breathing no nitric oxide, is repeated from 1 to 6 times a day. According to some embodiments, the cycles are repeated 5 times a day.

[216] In some embodiments, a cycle of intermittent breathing cycle- coordinated pulse delivery of nitric oxide, e.g., at nitric oxide-load of 80 ppm-hour per cycle, followed by 3.5 hours of breathing no nitric oxide, is repeated from 1 to 6 times a day. According to some embodiments, the cycles are repeated 5 times a day.

[217] According to some embodiments of the present invention, the regimen of 1-5 cycles of intermittent breathing cycle-coordinated pulse delivery of nitric oxide per day is carried out for 1 to 7 days, or from 2 to 7 days, or from 3 to 7 days, or for 1, 2, 3, 4 or 5 successive weeks. According to some embodiments of the present invention, the intermittent breathing cycle-coordinated pulse delivery inhalation is effected during a time period of 14 days. However, longer time periods of intermittent nitric oxide administration as described herein, are also contemplated.

[218] According to some embodiments, the intermittent inhalation comprises: (1) repetitive administration of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, continuously for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

[219] According to some embodiments, the intermittent inhalation comprises: (1) repetitive administration of 5 cycles per day of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

[220] According to embodiments of the present invention, the nitric oxide- containing gaseous mixture, which the subject inhales during the first time period, is generated in-situ in an inhalation device which is configured to respond to the subject's breathing cycle such that nitric oxide is mixed into the inhalant in one or more pulses when the subject breaths in at a high rate, namely at the inhalation period of the breathing cycle. This mode of administration of nitric oxide by inhalation is referred to herein as "breathing cycle-coordinated pulse delivery inhalation".

[221] In the context of embodiments of the present invention, the term

"pulse" refers to a mode of administering nitric oxide, which is introduced into the inhalant in interrupted and concentrated doses during a predetermined period of time, referred to herein as the "pulse delivery period", wherein each pulse, effected during the pulse delivery period, spans a predetermined period of time, referred to herein as the "pulse-on period", and interrupted by a "pulse-off period".

[222] According to embodiments of the present invention, the pulse delivery period starts during the inhalation period, after a period of time which is referred to herein as the "pulse delay period". According to some embodiments of the present invention, the pulse delivery period is typically shorter than the inhalation period, and the time between the end of the pulse delivery period and the end of the inhalation period is referred to herein as the "pulse cessation period".

[223] According to some embodiments of the present invention, the inhalation device for delivering the breathing cycle-coordinated pulse delivery inhalation of gashouse nitric oxide is configured to detect the various phases of the breathing cycle, namely the onset of the inhalation and the exhalation periods, and can therefore coordinate the pulses with the breathing cycle such that the pulse delay period is coordinated to start as soon as the rate of intake increases at the onset of the inhalation period, and the pulse cessation period is coordinated to start with as soon as the rate of intake decreases close to the end of the inhalation period.

[224] In some embodiments, the length of the various time periods in the breathing cycle-coordinated pulse delivery inhalation scheme is determined and/or calculated relative to the duration of the breathing cycle, namely in percent of the total duration of the breathing cycle, or parts thereof. For example, the duration of the inhalation period is determined by sensing the flow rate of the inhalant, and the pulse delay period is automatically set to 20 % of the inhalation period. Consequently, the pulse delivery period can be set to 60 % of the inhalation period, and the pulse cessation period is the remaining 20 % of the inhalation period. The number of pulses, namely the pulse-on and pulse-off periods, can be set similarly according to the duration of the pulse delivery period. For example, the number of pulses can be set to one, namely a pulse that spans the entire duration of the pulse delivery period. This example may be suitable for a subject experiencing shortness of breath or any difficulty in respiration. Alternatively, in cases where the subject is breathing normally, the pulse-on period is set to 200-300 milliseconds (ms), and the pulse-off period is set to 100 ms, while the number of pulses is automatically set by the duration of pulse delivery period which is derived from the measured inhalation period.

