Nitric oxide inhalation therapy for infants with bronchiolitis

Abstract

To provide a method for treating bronchiolitis in infants.SOLUTION: The method comprising repeatedly administering to the infant a gas mixture comprising nitric oxide in a concentration of about 144 to about 176ppm for a first period of time, followed by a gas mixture that does not include nitric oxide for a second period of time; The administration is repeated for a time sufficient to a) reduce the length of hospitalization required to achieve 92% or greater oxygen saturation in room air, b) reduce the length of hospitalization required to achieve a clinical score of 5 or less, c) reduce the length of hospitalization required to discharge from hospital, or d) any combination thereof, as compared to an infant not subjected to the repeated administration of nitric oxide.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention claims priority to U.S. Provisional Patent Application No. 62 / 215,809, filed September 9, 2015, and U.S. Provisional Patent Application No. 62 / 220,321, filed September 18, 2015, both of which are incorporated herein by reference in their entireties. [Background technology]

[0002] FIELD OF THE INVENTION Some embodiments relate to therapies, methods, and devices for treating bronchiolitis in infants and young children.Some embodiments relate to therapies, methods, and devices for potentiating antimicrobial agents and / or sensitizing antimicrobial-resistant microorganisms to antimicrobial treatment. Summary of the Invention

[0003] The following embodiments and aspects of the present specification are described and illustrated in connection with systems, tools and methods, which are meant to be exemplary and illustrative, and not limiting in scope.

[0004] Among these disclosed utilities and improvements, other objects and advantages of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. While detailed embodiments of the present invention are disclosed herein, it should be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Furthermore, each of the examples shown in connection with various embodiments of the present invention are intended to be illustrative and not limiting.

[0005] Throughout this specification and claims, the following terms have the meanings clearly associated therewith, unless the context clearly dictates otherwise. As used herein, the phrases "in one embodiment" and "in some embodiments" do not necessarily refer to the same embodiment, but they can. Additionally, as used herein, the phrases "in another embodiment" and "in some other embodiments" do not necessarily refer to different embodiments, but they can. Thus, as described below, various embodiments of the invention can be readily combined without departing from the scope or spirit of the invention.

[0006] Additionally, as used herein, the term "or" is an inclusive "or" operator and is synonymous with the term "and / or" unless the context clearly indicates otherwise. The term "based on" is not exclusive and may be based on additional unexplained factors unless the context clearly indicates otherwise. Additionally, throughout the specification, "a," "an," and "the" include plural meanings. The meaning of "in" includes "in" and "on."

[0007] In some embodiments, the present invention is a method for treating bronchiolitis in an infant in need thereof, comprising repeatedly administering to the infant a gas mixture comprising nitric oxide at a concentration of about 144 to about 176 ppm for a first period, followed by a gas mixture without nitric oxide for a second period, wherein the administration is repeated for a period sufficient to a) reduce the length of hospital stay required to achieve oxygen saturation of 92% or greater on room air compared to infants not administered the repeated administration of nitric oxide; b) reduce the length of hospital stay required to achieve a clinical score of 5 or less compared to infants not administered the repeated administration of nitric oxide; c) reduce the length of hospital stay required to be discharged compared to infants not administered the repeated administration of nitric oxide; or d) any combination thereof.

[0008] In some embodiments, the first period of time is 30 minutes and the second period of time is from about 3 to about 5 hours.

[0009] In some embodiments, administration is repeated six times daily.

[0010] In some embodiments, the nitric oxide is administered repeatedly for a period of about 1 day to 3 weeks.

[0011] In some embodiments, the nitric oxide is administered repeatedly for 5 days.

[0012] In some embodiments, the method further comprises monitoring at least one on-site oxygen measurement parameter in the infant, wherein the on-site parameter is selected from the group consisting of oxyhemoglobin saturation (SpO2), methemoglobin (SpMet), perfusion index (PI), respiratory rate (RRa), oxyhemoglobin saturation (SpO2), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI).

[0013] In some embodiments, the method further comprises monitoring at least one additional on-site spirometry parameter in the infant, wherein the at least one additional on-site parameter is selected from the group consisting of forced expiratory volume (FEV1), maximum mid-expiratory flow (MMEF), diffusing capacity of the lungs for carbon monoxide (DLCO), forced vital capacity (FVC), total lung capacity (TLC), and residual volume (RV).

[0014] In some embodiments, the method further comprises monitoring at least one on-site parameter in the gas mixture inhaled by the infant, wherein the on-site parameter is selected from the group consisting of end-tidal CO2 (ETCO2), nitrogen dioxide (NO2), nitric oxide (NO), serum nitrite / nitrate, and fractional inspired oxygen (FiO2).

[0015] In some embodiments, the method further comprises monitoring at least one off-site body fluid parameter in the infant, the parameter being selected from the group consisting of bacterial and / or fungal load, urine nitrite, blood methemoglobin, blood pH, clotting factors, blood hemoglobin, hematocrit, red blood cell count, white blood cell count, platelet count, vascular endothelial activating factor, renal function, electrolytes, pregnancy hormones, serum creatinine, and liver function.

[0016] Some embodiments of one aspect of the present invention provide methods of treating a subject having a medical condition associated with a pathogenic microorganism, the methods comprising: (i) administering to the subject an enhancing-effective amount of nitric oxide; and (ii) administering to the subject a therapeutically effective amount of an antimicrobial agent, wherein the antimicrobial agent is other than nitric oxide.

[0017] In some embodiments, the potentiating effective amount is less than the therapeutically effective amount of nitric oxide against the pathogenic microorganism.

[0018] In some embodiments, the potentiating effective amount is less than 1 MIC unit of nitric oxide against a pathogenic microorganism.

[0019] In some embodiments, the pathogenic microorganism is resistant to the antimicrobial agent prior to administration of an enhancing amount of nitric oxide.

[0020] In some embodiments, resistance to pathogenic microorganisms is innate or acquired.

[0021] In some embodiments, the antimicrobial agent is inactive when used by itself (without administration of NO) against pathogenic microorganisms.

[0022] Some embodiments of one aspect of the present invention provide methods of treating a subject having a medical condition associated with a pathogenic microorganism in which antimicrobial resistance has emerged following treatment of the subject with an antimicrobial agent, the method being carried out by (i) administering a re-sensitizing effective amount of nitric oxide to the subject after treatment with the antimicrobial agent and the emergence of antimicrobial resistance, and (ii) administering a therapeutically effective amount of an antimicrobial agent to the subject, wherein the antimicrobial agent is other than nitric oxide and the re-sensitizing effective amount of nitric oxide is less than the therapeutically effective amount of nitric oxide for the microorganism.

[0023] According to some embodiments of one aspect of the present invention, a method for sensitizing or re-sensitizing a microorganism to an antimicrobial agent is provided, comprising: (i) contacting the microorganism with a sensitizing or re-sensitizing effective amount of nitric oxide; and (ii) contacting the microorganism with a therapeutically effective amount of an antimicrobial agent, wherein the antimicrobial agent is other than nitric oxide.

[0024] In some embodiments, the sensitizing or re-sensitizing effective amount is less than the therapeutically effective amount of nitric oxide against the pathogenic microorganism.

[0025] In some embodiments, the effective sensitizing or re-sensitizing amount is less than 1 MIC unit of nitric oxide against the pathogenic microorganism.

[0026] In some embodiments, contacting the microorganism with nitric oxide comprises administering a sensitizing or re-sensitizing amount of nitric oxide to a subject having a medical condition associated with the microorganism.

[0027] In some embodiments, the methods disclosed herein further comprise administering to the subject an antimicrobial agent.

[0028] In some embodiments, (i) is performed before (ii).

[0029] In some embodiments, (ii) is performed before (i).

[0030] In some embodiments, (i) is performed simultaneously with (ii).

[0031] Some embodiments of one aspect of the present invention provide pharmaceutical compositions comprising a sensitizing or re-sensitizing effective amount of nitric oxide, a therapeutically effective amount of an antimicrobial agent, and at least one pharmaceutically acceptable carrier.

[0032] In some embodiments, the pharmaceutical compositions presented herein are in a multi-part dosage form, wherein the nitric oxide is in a first part and the antimicrobial agent is in a second part.

[0033] Some embodiments of one aspect of the present invention provide a pharmaceutical composition unit dosage form comprising a therapeutically effective amount of an antimicrobial agent in a first unit dosage form and a sensitizing or re-sensitizing effective amount of nitric oxide in a second unit dosage form, wherein the sensitizing or re-sensitizing effective amount is an amount that achieves sensitization or re-sensitization of a pathogenic microorganism to the antimicrobial agent, and wherein the sensitizing or re-sensitizing effective amount is less than the therapeutically effective amount of nitric oxide for the pathogenic microorganism.

[0034] Some embodiments of one aspect of the present invention provide a pharmaceutical kit comprising packaging material and a therapeutically effective amount of an antimicrobial agent packaged in the packaging material, the kit being labeled for treating a medical condition associated with a pathogenic microorganism and / or for sensitizing or re-sensitizing a pathogenic microorganism to the antimicrobial agent upon co-administration to a treated subject of a sensitizing or re-sensitizing amount of nitric oxide.

[0035] In some embodiments, the kit further comprises a sensitizing or re-sensitizing effective amount of nitric oxide, wherein the antimicrobial agent and the nitric oxide are packaged separately within the kit.

[0036] In some embodiments of the methods, compositions, unit dosage forms or kits provided herein, the therapeutically effective amount of the antimicrobial agent is less than 1 MIC against a pathogenic microorganism.

[0037] In some embodiments of the methods, compositions, unit dosage forms, or kits provided herein, nitric oxide is administered or contacted in the form of gaseous nitric oxide or a nitric oxide-releasing compound.

[0038] In some embodiments of the methods, compositions, unit dosage forms, or kits presented herein, the nitric oxide is administered or contacted with the microorganism by inhalation.

[0039] The present invention will be further described with reference to the accompanying drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention. Moreover, some features may be exaggerated to particularly show details of the components.

[0040] The drawings constitute a part of this specification and include illustrative embodiments of the present invention and illustrate various objects and features thereof. Moreover, the drawings are not necessarily to scale, and some features may be exaggerated to show details of particular components. Furthermore, any measurements, specifications, etc. shown in the drawings are intended to be illustrative and not limiting. Therefore, specific structural and functional details disclosed herein should not be construed as limiting, but merely as a representative basis for teaching those skilled in the art how to variously use the present invention. The drawings are now described. [Brief explanation of the drawings]

[0041] [Figure 1] FIG. 1 illustrates a test plan according to some embodiments of the present invention. [Figure 2] 1 shows the composition of infants treated according to some embodiments of the present invention, where N=number of infants, ITT=intention-to-treat subgroup, and PP=per-protocol subgroup. [Figure 3]

[0023] Figures 1A-1C show methemoglobin levels in infants treated according to some embodiments of the present invention. Panel A: Mean (±SE) MetHb levels over time for first treatment (ITT, N=43). Panel B: Mean (±SE) metHb levels pre- and end-of-treatment by treatment number in the NO treatment group (ITT). ITT = intention-to-treat subgroup, MetHb = methemoglobin, N = number of infants in each treatment number, SE = standard error. [Figure 4] FIG. 1 shows NO2 levels at first dose of NO in infants treated with some embodiments of the present invention. [Figure 5] Figure 1 shows the median length of hospitalization (LOS) observed in infants treated according to some embodiments of the invention by treatment group and subgroup. Panel A: ITT analysis (n=43). Panel B: ITT with LOS<24 hours (n=16). Panel C: ITT with LOS>24 hours (n=27). [Figure 6] Figure 1 shows Kaplan-Meier analyses of LOS by treatment group and subgroup (ITT and PP) in infants treated according to some embodiments of the invention. Panel A: Time to discharge, ITT (n=43). Panel B: Time to discharge, LOS > 24 hours subgroup (n=27). Panel C: Time to discharge, PP analysis (n=39). Panel D: Time to discharge, PP > 24 hours (n=24). [Figure 7]Figure 1 shows a Kaplan-Meier analysis of time to first sustained O2 saturation by treatment group and subgroup (ITT and PP) in infants treated according to some embodiments of the invention. Panel A: Time to first sustained O2 saturation, ITT population (n=42). One infant (control group) was excluded from the analysis because this infant had an SpO2 of 92%. Panel B: Time to first sustained O2 saturation, ITT (n=24) with LOS > 24 hours. Panel C: Time to first sustained O2 saturation, PP population (n=38). One infant (control group) was excluded from the analysis because this infant had an SpO2 of 92%. Panel D: Time to first sustained O2 saturation, PP (n=24) with LOS > 24 hours. [Figure 8] Figure 1 shows a Kaplan-Meier analysis of time to clinical score ≦5 by treatment group and subgroup (ITT and PP) in infants treated according to some embodiments of the invention. Panel A: Time to achieve clinical score ≦5, ITT population (n=43). Panel B: Time to achieve clinical score ≦5, ITT (n=27) with LOS >24 hours. Panel C: Time to achieve clinical score ≦5, PP population (n=39). Panel D: Time to achieve clinical score ≦5, PP (n=24) with LOS >24 hours. [Figure 9] FIG. 1 is a plot showing the antimicrobial activity of nitric oxide against Serratia marcescens, as described in the Background Art, demonstrating the latency of antimicrobial activity as a function of the time required to exhaust the chemical defense mechanisms of the microorganism. DETAILED DESCRIPTION OF THE INVENTION

[0042] Treatment of Bronchiolitis: Inflammation is the body's primary or secondary response to cell damage, infection, or the presence of foreign substances. As a primary cause, inflammation is associated with numerous diseases and disorders and can also lead to system deterioration and failure, and if left untreated, can cause secondary symptoms. Aside from the typical symptoms of inflammation, such as fever, swelling, and pain, inflammation can also be diagnosed by monitoring certain endogenous factors or inflammatory biomarkers, the amount of which in the body is an indicator of the severity and stage of inflammation.

[0043] Bronchiolitis is defined as an infection of the small airways. It is also the most common manifestation of acute lower respiratory tract infection (ALRI) in infancy and is the leading cause of childhood mortality worldwide. Viral bronchiolitis is currently the most common cause of pediatric hospitalization in the United States, accounting for nearly 20% of all causes of hospitalization in infants. Viral etiologies are the predominant cause, and among respiratory viruses, respiratory syncytial virus (RSV) is thought to be the most important viral pathogen causing ALRI in infants. The disease is primarily prevalent in the first year of life. Clinical signs and symptoms are consistent with hypoxia, dyspnea, coryza, anorexia, asthma, wheezing and crepitus on auscultation, and occasionally respiratory failure.

[0044] In one embodiment, the present invention administers nitric oxide to infants, where the administration is short-term high-level nitric oxide, which improves lung function in infants with bronchiolitis, while not producing other signs of lung damage or adverse effects.

[0045] Throughout this specification, when the term "nitric oxide" is used in the context of inhalation, it should be understood that nitric oxide is inhaled in its gaseous state.

[0046] Some embodiments of the present invention provide methods of treating bronchiolitis in an infant in need thereof (e.g., an infant having or diagnosed with bronchiolitis). Diagnosis of bronchiolitis may be made by methods known in the art, including those described in the Examples section below.

[0047] The methods described herein include subjecting an infant to intermittent inhalation of a gas mixture comprising nitric oxide or any combination thereof, as described in any one of the embodiments relating to intermittent inhalation.

[0048] According to embodiments of the present invention, methods of treating bronchiolitis include any effective therapeutic effect demonstrated in bronchiolitis patients, including, for example, improvement in bronchiolitis symptoms (e.g., improved lung function), improvement in medical conditions associated with bronchiolitis (e.g., reduced microbial infections associated with bronchiolitis, reduced load of pathogenic microorganisms associated with bronchiolitis, reduced inflammation), and reduced length of hospital stay in infants.

[0049] In some embodiments, the methods of treating bronchiolitis described herein are contemplated as methods of treating infants and young children with bronchiolitis, and include methods of improving symptoms of bronchiolitis (e.g., improving lung function), improving medical conditions associated with bronchiolitis (e.g., reducing microbial infections associated with bronchiolitis, reducing the amount of pathogenic microorganisms associated with bronchiolitis, reducing inflammation), and shortening the length of hospital stay for patients.

[0050] In terms of the efficacy of treating bronchiolitis in infants, it is generally accepted that pulmonary function is one of the simplest and most direct markers for improving bronchiolitis symptoms, and therefore improvement of pulmonary function in infants represents effective treatment of infants with bronchiolitis.

[0051] Without being bound by any particular theory, nitric oxide delivered in exogenous gaseous form readily enters the pulmonary system and acts by pulmonary vasodilation, reducing pathogenic microbial load, reducing inflammation, and alleviating other clinical symptoms.

[0052] In some embodiments, the methods described herein, in any one of the embodiments and in any combination thereof, are practiced by improving one or more physiological parameters in an infant with bronchiolitis complicated by a medical condition associated with bronchiolitis, where improvement in any of these parameters is indicative of a beneficial effect of treatment with intermittent inhaled nitric oxide, in accordance with any one of the embodiments described herein.

[0053] According to some embodiments of the present invention, the method is carried out by improving at least one pulmonary function (spirometry parameter), such as, but not limited to, forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), FEV1 / FVC ratio or FEV1%, and forced expiratory flow (FEF).

[0054] The spirometric parameter forced vital capacity (FVC) is the volume of air measured in liters that is forcibly expired after maximal inspiration and constitutes the most fundamental maneuver in respiratory function testing.

[0055] The spirometry parameter forced expiratory volume in the first second (FEV1) is the volume of air forcibly exhaled in 1 second after maximal inspiration. The mean value of FEV1 depends primarily on gender and age, while values ​​between 80% and 120% of the mean are considered normal. The predicted normal value of FEV1 can be calculated on-site and depends on age, gender, height, weight, and ethnicity, as well as the research study on which these are based.

[0056] The spirometry parameter FEV1 / FVC ratio (FEV1%) is the ratio of FEV1 to FVC, which is approximately 75-80%. Predicted FEV1% is defined as the patient's FEV1% divided by the mean FEV1% in the appropriate population.

[0057] The spirometric parameter forced expiratory flow (FEF) is the flow rate (or rate) of air expelled from the lungs during the middle portion of a forced expiration. It can be obtained at discrete times and is generally calculated as the residual portion of the forced vital capacity (FVC), i.e., 25% of the FVC (FEF 25 ), 50% of FVC (FEF 50 ) or 75% of FVC (FEF 75 ), which can also be taken as the average of flow during an interval and is generally bounded by a specific residual fraction of the FVC, usually between 25 and 75% (FEF 25~75 ) Measured values ​​ranging from 50-60% to 130% of the mean 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 on which they are based. Recent studies have shown that FEF 25~75 % or FEF 25~50 These findings suggest that FEV1% may be a more sensitive parameter than FEV1 in detecting obstructive small airway disease. However, in the absence of concomitant changes in standard markers, differences in mid-range expiratory flow rates may not be specific enough to be useful, and current practice guidelines recommend continuing to use FEV1, VC, and FEV1 / VC as indices of obstructive disease.

[0058] In some embodiments, other spirometry parameters may be used to track the progress and efficacy of bronchiolitis treatment with intermittent inhalation of 160 ppm nitric oxide, as defined and described herein below, and / or to track safety parameters of the treatment.

[0059] In some embodiments, FEV1 is monitored as an on-site parameter, as defined below, and is an indicator of the beneficial effect of the intermittent inhalation of nitric oxide provided herein. Generally, an increase in FEV1 is considered a desirable effect in infants with bronchiolitis, where an increase of at least 3% of the patient's baseline FEV1 (before treatment begins) is considered a significant improvement. In some embodiments, the method is carried out such that FEV1 increases by at least 3, 5, 10, 15, or 20% during and / or after intermittent inhalation of nitric oxide (e.g., during and / or after the entire period during which intermittent inhalation of nitric oxide is administered), as described herein.

[0060] According to an embodiment of the present invention, the method is carried out to reduce the pathogenic microbial load in infants by at least one log during intermittent inhalation therapy.