[225] In some embodiments, the pulse delay period ranges from 0 ms to

2500 ms. Alternatively, in some embodiments, the pulse delay period ranges from 0 % to 80 % of the inhalation period.

[226] In some embodiments, the pulse cessation period ranges from 0 ms to

2500 ms. Alternatively, in some embodiments, the pulse cessation period ranges from 80 % to 0 % of the inhalation period.

[227] In some embodiments, each the pulse-on periods individually ranges from 100 ms to 5000 ms. Alternatively, each the pulse-on periods individually ranges from 10 % to 100 % of the inhalation period. [228] In some embodiments, each the pulse-off period individually ranges from 0 ms to 2500 ms. Alternatively, each the pulse-off periods individually ranges from 0 % to 200 % of the pulse-on period.

[229] In some embodiments, the method is based on a single pulse per inhalation period. In some embodiments, the single pulse is effected such that the pulse delivery period starts essentially as the inhalation period starts (pulse delay period is essentially zero), and ends essentially as the inhalation period ends (pulse cessation period is essentially zero). In other embodiments the method is effected by using a single pulse that starts after the inhalation period starts, and ends before the inhalation ends.

[230] In some embodiments, the coordination of pulse delivery is set to deliver more than one pulse in succession during the pulse delivery period, until the device senses a decrease in the rate of intake close to the end of the inhalation period. In such embodiments, the device is set to interrupt each pulse-on period with a pulse- off period. In some embodiments, the device is set to deliver a predetermined number of pulses that ranges from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 1 to 6, from 1 to 7, from 1 to 8, from 1 to 9, from 1 to 10, or from 1 to any number of pulses that can take place within the pulse delivery period as determined by any given breathing cycle. It is further noted that each of the pulses may span a different pulse- on period and be interrupted by a pulse-off period of different lengths.

[231] The concentration of nitric oxide in the nitric oxide-containing gaseous mixture is controlled by the concentration of nitric oxide is introduced into the inhalant, the output by which nitric oxide is introduced into the inhalant, the duration of the pulse-on period and the number of pulses introduced into the inhalant during the pulse delivery period. According to some embodiments of the present invention, during the pulse delivery period the inhalant is essentially a nitric oxide-containing gaseous mixture which contains at least 160 ppm nitric oxide, or nitric oxide-load of 80 ppm-hour per cycle, while during the pulse delay period and the pulse cessation period the inhalant is essentially devoid of nitric oxide.

[232] According to some embodiments, the method is affected by using more than one pulse, wherein the inhalant, which is produced by each of the pulses, delivers to the patient a different concentration of nitric oxide. For example, the method may be carried out by administering to the patient, during the pulse delivery period, three pulses, such that the inhalant that stems from the first pulse is characterized by an nitric oxide concentration of 160 ppm, the inhalant that stems from the second pulse is characterized by an nitric oxide concentration of 80 ppm, and the inhalant that stems from the first pulse is characterized by an nitric oxide concentration of 100 ppm. Hence, at least one pulse effects a concentration of at least 160 ppm. In other examples, some of the pulses may deliver an inhalant characterized by an nitric oxide concentration of more than 160 ppm.

[233] Alternatively, the number of pulses, the concentration of nitric oxide in each of the pulses, and the duration of the first time period during which pulses are generated, are configured to deliver an nitric oxide-load per cycle of 80 ppm-hour.

[234] As presented hereinabove, breathing cycle-coordinated pulse delivery inhalation allows the introduction of high concentrations of nitric oxide essentially during the periods of time in which the subject inhales at the highest in-breathing rate, thereby minimizing exposure of parts of the respiratory tract to high concentrations of nitric oxide. For example, since nitric oxide is introduced in pulses after the beginning of the inhalation period and before the end of the inhalation period, parts of the upper respiratory tract, the trachea and the some of the respiratory tree in the lungs which are not rich with alveolor capillaries, are only briefly exposed to high concentrations of nitric oxide due to the rate of inhalant intake, while the alveoli are exposed to this high concentrations of nitric oxide for a longer period of time.

[235] According to some embodiments of the present invention, subjecting the subject to the method described herein is carried out by use of an inhalation device which can be any device which can deliver the mixture of gases containing nitric oxide, including but not limited to breathing cycle-coordinated pulse delivery to a respiratory organ of the subject. An inhalation device, according to some embodiments of the present invention, includes, without limitation, a stationary inhalation device comprising tanks, gauges, tubing, a mask, controllers, values and the likes; a portable inhaler (inclusive of the aforementioned components), a metered- dose inhaler, a respiration machine/system and an intubated inhalation/respiration machine/system.

[236] Exemplary inhalation devices which may be suitable for the execution of any embodiment of any of the methods described herein, are provided, for example, by U.S. Provisional Patent Application Nos. 61/876,346 and 61/969,201, and U.S. Patent Nos. 6,164,276 and 6,109,260, the contents of which are hereby incorporated by reference. Commercial inhalation devices which may be suitable for the execution of any of the methods described herein, include the INOpulse® DS-C developed by Ikaria Australia Pry Ltd, or the Ohmeda INOpulse Delivery System by Datex-Ohmeda.

[237] An inhaler, according to some embodiments of the present invention, can generate spirometry data and adjust the treatment accordingly over time as provided, for example, in U.S. Patent No. 5,724,986 and WO 2005/046426, the contents of which are hereby incorporated by reference. The inhaler can modulate the subject's inhalation waveform to target specific lung sites. According to some embodiments of the present invention, a portable inhaler can deliver both rescue and maintenance doses of nitric oxide at subject's selection or automatically according to a specified regimen.

[238] According to some embodiments of the present invention, an exemplary inhalation device may include a delivery interface adaptable for inhalation by a human subject. According to some embodiments of the present invention, the delivery interface includes a mask or a mouthpiece for delivery of the mixture of gases containing nitric oxide to a respiratory organ of the subject.

[239] According to some embodiments of the present invention, the inhalation device further includes a nitric oxide analyzer positioned in proximity to the delivery interface for measuring the concentration of nitric oxide, oxygen and nitrogen dioxide flowing to the delivery interface, wherein the analyzer is in communication with the controller.

[240] It is expected that other methods for treating an inflammatory disease or disorder by intermittent inhalation of nitric oxide at 160 ppm or more will be developed and the scope of the term treating an inflammatory disease or disorder by intermittent inhalation of nitric oxide is intended to include all such new technologies a priori.

[241] As used herein the term "about" refers to ± 10 %.

[242] The terms "comprises", "comprising", "includes", "including",

"having" and their conjugates mean "including but not limited to".

[243] The term "consisting of means "including and limited to". [244] The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[245] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[246] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[247] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

[248] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[249] As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. [250] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[251] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[252] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

[253] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

EXAMPLE 1

Treatment of CF Patients with Progressive Lung Disease Caused by

Mycobacterium abscessus by nitric oxide inhalation

[254] The purpose of this study was to evaluate the effect of NO treatment according to some embodiments of the present invention on the clinical, physiological, bacteriological and inflammatory markers in CF patients with progressive lung disease due to Mycobacterium abscessus. The study will utilize a prospective, open-label interventional study comparing outcome parameters before after the first and at end of NO treatment.

Inclusion Criteria:

[255] CF patients with significant unresponsive lung disease due to

Mycobacterium abscessus. Sputum smears and mycobacterial cultures will be performed using approved techniques. Identification of "M abscessus" will be based on rpoB and hsp65 sequencing; multilocus sequencing analysis will be performed in cases of discrepancy between rpoB and hsp65 sequence data

[256] Willingness to sign written informed consent by the patient or legal guardian as applicable.

[257] Ability to understand and comply with study requirements.

[258] Age older than 12 years. NO will be provided on companionate basis on a'named-patient' basis. Patients will be allowed to receive other antimicrobial agents in combination with NO.