[0061] The term "log unit" used herein to describe changes in the load of pathogenic microorganisms is also known as "log reduction" or "log increase," and is a mathematical term used to indicate the relative number of live microorganisms removed from a system by implementing the method of intermittent inhalation of nitric oxide, as described herein. For example, a 5-log reduction means that the number of microorganisms is reduced by 100,000 times; that is, if a sample contains 100,000 microorganisms, a 5-log reduction will reduce the number of microorganisms to 1. Thus, a 1-log reduction means that the number of pathogenic microorganisms is reduced by 10 times, a 2-log reduction means that the number of pathogens is reduced by 100 times, a 3-log reduction means that the number of pathogens is reduced by 1000 times, a 4-log reduction means that the number of pathogens is reduced by 10,000 times, and so on.

[0062] Bronchiolitis is generally associated with an inflammatory condition in at least one body part, such as the lungs, or with acute, chronic, localized, or systemic inflammation caused by one or more medical conditions, including, but not limited to, pathogen infection. Inflammation in infants with bronchiolitis can also be considered a secondary symptom to bronchiolitis (a medical condition associated with bronchiolitis). According to some embodiments of the present invention, the method is carried out by reducing the level of inflammation associated with bronchiolitis.

[0063] The reduction of inflammation associated with bronchiolitis is generally considered to be the beneficial effect of treating bronchiolitis.Similarly, the reduction of the amount of inflammatory biomarkers associated with bronchiolitis can be considered as an indicator of the efficacy of the method of treating infants with bronchiolitis provided herein.

[0064] In the context of some embodiments of the present invention, inflammation or inflammatory markers associated with bronchiolitis include, but are not limited to, serum / blood levels of C-reactive protein (CRP), interleukins such as IL-6 and IL-1β, alpha-1-antitrypsin (AAT), haptoglobin, transferrin, various immunoglobulins, granzyme B (GzmB), chemokine CC motif ligand 18 (CCL18 / PARC), surfactant protein D (SP-D), lipopolysaccharide (LPS) binding protein, and soluble cluster classification 14 (sCD14).

[0065] The term "cytokine", as used in the context of embodiments of the present invention, includes chemokines, interferons, interleukins, lymphokines and tumor necrosis factors.

[0066] A brief description of four non-limiting example inflammatory biomarkers associated with bronchiolitis follows.

[0067] Tumor necrosis factor alpha (TNFα) signals the body to migrate neutrophilic white blood cells to the site of infection or injury. TNFα is known as a cytokine, or cell signaling protein. TNFα acts like a "first responder" in emergencies by signaling the body to where the most damage is, allowing the immune system to respond effectively and send neutrophils to the scene.

[0068] Nuclear factor kappa B (NFkB) is a transcription factor protein that acts as a switch on specific genes. When NFkB is able to enter the nucleus, which it does with the help of TNFα, it turns on genes that allow cells to grow, mature, and avoid destruction by apoptosis (programmed cell death). This allows white blood cells to replicate and exert their activity in clearing infected or damaged areas. NFkB acts like a priority setting on the lines of communication by opening all available channels for the most rapid response.

[0069] Interleukin-6 (IL-6) is a cytokine that causes neutrophils to destroy themselves and attract another type of white blood cell, monocytes, to the area of ​​infection or injury instead. Monocytes create macrophages that sweep away debris and pathogens through phagocytosis, the process by which macrophages break down dead cells and other particles whole.

[0070] C-reactive protein (CRP) is a "pattern recognition receptor" protein, meaning it marks recognized debris for removal; it is produced by the liver in response to IL-6 levels and binds to the surface of dead or dying cells, and also to certain types of bacteria. CRP acts as a form of signal for macrophages to ingest something through phagocytosis, thereby aiding in the eventual removal of debris during inflammation.

[0071] According to some embodiments, monitoring the concentration of inflammatory biomarkers associated with bronchiolitis is useful for determining the progress and effectiveness of treatment for inflammation associated with bronchiolitis, hi some embodiments, the concentration of the biomarkers associated with bronchiolitis in serum taken from the infant is reduced by at least 3, 5, 10, 15, 20, 30, 35, 40, 50, or at least 60% during treatment, based on the baseline blood concentration in the infant before treatment began.

[0072] In some embodiments, the biomarker associated with bronchiolitis is CRP, and the blood level of CRP is reduced by at least 3, 5, 10, 15, 20, 30, 35, 40, 50, or at least 60% during intermittent inhalation treatment compared to the baseline level in the infant before treatment began.

[0073] In some embodiments, the biomarker associated with bronchiolitis is a cytokine, such as, but not limited to, TNFα, IL-1β, IL-6, IL-8, IL-10, and / or IL-12p70, and the blood concentration of the cytokine is reduced by at least 3, 5, 10, 15, 20, 30, 35, 40, 50, or at least 60% compared to the baseline concentration in the infant before treatment began. In some embodiments, the cytokines used as inflammatory biomarkers in the methods provided herein are IL-6 and IL-1β.

[0074] Some embodiments of the present invention provide methods for reducing the burden of pathogenic microorganisms in infants by subjecting the infant to intermittent inhalation of a gas mixture comprising nitric oxide at a concentration of at least 160 ppm.

[0075] In some embodiments, the pathogenic microorganism causes a microbial infection associated with bronchiolitis, as described herein. According to some embodiments, the pathogenic microorganism is selected from the group consisting of P. alcaligenes, non-mucoid and mucoid Pseudomonas aeruginosa, A. fumigatus, Streptococcus aureus, Haemophilus influenzae, Burkholderia cepacia complex, Klebsiella pneumoniae, Escherichia coli, methicillin-resistant Streptococcus aureus (MRSA), methicillin-susceptible Streptococcus aureus (MSSA), Stenotrophomonas maltoferia, Achromobacter species, Achromobacter xylosoxidans, and nontuberculous mycobacteria (NTM) species.

[0076] According to some embodiments, the pathogenic microorganism is selected from the group consisting of P. alcaligenes, methicillin-susceptible Streptococcus aureus (MSSA), Achromobacter species, A. fumigatus, non-mucoid Pseudomonas aeruginosa, and mucoid Pseudomonas aeruginosa.

[0077] As discussed above, in some embodiments, the pathogenic microbial load is reduced by at least one log unit during intermittent inhalation by the presently claimed methods.

[0078] Some embodiments of the present invention provide methods for reducing the amount of inflammatory biomarkers associated with bronchiolitis in infants by subjecting the infant to treatment with intermittent inhalation of a gas mixture containing nitric oxide at a concentration of at least 160 ppm.

[0079] According to some embodiments, inflammatory biomarkers associated with bronchiolitis, and / or changes in their normal physiological concentrations, are associated with complications and other medical conditions associated with cystic fibrosis and / or bronchiolitis. Reducing the concentration of inflammatory biomarkers associated with bronchiolitis in infants with bronchiolitis is an indication for treating inflammation (as a secondary medical condition).

[0080] In some embodiments, the inflammatory biomarker associated with bronchiolitis is targeted for reduction by the claimed methods and is selected from the group consisting of C-reactive protein (CRP), cytokines, alpha-1-antitrypsin (AAT), haptoglobin, transferrin, immunoglobulins, granzyme B (GzmB), chemokine CC motif ligand 18 (CCL18 / PARC), surfactant protein D (SP-D), lipopolysaccharide (LPS) binding protein, and soluble cluster classification 14 (sCD14).

[0081] In some embodiments of this aspect of the invention, the inflammatory biomarker associated with bronchiolitis is C-reactive protein (CRP), and the intermittent inhalation results in a reduction of at least 3, 5, 10, 15, 20, 30, 35, 40, 50, or at least 60% compared to the baseline concentration of the biomarker in the patient.

[0082] In some embodiments of this aspect of the invention, the inflammatory biomarker associated with bronchiolitis is a cytokine and is selected from the group consisting of TNFα, IL-1β, IL-6, IL-8, IL-10, and IL-12p70. In some embodiments, the inflammatory biomarkers are IL-6 and IL-1β. The reduction in cytokine concentration as a result of treatment is at least 3, 5, 10, 15, 20, 30, 35, 40, 50, or at least 60% compared to the baseline concentration of the biomarker in the patient.

[0083] Intermittent inhalation As indicated above, any of the methods provided herein involve subjecting an infant to intermittent inhalation of a gas mixture containing nitric oxide at a concentration of at least 160 ppm.

[0084] The term "intermittent" is used herein and in the art as the opposite of "continuous" to mean starting and stopping an activity and / or performing an activity at intervals.

[0085] "Intermittent inhalation" means that the infant intermittently inhales a gas mixture containing a designated concentration of nitric oxide, such that the volume of the gas mixture inhaled does not change significantly during the intermittent inhalation, but the chemical composition of the mixture varies according to a predetermined dosing regimen, as described later in this specification. The infant thus inhales a gas mixture containing nitric oxide at a concentration of at least 160 ppm for predetermined periods of time, and between these periods the infant inhales a gas mixture substantially devoid of nitric oxide (e.g., ambient air or another nitric oxide-free mixture).

[0086] Throughout this specification, "nitric oxide-containing gas mixture" or "gas mixture comprising nitric oxide" is used to describe a gas mixture containing at least 160 ppm of nitric oxide. Nitric oxide-containing gas mixtures may contain 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm, and even higher concentrations of nitric oxide. Other gas mixtures referred to herein contain less than 160 ppm of nitric oxide or are substantially devoid of nitric oxide, as defined herein.

[0087] "Substantially devoid of nitric oxide" means 50 ppm or less, 40 ppm or less, 30 ppm or less, 20 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, and ppb or less, including the complete absence of nitric oxide.

[0088] According to some embodiments of the invention, the intermittent inhalation comprises one or more cycles, each cycle comprising a first period of continuous inhalation of a gas mixture comprising nitric oxide at a particular high concentration (e.g., at least 160 ppm), followed by a second period of inhalation of a gas mixture substantially devoid of nitric oxide. According to some embodiments of the invention, during the second period, the infant may inhale ambient air or a controlled gas mixture substantially devoid of nitric oxide, as defined herein.

[0089] In some embodiments, the first period of time spans 10 to 45 minutes, or 20 to 45 minutes, or 20 to 40 minutes, and in some embodiments, about 30 minutes.

[0090] According to some embodiments of the present invention, the second period of time ranges from 3 to 5 hours, or 3 to 4 hours, and according to some embodiments, the second period of time spans about 3.5 hours.

[0091] According to some embodiments of the present invention, this inhalation regimen is repeated 1 to 6 times over a 24 hour period, depending on the first and second periods.

[0092] In some embodiments, a cycle of intermittent nitric oxide delivery, e.g., 30 minutes of 160 ppm followed by 3.5 hours of nitric oxide-free breathing, is repeated 1-6 times per day. In some embodiments, the cycle is repeated 5 times per day. Alternatively, the cycle is repeated 3 times per day.

[0093] According to some embodiments of the invention, a 1-5 times per day dosing regimen is administered for 1-21 days, or 2-14 days, or 3-10 days. According to some embodiments of the invention, intermittent inhalation is administered for 2 weeks. However, longer-term intermittent nitric oxide administration is also contemplated, as described herein.

[0094] safety As discussed above, intermittent inhalation of 160 ppm 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 infants as a measure of safety in the methods presented herein, while noting that no significant adverse changes in the monitored parameters occurred. According to any one of the embodiments of the present invention, intermittent inhalation is administered while monitoring one or more physiological parameters in the infant.

[0095] In some embodiments, the methods disclosed herein are performed while monitoring various parameters related to maintaining the desired dosage and dosing regimen, related to the safety of the treatment, and related to the efficacy of the treatment.

[0096] According to any one of the embodiments of the present invention, the method is carried out while monitoring one or more physiological parameters in the infant as a measure of safety of the methods described herein, and noting that no significant adverse changes occur in the monitored safety parameters.

[0097] In some embodiments, the methods are performed while maintaining safety by including noninvasive monitoring of body fluid chemistries, such as perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (SpO2 / SaO2 / DO), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), as measured by pulse oximetry. These physiological parameters are commonly known in the art.

[0098] Other parameters that are also monitored as safety measurements in the disclosed methods, according to some embodiments thereof, are off-site physiological parameters, which are typically measured by collecting body samples using non-invasive (e.g., urine, feces, or sputum samples) and invasive (e.g., blood or biopsy) methods.

[0099] For example, off-site physiological parameters commonly measured by invasive methods include serum nitrite / nitrate (NO2 - / NO3 - ), blood hemoglobin, complete blood count (CBC), blood chemistry / biochemistry (electrolytes, kidney and liver function tests, etc.) and coagulation tests.

[0100] Off-site physiological parameters commonly measured by non-invasive methods include nitrite / nitrate (NO2 - / NO3 - ), urine pregnancy test, and bacterial and fungal loads in sputum, urine, or feces.

[0101] In some embodiments, the method is performed while maintaining safety measures, including controlling the inhaled gas mixture and monitoring exhaled gases, by standard means for on-site monitoring and control of the content and / or flow rate of the mixture to which the infant is provided or delivered through the delivery interface, and / or by on-site monitoring of exhaled gases and controlling uptake by real-time feedback. In some embodiments, the method is performed while monitoring the concentrations of nitric oxide, O, CO, and NO in the gas mixture to which the infant is exposed or exhaled.

[0102] In some embodiments, the concentration of nitric oxide in the nitric oxide-containing gas mixture is controlled so that it does not deviate from the predetermined concentration by more than 10%. For example, the method is performed with the concentration of nitric oxide set at 160 ppm without substantially exceeding the boundary between 144 ppm and 176 ppm.

[0103] Similarly, the NO2 content in the nitric oxide-containing mixture is controlled to maintain the concentration of NO2 below 5 ppm.

[0104] Additionally, the amount of oxygen in the nitric oxide-containing gas mixture is controlled so that the O2 concentration in the mixture is in the range of about 20 to about 25%.

[0105] Alternatively, or additionally, the amount of oxygen in the nitric oxide-containing gas mixture is controlled so that the fractional inspired oxygen (FiO2) is in the range of about 20 to about 100%.

[0106] The phrase "inspired oxygen concentration ratio" or "FiO2," as used herein, refers to the ratio or percentage of oxygen in a given gas sample. For example, ambient air at sea level contains 20.9% oxygen, which corresponds to an FiO2 of 0.21. Oxygen-enriched air has an FiO2 greater than 0.21 and up to 1.00, meaning 100% oxygen. In the context of embodiments of the present invention, the FiO2 is maintained below 1 (less than 100% oxygen).

[0107] According to some embodiments, the fractional fraction of inspired oxygen (FiO2) in the nitric oxide-containing gas mixture is 0.20. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.25. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.3. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.35. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.4. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.45. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.5. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.55. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.6. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.65. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.7. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.75. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.8. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.85. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.9. In an alternative embodiment, the FiO2 in the nitric oxide-containing gas mixture is 0.95.

[0108] In some embodiments, the nitric oxide-containing gas mixture is prepared by mixing a stock supply of nitric oxide with air to dilute the stock supply of nitric oxide to the desired concentration. In some embodiments, the stock supply of nitric oxide is mixed with air and oxygen to maintain an FiO2 above 0.20. The ratio of nitric oxide, air, and / or oxygen may be varied to obtain the desired nitric oxide concentration and FiO2.

[0109] The phrase "end-tidal CO2" or "ETCO2," as used herein, refers to the partial pressure or maximum concentration of carbon dioxide (CO2) at the end of exhaled breath, expressed as a percentage of CO2 or pressure units of mmHg. Normal values ​​in humans range from 5-6% CO2, which corresponds to 35-45 mmHg. Because CO2 diffuses from the lungs into exhaled air, ETCO2 values ​​reflect cardiac output (CO2), and pulmonary blood flow as a gas is transported to the right side of the heart by the venous system and then pumped to the lungs by the right ventricle. A device called a capnometer measures the partial pressure and maximum concentration of CO2 at the end of exhalation. In the context of an embodiment of the present invention, a capnometer is used to monitor ETCO2 levels and provide warning feedback if ETCO2 exceeds 60 mmHg.

[0110] Respiratory NO, NO2, and O2 concentrations (both inhaled and exhaled, inspiratory and expiratory gases) are typically monitored continuously by sampling from a mouthpiece sample port placed on an inhalation mask equipped with an electrochemical analyzer. In the context of embodiments of the present invention, safety considerations require absolute minimization of the number of occasions during nitric oxide administration where NO2 concentrations exceed 5 ppm, nitric oxide concentration fluctuations exceed 10%, and FiO2 / O2 concentrations fall below 20%.

[0111] It is known that a sudden rise in inflammatory biomarkers may be associated with a phenomenon called a "cytokine storm," which has been observed in infants receiving inhaled nitric oxide therapy. Thus, monitoring inflammatory biomarkers while performing the methods described herein, according to embodiments of the present invention, has an additional role in safety considerations for the methods, where the absence of a significant increase in inflammatory markers is an indicator of safety.

[0112] In some embodiments, monitoring one or more physiological parameters is performed by non-invasive and / or mildly invasive measurements.

[0113] In some embodiments, monitoring physiological parameters in an infant is performed by on-site measurement and analysis techniques based on samples collected singly, continuously or periodically on-site from the infant in real time at the infant's bedside, and / or off-site measurement and analysis based on samples collected singly or periodically from the infant that are sent for processing off-site that provides results and analysis at a later time.

[0114] In the context of some embodiments of the present invention, the phrase "on-site measurement and analysis technology" or "on-site technology" refers to monitoring technology that informs a physician of certain physiological parameters of an infant in real time, without the need to send samples or raw data to an off-site facility for analysis. On-site technologies are often non-invasive, although some rely on sampling from an invasive medical device, such as a breathing tube, a drain tube, an intravenous catheter, or a subcutaneous port or any other implantable probe. Thus, the phrase "on-site parameter," as used herein, refers to a physiological parameter that can be obtained with online technology.

[0115] Besides the obvious advantages of real-time, on-site measurement of physiological parameters, which are primarily manifested in the physician's ability to quickly and manually respond to any significant changes therein, data obtained from real-time, online measurement of physiological parameters can be fed to a device and used for real-time feedback control of the device. In the context of embodiments of the present invention, the term "real-time" also relates to systems that update and respond to information almost instantaneously as they receive it. Such real-time feedback can be used to ensure compliance with treatment regimens and / or to automatically act immediately in response to significant deviations from parameters acceptable as safety measures.

[0116] Thus, in accordance with embodiments of the present invention, the term "on-site parameters" refers to physiological and / or mechanical and / or chemical data that is available and can be used or considered at or near the infant's location (e.g., bedside) in a relatively short time, i.e., the time between sampling, testing, processing, and displaying / using the data is relatively short. "On-site parameters" can be obtained from sampling to use in, for example, 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. For example, the time required to obtain on-site parameters through a technique known as pulse oximetry is nearly instantaneous; once the device is in place and configured, data related to, for example, peripheral oxygen saturation of an infant is available from sampling to use in less than 1 second.

[0117] In the context of some embodiments of the present invention, the phrase "off-site measurement and analysis technology" or "off-site technology" refers to technology in which samples or raw data are sent to an offline, and typically off-site, facility and provided with information regarding a given physiological parameter of an infant after undergoing offline analysis, sometimes hours or days after the sample is obtained. Off-site technologies are often based on samples collected by mildly invasive techniques, such as blood sampling to monitor inflammatory cytokine plasma levels, and invasive techniques such as biopsy, catheterization, or drainage tubes, although some off-site technologies rely on non-invasive sampling and off-site analysis, such as offline urine and fecal chemistry. The phrase "off-site parameter," as used herein, refers to a physiological parameter that can be obtained by off-site laboratory technology.

[0118] Thus, in accordance with embodiments of the present invention, the term "off-site parameters" refers to physiological and / or mechanical and / or chemical data that can be obtained and used or considered over a relatively long period of time, i.e., the time span over the stages of sampling, testing, processing, and displaying / using the data is long compared to on-site parameters. Thus, "off-site parameters" can be obtained 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.

[0119] "Off-site parameters" are typically obtained by subjecting a sample to chemical, biological, mechanical or other treatment, which is typically performed in a laboratory and is therefore not performed "on-site," i.e., near or adjacent to the infant's location.

[0120] Non-invasive measurements for monitoring various physiological parameters include, but are not limited to, sputum, urine, and fecal sampling, pulse oximetry, non-intubation respiratory analysis, and / or capnometry. Invasive measurements for monitoring various physiological parameters include, but are not limited to, blood sampling, continuous blood gas and metabolite analysis, and in some embodiments, intubation respiratory analysis and transcutaneous monitoring measurements. Severely invasive measurements include bipsies and other surgical procedures.