Exclusion Criteria:

[259] Breastfeeding or pregnancy as evidenced by a positive blood pregnancy test.

[260] Receiving any investigational agent within 4 weeks prior to screening visit. [261] Standards of care would be maintained during the study. The patients will be hospitalized for 5days. During these 5 days (day 1-5) they will receive NO 160ppm in 40% oxygen intermittently (30 minutes every 4 hours). Day 6-15 they will receive ambulatory treatment either at home or at our Pediatric Pulmonary Institute (according to preference). Day 6-10 three inhalations per day, and day 11-15 twice day.

[262] During treatment vital signs will be recorded. Additionally, the patients will have CO- oximeter (Masimo) and oxygen saturation and methemoglobin will be recorded noninvasive by a finger probe. Any increase in methemoglobin to above 5% for longer than 2 minutes will lead to cessation of treatment until the methemoglobin level will decrease to less than 5%. The system measures gases concentrations before inhaling, any N02 concentration above 5ppm will lead to cessation until baseline levels will be recorded. All adverse events will be recorded and reported. An adverse event is the appearance to or worsening of any undesirable sign, symptom, or medical condition occurring after starting NO even if the event is not considered to be related to the therapy. Medical conditions/diseases present before starting NO will be considered adverse events if they worsen after starting NO. Abnormal laboratory values or test results will be considered adverse events if they induce clinical signs or symptoms, are considered clinically significant, or require therapy. All adverse events will be recorded with the following information:

[263] 1. The severity grade (mild, moderate, severe)

[264] 2. Its relationship to the study drug(s) (suspected/not suspected)

[265] 3. Its duration (start and end dates or if continuing at final exam)

[266] 4. Whether it constitutes a serious adverse event (SAE)

[267] All adverse events will be treated appropriately and according to the discretion of the physician. To ensure patient safety, every serious adverse event (SAE), regardless of suspected causality, occurring after the patient has provided informed consent and until 30 days after the patient has stopped study participation (defined as time of last dose of study drug taken or last visit whichever is later) will be reported to AIT within 24 hours. Any SAEs experienced after this 30-day period will also be reported to AIT if the investigator suspects a causal relationship to the study drug.

[268] Outcome parameters will include: Pulmonary function tests, sputum culture and density and circulating and sputum /serum markers of inflammation. Evaluation will be carried at base line, at day 5 , day 10, day 15and day 45 (30 days after completion of treatment, Laboratory evaluation will be carried out blinded. The parameters that will be evaluated are:

[269] Two CF patients (age 13 & 20 years) with persistent aggressive

MABSC were identified, and the elder patient was treated according to some embodiments of the present invention. The older patient had shown positive MABSC cultures for more than 5 years. Additionally, the older patient had shown rapidly progressive changes in CT and rapid deterioration in pulmonary functions tests. See Figure 1, where the patient's FEVi declined over time (Aug 2014: 2.25 (82%); May 2015 2.25 (80%); June 2015 1.76 (62%); Nov 2015 1.46 (52%)). Repeated antibiotic interventions, including all known protocols (5 drugs and IV line) resulted in no improvement, and the patient suffered significant side effects.

[270] The younger patient had shown positive MABSC cultures for more than 6 months. In addition, the patient was hospitalized, to treat the side effects of linezolid. Prior to treatment according to some embodiments of the present invention, the patient was receiving IV meropenem, inhaled amikacin, moxifioxacin, and azenil. The younger patient's condition continued to decline.