[0121] The term "pulse oximetry" refers to the measurement of blood pressure by tracing the light absorption properties of hemoglobin through the skin (finger, earlobe, etc.) and determining the oxygenated and non-oxygenated species of hemoglobin, as well as hemoglobin species bound to other molecules such as carbon monoxide (CO), and iron in the hemoglobin group as Fe 3+ Pulse oximetry is a noninvasive, on-site technique that measures physiological parameters related to respiration based on spectroscopic differences observed in the ferric state of methemoglobin. Physiological parameters that can be measured by pulse oximetry include, for example, SpO2, SpMet, and SpCO.

[0122] The phrase "non-intubation breath analysis," as used herein, refers to a group of non-invasive, on-site techniques, such as spirometry and capnography, that provide measurements of physiological lung function and respiratory gas chemistry by sampling inspiratory / expiratory airflow or by directing the infant's breath into a detector, all without penetrating the infant's airways or other openings or piercing the skin at any stage.

[0123] As used herein, the term "spirometry" refers to the combined measurement of respiratory-related parameters and pulmonary function by a non-invasive, on-site spirometer. Exemplary spirometry parameters that may be used in the context of some embodiments of the present invention are listed below:

[0124] The spirometry parameter tidal volume (TV) is the amount of air normally inhaled and exhaled at rest, with normal values ​​based on a subject's ideal body weight.

[0125] The spirometric parameter total lung capacity (TLC) is the maximum volume of air present in the lungs.

[0126] The spirometric parameter vital capacity (VC) is the maximum volume of air that can be expelled from the lungs after maximal inspiration and is equal to the sum of the inspiratory reserve, tidal volume, and expiratory reserve volume.

[0127] The spirometry parameter static vital capacity (SVC) is the volume of air that can be inhaled as deeply as possible and then completely exhaled, and measures how deeply an infant can breathe.

[0128] The spirometric parameter forced vital capacity (FVC) is the volume of air, measured in liters, that is forcibly expired after a maximal inspiration and constitutes the most fundamental measure in spirometry testing.

[0129] The spirometry parameter forced expiratory volume in the first second (FEV1) is the volume of air forcibly exhaled in 1 second after maximal inspiration. The mean value of FEV1 depends primarily on gender and age, while values ​​between 80% and 120% of the mean are considered normal. Predicted normal values ​​for FEV1 can be calculated on-site and depend on age, gender, height, weight, and ethnicity, as well as the research study on which they are based.

[0130] The spirometry parameter FEV1 / FVC ratio (FEV1%) is the ratio of FEV1 to FVC and is approximately 75-80% in adults. Predicted FEV1% is defined as the patient's FEV1% divided by the mean FEV1% in the appropriate population.

[0131] The spirometric parameter forced expiratory flow (FEF) is the flow rate (or rate) of air expelled from the lungs during the middle portion of a forced expiration. It can be obtained at discrete times and is generally calculated as the residual portion of the forced vital capacity (FVC), i.e., 25% of the FVC (FEF 25 ), 50% of FVC (FEF 50 ), or 75% of FVC (FEF 75 ), which can also be taken as the average of flow during an interval and is generally bounded by a specific residual fraction of the FVC, usually between 25 and 75% (FEF 25~75 %). Values ​​measured in the range of 50-60% to 130% of the mean 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 on which they are based. Recent studies have shown that FEF 25~75 % or FEF 25~50 These findings suggest that FEV1% may be a more sensitive parameter than FEV1 in detecting obstructive small airway disease. However, in the absence of concomitant changes in standard markers, differences in mid-range expiratory flow rates may not be specific enough to be useful, and current practice guidelines recommend continuing to use FEV1, VC, and FEV1 / VC as indices of obstructive disease.

[0132] The spirometry parameter negative inspiratory flow (NIF) is the maximum force that the chest muscles can exert in breathing, and the value indicates the status of the respiratory muscles.

[0133] The spirometry parameter MMEF or MEF refers to the maximum (mid-expiratory) flow rate, which is the peak expiratory flow rate obtained from a flow curve and is measured in liters per second. MMEF is related to peak expiratory flow (PEF), which is commonly measured by a peak flow meter and is given in liters per minute.

[0134] The spirometry parameter peak expiratory flow (PEF) refers to the maximum flow rate (or rate) achieved during a maximally forced expiration initiated with a maximal inspiration, measured in liters per minute.

[0135] The spirometry parameter carbon monoxide diffusing capacity (D L Nitric oxide (NO) refers to the uptake of nitric oxide from a single inspiration over a standard period of time (usually 10 seconds). Utilizing on-site computing equipment, the D is adjusted for hemoglobin content, anemia, pulmonary hemorrhage, and the altitude and / or atmospheric pressure at which the measurement was taken. L CO can be corrected.

[0136] The spirometry parameter maximal ventilation (MVV) is a measure of the maximum volume of air that can be inhaled and exhaled within one minute. This parameter is typically measured over a 15-second period and then extrapolated to a 1-minute value expressed as liters per minute. Average values ​​for men and women are 140-180 and 80-120 liters per minute, respectively.

[0137] The spirometry parameter static lung compliance (Cst) refers to the change in lung volume at any given pressure. Static lung compliance is perhaps the most sensitive parameter for detecting abnormal lung function. Cst is considered normal if it is 60-140% of the mean value for the same reference population.

[0138] The spirometry parameter forced expiratory time (FET) measures the length of expiration in seconds.

[0139] The spirometric parameter static vital capacity (SVC) is the maximum volume of air that can be slowly exhaled after a slow maximal inspiration.

[0140] The spirometry parameter static intrinsic positive end-expiratory pressure (static PEEPi) is measured as the plateau airway opening pressure during airway obstruction.

[0141] The spirometric parameter maximal inspiratory pressure (MIP) is a measure of the maximum amount of negative pressure a person can generate during inspiration, expressed in centimeters of water pressure (cmH2O) and measured manometrically, and serves as an index of diaphragm strength and an independent diagnostic parameter.

[0142] The term "capnography" refers to a technique for monitoring the concentration or partial pressure of carbon dioxide (CO2) in respiratory gases. End-tidal CO2, or ETCO2, is a parameter that can be measured by capnography.

[0143] Gas detection technology is integrated into many medical and other industrial devices, enabling quantitative measurement of the chemical composition of gas samples flowing through or captured therein. In the context of embodiments of the present invention, such gas chemical measurements are part of an on-site, non-invasive combination of testing, controlled, and monitored activities in the methods presented herein. Gas detectors, as well as gas mixing devices and regulators, are used to measure and control parameters such as inspired oxygen fraction (FiO2) and nitric oxide concentration in the inhaled gas mixture.

[0144] According to some embodiments of the present invention, measurements of vital signs such as pulse, blood pressure, respiration rate and temperature are considered part of the combination of on-site and non-invasive measurements.

[0145] The term "Integrated Pulmonary Index" or IPI refers to a patient's pulmonary index, which uses information about inhaled / exhaled gases from capnography and information about gases dissolved in the blood from pulse oximetry to provide a single value describing the patient's respiratory status. The IPI is obtained through on-site, noninvasive technology and integrates four key physiological parameters provided by patient monitoring (end-tidal CO2 and respiratory rate measured by capnography, and pulse rate and blood oxygenation SpO2 measured by pulse oximetry) and uses this information with an algorithm to derive an IPI score. The IPI provides a simple, real-time (on-site) indicator of a patient's overall ventilatory status as an integer (score) ranging from 1 to 10. The IPI score does not replace the patient's respiratory parameters but can be used to rapidly assess a patient's respiratory status and determine the need for additional clinical evaluation or intervention.

[0146] In accordance with some of the embodiments described herein, the physiological or chemical parameters monitored may include one or more of the following parameters: perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (SpO2), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), and / or serum nitrite / nitrate (NO2). - / NO3 - ), serum or urinary nitrite / nitrate (NO2 - / NO3 - ) and at least one off-site parameter selected from the group consisting of blood hemoglobin.

[0147] In accordance with some of the embodiments described herein, the physiological or chemical parameters monitored may include one or more of the following parameters: perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (SpO2), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), and / or serum nitrite / nitrate (NO2). - / NO3 - ), and at least one off-site parameter selected from the group consisting of skin salinity.

[0148] According to some of the embodiments described herein, the method is performed while monitoring at least one or more of the following on-site parameters in the gas mixture inhaled by the infant: end-tidal CO2 (ETCO2), nitrogen dioxide (NO2), nitric oxide (NO), and fractional inspired oxygen (FiO2).

[0149] The physiological or chemical parameters monitored by some of the embodiments described herein further include one or more of the following parameters: urinary nitrogen dioxide output (urinary nitrite output) (offline parameter); vital signs selected from the group consisting of pulse, blood pressure, respiratory rate, and body temperature (offline parameter); hematological markers such as, but not limited to, hemoglobin level, hematocrit level, red blood cell count, white blood cell count, differential white blood cell count, and platelet count (offline parameter); coagulation parameters such as, but not limited to, prothrombin time (PT), prothrombin ratio (PR), and international normalized ratio (INR) (offline parameter); liver function markers selected from the group consisting of serum creatinine level (offline parameter), aspartate aminotransferase (AST) level, serum glutamic oxaloacetic transaminase (SGOT) level, alkaline phosphatase level, and gamma-glutamyltransferase (GGT) level (offline parameter); and vascular endothelial activating factors selected from the group consisting of Ang-1, Ang-2, and the Ang-2 / Ang-1 ratio (offline parameter).

[0150] It is known that a sudden rise in inflammatory biomarkers can be associated with a phenomenon called a "cytokine storm," which has been observed in infants receiving inhaled nitric oxide therapy. Thus, monitoring inflammatory biomarkers while performing the method as described herein, according to embodiments of the present invention, has an additional role in safety considerations for the method, where the absence of a significant increase in inflammatory markers is an indicator of safety.

[0151] According to some embodiments herein, the methods disclosed herein do not show significant changes in the amounts of physiological parameters and biomarkers monitored for safety and efficacy of the treatment as set forth above.

[0152] In the context of this embodiment, a change in a parameter or biomarker amount is considered large if the value of an observation (measurement, test result, reading, calculation, etc.) or group of observations deviates significantly from normal levels, for example, deviates by about two times the upper limit of normal levels.

[0153] A "normal" level of a parameter or a "normal" level of a biomarker is referred to herein as a baseline value or simply a "baseline." In the context of this embodiment, the term "baseline" is defined as a range of values ​​that has been statistically determined from numerous observations and / or measurements collected over several years of medical practice for the general human population, a specific subset thereof (cohort), or possibly a specific individual. A baseline is a parameter / biomarker-specific value that is generally and medically accepted in the art as normal for infants under a particular health condition. These baseline or "normal" values, and the means for measuring these normal values, are known in the art. Alternatively, a baseline value can be measured from or in a specific infant using well-known and accepted methods, procedures, and technical means prior to performing the methods described herein. A baseline is therefore associated with a tolerance value, or a range of acceptable error, and is determined in conjunction with the measurement of a parameter / biomarker. In other words, a baseline is a range of acceptable values ​​that defines the range of observations that are considered "normal." The width of a baseline, or the difference between its upper and lower limits, is referred to as the "baseline range," and the difference from the middle 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.

[0154] In the context of this embodiment, a significant variation in an observation for a parameter / biomarker of interest is a variation that deviates from a predetermined acceptable baseline by more than 2 acceptable deviation units (2 ADU). For example, for a 4-8 baseline (characterized by a baseline range of 4 and an acceptable deviation unit of 2), 10 observations deviate from the baseline by 1 acceptable deviation unit, or 1 ADU. Alternatively, a variation is considered large if it is more than 1.5 ADU, more than 1 ADU, or more than 0.5 ADU.

[0155] In the context of the present embodiment, a "statistically significant observation" or a "statistically significant deviation from baseline" is one that does not arise as a result of random factors, error, or chance.

[0156] It is noted that for some parameters / biomarkers or groups of parameters / biomarkers, the significance of their changes may be context-dependent, biological system-dependent, medical case-dependent, infant-dependent, and even measurement device-dependent, i.e., a particular parameter / biomarker may require or require stricter or looser criteria for determining whether its reading should be considered significant. It is noted herein that in certain examples, some parameters / biomarkers may not be measurable due to the patient's condition, age, or other reasons. In such cases, the method is performed while monitoring other parameters / biomarkers.

[0157] Deviation from baseline is thus defined as a statistically significant variation in the value of a parameter / biomarker measured during and / or after all or part of the administration of a dosing regimen described herein, compared to the corresponding baseline for that parameter / biomarker. It is noted herein that the observations of some parameters / biomarkers may vary for a number of reasons, and that the measurement of significant variation therein should take such events into account and correct for the appropriate baseline.

[0158] Monitoring methemoglobin and serum nitrite concentrations is accepted in the art as essential for monitoring the safety of inhaled nitric oxide in infants, but to date there is no clear indication that methemoglobin and serum nitrite concentrations remain largely unchanged during inhaled nitric oxide by infants.

[0159] According to some embodiments of the invention, the method comprises monitoring and / or improving at least one of the aforementioned parameters / biomarkers.

[0160] According to some embodiments, the parameter monitored is methemoglobin concentration.

[0161] When methemoglobin concentrations can be measured using noninvasive measurements, the parameter of peripheral saturation of methemoglobin (SpMet) is used to monitor the stability, safety, and efficacy of the methods described herein. Thus, according to some embodiments of the present invention, the parameter monitored is SpMet, and during and after administration, the amount of SpMet does not exceed 5%, and preferably does not exceed 1%. As shown in the Examples section below, the amount of SpMet in infants undergoing the methods described herein does not exceed 1%.

[0162] According to some embodiments, the parameter monitored is serum nitrate / nitrite concentration.

[0163] High nitrite and nitrate concentrations in infant serum are associated with nitric oxide toxicity, and therefore serum nitrite / nitrate concentrations are used to detect adverse events in the methods presented herein. According to some embodiments of the present invention, the parameters tested are serum nitrite / nitrate, which are monitored during and after treatment, with acceptable levels of serum nitrite being less than 2.5 micromoles / liter and serum nitrate being less than 25 micromoles / liter.

[0164] According to some of the embodiments described herein, the methods are performed while monitoring at least one, at least two, or all on-site parameters including perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (SpO2 / SaO2 / DO), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI), and / or while monitoring at least one or all off-site parameters including serum nitrite / nitrate concentrations.

[0165] According to some of the embodiments described herein, the methods are performed while monitoring at least one, at least two, or all on-site parameters in the gas mixture inhaled by the infant, including end-tidal CO2 (ETCO2), nitrogen dioxide (NO2), nitric oxide (NO), and fractional inspired oxygen (FiO2).

[0166] According to some of the embodiments described herein, the method is performed while monitoring at least one, at least two, or all on-site and / or off-site safety parameters related to nitric oxide inhalation, such as methemoglobin production, and while monitoring at least one, at least two, or all on-site and / or off-site efficacy parameters.

[0167] According to some of the embodiments described herein, the methods are performed while monitoring at least one, at least two, or all on-site and / or off-site safety parameters related to nitric oxide inhalation, e.g., methemoglobin production, and while monitoring at least one, at least two, or all on-site and / or off-site efficacy parameters related to CF symptoms, including lung function and / or inflammatory biomarkers.

[0168] According to some of the embodiments described herein, the method is performed while monitoring at least one, at least two, or all on-site and / or off-site safety parameters related to nitric oxide inhalation, such as methemoglobin production, and while monitoring at least one, at least two, or all on-site and / or off-site efficacy parameters related to bronchiolitis symptoms, including lung function and / or inflammatory biomarkers.

[0169] According to some of the embodiments described herein, the method can be used to measure forced expiratory volume (FEV1), maximum mid-expiratory flow (MMEF), diffusing capacity of the lung for carbon monoxide (D L This is performed while monitoring at least one, at least two, or all on-site pulmonary function parameters (spirometry parameters), such as CO, forced vital capacity (FVC), total lung capacity (TLC), and residual volume (RV).

[0170] For example, the method according to some embodiments is performed while monitoring SpMet as an on-site parameter. Alternatively, the method is performed while monitoring SpMet and ETCO2 as on-site parameters. Alternatively, the method is performed while monitoring SpMet, ETCO2, and SpO2 as on-site parameters.

[0171] Alternatively, the method according to some embodiments may use SpMet and NO2 as one on-site parameter. - / NO3 - The method is performed while monitoring one off-site parameter, such as blood or urine concentrations of ATP. Alternatively, the method is performed while monitoring SpMet and SpO2 as on-site parameters and serum nitrite / nitrate concentrations as off-site parameters. Alternatively, the method is performed while monitoring SpMet as an on-site parameter and plasma inflammatory biomarkers (for efficacy) and serum nitrite / nitrate concentrations as off-site parameters. Alternatively, the method is performed while monitoring SpO2 as an on-site parameter and bacterial load and serum nitrite / nitrate concentrations as off-site parameters. Alternatively, the method is performed while monitoring SpO2 as an on-site parameter and plasma inflammatory biomarkers and pulmonary function parameters such as FEV1.

[0172] Alternatively, the method is performed while monitoring SpMet, FEV1 and SpO2 as on-site parameters, and plasma inflammatory biomarkers and serum nitrite / nitrate concentrations as off-site parameters.

[0173] According to some of the embodiments described herein, the method is performed while monitoring at least one, at least two, or all on-site parameters including SpMet, FEV1, and SpO2, and / or while monitoring at least one or all off-site parameters including serum nitrite / nitrate concentrations and plasma inflammatory biomarkers, and while further monitoring one or more, and any combination of, urinary NO2 concentration (off-site parameter), vital signs (on-site parameter), pulmonary function (on-site parameter), hematological markers (off-site parameter), coagulation parameters (off-site parameter), serum creatinine concentration (off-site parameter), renal function markers (off-site parameter), liver function markers (off-site parameter), vascular endothelial activating factor (off-site parameter).

[0174] According to some of the embodiments described herein, the method is performed while monitoring at least one, at least two, or all on-site chemical parameters in the inhaled gas mixture, such as FiO2 and NO2.

[0175] It is noted herein that in any of the foregoing embodiments, the method is performed in the absence of significant variation in any one or more or all of the monitored parameters described herein.

[0176] According to some embodiments of the present invention, the method is carried out while monitoring the urinary nitrite concentration so that the urinary nitrite concentration does not change substantially during and after carrying out the method provided herein. It is noted herein that urinary nitrite may fluctuate for several known reasons, and the measurement of significant fluctuations therein should take such events into account and correct for the appropriate baseline accordingly.

[0177] According to some embodiments of the present invention, hematological markers such as hemoglobin concentration, hematocrit, red blood cell count, white blood cell count, white blood cell differential count, and platelet count remain substantially unchanged during and after performing the methods provided herein.

[0178] According to some embodiments of the present invention, vascular endothelial activators such as Ang-1, Ang-2, and the Ang-2 / Ang-1 ratio, as well as serum creatinine concentration and various liver function markers such as aspartate aminotransferase (AST) concentration, serum glutamic oxaloacetic transaminase (SGOT) concentration, alkaline phosphatase concentration, and gamma-glutamyltransferase (GGT) concentration, remain substantially unchanged during and after performing the methods presented herein.

[0179] Infant oxygenation can be assessed by measuring the infant's peripheral oxygen saturation (SpO2). This parameter is an assessment of oxygen saturation levels and is commonly measured using non-invasive measurements such as a pulse oximeter device. Thus, according to some embodiments of the present invention, the parameter monitored during and after administration is SpO2, and the amount of SpO2 is greater than about 89%.

[0180] According to some embodiments of the present invention, various vital signs such as pulse, blood pressure, respiratory rate and temperature, as well as various coagulation parameters (off-site parameters) such as prothrombin time (PT), prothrombin ratio (PR) and international normalized ratio (INR) remain substantially unchanged during and after the methods presented herein. It is noted that these parameters are taken as indicators that the infant's overall health has not deteriorated as a result of the medical condition and / or treatment.

[0181] According to some embodiments, the aforementioned general health indicators show improvement during and after performing the methods provided herein, indicating that the treatment is effective for the infant.

[0182] Thus, according to some embodiments of the present invention, the methods disclosed herein are carried out such that the general health indicators described herein at least remain constant or improve.

[0183] Administration Method and Inhalation Device Infants are subjected to inhalation by active or passive means.

[0184] "Active means" means that the gas mixture is administered or delivered to the respiratory tract of the infant. This may be done, for example, by means of an inhalation device having a delivery interface adapted for use in a human respiratory system. For example, the delivery interface may be intermittently placed in the infant's respiratory system, so that when it is removed, the infant inhales ambient air or any other gas mixture lacking nitric oxide, as defined herein.

[0185] "Passive means" means that the infant inhales a gas mixture containing the indicated dose of nitric oxide without the use of a device to deliver the gas mixture to the airways.

[0186] For example, an infant may be exposed to 160 ppm or more of nitric oxide on an intermittent dosing regimen by passing in and out of an atmosphere-controlled enclosure filled with a nitric oxide-containing mixture of gases discussed herein, or by filling and emptying an atmosphere-controlled enclosure in contact with the infant's airways.

[0187] According to some embodiments of the present invention, in any of the treatment methods presented herein, nitric oxide administration may be performed by an inhalation device, including but not limited to, a stationary inhalation device, a portable inhaler, a metered dose inhaler, and an intubated inhaler.

[0188] Inhalers, according to some embodiments of the present invention, can generate spirometry data over time and adjust therapy accordingly, as presented, for example, in U.S. Patent No. 5,724,986 and WO 2005 / 046426. Inhalers can adjust a subject's inhalation waveform to target specific lung sites. According to some embodiments of the present invention, portable inhalers can deliver both spike and maintenance doses of nitric oxide automatically, at the infant's choice or according to a specific dosing regimen.

[0189] In accordance with some embodiments of the present invention, an exemplary inhalation device may include a delivery interface that is adaptable for inhalation by an infant.

[0190] According to some embodiments of the present invention, the delivery interface comprises a mask or mouthpiece for delivery of a gas mixture comprising nitric oxide to the infant's respiratory tract.

[0191] According to some embodiments of the present invention, the inhalation device further includes a nitric oxide analyzer positioned proximal to the delivery interface for measuring the concentrations of nitric oxide, oxygen, and nitrogen dioxide flowing into the delivery interface, wherein the analyzer is connected to the controller.

[0192] According to some embodiments of the present invention, subjecting an infant to the methods described herein is carried out through the use of an inhalation device, which may be any device capable of delivering a nitric oxide-containing gas mixture to the infant's respiratory system. Inhalation devices, according to some embodiments of the present invention, include, but are not limited to, stationary inhalation devices including tanks, gauges, tubing, masks, controllers, valves, etc., portable inhalers (including the aforementioned components), metered-dose inhalers, controlled-atmosphere enclosures, breathing apparatus / systems, and intubated inhalation / breathing apparatus / systems. Controlled-atmosphere enclosures include, but are not limited to, head enclosures (bubbles), whole-body enclosures, or chambers, and the atmosphere filling the enclosure can be controlled by flow rate, continuous or intermittent content exchange, or any other method of controlling the gas mixture content.

[0193] According to some embodiments of the present invention, intermittent inhalation is achieved by intermittently exposing the infant to a gas mixture (inhalant) via respiratory cycle coordinated pulse delivery, the gas mixture containing nitric oxide at a designated concentration (a nitric oxide-containing gas mixture). This mode of inhalation is referred to herein as intermittent respiratory cycle coordinated pulse delivery inhalation.

[0194] According to an alternative aspect of some embodiments of the present invention, there is provided a method of treating bronchiolitis in an infant, comprising subjecting the infant to intermittent inhalation of an inhalant, wherein the intermittent inhalation comprises at least one cycle of breath-cycle-coordinated pulsed delivery inhalation for a first period of time, followed by inhalation that is substantially free of nitric oxide for a second period of time, wherein the breath-cycle-coordinated pulsed delivery inhalation is configured to deliver approximately 80 ppm·hr of nitric oxide in at least one cycle.

[0195] In the context of method invention embodiments, the term "nitric oxide burden" (NO burden) refers to the specific cumulative amount of nitric oxide to which a subject or pathogen is exposed during inhalation therapy (e.g., treatment according to the claimed invention), which is estimated in ppm·hr, i.e., the average concentration of nitric oxide in the inhalant multiplied by the total time of exposure. Nitric oxide burden can be estimated per cycle of therapy (NO burden / cycle) or per unit of time, such as per day (daily NO burden).

[0196] According to some embodiments of the present invention, intermittent delivery of nitric oxide to an infant is performed such that the subject inhales nitric oxide at a daily nitric oxide loading dose in the range of 600-2000 ppm·hr, wherein the intermittent delivery is performed such that the daily nitric oxide loading dose is inhaled over one session of uninterrupted administration.

[0197] According to some embodiments of the present invention, intermittent delivery is performed so that a daily nitric oxide loading is inhaled in one or more sessions of intermittent breath-cycle-coordinated pulsed delivery inhalation, with each cycle having a nitric oxide loading / cycle of at least about 80 ppm·hr. Such a nitric oxide loading / cycle can be achieved, for example, by configuring pulses to deliver an inhalant having 160 ppm nitric oxide for 30 minutes (first period) during one cycle. Note that other concentrations and other first periods resulting in a nitric oxide loading of at least 80 ppm·hr / cycle are also contemplated and encompassed by embodiments of the present invention.

[0198] "Intermittent breath-cycle-coordinated pulsed delivery inhalation" refers to an infant being intermittently provided with a gas mixture containing a designated concentration of nitric oxide, thereby inhaling such nitric oxide-containing gas mixture two or more times by breath-cycle-coordinated pulsed delivery with an interval between each inhalation. The infant may then inhale the nitric oxide-containing gas mixture, then stop inhaling the nitric oxide-containing gas mixture by breath-cycle-coordinated pulsed delivery and instead inhale a gas mixture (e.g., air) without the designated concentration of nitric oxide, then again inhale the nitric oxide-containing gas mixture by breath-cycle-coordinated pulsed delivery, and so on.

[0199] In some embodiments of this aspect of the invention, a "nitric oxide-containing gas mixture" is used to describe a gas mixture containing at least 160 ppm of nitric oxide. Nitric oxide-containing mixtures may contain 160 ppm, 170 ppm, 180 ppm, 190 ppm, 200 ppm, and even higher concentrations of nitric oxide. Other gas mixtures referred to herein contain less than 160 ppm of nitric oxide or are substantially devoid of nitric oxide, as defined herein.

[0200] In some embodiments, "nitric oxide-containing gas mixture" describes a gas mixture that delivers 80 ppm·hr of nitric oxide.

[0201] "Substantially devoid of nitric oxide" means 50 ppm or less, 40 ppm or less, 30 ppm or less, 20 ppm or less, 10 ppm or less, 5 ppm or less, 1 ppm or less, and ppb or less, including the complete absence of nitric oxide.

[0202] According to some embodiments of the present invention, the intermittent breath-cycle coordinated pulse-delivery inhalation comprises one or more cycles, each cycle comprising a breath-cycle coordinated pulse-delivery inhalation of a gas mixture containing nitric oxide at a particular concentration (e.g., at least 160 ppm) for a first period, followed by a second period of inhalation of a gas mixture without nitric oxide, also referred to herein as nitric oxide / cycle. According to some embodiments of the present invention, during the second period, the infant may inhale ambient air or a controlled gas mixture substantially devoid of nitric oxide, as defined herein.

[0203] In some embodiments, the first period of time spans 10 to 45 minutes, or 20 to 45 minutes, or 20 to 40 minutes, and in some embodiments, about 30 minutes.

[0204] According to some embodiments of the present invention, the second period of time ranges from 3 to 5 hours, or 3 to 4 hours, and according to some embodiments, the second period of time spans about 3.5 hours.

[0205] According to some embodiments of the present invention, this inhalation regimen is repeated 1 to 6 times over a 24 hour period, depending on the first and second periods.

[0206] In some embodiments, a cycle of intermittent breath-cycled coordinated pulsed delivery inhalation of nitric oxide, e.g., 160 ppm for 30 minutes followed by 3.5 hours without nitric oxide, is repeated 1-6 times per day, in some embodiments, 5 times per day.

[0207] In some embodiments, a cycle of intermittent breathing-cycled coordinated pulsed delivery inhalation of nitric oxide, e.g., a nitric oxide loading dose of 80 ppm·hr / cycle, followed by 3.5 hours without breathing nitric oxide, is repeated 1-6 times per day. In some embodiments, the cycle is repeated 5 times per day.

[0208] According to some embodiments of the invention, a dosing regimen of 1 to 5 cycles of intermittent, breath-cycle-coordinated, pulsed delivery of nitric oxide per day is administered for 1 to 7 days, or 2 to 7 days, or 3 to 7 days, or for 1, 2, 3, 4, or 5 consecutive weeks. According to some embodiments of the invention, the intermittent, breath-cycle-coordinated, pulsed delivery inhalation is administered for a period of 14 days. However, longer periods of intermittent, breath-cycle-coordinated, pulsed delivery of nitric oxide are also contemplated as described herein.

[0209] According to an embodiment of the present invention, the nitric oxide-containing gas mixture inhaled by the infant during the first time period is produced in situ in an inhalation device that is configured to respond to the infant's breathing cycle so that nitric oxide is mixed into the inhalant in one or more pulses when the infant breathes at a rapid rate, i.e., during the inhalation portion of the breathing cycle. This mode of administration of nitric oxide by inhalation is referred to herein as "breath cycle-coordinated pulsed delivery inhalation."

[0210] In the context of embodiments of the present invention, the term "pulse" refers to a manner of administering nitric oxide in which the nitric oxide is introduced into an inhalant in intermittent concentrated doses over a predetermined period of time, referred to herein as a "pulse delivery period," wherein each pulse delivered during the pulse delivery period lasts for a predetermined period of time, referred to herein as a "pulse-on period," and is interrupted by a "pulse-off period."

[0211] According to embodiments of the present invention, the pulse delivery period begins during the inhalation period, after a period referred to herein as the "pulse delay period." According to some embodiments of the present invention, the pulse delivery period is generally 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 interruption period."

[0212] According to some embodiments of the present invention, an inhalation device for delivering breath-cycle coordinated pulse delivery inhalation of gashouse nitric oxide is configured to detect various stages of the breath cycle, i.e., the start of the inhalation and exhalation periods, and thus coordinate the pulses with the breath cycle such that a pulse delay period is coordinated to begin as soon as the intake rate increases at the start of the inhalation period, and a pulse pause period is coordinated to begin as soon as the intake rate decreases towards the end of the inhalation period.

[0213] In some embodiments, the lengths of various periods in a respiratory cycle-coordinated pulse delivery inhalation plan are determined and / or calculated relative to the respiratory cycle duration, i.e., the percentage of the entire duration of the respiratory cycle or a portion thereof. For example, the duration of the inhalation phase is determined by sensing the inhalant flow rate, and the pulse delay phase is automatically set to 20% of the inhalation phase. As a result, the pulse delivery phase can be set to 60% of the inhalation phase, and the pulse off phase is the remaining 20% ​​of the inhalation phase. The pulse number, i.e., the pulse on and pulse off periods, can similarly be set according to the duration of the pulse delivery phase. For example, the pulse number can be set to 1, i.e., a pulse for the entire duration of the pulse delivery phase. This example may be appropriate for infants with shortness of breath or other breathing difficulties. Alternatively, if the infant 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 pulse number is automatically set according to the duration of the pulse delivery phase obtained from the measured inhalation period.

[0214] In some embodiments, the pulse delay period is in the range of 0 to 2500 ms. Alternatively, in some embodiments, the pulse delay period is in the range of 0 to 80% of the inhalation period.

[0215] In some embodiments, the pulse pause is in the range of 0 to 2500 ms, or in some embodiments, the pulse pause is in the range of 80 to 0% of the inhalation period.

[0216] In some embodiments, each pulse-on period is individually in the range of 100-5000 ms. Alternatively, each pulse-on period is individually in the range of 10-100% of the inhalation period.

[0217] In some embodiments, each pulse-off period individually ranges from 0 to 2500 ms. Alternatively, each pulse-off period individually ranges from 0 to 200% of the pulse-on period.

[0218] In some embodiments, the method is based on a single pulse per inhalation phase. In some embodiments, the pulse delivery phase is performed such that it begins substantially when the inhalation phase begins (the pulse delay phase is substantially zero) and ends substantially when the inhalation phase ends (the pulse pause phase is substantially zero). In other embodiments, the method is performed by using a single pulse that begins after the inhalation phase begins and ends before the inhalation ends.

[0219] In some embodiments, the coordination of pulse delivery is configured to deliver more than one pulse consecutively during a pulse delivery phase until the device senses a slowing of the intake rate near the end of the inhalation phase. In such embodiments, the device is configured to interrupt each pulse-on period with a pulse-off period. In some embodiments, the device is configured to deliver a predetermined number of pulses ranging from 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, or 1 to any number of pulses that can be implemented within a pulse delivery period determined by any given respiratory cycle. It is further noted that each pulse may span a different pulse-on period and be interrupted by a pulse-off period of different length.

[0220] The concentration of nitric oxide in the nitric oxide-containing gas mixture is controlled by the concentration of nitric oxide introduced into the inhalant, the output at which nitric oxide is introduced into the inhalant, the duration of the pulse-on phase, and the number of pulses introduced into the inhalant during the pulse-delivery phase. According to some embodiments of the present invention, during the pulse-delivery phase, the inhalant is a nitric oxide-containing gas mixture that substantially contains at least 160 ppm nitric oxide, or a nitric oxide loading of 80 ppm·hr per cycle, while during the pulse delay and pulse-off phases, the inhalant is substantially devoid of nitric oxide.

[0221] In some embodiments, the method is performed using more than one pulse, wherein the inhalant produced by each pulse delivers a different concentration of nitric oxide to the patient. For example, the method may be performed by administering three pulses to an infant during a pulse delivery period, such that the inhalant from the first pulse is characterized by a nitric oxide concentration of 160 ppm, the inhalant from the second pulse is characterized by a nitric oxide concentration of 80 ppm, and the inhalant from the first pulse is characterized by a nitric oxide concentration of 100 ppm. Thus, at least one pulse results in a concentration of at least 160 ppm. In other examples, some of the pulses may deliver an inhalant characterized by a nitric oxide concentration greater than 160 ppm.

[0222] Alternatively, the number of pulses, the concentration of nitric oxide in each pulse, and the duration of the first period during which the pulses occur are configured to deliver a nitric oxide loading dose of 80 ppm·hr per cycle.

[0223] As shown above, respiratory cycle-coordinated pulsed delivery inhalation allows for the introduction of substantially high concentrations of nitric oxide during periods when infants inhale at their highest inhalation-breathing rates, thereby minimizing the exposure of portions of the respiratory tract to high concentrations of nitric oxide. For example, because nitric oxide is introduced after the beginning of the inhalation phase and before the end of the inhalation phase, portions of the upper respiratory tract, the trachea, and some of the respiratory tree in the lungs that are not rich in alveolar capillaries, are only briefly exposed to high concentrations of nitric oxide depending on the inhalant uptake rate, while the alveoli are exposed to high concentrations of nitric oxide for a longer period of time.

[0224] According to some embodiments of the present invention, subjecting an infant to the methods described herein is carried out by use of an inhalation device, which may be any device capable of delivering a nitric oxide-containing gas mixture, including but not limited to, respiratory cycle-coordinated pulse delivery to the subject's respiratory system. Inhalation devices, according to some embodiments of the present invention, include, but are not limited to, stationary inhalation devices including a tank, gauge, tubing, mask, controller, valves, etc.; portable inhalers (including the aforementioned components), metered-dose inhalers, breathing apparatus / systems, and intubated inhalation / breathing apparatus / systems.

[0225] Exemplary inhalation devices that may be suitable for carrying out any embodiment of any of the methods described herein are provided, for example, by U.S. Provisional Patent Applications 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 incorporated herein by reference. Commercially available inhalation devices that may be suitable for carrying out any of the methods described herein include the INOpulse® DS-C, developed by Ikaria Australia Pty Ltd, or the Ohmeda INOpulse Delivery System by Datex-Ohmeda.

[0226] Inhalers, according to some embodiments of the present invention, can generate respiratory function data and adjust therapy over time, as provided, for example, in U.S. Pat. No. 5,724,986 and WO 2005 / 046426, the contents of which are incorporated herein by reference. The inhaler can adjust the subject's inhalation waveform to target specific lung sites. According to some embodiments of the present invention, the portable inhaler can deliver both spike and maintenance doses of nitric oxide at the subject's option or automatically according to a specific dosing regimen.

[0227] In accordance with some embodiments of the present invention, an exemplary inhalation device may include a delivery interface that is adaptable for inhalation by an infant. In accordance with some embodiments of the present invention, the delivery interface includes a mask or mouthpiece for delivery of a gas mixture including nitric oxide to the infant's respiratory tract.

[0228] According to some embodiments of the present invention, the inhalation device further includes a nitric oxide analyzer positioned proximal to the delivery interface for measuring the concentrations of nitric oxide, oxygen, and nitrogen dioxide flowing to the delivery interface, wherein the analyzer is connected to the controller.

[0229] It is expected that other methods for treating inflammatory diseases or disorders with intermittent inhalation of nitric oxide at 160 ppm or greater will be developed, and the scope of the phrase treating inflammatory diseases or disorders with intermittent inhalation of nitric oxide is intended a priori to include all such new technologies.

[0230] As used herein, the term "about" refers to ±10%.

[0231] The terms "include," "including," "contain," "containing," "having," and their cognates mean "including but not limited to."

[0232] The term "consisting of" means "including and limited to."

[0233] 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 substantially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0234] Throughout this application, various embodiments of the present invention may be presented in a range format. The range format is understood to be merely for convenience and brevity and should not be construed as an indefinite limitation on the scope of the present invention. Accordingly, a range description should be construed as including all specifically disclosed subranges, as well as individual numerical values ​​within that range. For example, a range description such as 1 to 6 should be construed as including specifically disclosed subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, and 3 to 6, as well as individual numerical values ​​within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0235] Whenever a numerical range is given herein, it is meant to include any recited numbers (partial or whole) within the stated range. The phrases "ranging from" a first designated number and a second designated number, and "ranging from" a first designated number to a second designated number, are interchangeable when used herein and are meant to include the first and second designated numbers and all partial or whole numbers therebetween.

[0236] As used herein, the term "method" refers to ways, means, techniques, and procedures for accomplishing a given task, and includes, but is not limited to, those ways, means, techniques, and procedures known by those of skill in the chemical, pharmacological, biological, biochemical, and medical arts, or readily developed from known ways, means, techniques, and procedures.

[0237] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing or reversing the progression of the condition, substantially ameliorating the clinical or cosmetic manifestations of the condition, or substantially preventing the clinical or cosmetic manifestations of the condition.

[0238] Where reference is made to a particular sequence listing, such reference also encompasses sequences that are substantially identical to the complementary sequence, including minor sequence variations resulting from sequencing errors, cloning errors, or other mutations that result in base substitutions, deletions, or additions, where the frequency of such variations is less than 1 in 50 nucleotides, or less than 1 in 100 nucleotides, or less than 1 in 200 nucleotides, or less than 1 in 500 nucleotides, or less than 1 in 1000 nucleotides, or less than 1 in 5,000 nucleotides, or less than 1 in 10,000 nucleotides.

[0239] Antimicrobial Enhancement: In some embodiments, the present invention provides methods for enhancing antimicrobial agents (e.g., antibiotics and antifungals) used in the treatment of medical conditions and diseases associated with pathogenic microorganisms (e.g., bacteria or fungi), the methods comprising exposing pathogenic microbial cells to NO, thereby rendering these cells more susceptible to the antimicrobial agent. Without being bound by any particular theory, the methods disclosed herein utilize exposure of pathogenic microbial cells to NO to deplete their natural defense mechanisms against the antimicrobial agent, thereby rendering the pathogenic microorganisms more susceptible to the antimicrobial agent and / or less able to resist the antimicrobial agent.

[0240] In some embodiments, the methods for enhancing antimicrobial agents presented herein involve depleting thiols in target cells by exposing them to nitric oxide. This method of NO exposure allows for the use of broad-spectrum antibiotics and antimicrobials not only against drug-susceptible pathogens, but also against drug-resistant (MDR or XDR) microbial strains that have accumulated mutations in proteins previously targeted by specific drugs. This method of NO exposure also broadens the effectiveness of antibiotics and antimicrobials against pathogens not identified as sensitive to these drugs prior to exposure to NO, i.e., turning narrowly acting antimicrobials into broad-spectrum antimicrobials.