[271] The 19 year old patient was hospitalized and treated as follows: the patient received a total of 5 high dose (160ppm) NO intermittent treatments a day (30 minutes every 4 hours), with 0₂-enriched air. Next, on days 6-7, the patient received a total of 2 high dose (160ppm) NO intermittently treatments a day (30 minutes every 4 hours) as an out patient. Next, on days 8-12, the patient received a total of 3 high dose (160ppm) NO intermittently treatments a day (30 minutes every 4 hours) as an outpatient. Delivered NO, N0₂ and 0₂ concentrations were continuously monitored using dedicated gas analyzers. Safety measures, included % methemoglobin (MetHb), Oxygen saturation (Sp0₂), bleeding episodes, vital signs, and any other adverse event (AE). M. abscessus load was evaluated by direct microscopy, culturing, and quantitative real-time PCR procedures. The preliminary results are shown below:

[272] The 13 year old patient was treated as follows: the patient received a total of 5 high dose (160ppm) NO intermittent treatments a day (30 minutes every 4 hours), with ( enriched air. Next, on days 6-7, the patient received a total of 2 high dose (160ppm) NO intermittently treatments a day (30 minutes every 4 hours) as an out patient. Next, on days 8-12, the patient received a total of 3 high dose (160ppm) NO intermittently treatments a day (30 minutes every 4 hours) as an outpatient. Delivered NO, N0₂ and 0₂ concentrations were continuously monitored using dedicated gas analyzers. Safety measures, included % methemoglobin (MetHb), Oxygen saturation (Sp0₂), bleeding episodes, vital signs, and any other adverse event (AE). M. abscessus load was evaluated by direct microscopy, culturing, and quantitative real-time PCR procedures.

[273] Both patient continued to receive antibiotic therapy during the treatment regimen disclosed above. No serious adverse events were reported, and the 19 year old subject stated that they "felt much better". Referring to Figures 2, 3, and 5, the 19 year old patient's FEVi and mean perfusion index appeared to increase as a result of the treatment outlined above. Furthermore, the patient's LCI and CRP decreased (see Figures 4 and 6).

[274] Peak MetHb levels were 4.5 and 4.7 % and peak N0₂ level were 3.2, and 4.4, respectively. In both patients, within 3 days sputum volume increased significantly. FEVi increased in the 19 year old patient by 9% and sustained until two weeks after treatment ended. FEVi in the 13 year old patient remained stable during treatment. Lung Clearance Index (LCI) remained stable in both patients. Six min walk increase in the 13 year old patient from 515 meters to 680 meters after 3 weeks. Elevated CRP levels was observed only in the 19 year old patient and decreased from 80 to 38. M. abscessus load decreased in 19 year old patient after one week (25 treatments) by 10-100 fold, but returned to baseline after 2 weeks of decrease inhalations rate to twice daily. Sputum cultures remained positive for abscessus.

[275] All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

CLAIMS What is claimed is:

1. A method for treating cystic fibrosis in a human subject in need thereof,

wherein the method comprises:

repeatedly administering to the human subject a gas mixture comprising nitric oxide at a concentration from about 144 to about 176 ppm for a first period of time, followed by a gas mixture containing no nitric oxide for a second period of time,

wherein the administration is repeated for a time sufficient to:

a) reduce the level of at least one inflammatory biomarker in the human subject when compared to the level of the inflammatory biomarker prior to the administration;

b) reduce the microbial density by 1 to 2 log units as measured by colony forming units in the human subject when compared to the microbial density prior to the administration; or

c) a combination thereof.

2. The method of claim 1, wherein the human subject suffers from a microbial infection associated with cystic fibrosis.

3. The method of claim 2, wherein the microbial infection is caused by a pathogenic microorganism.

4. The method of claim 3, wherein the pathogenic microorganism is selected from the group consisting of Pseudomonas alcaligenes, non-mucoid and mucoid Pseudomonas aeruginosa, Aspergillus fumigates, Staphylococcus aureus, Haemophilus influenza, Burkholderia cepacia complex, Klebsiella pneumonia, Escherichia coli, methicillin- resistant Staphylococcus aureus (MRSA), methicillin- sensitive Staphylococcus aureus (MSSA), Stenotrophomonas maltophilia, Achromobacter spp., Achromobacter xylosoxidans, Achromobacter ruhlandii, Achromobacter piechaudii, non-tuberculous mycobacteria (NTM) species, non-mucoid Pseudomonas aeruginosa, Mycobacterium abscessus complex (MABSC), mucoid Pseudomonas aeruginosa, and Acinetobacter baumannii.