[0241] In some embodiments, the methods provided herein are also useful for preventing, reducing, and / or eliminating the formation or persistence of microbial biofilms by exposing biofilms to nitric oxide in combination with antimicrobial or antibiofilm-forming agents (ABF agents). This method of NO exposure increases the efficacy of antibiotics, antimicrobial agents, and ABF agents to reduce and / or eliminate microbial biofilms that are little or not affected by them without exposure to NO. Thus, throughout this specification, references to the treatment of medical conditions associated with pathogenic microorganisms are meant to encompass treatment for the formation of microbial biofilms. For example, the methods provided herein can be effectively used to treat tuberculosis caused by Mycobacterium tuberculosis (Mtb) that has acquired point mutations that render Mtb resistant to first-line antibiotics within several years. Furthermore, because these bacteria are inherently resistant to many readily available antimicrobial agents already used effectively against other bacterial infections, the methods presented here can attenuate the inherent resistance of Mtb by depleting its native MSH levels, thereby demonstrating the feasibility of using these readily available antimicrobial agents against novel MDR and XDR TB strains.

[0242] The present invention, in some embodiments thereof, relates to pharmaceutical antimicrobial treatments, and more particularly, but not exclusively, to methods of potentiating antimicrobial agents and / or sensitizing or re-sensitizing antimicrobial-resistant microorganisms to antimicrobial treatment, thereby treating various conditions associated with pathogenic microorganisms, and potentiating antimicrobial agents to prevent or eradicate the formation of microbial biofilms.

[0243] The utility of currently practiced antimicrobial agents and therapies is severely limited, primarily by the development of resistance to these antimicrobial agents. The present inventors have surprisingly discovered that nitric oxide exhibits antimicrobial sensitization and / or resensitization activity and is further advantageously characterized as an effective enhancer of antimicrobial agents, even when used at concentrations lower than the effective antimicrobial concentrations, i.e., at concentrations and exposure times at which nitric oxide does not eradicate microorganisms. This enhancement by nitric oxide allows, for example, the antimicrobial agent to eradicate microorganisms at concentrations lower than those typically eradicated by the antimicrobial agent without nitric oxide enhancement and / or at shorter exposure times than those typically eradicated by the antimicrobial agent without nitric oxide enhancement.

[0244] It is known in the art that microbial resistance to an antimicrobial agent typically develops in a population of subjects treated with the antimicrobial agent when multiple generations of target microorganisms infecting that population are exposed to the antimicrobial agent. During that exposure, surviving resistant cells (which become resistant and where resistance has developed) can infect other subjects. Treatment of these subjects infected with the resistant microorganism with the same antimicrobial agent is no longer effective. Note that in some instances, resistance can also develop in a single subject during antimicrobial treatment, typically when the treatment is prolonged and / or at a suboptimal dose of the drug. According to some embodiments of the present invention, combined treatment using nitric oxide and an antimicrobial agent, as presented herein, allows treatment of a microorganism with the same antimicrobial agent even when the microorganism has already developed resistance to that same antimicrobial agent. The provided method also prevents selective pressure against resistant microorganisms.

[0245] The current use of antimicrobial agents is also very limited in combating microbial biofilms, primarily due to the protection conferred to microbial cells by their secreted coating of extracellular polysaccharides. Exposure of microbial biofilms to nitric oxide, in combination with exposure of the biofilm to an antimicrobial agent, can be beneficial against microbial biofilms compared to the antibiofilm efficacy of the antimicrobial agent when used without nitric oxide.

[0246] Nitric oxide, when administered with antibiotics, has been found to be highly effective in eradicating resistant microorganisms and has been shown to be able to resensitize microorganisms that have become resistant to antibiotics, effectively eradicating the bacteria when the same antibiotic is used. Nitric oxide, when used in combination with antibiotics, can also be used to prevent the emergence of resistance in microorganisms that are expected to develop resistance to the antibiotic by preventing selection pressure against the resistant microorganisms.

[0247] Nitric oxide is therefore highly useful in treating conditions associated with resistant microorganisms and in reducing or eradicating microbial biofilms by being (i) effective when administered in combination with otherwise ineffective antimicrobial treatments, (ii) effective in preventing the emergence of resistance to antimicrobial agents when administered in combination with those antimicrobial agents, and (iii) effective in re-sensitizing microorganisms to antimicrobial agents used when resistance to those agents emerges.

[0248] Nitric oxide is also very useful in reducing or eradicating microbial biofilms when administered or applied in combination with otherwise ineffective anti-biofilm treatments. Thus, in the context of embodiments of the present invention, the term "antimicrobial agent" is meant to encompass anti-biofilm agents or is used synonymously with the term "anti-biofilm agent."

[0249] Thus, the present invention provides a method for rendering microorganisms more susceptible to antimicrobial agents that are no longer effective against them or that have lost their effectiveness, by exposing the microorganisms to nitric oxide. Without being bound by any particular theory, it is speculated that exposing microorganisms to NO in accordance with the method embodiments presented herein weakens the microorganism's first line of defense against xenobiotics, including free radicals, toxins, antibiotics, and other antimicrobial agents. This first line of defense is based on the presence of low-molecular-weight thiols in target cells, and therefore the beneficial effects of the methods presented herein result from reducing and depleting the amount of these thiols with nitric oxide, thereby rendering target cells more susceptible to a broad range of antimicrobial agents.

[0250] method Generally, embodiments of the present invention use the antimicrobial-potentiating activity of nitric oxide, which has been previously described herein in the context of depleting microbial defense mechanisms. The phrase "antimicrobial-potentiating activity," as used herein in the context of embodiments of the present invention, defines a characteristic of a method involving three entities: (i) nitric oxide, (ii) an antimicrobial agent, and (iii) a microorganism, where the microorganism is known to be insensitive (not sensitive, resistant) to the antimicrobial agent, or was sensitive to the antimicrobial agent but has become or can become resistant to the antimicrobial agent, in the sense that it is no longer sensitive to the antimicrobial agent as a result of the development of resistance to the antimicrobial agent in that strain of the microorganism. Thus, in the context of embodiments of the present invention, the presence of antimicrobial-potentiating activity allows nitric oxide to act synergistically and / or to impart efficacy to the antimicrobial agent against the microorganism and / or to potentiate and / or repotentiate the antimicrobial agent by sensitizing or resensitizing the microorganism to the antimicrobial agent.

[0251] It is noted herein that, in the context of this method, as well as in all other aspects of the invention presented herein, microbial resistance to any of the target antimicrobial agents can be congenital or acquired; i.e., the microorganism may be inherently non-susceptible to the antimicrobial agent due to its chemical and biological properties, or may become non-susceptible to the antimicrobial agent as a result of exposure of a previous generation of microorganisms to the antimicrobial agent. For example, antimicrobial resistance may emerge to an antimicrobial agent in a population of subjects with a medical condition as a result of treating the population of subjects with that antimicrobial agent. Subjects infected with these resistant microorganisms can be treated with the same antimicrobial agent by practicing the methods presented herein, i.e., by administering a therapeutically effective amount of nitric oxide in combination with the antimicrobial agent in an amount effective to potentiate, sensitize, or re-sensitize. The methods presented herein are effective in such instances, regardless of the mechanism by which the microorganism acquired resistance to the antimicrobial agent.

[0252] Thus, according to aspects of an embodiment of the present invention, there is provided a method of treating a subject having a medical condition associated with a pathogenic microorganism by the steps of (i) administering to the subject an enhancing-effective amount of nitric oxide or a nitric oxide-releasing compound, and (ii) administering to the subject a therapeutically effective amount of an antimicrobial agent.

[0253] In some antimicrobial treatments, resistance of the target microorganism often becomes evident following the discovery that a commonly effective antimicrobial agent is no longer effective against it. Accordingly, another aspect of an embodiment of the present invention provides a method of treating a subject having a medical condition associated with a pathogenic microorganism in which antimicrobial resistance becomes evident after treating the subject with the antimicrobial agent, the method comprising the steps of: (i) administering a re-sensitizing effective amount of nitric oxide to the subject after treatment with the antimicrobial agent and the antimicrobial resistance becoming evident; and (ii) administering a therapeutically effective amount of the antimicrobial agent to the subject.

[0254] According to embodiments of this aspect of the invention, the antimicrobial agent is not nitric oxide, and the resensitizing effective amount of nitric oxide is less than the therapeutically effective amount of nitric oxide against the microorganism.

[0255] "Enhancement" means that a microorganism that is not susceptible or only slightly susceptible (non-susceptible) to an antimicrobial agent (i.e., a narrow-spectrum antibiotic) becomes susceptible (sensitive) to the antimicrobial agent. In such cases, the methods provided herein can be said to render some antimicrobial agents effective against some microbial species that would not be susceptible to the antimicrobial agent if the antimicrobial agent were used conventionally, i.e., without step (i) of the methods provided herein.

[0256] "Resensitization" or "repotentiation" means that a microorganism that is normally susceptible to treatment with an antimicrobial agent is found to be resistant to such treatment for some reason and becomes susceptible again to such treatment.

[0257] As used herein, the phrase "therapeutically effective amount" describes the amount of active ingredient administered that is required to substantially reduce or substantially eradicate a microorganism in a subject, and thus is administered in an amount that is harmful to the target microorganism, i.e., a microbicidal concentration or a concentration that substantially inhibits the growth of, or in some embodiments substantially eradicates, the microorganism in the subject, thereby alleviating to some extent one or more symptoms of a condition caused by the microorganism. In the context of this embodiment, the phrase "therapeutically effective amount" describes the amount of antimicrobial agent administered and / or readministered in combination with nitric oxide, which is generally lower than the amount required to achieve a similar result without the combination with nitric oxide. As used herein, "therapeutically effective amount" also encompasses the duration of exposure of the microorganism to a given antimicrobial agent.

[0258] As used herein, the phrase "enhancing-effective amount" describes an amount of nitric oxide sufficient to impart antimicrobial enhancing activity, thereby enhancing an antimicrobial agent against a microorganism or sensitizing a microorganism to an antimicrobial agent. An enhancing-effective amount of nitric oxide is defined as insufficient to eradicate the microorganism in a subject (killing at least 50%, 70%, 80%, or 100% of the microorganisms).

[0259] As used herein, the phrase "resensitizing-effective amount" describes an amount of nitric oxide that is sufficient to reverse resistance that has emerged in a microorganism to an antimicrobial agent. A resensitizing-effective amount of nitric oxide is defined as insufficient to eradicate the microorganism in a subject (killing at least 50%, 70%, 80%, or 100% of the microorganism). In some embodiments, the phrase "resensitizing-effective amount" describes an amount of nitric oxide that is sufficient to reverse or prevent the emergence of resistance in a pathogenic microorganism that results in a medical condition.

[0260] It should be noted that, although nitric oxide may inherently exhibit microbicidal activity, a potentiating or resensitizing effective amount of nitric oxide is substantially different from a microbicidal amount (therapeutically effective amount) of nitric oxide in the sense that, when used exclusively in the absence of another antimicrobial agent, in accordance with embodiments of the present invention, a potentiating or resensitizing effective amount of nitric oxide would not be expected to be sufficient to cause disruption or disruption to the life cycle of a target microorganism. In other words, in the context of embodiments of the present invention, a potentiating, sensitizing, or resensitizing effective amount of nitric oxide is generally lower than a therapeutically effective amount of nitric oxide.

[0261] According to some embodiments of the present invention, nitric oxide may exhibit antimicrobial therapeutic activity against pathogenic microorganisms. The potentiating or resensitizing effective amount of nitric oxide is generally lower than the therapeutically effective amount of nitric oxide when used as an antimicrobial agent against the microorganisms causing the condition being treated.

[0262] Therefore, according to some embodiments of the present invention, the potentiating or resensitizing effective amount of nitric oxide is lower than the therapeutically effective amount of nitric oxide against the microorganism to be eradicated when / if nitric oxide is administered by itself.

[0263] Antimicrobial efficacy is often referred to in terms of minimum inhibitory concentration units, or MIC units. MIC is the lowest concentration of an antimicrobial capable of inhibiting microbial growth after an incubation period, typically 24 hours, and is typically measured in micromoles (μM) or micrograms per milliliter (μg / ml). MIC values ​​are used as a diagnostic criterion to assess microbial resistance to antimicrobials and to monitor the activity of targeted antimicrobials. MICs are measured by standard laboratory methods, which are described and illustrated in the Examples section below. Standard laboratory methods typically follow standard guidelines from reference organizations such as the Clinical and Laboratory Standards Institute (CLSI), the British Society for Antibiotic Therapy (BSAC), or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). In clinical practice, minimum inhibitory concentrations are used to determine the amount of antimicrobial a subject receives as well as the type of antimicrobial used.

[0264] Thus, in some embodiments, an effective nitric oxide enhancing or resensitizing amount is less than 1 MIC of nitric oxide. In some embodiments, an effective nitric oxide enhancing or resensitizing amount is in the range of 1 to 1 / 10 MIC. In some embodiments, an effective nitric oxide enhancing or resensitizing amount is in the range of 1 / 2 to 1 / 8 MIC.

[0265] medical conditions In the context of embodiments of the present invention, the phrase "medical condition associated with a pathogenic microorganism" is meant to encompass any medical condition that is caused, directly or indirectly, by the presence of a microorganism in or on a subject. The phrase "medical condition associated with a pathogenic microorganism" therefore encompasses conditions associated with prokaryotes, gram-negative bacteria, gram-positive bacteria, eubacteria, archaea, eukaryotes, yeast, fungi, algae, protozoa, and / or other parasites.

[0266] Medical conditions associated with pathogenic microorganisms, according to embodiments of the present invention, include infection, infestation, contamination, and transmission by or of pathogenic microorganisms. Generally, disease-causing infection is the invasion of a pathogenic microorganism into the tissues of a host organism. The invasion of body tissues by parasites and other more highly pathogenic organisms is commonly referred to as an invasion.

[0267] Invading organisms, such as bacteria, produce toxins that damage host tissues and interfere with normal metabolism; some toxins are actually enzymes that break down host tissues. Other bacterial substances can inflict this damage by destroying the host's phagocytes, making the body more susceptible to infection by other pathogenic microorganisms. Substances produced by many invading organisms cause allergic hypersensitivity in the host. Infections can be spread through respiratory droplets, direct contact, contaminated food, or vectors such as insects. They can also be transmitted sexually and from mother to fetus.

[0268] Diseases caused by bacterial infections include, but are not limited to, actinomycosis, anthrax, aspergillosis, bacteremia, bacterial skin diseases, bartonella infections, botulism, brucellosis, burkholderia infections, campylobacter infections, candidiasis, feline diseases, chlamydia infections, cholera, clostridial infections, coccidioidomycosis, cryptococcosis, Dermatomycosis, diphtheria, ehrlichiosis, epidemic cystitis, Escherichia coli infection, Fusobacterium infection, gangrene, general infections, general mycoses, gonorrhea, gram-negative bacterial infections, gram-positive bacterial infections, histoplasmosis, impetigo, Klebsiella infection, Legionnaires' disease, leprosy, leptospirosis, listeria infection, Lyme disease, malaria, maderomosis, melidioidomycosis, mycobacterium infection, mycoplasma infection, necrotizing fasciitis, nocardia infection, onychomycosis, Commonly includes asthma, pneumococcal infections, pneumonia, pseudomonal infections, Q fever, rat bite fever, relapsing fever, rheumatic fever, rickettsial infections, Rocky Mountain fever, salmonellosis, spotted fever, scrub typhus, sepsis, sexually transmitted bacterial diseases, staphylococcal infections, streptococcal infections, surgical site infections, tetanus, tick-borne diseases, tuberculosis, tularemia, typhoid fever, urinary tract infections, vibrio infections, yaws, yersiniosis, Yersinia pestis infections, zoonotic diseases and zygomycosis.

[0269] Examples of microbial infections that are effectively treated by the methods provided herein include, but are not limited to, typical gram-positive infections, such as staphylococcal and streptococcal infections, which can be treated with nitric oxide in combination with penicillins or cephalosporins. Typical gram-negative infections, such as Klebsiella, Escherichia coli, and Pseudomonas species infections, can be treated with nitric oxide in combination with penicillins, cephalosporins, and quinolones.

[0270] Medical conditions associated with fungi (fungal infections) treatable by the methods and compositions presented herein include, but are not limited to, endemic fungal infections, opportunistic fungal infections, histoplasmosis associated with Histoplasma capsulatum, coccidioidomycosis associated with Coccidioides immitis, blastomycosis associated with Blastomyces dermatitidis, paracoccidioidomycosis associated with Paracoccidioides brasiliensis, candidiasis associated with Candida species, aspergillosis associated with Aspergillus species, mucormycosis associated with Mucor species, infections associated with Absidia, infections associated with Rhizopus species, and cryptococcosis associated with Cryptococcus neoformans.

[0271] As stated above, treating a medical condition by the methods presented herein, as well as all other aspects of the present invention, is meant to encompass the prevention, reduction, or eradication of microbial biofilms.

[0272] Synergy According to an embodiment of the present invention, the therapeutically effective amount of the antimicrobial agent administered in step (ii) is significantly lower than the effective amount of the antimicrobial agent administered without performing step (i), as demonstrated herein. In other words, the efficacy of the antimicrobial agent is significantly increased as a result of administering nitric oxide to the subject, thereby constituting a synergistic effect of nitric oxide treatment. Although nitric oxide may have already disappeared from the subject's system or from the cells of the pathogenic microorganism, the effect of nitric oxide on the microorganism's defense mechanisms still remains, thereby making the antimicrobial agent more effective against the microorganism.

[0273] Synergy is also demonstrated when using the methods presented herein against biofilms.

[0274] How the method is carried out Generally, the method is carried out by step (i), in which cells of a target microbial pathogen affecting a medical condition in a subject are exposed to nitric oxide in an amount and for a period of time sufficient to substantially reduce or deplete low molecular weight thiols in the target cells. The method is further carried out by step (ii), in which an antimicrobial agent is administered to the subject to substantially eradicate the pathogenic microorganism in the subject.

[0275] In some of the embodiments described herein, the period during which the subject is treated with NO, i.e., the period of step (i), corresponds to the latency period, which is defined as the period during which low molecular weight thiols in the target pathogenic cells are present in an amount that renders the cells, at least to some extent, resistant to the effects of the antimicrobial agent. The duration of the latency period depends on the species / strain of the target pathogenic cells, the amount, distribution, and localization of the pathogenic cells in the subject, the mode of administration of NO, and the effective concentration of NO administered to the subject. A list of latency periods measured in vitro for various bacterial species / strains, as well as a 2.5-fold reduction in microbial load (-2.5Log 10 ) and duration of microbial eradication (LD100) are shown in Table 4 in the following Examples section.

[0276] According to some embodiments, the duration of step (i) is substantially equal to the duration of the latency period. According to some embodiments, the duration of step (i) is at least 10%, 20%, 50% or at least 100% longer than the latency period, and in some embodiments, the latency period is less than 50%, 20% or 10% longer than the duration of step (i).

[0277] In some embodiments, step (i) is performed prior to the subsequent step (ii), thereby allowing a latency period to substantially pass before introducing the antimicrobial agent into the subject. In some embodiments, delivery and distribution of nitric oxide to target cells in the subject is performed after a pause between steps (ii) to allow a latency period to pass.

[0278] In some embodiments, step (i) is performed simultaneously with step (ii), thereby allowing the antimicrobial agent to be delivered and distributed throughout the subject during the latent period. In some embodiments, the administration and distribution of the antimicrobial agent is performed in step (ii) prior to step (i), thereby allowing the antimicrobial agent to reach the target cells at the end of the latent period.

[0279] The methods presented herein are carried out by administering NO to a subject in the form of gaseous NO (gNO) in a carrier gas or in the form of a nitric oxide (NO) donating / releasing compound, as those terms are defined herein.

[0280] Use of inhaled nitric oxide Because NO is a gas, it can be administered directly by inhalation. Modes of administration of gNO suitable in the context of this embodiment include local administration by exposing a subject (whole body or part thereof) to an NO-containing gas mixture, and systemic administration by inhalation of an NO-containing gas mixture.

[0281] In the context of embodiments of the present invention, the concentration of NO in the NO-containing gas mixture ranges from 80 to 5000 ppm, depending in large part on the mode of administration, although other concentration ranges are contemplated according to embodiments of the present invention. For example, the concentration of NO in the NO-containing gas mixture is at least about 80 ppm, 90 ppm, 100 ppm, 120 ppm, 140 ppm, 160 ppm, 180 ppm, 200 ppm, 240 ppm, 280 ppm, 320 ppm, 400 ppm, 500 ppm, or 1000 ppm.

[0282] When NO is administered by inhalation, it is important to take into consideration the toxicity of NO. For example, the safety of subjects can be achieved by administering NO by inhalation while monitoring the NO concentration in the inhalant and blood methemoglobin (SpMET) concentration, for example, maintaining a NO concentration of less than 5 ppm and preferably less than 2.5 ppm, and an SpMET concentration of no more than 5%, preferably no more than 1%.

[0283] According to some embodiments of the present invention, in which the NO-containing gas mixture is intended for inhalation by a subject, the term "NO-containing gas mixture" refers to a gas mixture of NO, oxygen, and air or nitrogen, which is characterized by predetermined, controlled, and constant concentrations of NO and O mixed together in a carrier gas (i.e., air or nitrogen).

[0284] According to some embodiments of the invention, step (i) may be performed in one or more cycles, where each cycle is characterized by the continuous inhalation of an NO-containing gas mixture at a particular NO concentration (e.g., about 80-200 ppm NO, or at least 160 ppm) for a first period, followed by the inhalation of air or a NO-free gas mixture for a second period. According to some embodiments of the invention, during the second period, the subject may inhale ambient air or a controlled gas mixture substantially devoid of NO, referred to herein as a carrier mixture.

[0285] In some embodiments, the first period of time lasts for 10 to 45 minutes, or 20 to 45 minutes, or 20 to 40 minutes, and in some embodiments, for about 30 minutes.

[0286] According to some embodiments of the present invention, the second period of time ranges from 3 to 5 hours, or 3 to 4 hours, and according to some embodiments, the second period of time spans about 3.5 hours.

[0287] According to some embodiments of the present invention, in which the NO-containing gas mixture is intended for local administration to a subject without inhaling the mixture, the term "NO-containing gas mixture" refers to a gas mixture of NO and a carrier gas characterized by a predetermined, controlled, constant concentration of NO.

[0288] Use of NO-releasing compounds In instances where NO administration via inhalation of gNO is ineffective, such as when nitric oxide does not reach target organs and / or biological systems and is associated with both biological and medical complications, including, for example, methemoglobinemia and direct lung injury, nitric oxide administration is carried out using a nitric oxide precursor or NO-releasing compound, as defined herein. Therefore, according to embodiments of the present invention, in step (i), NO is delivered to the site and generated by a prodrug. NO prodrugs are known as NO donors, which spontaneously generate NO under physiological conditions and / or are metabolized by enzyme metabolism to produce or release active NO. Therefore, according to embodiments of the present invention, NO donors, also referred to herein and in the prior art as NO prodrugs or NO-releasing compounds, are pharmacologically active substances that spontaneously release or are metabolized to NO or its redox congeners. In some embodiments, the NO-releasing compound spontaneously generates nitric oxide under physiological conditions.

[0289] Modes of administration of nitric oxide precursors in the form of NO-releasing compounds are suitable in the context of the present embodiments and include local and systemic administration, and include, but are not limited to, oral, rectal, intravenous, topical (ocular, vaginal, rectal, intranasal), intranasal, intradermal, transdermal, subcutaneous, intramuscular, intraperitoneal, by inhalation, or by intrathecal catheter.

[0290] As used herein, the term "nitric oxide (NO) donating / releasing compound" or "NO-releasing compound" refers to an organic or inorganic compound capable of releasing nitric oxide. In some embodiments, an NO-releasing compound is a small molecule, generally described as a molecule having a molecular weight of less than 600 g / mol.

[0291] Some classes, such as organic nitrates, have been used for decades as NO-releasing compounds in therapy.Some non-limiting examples of organic NO-releasing compounds include organic esters of nitric acid (nitrates), such as nitroglycerin, ethylene glycol dinitrate, isopropyl nitrate, glyceryl 1-mononitrate, glyceryl 1,2-dinitrate, glyceryl 1,3-dinitrate, nitroglycerin, butane-1,2,4-triol trinitrate, erythrityl tetranitrate, pentaerythrityl tetranitrate, and isosorbide mononitrate, and then include isosorbide 2-mononitrate, isosorbide 5-mononitrate, and isoprovide dinitrate.

[0292] Diazeniumdiolates, also known as "NONOates" (1-substituted diazen-1-ium-1,2-diolates, e.g., DETA NONOate), constitute another class of NO-releasing compounds containing an [N(O)NO]-functional group. NO-releasing compounds having a diazeniumdiolate group are disclosed as NO-releasing agents in, for example, U.S. Pat. Nos. 4,954,526, 5,039,705, 5,155,137, and 5,208,233, all of which are incorporated herein by reference. The advantage of these NO-releasing compounds is their wide range of half-lives, which depends on the structure of the amine bearing the diazeniumdiolate group.

[0293] NO-releasing C-based diazeniumdiolate molecules are disclosed, for example, in US Pat. Nos. 6,232,336, 6,511,991, and 6,673,338, all of which are incorporated herein by reference.

[0294] Non-diazeniumdiolate forms of NO-releasing compounds, including S-nitroso compounds, are also disclosed, for example, in U.S. Pat. Nos. 5,536,723 and 5,574,068, and C-nitroso compounds, for example, in U.S. Pat. No. 6,359,182, all of which are incorporated herein by reference.

[0295] NO-releasing compounds, according to embodiments of the present invention, include NO-releasing imidates, methanetrisdiazeniumdiolates, and bisdiazeniumdiolates derived from 1,4-benzoquinonedioxime, as well as the NO-releasing imidates and thioimidates disclosed in U.S. Pat. No. 6,673,338, incorporated herein by reference.

[0296] Other NO-releasing compounds suitable in the context of embodiments of the present invention are disclosed, for example, in European Patent No. 1004294, and U.S. Patent Nos. 7,569,559, 7,763,283, 7,829,553, 8,093,343, 8,101,589, and 8,101,658, all of which are incorporated herein by reference.

[0297] According to some embodiments, an effective potentiating or resensitizing amount of an NO-releasing compound ranges from about 0.01 to about 50 mg / kg body weight (mg of NO-releasing compound per kg of subject body weight).

[0298] pathogenic microorganisms Throughout this specification, the phrase "pathogenic microorganism" is used to describe any microorganism that can cause disease or disorder in higher organisms, such as mammals in general, and humans in particular. Pathogenic microorganisms can belong to any family of organisms, including, but not limited to, prokaryotes, gram-negative bacteria, gram-positive bacteria, eubacteria, archaea, eukaryotes, yeasts, fungi, algae, protozoa, and other parasites. Non-limiting examples of pathogenic microorganisms are Plasmodium falciparum and related Plasmodium parasites, Acanthamoeba and other free-living amoebae, Aeromonas hydrophila, Anisakis and related worms, and, but not limited to, Serratia species, Enterobacter species, Acinetobacter species, Acinetobacter baumannii, Ascaris lumbricoides, Bacillus cereus, Brevundimonas deminuta, Campylobacter jejuni, Clostridium botulinum, Clostridium perfringens, Cryptosporidium parvum, Cyclospora caetanensis, Diphyllobotorium, Entamoeba histolytica, certain strains of Escherichia coli, nematodes, Giardia lamblia , Klebsiella pneumoniae, Listeria monocytogenes, Schistosoma haematobium, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Plesiomonas shigelloides, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella, Serratia odorifera, Shigella, Staphylococcus aureus, Stenotrophomonas maltophilia, Streptococcus, Trichuris trichiura, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnifuscus and other vibrios, Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia christensenii.

[0299] Thus, a condition associated with a pathogenic microorganism refers to an infectious condition resulting from the presence of the microorganism in a subject. The infectious condition can be, for example, a bacterial infection, a fungal infection, a protozoan infection, or the like.

[0300] Treating conditions associated with pathogenic microorganisms describes measures to prevent, reduce, ameliorate, or eliminate the signs of an infectious disease. This treatment is generally accomplished by inhibiting the growth and / or eradicating the pathogenic microorganisms.

[0301] antimicrobial agents The term "antimicrobial agent," as used herein, includes all antimicrobial agents, but excludes nitric oxide as an antimicrobial agent per se. In accordance with the definition of microorganism provided above, the term "antimicrobial agent" includes antibiotics (also referred to herein as antibiotics) as well as antifungal agents, antiprotozoal agents, antiparasitic agents, and the like.

[0302] According to some embodiments, the antimicrobial agent is an antibiotic. Generally, but without being bound by any particular theory, the mechanism of antimicrobial activity of the antimicrobial agent according to embodiments of the present invention is different from the mechanism of activity of the polymer according to embodiments of the present invention.

[0303] It is noted that the phrase "antimicrobial agent" as used herein contemplates any combination of antimicrobial agents, and it is further noted that in the context of embodiments of the present invention, nitric oxide is excluded from the scope of the phrase "antimicrobial agent."

[0304] An example of some known combinations of antimicrobial agents that are considered "antimicrobial agents" in the context of embodiments of the present invention is the combination of a penicillin or cephalosporin with the addition of an aminoglycoside, such as gentamicin.

[0305] Non-limiting examples of antimicrobial agents suitable for use in this context of the present invention include, but are not limited to, mandelic acid, 2,4-dichlorobenzenemethanol, 4-[bis(ethylthio)methyl]-2-methoxyphenol, 4-epi-tetracycline, 4-hexylresorcinol, 5,12-dihydro-5,7,12,14-tetrazapentacene, 5-chlorocarbacrol, 8-hydroxyquinoline, acetarzole, acetylkitasamycin, acriflavine, alatrofloxacin, ambazone, amphomycin, amikacin, amikacin sulfate, amino Acridine, Calcium Aminosalicylate, Sodium Aminosalicylate, Aminosalicylic Acid, Ammonium Sulfobitinate, Amorolfine, Amoxicillin, Amoxicillin Sodium, Amoxicillin Trihydrate, Amoxicillin-Clavulanate Potassium Combination, Amphotericin B, Ampicillin, Ampicillin Sodium, Ampicillin Trihydrate, Ampicillin-Sulbactam, Apalcillin, Arbekacin, Aspoxicillin, Astromycin, Astromycin Sulfate, Abelmycin, Azanidazole, Azidamfenicol, A Didocillin, azithromycin, azlocillin, aztreonam, bacampicillin, bacitracin, bacitracin zinc, bekanamicillin, benzalkonium chloride, benzethonium chloride, benzoxonium chloride, berberine hydrochloride, biapenem, vibrocator, biclotimol, bifonazole, bismuth subsalicylate, bleomycin antibiotic complex, bleomycin hydrochloride, bleomycin sulfate, brodimoprim, bromochlorosalicylanilide, bronopol, broxquinoline, butenafine, butenafine hydrochloride, butoconazole, undecylenate Calcium, candicidin antibiotic complex, capreomycin, carbenicillin, carbenicillin disodium, carfecillin, carindacillin, carumonam, carzinophilin, caspofungin acetate, cephacetrile, cefaclor, cefadroxil, cephalexin, cephalexin hydrochloride, cephalexin sodium, cephaloglycin, cephaloridine, cephalothin, cephalothin sodium, cefamandole, cefamandole nafate, cefamandole sodium, cephapirin, cephapirin sodium, cefatrizine,Cefatrizine propylene glycol, cefazedone, cefazedone sodium salt, cefazolin, cefazolin sodium, cefbuperazone, cefbuperazone sodium, cefcapene, cefcapene pivoxil hydrochloride, cefdinir, cefditoren, cefditoren pivoxil, cefepime, cefepime hydrochloride, cefetamet, cefetamet pivoxil, cefixime, cefinenoxime, cefinetazole, cefinetazole sodium, cefininox, cefininox sodium, cefmorexin jh, cefodizime, cefodizime sodium, cefmorexin jh Fonicid, cefonicid sodium, cefoperazone, cefoperazone sodium, ceforanide, cefoselis sulfate, cefotaxime, cefotaxime sodium, cefotetan, cefotetan disodium, cefotiam, cefotiam hexetil hydrochloride, cefotiam hydrochloride, cefoxitin, cefoxitin sodium, cefozopran hydrochloride, cefpiramide, cefpiramide sodium, cefpirome, cefpirome sulfate, cefpodoxime, cefpodoxime proxetil, cefprozil, cefquinome, cephradine, cefroxadine, cefsulodin, ceftazidime cefteramu, cefteram, cefteram pivoxil, ceftezole, ceftibuten, ceftizoxime, ceftizoxime sodium, ceftriaxone, ceftriaxone sodium, cefuroxime, cefuroxime axetil, cefuroxime sodium, cetalkonium chloride, cetrimide, cetrimonium, cetylpyridinium, chloramine T, chloramphenicol, chloramphenicol palmitate, chloramphenicol sodium succinate, chlorhexidine, chlormidazole, chlormidazole hydrochloride, chloroxylenol, chlorphenesin, chloroxylenol Naldol, chlortetracycline, chlortetracycline hydrochloride, cyclacillin, ciclopirox, cinoxacin, ciprofloxacin, ciprofloxacin hydrochloride, citric acid, clarithromycin, potassium clavulanate, sodium clavulanate, clavulanic acid, clindamycin, clindamycin hydrochloride, clindamycin palmitate hydrochloride, clindamycin phosphate, clioquinol, cloconazole, cloconazole monohydrochloride, clofazimine, clofoctol, clometocillin, clomocycline, clotrimazole, cloxacillin,Cloxacillin sodium, colistin, colistin methanesulfonate sodium, colistin sulfate, cycloserine, dactinomycin, danofloxacin, dapsone, daptomycin, daunorubicin, DDT, demeclocycline, demeclocycline hydrochloride, dequalinium, dibekacin, dibekacin sulfate, dibromopropamidine, dichlorophen, dicloxacillin, dicloxacillin sodium, didecyldimethylammonium chloride, dihydrostreptomycin, dihydrostreptomycin sulfate, diiodohydroxyquinoline, dimetridazo ol, dipyrithione, dirithromycin, DL-menthol, D-menthol, dodecyltriphenylphosphonium bromide, doxorubicin, doxorubicin hydrochloride, doxycycline, doxycycline hydrochloride, econazole, econazole nitrate, enilconazole, enoxacin, enrofloxacin, eosin, epicillin, ertapenem sodium, erythromycin, erythromycin estolate, erythromycin ethyl succinate, erythromycin lactobionate, erythromycin stearate, ethacridine, ethacridine lactate, ethambutol Toll, ethanoic acid, ethionamide, ethyl alcohol, eugenol, exalamide, faropenem, fenticonazole, fenticonazole nitrate, phenathion, fleroxacin, flomoxef, flomoxef sodium, florfenicol, flucloxacillin, flucloxacillin magnesium, flucloxacillin sodium, fluconazole, flucytosine, flumequine, flurithromycin, flutrimazole, fosfomycin, fosfomycin calcium, fosfomycin sodium, framycetin, framycetin sulfate, flagyl , furazolidone, fusafungin, fusidic acid, fusidic acid sodium salt, gatifloxacin, gemifloxacin, gentamicin antibiotic complex, gentamicin cla, gentamicin sulfate, glutaraldehyde, gramicidin, grepafloxacin, griseofulvin, halazone, haloprogin, hetacillin, hetacillin potassium, hexachlorophene, hexamidine, hexetidin, hydragraphen, hydroquinone, hygromycin, imipenem, isepamicin, isepamicin sulfate, isoconazole, isoconazole nitrate, isoniazid,Isopropanol, itraconazole, josamycin, josamycin propionate, kanamycin, kanamycin sulfate, ketoconazole, kitasamycin, lactic acid, lanoconazole, lenampicillin, leucomycin A1, leucomycin A13, leucomycin A4, leucomycin AS, leucomycin A6, leucomycin A7, leucomycin A8, leucomycin A9, levofloxacin, lincomycin, lincomycin hydrochloride, linezolid, liranaftate, 1-menthol, lomefloxacin, lomefloxacin hydrochloride, loracarbef, Lymecycline, lysozyme, mafenide acetate, magnesium monoperoxophthalate hexahydrate, ethyl mecetronium sulfate, mecillinam, meclocycline, meclocycline sulfosalicylate, mepartricin, merbromin, meropenem, metharconium chloride, methampicillin, methacycline, methenamine, methyl salicylate, methylbenzethonium chloride, methylrosaniline chloride, methicillin, methicillin sodium, metronidazole, metronidazole benzoate, mezlocillin, mezlocillin sodium, miconazole, miconazole nitrate , micronomycin, micronomycin sulfate, midecamycin, minocycline, minocycline hydrochloride, miokamycin, myristalkonium chloride, mitomycin c, monendin, monendin sodium, molinamide, moxalactam, moxalactam disodium, moxifloxacin, mupirocin, mupirocin calcium, nadifloxacin, nafcillin, nafcillin sodium, naftifine, nalidixic acid, natamycin, neomycin a, neomycin antibiotic complex, neomycin C, neomycin sulfate, neticonazole, netilmicin , netilmicin sulfate, nifuratel, nifuroxazide, nifurtoinol, nifurzide, nimorazole, niridazole, nitrofurantoin, nitrofurazone, nitroxoline, norfloxacin, novobiocin, nystatin antibiotic complex, octenidine, ofloxacin, oleandomycin, omoconazole, orbifloxacin, ornidazole, ortho-phenylphenol, oxacillin, oxacillin sodium, oxiconazole, oxiconazole nitrate, oxoferrin, oxolinic acid, oxychlorosene, oxytetracycline,Oxytetracycline calcium, oxytetracycline hydrochloride, panipenem, paromomycin, paromomycin sulfate, pazufloxacin, pefloxacin, pefloxacin mesylate, penamecillin, penicillin G, penicillin G potassium, penicillin G sodium, penicillin V, penicillin V calcium, penicillin V potassium, pentamidine, pentamidine diisethionate, pentamidine mesilate, pentamycin, phenethicillin, phenol, phenoxyethanol, phenylmercuriborate, PHMB, phthalylsulfathiazole, picro Xidine, pipemidic acid, piperacillin, piperacillin sodium, piperacillin sodium-tazobactam sodium, piromidic acid, pivampicillin, pivcefalexin, pivmecillinam, pivmecillinam hydrochloride, policreslen, polymyxin antibiotic complex, polymyxin B, polymyxin B sulfate, polymyxin B1, polynoxylin, povidone-iodine, propamidine, propenidazole, propicillin, propicillin potassium, propionic acid, prothionamide, prothiophate, pyrazinamide, pyrimethamine, pyridomycin, pyrithione, pyroi Nitrilin, quinoline, quinupristin-dalfopristin, resorcinol, ribostamycin, ribostamycin sulfate, rifabutin, rifampicin, rifamycin, rifapentine, rifaximin, lithiamine, rokitamycin, rolitetracycline, losoxacin, roxithromycin, rufloxacin, salicylic acid, secnidazole, selenium disulfide, sertaconazole, sertaconazole nitrate, siccanin, sisomicin, sisomicin sulfate, sodium thiosulfate, sparfloxacin, spectinomycin, spectinomycin hydrochloride Syn, spiramycin antibiotic complex, spiramycin b, streptomycin, streptomycin sulfate, succinylsulfathiazole, sulbactam, sulbactam sodium, sulbenicillin disodium, sulbentin, sulconazole, sulconazole nitrate, sulfabenzamide, sulfacarbamide, sulfacetamide, sulfacetamide sodium, sulfachlorpyridazine, sulfadiazine, silver sulfadiazine, sulfadiazine sodium, sulfadiclamide, sulfadimethoxine, sulfadoxine, sulfaguanidine,Sulfalene, sulfamazone, sulfamerazine, sulfamethazine, sulfamethazine sodium, sulfamethizole, sulfamethoxazole, sulfamethoxazole-trimethoprim, sulfamethoxypyridazine, sulfamonomethoxine, sulfamoxole, sulfanilamide, sulfaperine, sulfa, Phenazole, sulfapyridine, sulfaquinoxaline, sulfasuccinamide, sulfathiazole, sulfathiourea, sulfatramide, sulfatriazine, sulfisomidine, sulfisoxazole, sulfisoxazole acetyl, sulfonamide, sultamicillin, sultamicillin tosylate, tacrolimus, talampicillin hydrochloride, teicoplanin A2 complex, teicoplanin A2-1, teicoplanin A2-2 , teicoplanin A2-3, teicoplanin A2-4, teicoplanin A2-5, teicoplanin A3, teicoplanin antibiotic complex, telithromycin, temafloxacin, temocillin, thenol, terbinafine, terconazole, terizidone, tetracycline, tetracycline hydrochloride, tetracycline metaphosphate, tetramethylthiuram monosulfide, tetroxoprim, thiabendazole, thiamphenicol, thiamine These include afenicol glycinate hydrochloride, thiomersal, thiram, thymol, tibezonium iodide, ticarcillin, ticarcillin-clavulanic acid mixture, ticarcillin disodium, ticarcillin monosodium, tilbroquinol, tilmicosin, thymidazole, tioconazole, tobramycin, tobramycin sulfate, tolciclate, trindate, tolnaftate, toloconium methylsulfate, toltrazuril, tosufloxacin, triclocarban, triclosan, trimethoprim, trimethoprim sulfate, triphenylstibine sulfide, troleandomycin, trovafloxacin, tylosin, tylothricin, undecoilium chloride, undecylenic acid, vancomycin, vancomycin hydrochloride, viomycin, virginiamycin antibiotic complex, voriconazole, xanthocillin, xivomol, and zinc undecylenate.