5. The method of claim 1, the first period of time is 30 minutes and the second period of time is from about 3 to about 5 hours.

6. The method of claim 1, wherein the administration is repeated 6 times per day.

7. The method of claim 1, wherein the nitric oxide is repeatedly administered for a period of time from about one day to three weeks.

8. The method of claim 1, wherein nitric oxide is repeatedly administered for 5 days.

9. The method of claim 1, the administration further comprises an intermittent inhalation, comprising: (1) repetitive administration of 5 cycles per day of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

10. The method of claim 1, wherein the administration further comprises an intermittent inhalation, comprising: (1) repetitive administration of 5 cycles per day of a cycle comprising inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours, for about 5 days, then (2) three cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours; then (3) two cycles per day, for 4 days, of a cycle consisting of inhalation of the mixture for a first time period of 30 min, followed by inhalation of no nitric oxide for a second time period of 4 hours.

11. The method of claim 1, wherein the at least one inflammatory biomarker is selected from the group consisting of C-reactive protein (CRP), TNFa, TNF RII, IL- 1β, IL-lra/IL-lF3, IL-2, IL-4, IL-5, IL-6, IL-8, CXCL8/IL-8, IL-10, IL-12 p70, IL- 17A, GM-CSF, ICAM-1, IFN-gamma, MMP-8, MMP-9, VEGF and IL-12p70, neutrophils, lymphocytes and eosinophils count, neutrophil elastase activity, alpha- 1- antitrypsin (AAT), haptoglobin, transferrin, an immunoglobulin, granzyme B (GzmB), eosinophil cationic protein (ECP), eotaxin, tryptase, chemokine C-C motif ligand 18 (CCL18/PARC), RANTES (CCL5), surfactant protein D (SP-D), lipopolysaccharide (LPS)-binding protein and soluble cluster of differentiation 14 (sCD14).

12. The method of claim 1, wherein the at least one inflammatory biomarker is C- reactive protein (CRP).

13. The method of claim 1, wherein the method further comprises monitoring at least one on-site oximetric parameter in the subject, the at least one on-site oximetric parameter being selected from the group consisting of: oxyhemoglobin saturation (Sp0₂); methemoglobin (SpMet); perfusion index (PI); respiration rate (RRa); oxyhemoglobin saturation (SpC^); total hemoglobin (SpHb); carboxyhemoglobin (SpCO); methemoglobin (SpMet); oxygen content (SpOC); and pleth variability index (PVI).

14. The method of claim 1, wherein the method further comprises monitoring at least one additional on-site spirometric parameter in the subject, the at least one additional on-site parameter being selected from the group consisting of: forced expiratory volume (FEVl); maximum mid-expiratory flow (MMEF); diffusing capacity of the lung for carbon monoxide (DLCO); forced vital capacity (FVC); total lung capacity (TLC); and residual volume (RV).

15. The method of claim 1, wherein the method further comprises monitoring at least one on-site parameter in the gas mixture inhaled by the subject, the on-site parameter being selected from the group consisting of: an end tidal C0₂ (ETCO₂); a nitrogen dioxide (NO₂), a nitric oxide (NO); a serum nitrite/nitrate; afraction of inspired oxygen (F1O₂); and any combination thereof.

16. The method of claim 1, wherein the method further comprises monitoring at least one off-site bodily fluid parameter in the subject, the parameter being selected from the group consisting of: a bacterial and/or fungal load; urine nitrite; blood methemoglobin; blood pH; a coagulation factor; a blood hemoglobin; a hematocrit ratio; a red blood cell count; a white blood cell count; a platelet count; a vascular endothelial activation factor; a renal function; an electrolyte; a pregnancy hormone; serum creatinine; a liver function; and any combination thereof.