[0306] Antifungal agents that may be usefully used in any other aspect of the invention include, but are not limited to, polyenes, amphotericin B, liposomal amphotericin, nystatin, and pimaricin; azoles, fluconazole, itraconazole, ketoconazole, itraconazole, voriconazole, posaconazole; aquinocandins such as anidulafungin, caspofungin, and micafungin; allylamines and morpholines such as naftifine and terbinafine and amorolfine; and antimetabolites such as 5-fluorocytosine.

[0307] In some embodiments, the antimicrobial agent is an antibiotic. Exemplary antibiotics include, but are not limited to, oxacillin, piperacillin, penicillin G, ciprofloxacin, erythromycin, tetracycline, gentamicin, vancomycin, and methicillin. These antibiotics are known to be associated with the emergence of resistance to them.

[0308] Pharmaceutical Composition In any of the methods described herein, nitric oxide, either as gaseous NO or in the form of a NO-releasing compound, and / or the antimicrobial agent may be administered as part of a pharmaceutical composition, which further comprises a pharmaceutically acceptable carrier, as described herein.

[0309] In embodiments in which step (i) and step (ii) of the above-described method are not performed simultaneously, the pharmaceutical composition comprises two or more parts, where at least one part comprises nitric oxide and another part comprises an antimicrobial agent.

[0310] In embodiments in which NO is inhaled or otherwise used as a gas and the antimicrobial agent is administered as a solid, liquid, paste, ointment, or suspension / emulsion, the pharmaceutical composition comprises at least one gaseous portion for the nitric oxide and a separate non-gaseous portion for the antimicrobial agent. The carrier in the portion containing the nitric oxide can be selected according to the mode of administration (inhalation or topical administration).

[0311] In embodiments in which NO is administered as the NO-releasing compound, the nitric oxide and / or antimicrobial agent can be administered via any route of administration, including, but not limited to, oral, inhaled, or parenteral, for example, by intravenous infusion or intraperitoneal, subcutaneous, intramuscular, or intravenous injection, or topically. Carriers for any part of the composition are selected to be appropriate for the chosen route of administration.

[0312] According to another aspect of the present invention, there is provided the use of an antimicrobial agent in the manufacture of a medicament, which further comprises nitric oxide, as described in the aforementioned methods of treatment, for treating a medical condition associated with a pathogenic microorganism. Alternatively, there is provided the use of nitric oxide in the manufacture of a medicament, which further comprises an antimicrobial agent, as described in the aforementioned methods of treatment, for treating a medical condition associated with a pathogenic microorganism.

[0313] According to embodiments of the present invention, the antimicrobial agent and / or amount thereof is selected such that when a potentiating or resensitizing effective amount of nitric oxide is used, the therapeutically effective amount of the antimicrobial agent is substantially lower than the therapeutically effective amount of the antimicrobial agent when used without nitric oxide. As in several other aspects presented herein, and according to some embodiments, nitric oxide may be used in combination with an antimicrobial agent, which may be administered simultaneously with or after administering nitric oxide.

[0314] Thus, according to another aspect of embodiments of the present invention, there is provided a pharmaceutical composition comprising as active ingredients a sensitizing or re-sensitizing effective amount of nitric oxide, a therapeutically effective amount of an antimicrobial agent, and a pharmaceutically acceptable carrier. According to some embodiments, the composition is packaged in packaging material and is identified in print in or on the packaging material for use in treating a medical condition associated with a pathogenic microorganism. According to other embodiments, the composition is packaged in packaging material and is identified in print in or on the packaging material for use in treating a medical condition associated with a resistant pathogenic microorganism, as described above.

[0315] As used herein, the phrase "pharmaceutical composition" or term "medicament" refers to a preparation of nitric oxide and one or more antimicrobial agents described herein with other chemical components, such as suitable pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration of nitric oxide and / or antimicrobial agents to a subject.

[0316] Hereinafter, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to organisms and does not abolish the biological activity and properties of the administered compound. Examples of non-gaseous carriers include, but are not limited to, air, nitrogen, argon, and other carrier gases that are substantially inert to nitric oxide and / or antimicrobial agents when administered as an inhalation powder, vapor, or gas. Examples of non-gaseous carriers include, but are not limited to, polyethylene glycol, saline, emulsions, and mixtures of organic solvents and water.

[0317] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0318] Techniques concerning pharmaceutical formulation and administration may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0319] Any portion of the pharmaceutical composition may be formulated for administration by one or more routes, depending on whether local or systemic treatment or administration is selected and the area to be treated. Administration may be oral, inhaled, or parenteral, such as by intravenous drip or intraperitoneal, subcutaneous, intramuscular, or intravenous injection, or topically (including ophthalmically, vaginally, rectally, intranasally).

[0320] Formulations for topical administration may include, but are not limited to, lotions, ointments, gels, creams, suppositories, drops, liquids, sprays, or powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like may be necessary or desirable.

[0321] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, unit dose packets, pills, caplets, or tablets. Thickeners, diluents, flavorings, dispersing aids, or binders may be desirable.

[0322] Formulations for parenteral administration may include sterile solutions which may also contain, but are not limited to, buffers, diluents and other suitable additives. Delayed-release compositions are contemplated for therapeutic use.

[0323] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0324] Any portion of the pharmaceutical composition for use in accordance with embodiments of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, including additives and adjuvants, to facilitate processing of any dosage form of nitric oxide and any dosage form of antimicrobial agent into a pharmaceutically usable preparation. The proper formulation will depend on the route of administration selected.

[0325] The toxicity and therapeutic efficacy of antimicrobial agents, as well as the potentiating and resensitizing efficacy of nitric oxide as described herein, can be measured by standard pharmaceutical procedures in laboratory animals, e.g., by measuring the MIC, EC for any combination of antimicrobial agent and nitric oxide. 50 ,I C 50 , L.D. 50 (lethal dose producing death in 50% of the animals tested) and / or LD 100 The data obtained from these activity measurements and animal studies can be used in formulating a range of dosage for use in humans.

[0326] Doses may vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration, and dosage can be selected by an individual physician in light of the patient's condition (see, for example, Fingl et al., 1975, "The Pharmacological Basis of Therapeutics," Ch. 1, p. 1). Generally, dosage is related to the potency of the active ingredient, which in the context of embodiments of the present invention is related to its minimum inhibitory concentration (MIC) and its specific pharmacokinetics and pharmacology in terms of absorption, distribution, metabolism, excretion, and toxicity (ADMETox) parameters. For antimicrobial agents, a therapeutically effective amount is often about 10 times the MIC of the antimicrobial agent. A potentiating or resensitizing amount of nitric oxide is less than 1 MIC unit relative to nitric oxide and any target microorganism, and a therapeutically effective amount of any target antimicrobial agent used in combination with nitric oxide as described herein may be 1 MIC unit or less relative to the antimicrobial agent and any target microorganism.

[0327] The amount of a composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0328] The compositions of the present invention may, if desired, be provided in a pack or dispensing device, such as an FDA (U.S. Food and Drug Administration)-approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as, but not limited to, a blister pack or a pressurized container (for inhalation). The pack or dispensing device may be accompanied by instructions for administration. The pack or dispensing device may also be accompanied by a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, the notice reflecting the official approval of the composition dosage form for human or animal administration. Such notice may, for example, be a label approved by the U.S. Food and Drug Administration for prescription drugs or an approved product insert. Compositions containing any dosage form of nitric oxide, either alone or in combination with an antimicrobial agent, formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as detailed herein.

[0329] Medicine Kit As noted above, nitric oxide is indicated for use in combination with an antimicrobial agent, and as further noted, the two active ingredients can be administered simultaneously as separate compositions or sequentially. Thus, there is an advantage in providing a health care provider or a self-administering subject with a kit containing all the required compositions in one package.

[0330] When in gas form, the pharmaceutical kit includes a container or canister containing gNO in a carrier gas, which is configured for inhalation or topical application.

[0331] Yet another aspect of the present invention provides a pharmaceutical kit comprising nitric oxide in any of the forms described herein and an individually packaged antimicrobial agent within packaging material, and the kit is labeled according to its intended use and includes instructions for carrying out its intended use, such as treating a medical condition associated with a pathogenic microorganism, or treating a medical condition associated with a resistant pathogenic microorganism as described above, and / or re-sensitizing a resistant pathogenic microorganism to an antimicrobial agent after the development of resistance to the antimicrobial agent.

[0332] According to an embodiment of this aspect, the kit includes a therapeutically effective amount of an antimicrobial agent that, for some reason, is ineffective against a particular microorganism when used without nitric oxide.

[0333] Unit Dosage Form As explained above, nitric oxide has unique properties that allow it to be used as a potentiating and / or resensitizing agent, which allow the use of antimicrobial agents at lower doses than would typically be practiced without nitric oxide.

[0334] Thus, according to another aspect of an embodiment of the present invention, there is provided an antimicrobial pharmaceutical composition unit dosage form, which comprises a therapeutically effective amount of an antimicrobial agent, which is intended for use in combination with nitric oxide.

[0335] Also provided are pharmaceutical unit dosage forms of nitric oxide, which contain a potentiating or resensitizing effective amount of nitric oxide in any of the dosage forms described herein. If in gaseous form, the unit dosage form of NO can be provided in a container or canister configured for inhalation or topical application.

[0336] The term "unit dosage form," as used herein, describes physically discrete units, each unit containing a predetermined quantity of one or more active ingredients calculated to produce the desired enhancing or resensitizing effect, in association with at least one antimicrobial agent and other pharmaceutically acceptable carriers, diluents, additives, and combinations thereof.

[0337] The single unit dosage forms described herein can be formulated for any of the modes of administration described herein.

[0338] According to an embodiment of this aspect, the pharmaceutical composition unit dosage form of the antimicrobial agent comprises a therapeutically effective amount of an antimicrobial agent that, for some reason, is not effective against a particular microorganism when used without nitric oxide.

[0339] In some embodiments of all aspects of the invention, nitric oxide is used in an amount lower than its MIC. In some embodiments, the amount of nitric oxide in the unit dosage form is from about 1 MIC unit to about 1 / 10 MIC unit of nitric oxide, as described herein. In some embodiments, the unit dosage form contains 0.5 to 20 MIC units of antimicrobial agent, or 1 / 2 MIC unit, 2 / 3 MIC unit, 3 / 4 MIC unit, 1 MIC unit, 2 MIC unit, 5 MIC unit, 10 MIC unit, and more of antimicrobial agent.

[0340] It will be recognized that, for ease of understanding, certain features of the invention are described in the context of separate embodiments, but may also be provided in combination in a single embodiment. Conversely, various features of the invention that are, for brevity's sake, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as appropriate in any other described embodiment of the invention. Particular features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is ineffective without those elements.

[0341] While numerous embodiments of the present invention have been described, it is understood that these embodiments are illustrative only and not limiting, and that numerous variations may become apparent to those skilled in the art. Furthermore, the various steps may be performed in any desired order (and any desired steps may be added and / or any desired steps may be eliminated).

[0342] Various embodiments and aspects of the present invention as delineated above and as claimed in the claims below find experimental support in the following examples.

[0343] example Example 1: Treatment of bronchiolitis with inhaled nitric oxide The study was conducted at Soroka University Medical Center in southern Israel and was approved by the Institutional and National Human Ethics Committee. A detailed study summary is included in the online supplement. The study is registered under clinical trial number NCT01768884.

[0344] The study was a randomized, prospective, single-center, double-blind trial of hospitalized infants aged 2 to 11 months with acute bronchiolitis (Figure 1). Inclusion and exclusion criteria are summarized in Table 1.

[0345] Subjects were screened within 4 hours of admission and randomized (1:1) to receive intermittent inhalation of 160 ppm NO with standard care (NO group) or intermittent inhalation of an O2 / air mixture with standard care (control group) for up to 25 inhalations.

[0346] Standard supportive care included humidified oxygen, nasal suction when needed, and hydration (oral, intravenous, or nasogastric fluids). The use of other concomitant medications was permitted according to the general practice of the ward.

[0347] Nitric oxide treatment: Subjects voluntarily inhaled 160 ppm NO via a face mask in fixed-flow mode. Nitric oxide (Maxima, Israel) at 800 ppm (0.08%) NO equilibrated with 99.999% pure nitrogen (N) was titrated into the O2 / air inspiratory delivery line. Inhaled NO, NO2, and O2 concentrations were continuously monitored in the patient's breathing circuit using a dedicated gas analyzer (AeroNox, International medical, USA).

[0348] Measuring Results Primary outcomes: Safety measures including %MetHb and NO2 production, bleeding episodes, and any adverse events (AEs) associated with NO treatment. The study thresholds for NO2 and MetHb were set at 5 ppm and 5%, respectively.

[0349] The primary outcome was the safety and tolerability of intermittent inhalation of 160 ppm NO administered to 2- to 11-month-old neonates with acute bronchiolitis. Safety measures included %MetHb (safety thresholds were determined as >5% for MetHb and >5 ppm for NO) and other AEs associated with NO treatment. Tolerability measures included the proportion of subjects who discontinued the study prematurely for any reason and the proportion of subjects who discontinued treatment prematurely due to serious SAEs.

[0350] Secondary outcomes: Efficacy parameters: LOS calculated in hours starting from the first inhalation until 1) oxygen saturation (SpO2) ≥ 92% on room air, 2) clinical score ≤ 5 (15, 16), and 3) blinded physician decision of "ready for discharge."

[0351] Secondary outcomes were efficacy measures and included LOS, time to achieve ≥ 92% SaO2, and time to achieve a clinical score of ≤ 5. LOS in hours was defined as the time between first treatment and the time the infant was "ready for discharge," defined as: 1. room air saturation ≥ 92%, 2. clinical severity sign score ≤ 5 (Table E1), 3. clinical decision made by a blinded physician.

[0352] A severity sign score was used to determine the severity of each infant (Table E1). The score consisted of four components: respiratory rate, accessory muscle use, wheezing and crepitus on auscultation, and room air oxygen saturation (SpO2). Each component was given 0 to 3 points, for a total possible score of 12. Infants with a score <6 were determined to be mild and were not included in the study, while infants with a score >10 were determined to be very severe and were also excluded.

[0353] Exam Overview Study staff were divided into "blinded" and "unblinded" groups. Unblinded staff administered the inhalation to the infants and monitored %MetHb, %SpO2 (co-oximeter, RAD57 / RAD87, Masimo Corporation, USA), fractional inspired oxygen O2 (FiO2), NO, and NO2 concentrations. The blinded group included the investigator and all other staff directly involved in the patient's care. Safety thresholds for NO2 and MetHb were set at 5 ppm and 5%, respectively. It should be emphasized that both NO / O2 (NO treatment) and O2 / air mixture (control) treatments were given via the same device, and therefore patients were blinded to the treatment group.

[0354] Each patient received five inhalations of NO (treatment group) or oxygen (control group) per day with standard supportive care, for a maximum of 25 inhalations (based on phase I safety data). Each subject was examined and evaluated every morning (9:00 AM and 3:00 PM) by a blinded pediatrician using a critical signs score. Treatment was discontinued when SpO2 reached 92% on room air, the score was ≤5, and the patient was assessed as "ready for discharge."

[0355] Initial assessment included disease severity determination by clinical score (Table E1). (For a more detailed description of the clinical score, see the online supplement.) Subjects were examined and evaluated twice daily using the score. Follow-up visits were conducted on days 14, 21, and 30 from the day of admission. (For more detailed study monitoring, see the online supplement.)

[0356] statistical analysis Data were processed and analyzed by an independent group of statistical experts using SAS® version 9.1 (SAS Institute, Cary, North Carolina). Paired t-tests were applied to test for changes from baseline for quantitative variables, two-sample t-tests / nonparametric Wilcoxon rank sum tests or median tests were used to analyze differences between study groups in quantitative parameters, chi-square tests were applied to test for differences in the frequencies of categorical variables between study groups, Kaplan-Meier survival function curves were applied to test for differences between study groups regarding efficacy endpoints, and Cox models were applied for comparative analysis of Kaplan-Meier curves and hazard ratio assessments.

[0357] Based on preclinical studies, the antimicrobial therapeutic effect of NO is predicted to require approximately 2.5 hours of exposure (i.e., 24-hour treatment), so a post-hoc subgroup analysis of subjects with LOS ≤ 24 hours and > 24 hours was also performed for the key post-hoc secondary endpoint. One-third of subjects were discharged from the hospital in less than 24 hours. A longer LOS is predicted to correlate with higher disease severity, and therefore any therapeutic effect should be more pronounced in the subgroup with an LOS > 24 hours.

[0358] LOS was calculated from the time from the first inhaled treatment to "dischargeable," defined as the physician's decision to discharge. "Dischargeable" time was obtained from the final applicable clinical score and from the subject's medical record in special circumstances (i.e., for subjects who did not achieve a clinical score ≤5 during the study and for subjects who remained in the hospital due to a suspected bronchiolitis-related event).

[0359] Time to achieve ≥92% room air SpO2 (improved oxygenation) to discharge was calculated from the first time an SpO2 of ≥92% room air was sustained from first treatment to discharge. Time to clinical score ≤5 was calculated from first inhalation to the first time the subject achieved a clinical score ≤5.

[0360] All measured variables and derived parameters were tabulated with descriptive statistics. Categorical variables were presented in summary tables including sample size, absolute and relative frequencies by study group and overall.

[0361] Continuous variables were summarized by study group in tables including sample size, arithmetic mean, SD, standard error, median, minimum, and maximum.

[0362] The following statistical tests were used in analyzing the data presented in this study: 1. Paired t-tests were applied to test the statistical significance of changes from baseline for quantitative variables within each study group. 2. Two-sample t-tests, nonparametric Wilcoxon rank sum tests, or median tests were used as appropriate to analyze differences between study groups in quantitative parameters. 3. Chi-square tests were applied to test the statistical significance of differences in the frequencies of categorical variables between study groups. 4. Survival analysis using Kaplan-Meier survival function curves was applied to test the statistical significance of differences between study groups in the following endpoints: LOS from first inhalation to discharge availability, time to achieve 92% saturation to discharge, and time to achieve a clinical score ≦5. The Cox model was applied for comparative analysis of Kaplan-Meier curves. Hazard ratios were evaluated by Cox regression models.

[0363] All tests applied were two-sided, and a P value of 5% or less was considered statistically significant. Data were analyzed using SAS® version 9.1 (SAS Institute, Cary, NC).

[0364] Post-hoc subgroup analyses of subjects with LOS ≤ 24 hours and > 24 hours were also conducted for the key post-hoc secondary endpoint. These post-hoc analyses were conducted because, based on preclinical studies, the antiviral / antimicrobial therapeutic effect of NO is predicted to require at least 2.5 hours of exposure (i.e., 24-hour treatment). Approximately one-third of subjects were discharged from the hospital in less than 24 hours. Subjects with LOS < 24 hours were considered to have "very mild disease," and their improvement was unlikely to be related to any treatment. A longer LOS is predicted to correlate with greater disease severity; therefore, any treatment effect should be more pronounced in the subgroup with LOS > 24 hours.

[0365] The planned sample size was 40 subjects, 20 in each study arm. Considering an expected dropout rate of approximately 10%, we planned to recruit 44 subjects to have a sample size of 40 patients completing the study.

[0366] result subject A total of 63 infants were screened (Figure 2), and 20 parents declined consent, so 43 subjects were randomly assigned to the "intention-of-treat" group (ITT), with 21 in the NO group and 22 in the control (O2 / air) group. The "per protocol" (PP) group included 19 subjects (90.5%) in the NO group and 20 subjects (90.0%) in the control group.

[0367] Demographic and baseline characteristics Treatment groups were well matched for demographic and baseline characteristics (sex, ethnicity, age, screening weight, gestational age at birth, and screening MetHb levels) (Table 2). The mean (±standard deviation [SD]) age was 4.8±2.3 months and 5.6±2.8 months in the NO and control groups, respectively. The mean baseline MetHb levels were 0.7±0.4% and 0.7±0.30%, respectively, and the mean clinical scores were 7.9±1.1 and 8.1±1.3. All tested infants in both groups had moderate bronchiolitis severity according to the scores.

[0368] In both treatment groups, the majority of subjects tested positive for RSV (71.4% and 63.6% in the NO and control groups, respectively). Other viruses detected included coronavirus (4 per group), adenovirus (2 in the control group), metapneumovirus (2 in the NO group and 1 in the control group), and influenza A (6 in the control group). Demographic and baseline characteristics were similar in the LOS > 24 h and LOS ≤ 24 h subgroups (Table E2).

[0369] A total of 156 NO inhalations were administered, and 198 O2 / air mixtures were given to the control group. The mean number of inhalations was lower in the NO group (7.4 ± 3.2, maximum 16) compared with the control group (9.0 ± 6.5, maximum 25).

[0370] Concomitant medications All subjects in both treatment groups had one or more concomitant medications, and the treatment groups were well balanced with regard to the overall frequency and type of concomitant medications. The most common medication types were: beta-agonists, paracetamol, atropine-like substances, hypertonic saline, systemic steroids, and antibiotics (Table E3).

[0371] Safety evaluation AEs were reported in 23 subjects (53.5%): 22 AEs in 10 subjects (47.6%) in the NO group and 22 AEs in 13 subjects (59.1%) in the control group ( Table 3 , E4).

[0372] AEs considered possibly related to NO treatment were MetHb > 5%, elevated NO2 > 5 ppm, and bleeding (17). AEs considered possibly or possibly related to inhaled treatment were reported in 5 (23.8%) and 2 (9.1%) subjects in the NO and control groups, respectively. Serious AEs (SAEs) were reported in 4 (19.0%) and 4 (18.2%) subjects in the NO and control groups, respectively. There were no treatment-related SAEs in the NO group compared with 1 in the control group. There were no bleeding events or deaths during the study.

[0373] Primary safety endpoint - Proportion of MetHb associated with inhaled NO In the NO group, six subjects (28.5%) had MetHb measurements >5% during the study treatment period, and in three of these subjects, values ​​>5% were observed more than once (the maximum was 5.6% in two subjects). MetHb levels increased with each NO inhalation, peaking at the end of the inhalation (mean 3.3 ± 0.9%) and then gradually decreasing to approach pretreatment levels (Fig. 3a). Comparing MetHb concentrations before and at the end of inhalation, there was no cumulative effect of MetHb concentrations over the treatment period (Fig. 3b).

[0374] One subject in the NO group experienced an increased NO concentration >5 ppm (5.5 ppm) once. The mean peak NO2 at the end of the first inhalation for 21 infants was 1.55 (SD = 0.55) ppm, which is well below the safety threshold of 5 ppm (Figure 4).

[0375] Tolerability: Four subjects discontinued the study / study treatment: two (9.5%) in the NO group and two (9.1%) in the control group. Two subjects (one in each group) discontinued treatment due to parental withdrawal of consent or parental noncompliance, a third subject (NO group) discontinued study due to a secondary AE of MetHb >5%, and a fourth subject (control group) discontinued treatment and was transferred to the pediatric intensive care unit due to an SAE of respiratory failure.

[0376] Secondary Outcome - Efficacy Evaluation Length of stay (LOS): In the intention-to-treat (ITT) analysis, 43 subjects were included. The mean ± SD LOS was 43.3 ± 32.95 hours in the NO group compared with 50.0 ± 46.2 hours in the control group (P = 0.86). When LOS was analyzed including the 16 infants with mild bronchiolitis who were discharged within <24 hours, the median LOS was 40 hours and 24.5 hours in the NO and control groups, respectively (P = 0.65). When a post-hoc analysis (ITT) was performed based on LOS > 24 hours and LOS ≤ 24 hours, the median LOS was significantly shorter in the NO group (41.92 hours) compared with the control group (62.50 hours) (P = 0.014) (Figure 5). The ITT and PP Kaplan-Meier analyses for LOS are illustrated in Figure 6.

[0377] Time to first 92% O2 saturation sustained until discharge: Time to first 92% O2 saturation sustained until discharge was 35.50 ± 33.73 hours in the NO group versus 45.75 ± 44.43 hours in the control group in the ITT (N = 42) study (P = 0.517). Kaplan-Meier analysis of LOS > 24 hours for PP showed a statistically significant difference in favor of the NO group (HR = 0.358, 95% CI = 0.139, 0.921, P = 0.028) (Figure 7).

[0378] Time to clinical score ≤5: Analysis for intention-to-treat (N = 43) showed a shorter, but not statistically significant, mean time to clinical score ≤5 in the NO group (32.83 ± 30.61 h) compared with 43.10 ± 43.91 h in the control group (P = 0.621). However, based on Kaplan-Meier analysis of subjects with LOS > 24 h, a statistically significant difference was found in favor of the NO group (HR = 0.391, 95% CI: 0.161, 0.949, log-rank P = 0.033) (Figure 8A and B). Kaplan-Meier analysis for PP also showed a statistically significant difference in favor of the NO group for subjects with LOS > 24 h (HR = 0.273, 95% CI: 0.093, 0.799, log-rank P = 0.013) (Figure 8C and D).

[0379] Consideration The primary results of this study in 43 infants aged 2 to 11 months with bronchiolitis were safe, tolerable, and promising. Although efficacy was not the focus of the study, secondary efficacy outcomes were evaluated. We found no statistically significant differences in safety or tolerability between NO and control treatment groups (ITT, PP). In the subgroup of subjects with LOS >24 hours (>2.5 hours of NO exposure), post-hoc analyses demonstrated a statistically significant clinical advantage of NO over standard care in terms of reduced LOS, time to SpO2 ≥92% on room air, and time to clinical score ≤5.

[0380] NO plays an important role in various biological functions, including smooth muscle vasodilation, neurotransmission, anti-inflammatory effects, regulation of wound healing, and immune responses to infection, such as bactericidal activity directed against various organisms. NO also acts as an antiviral agent, including inhibition of herpes simplex virus type 1. Several mechanisms and pathways are thought to explain NO's ability to eliminate viral infections: inhibition of viral proteinases and ribonucleotide reductases, transcription factors required for RNA entry into host cells, viral infection, and viral protein accumulation, and viral replication during the first stage of the viral replication cycle, viral release from infected cells, and modulation of the host response to infection.

[0381] NO is approved for the treatment of term and near-term neonates at a dose of 20 ppm, maintained by continuous delivery for up to 14 days. Doses up to 80 ppm have been used during clinical trials (FDA approval of NDA20-86INOmax nitric oxide gas, 1999).

[0382] Safety concerns with inhaled NO therapy include MethHb accumulation, NO2 production, and bleeding. Inhaled nitric oxide can combine with hemoglobin to form nitrosylhemoglobin, which is rapidly oxidized to methemoglobin (metHb). Cyanosis does not appear until metHb concentrations reach 15–20%, and clinical signs of hypoxia are generally not significant at hemoglobin concentrations below approximately 30%. In neonatal studies, MethHb concentrations of 5–10% were managed by halving the concentration of NO until they decreased to concentrations below 5%. Furthermore, the corresponding increase in MethHg% during the study confirmed that sufficient NO was present in the airways to be absorbed into the bloodstream and metabolized.

[0383] In the neonatal study, inhaled NO was discontinued when NO2 exceeded 7 ppm. At high doses, the primary toxicological effect of NO2 is pulmonary edema (Centers for Disease Control, 1988). However, in previous studies, NO doses below 80 ppm did not result in either significant elevations in measured NO2 levels or clinical evidence of NO2 toxicity. Similarly, in the present study, 160 ppm NO inhaled for 30 minutes five times per day was associated with neither significant elevations in NO2 nor clinical evidence of toxicity.

[0384] The intermittent administration method was selected to minimize the potential for adverse events while maximizing the additional therapeutic benefits of NO's antiviral and antibacterial effects, as well as its anti-inflammatory and vasodilator properties, and further promote airway clearance. The results of this study, showing no MetHb accumulation, no significant NO2 elevation events, and no bleeding episodes, support the rationale for intermittent 160 ppm inhaled therapy in humans. The dose and time requirements for NO as an antibacterial agent were measured and shown to be effective in both planktonic suspensions and biofilms. Treatment of influenza virus or infected cells with intermittent (30 min every 4 h) 160 ppm exogenous gaseous NO reduced not only viral replication but also its infectivity in a Madin-Darby canine kidney (MDCK) cell model of infection. Inhalation of 160 ppm NO for 30 min, 5 times daily for 5 consecutive days, is safe and well tolerated in healthy individuals.

[0385] Although this study did not focus on efficacy, it did evaluate secondary outcomes related to efficacy. Based on previous in vitro and animal studies demonstrating that at least 2.5 hours of NO exposure was required for antimicrobial efficacy, a post-hoc analysis was conducted comparing NO treatment with control in a subgroup of infants who remained hospitalized for >24 hours (>5 30-minute treatments). In this subgroup, the efficacy differences were statistically significant in favor of the NO group: shorter LOS (ITT), shorter time to score ≤5 (ITT), and time to SpO2 ≥92%.

[0386] The average hospital stay in infants with bronchiolitis is short, making it difficult to demonstrate significant reductions in outcomes such as viral load. We selected LOS in hours as the primary efficacy outcome. This outcome has been used in several recent studies (11, 23, 24). LOS depends on two important outcome measures: the rate of clinical improvement and the need for oxygen therapy. Severity scoring systems have high power to detect clinical differences between groups, especially when a placebo is used (11). The modified Tal score (15) has been shown to have internal consistency and excellent interrater reliability (16). We recently conducted an internal validation study of the modified Tal score, scoring 50 infants twice daily during their hospital stay, with highly significant intraclass correlation coefficients among 17 pediatricians with different levels of experience (unpublished data).

[0387] No specific treatment has yet been approved for RSV bronchiolitis. Recently, several selective RSV proviral compounds have been identified in clinical trials (25). Currently, only supportive care, such as oxygen, fluids, and nasopharyngeal suction, is recommended (12). Our preliminary results therefore suggest that inhaled NO may provide a valid tool for improving RSV outcomes. The therapeutic potential of inhaled NO suggested in this study needs to be further tested in larger, double-blind, placebo-controlled trials focused on both safety / tolerability and multiple efficacy endpoints.

[0388] In conclusion, the safety and tolerability of intermittent inhaled 160 ppm NO treatment were compared with standard supportive care in this study of hospitalized infants with acute bronchiolitis. A secondary exploratory analysis of the subgroup of subjects with LOS >24 hours demonstrated statistically significant therapeutic benefits of NO versus standard supportive care in terms of reduction in LOS and time to 92% saturation, as well as accelerated clinical improvement. Although the study sample was small, the efficacy results were promising and support the antiviral potential of intermittent 160 ppm inhaled NO in LRTI. Larger studies are needed to confirm the beneficial effects of inhaled NO in viral bronchiolitis.

[0389] Example 2: Antimicrobial Potential: Background Art To determine the effect of 200 ppm gNO on microorganisms, the response of bacteria isolated from patients with medical conditions associated with these bacteria to exposure to 200 ppm gNO was measured in clinically infectious (10 5 )10E 5 The gNO-exposed organisms were tested in a series of experiments at concentrations of CFU / ml [Chris C. Miller, PhD thesis, University of British Columbia, Canada, 2004]. Each experiment was performed at least once with a minimum of two samples per time point. In all experiments, the control (air-exposed) had a survival rate of 100% or greater over the test period compared to the initial inoculum. The reduction in CFU / ml for each gNO-exposed organism was 4-6 log units, indicating a significant bactericidal effect of 200 ppm gNO.

[0390] For each bacterium, a survival curve in CFU / ml was plotted as a function of time. Each graph plotted the survival curve for the bacterium at 200 ppm gNO and the control (air exposure).

[0391] Briefly, 5 x 10 cells of the exemplary bacterium Serratia marcescens 5The cells were suspended in 2 mL of saline in test tubes and exposed to 200 ppm NO in nitrogen at a flow rate of 1 L / min. Viability was measured by determining the number of bacterial cells remaining in the culture every hour by viable count and serial dilution, and the results are summarized in Figure 9. Figure 9 represents a plot, as explained in the Background section above, showing the antimicrobial activity of nitric oxide against Serratia marcescens, and the latency period of antimicrobial activity, which is attributed to the time required to exhaust the microorganism's chemical defense mechanisms. Here, curve 11 is the cell count as a function of exposure time to 200 ppm NO, curve 12 is the control situation (exposure of bacteria to air), point 13 represents the lethal dose (LD100) that kills 100% of the bacteria, point 14 represents the latency period (LP1), defined as the bacterial population is stable or a decrease of less than one log, and point 15 represents a decrease of -2.5 Log 10 Point 16 marks the end of the latent period, point 18 marks the point at which bacteria continue to die and do not recover even when NO is discontinued, and point 20 corresponds to a 50% drop in colony forming units (CFU). 50 ), while point 22 indicates CFU 100 and point 24 indicates 1 logarithmic unit.

[0392] Similar data were collected and plotted for S. aureus (ATCC 25923), P. aeruginosa (ATCC 27853), MRSA, and clinical strains of S. aureus, S. marcescens, Klebsiella pneumoniae, S. maltophilia, Enterobacter aerogenes, Acinetobacter baumannii, group B streptococcus, and Escherichia coli. Other microorganisms tested were multidrug-resistant strains of Candida albicans, Mycobacterium smegmatis, and Pseudomonas aeruginosa, and the results are summarized in Table 4.

[0393] Table 4 shows the latency periods measured in vitro for various bacterial species / strains, as well as the 2.5-fold reduction in microbial load (-2.5 Log 10 ) and microbial eradication (LD 100 ) indicates the period during which it was permitted.

[0394] Example 3: High-throughput synergy screening To screen for antimicrobial agents that exhibit high potentiation upon exposure of target cells to nitric oxide, a high-throughput synergy screening (HTSS) assay of an antimicrobial library was performed using pathogenic microorganisms that cause the specific disease to be treated. The pathogenic microorganisms were grown on agar media in the presence or absence of subinhibitory concentrations (sub-MIC amounts) of nitric oxide or NO-releasing compounds.

[0395] Although some antimicrobial agents in the library may exhibit antimicrobial activity without the presence of NO, assays are performed comparing the zone of inhibition in the presence or absence of nitric oxide. This comparison allows for the identification of antimicrobial agents that become more active in the presence of nitric oxide. The growth kinetics of the pathogenic microorganisms being tested are monitored in the presence of a range of concentrations of nitric oxide to identify the minimum inhibitory concentration of the antimicrobial agent.

[0396] We use the "checkerboard assay" (Ramon-Garcia Set. et al., AAAC, 2011), a standard method used to rigorously confirm the synergistic activity of antibiotics. The assay is performed on cultures growing in 96-well plates. A two-dimensional array of serial concentrations of two test compounds (nitric oxide and a specified antimicrobial agent) is then introduced to the logarithmically growing culture. Calculations based on the relative MIC concentrations in wells representing various ratios of the two compounds are used to demonstrate that the pairwise combination of drugs exerts an inhibitory effect (synergism) that exceeds the sum of their individual effects.

[0397] Example 4: Treatment regimen Respiratory infections in human subjects associated with respiratory pathogens such as bacteria can be treated using the methods presented herein, namely, using a combination of an appropriate antibiotic and nitric oxide (NO).

[0398] Two groups of patients are compared: those with a respiratory infection such as pneumonia who are treated with intermittent inhalation of, say, 160 ppm NO three times a day for about 10 days in combination with standard antibiotics, and a group of patients who are diagnosed with the same type of pneumonia but are treated with standard antibiotic therapy alone.

[0399] Patients treated with a combination of NO and standard antibiotic therapy should show significant improvements in clinical parameters such as body temperature (more rapid normalization), oxygen consumption, respiratory rate, and pulmonary function compared to patients treated with standard antibiotics alone.

[0400] Patients treated with a combination of NO and standard antibiotic therapy should show significant improvement in overall clinical scores based on the parameters tested compared to patients treated with standard antibiotics alone.

[0401] Furthermore, the respiratory bacterial flora should be altered by the indicated treatment with a reduction in the pathogenic microbial population.

[0402] Patients treated with a combination of NO and standard antibiotic therapy should experience a shorter length of hospital stay in days, fewer exacerbations in patients requiring admission to intensive care units for respiratory failure, and / or reduced antibiotic use overall.

[0403] Publications cited throughout this document are hereby incorporated by reference in their entirety. While various aspects of the present invention have been described above with reference to examples and preferred embodiments, it will be recognized that the scope of the present invention is to be determined not by the foregoing description but rather by the following claims, appropriately interpreted under the principles of patent law.

[0404] [Table 1] [Table 2] TIFF2026027384000004.tif65164 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8

Claims

1. 1. A method for treating bronchiolitis in an infant in need thereof, said method comprising repeatedly administering to said infant a gas mixture comprising nitric oxide at a concentration of about 144 to about 176 ppm for a first period of time, followed by a gas mixture without nitric oxide for a second period of time; The administration a) a reduction in the length of hospital stay required to achieve an oxygen saturation of 92% or greater on room air compared to infants not subjected to said repeated administrations of nitric oxide; b) a reduction in the length of hospital stay required to achieve a clinical score of 5 or less compared to infants not subjected to said repeated administrations of nitric oxide; c) a reduction in the length of hospital stay required before discharge compared to infants not subjected to said repeated administrations of nitric oxide; or d) any combination of these; The method is repeated a sufficient number of times.

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

3. 10. The method of claim 1, wherein the administration is repeated six times daily.

4. 10. The method of claim 1, wherein the nitric oxide is administered repeatedly for a period of about 1 day to 3 weeks.

5. 5. The method of claim 4, wherein the nitric oxide is administered repeatedly for 5 days.

6. The method further comprises monitoring at least one on-site oximetry parameter in the infant, the on-site parameter being oxyhemoglobin saturation (SpO ２ ), methemoglobin (SpMet), perfusion index (PI), respiration rate (RRa), oxyhemoglobin saturation (SpO2), total hemoglobin (SpHb), carboxyhemoglobin (SpCO), methemoglobin (SpMet), oxygen content (SpOC), and pleth variability index (PVI).

7. 10. The method of claim 1, wherein the method further comprises monitoring at least one additional on-site spirometry parameter in the infant, wherein the at least one additional on-site parameter is selected from the group consisting of forced expiratory volume (FEV1), maximum mid-expiratory flow (MMEF), diffusing capacity of the lungs for carbon monoxide (DLCO), forced vital capacity (FVC), total lung capacity (TLC), and residual volume (RV).

8. The method further comprises monitoring at least one on-site parameter in the gas mixture inhaled by the infant, the on-site parameter being end-tidal CO ２ (ETC ２ ), nitrogen dioxide (NO ２ ), nitric oxide (NO), serum nitrite / nitrate, and fractional inspired oxygen (FiO ２ 2. The method of claim 1, wherein the compound is selected from the group consisting of:

9. 10. The method of claim 1, wherein the method further comprises monitoring at least one off-site body fluid parameter in the infant, the parameter being selected from the group consisting of bacterial and / or fungal load, urinary nitrite, blood methemoglobin, blood pH, clotting factors, blood hemoglobin, hematocrit, red blood cell count, white blood cell count, platelet count, vascular endothelial activating factor, renal function, electrolytes, pregnancy hormones, serum creatinine, and liver function.

Images