WO2007146311A2 - Compositions and methods for protecting newborn lung development with ethyl nitrite - Google Patents

Abstract

The present invention provides methods of diagnosing, treating or preventing hyperoxia-induced lung disease using the compositions of the invention. The present invention also provides methods of reducing hyperoxia-induced lung inflammation, hyperoxia-induced NF-ϰB activation in the lung and hyperoxia-induced cytokine expression in the lung using the compositions of the invention.

Description

COMPOSITIONS AND METHODS FOR PROTECTING NEWBORN LUNG DEVELOPMENT WITH ETHYL NITRITE

BACKGROUND Nitric oxide and its biological target molecules regulate important pathways in postnatal lung development, including alveolar development (Ballard et al., Pediatr Res. 59(1): 157-62 (2006)) and pulmonary vascular development, (Lin et al., Pediatr Res 58(1):22- 9 (2005)) both of which are impaired in bronchopulmonary dysplasia (BPD). Inhaled nitric oxide has been shown to rescue hyperoxia-impaired alveolar development in newborn rats, in part through its effects on pulmonary vascular development (Lin et al., Pediatr Res 58(1):22- 9 (2005)). Clinical trials using inhaled nitric oxide have aimed to prevent BPD in very premature infants, but published trials have shown conflicting results (Schreiber et al., N Engl yMe</ 349(22):2099-107 (2003); Van Meuτs et &l., N EnglJ Med 353(\):U-22 (2005)). Nitric oxide mechanisms of action may include inhibiting inflammation, in part by inactivation of nuclear factor kappa B (NF-κB) (Marshall and Stamler, Biochemistry 40(6):1688-93 (2001)).

Kinsella and colleagues showed that inhaled nitric oxide partly prevented inflammatory responses in premature lambs treated with supplemental oxygen and mechanical ventilation designed to mimic clinical care of extremely premature newborns (Kinsella et al., Chest 116(1 Suppl): 15S- 16S ( 1999)). Inflammation is believed to be a central mechanism in the pathogenesis of BPD, (Auten and Ekekezie II, Pediatr Pulmonol 35(5):335-41 (2003)) and previous studies have shown that preventing inflammation in a hyperoxia-exposed newborn rat model protects against impaired alveolar development and lung function (Yi et al., Am J Respir Crit Care Med 170(11 ): 1188-96 (2004); Auten et al., Am J Physiol Lung Cell MoI Physiol 28 l(2):L336-44 (2001)). Nitric oxide has been implicated in both pro- and anti-inflammatory roles. Some of the maladaptive pro-inflammatory effects have been attributed to formation of higher-order nitrogen oxides (Janssen-Heininger et al., Am J Respir Crit Care Med 166(12 Pt 2):S9-S16 (2002)).

Under physiologic conditions, however, nitric oxide is exceedingly unstable, reacting essentially instantaneously with oxygen, superoxide anion, and redox metals (Lancaster et al., Proc. Natl. Acad. Sci. USA 87: 1223-1227 (1990); Ignarro et al., Circ. Res. 65:1-21 (1989); and Gryglewski et al., Nature 320:454-456 (1986)). This fact has lead to the supposition that, in order to exert its effects, nitric oxide must be stabilized in vivo in a form that preserves its biological activity (Stamler, Proc. Natl. Acad. Sci. USA , 89(1): 444-448 (1992). Introduction of nitric oxide into biological tissue can also result in significant adverse effects, which occur as a direct result of the particular chemical reactivity of the uncharged nitric oxide radical (NO^'). These adverse effects create impediments to nitric oxide therapy which generally involves administration of NO^", particularly via reactions with oxygen and superoxide. For example, the reaction between NO^", and O₂ or reactive O2 species which are present in high concentrations in many tissues, generates highly toxic products, such as NO₂ and peroxynitrite. These reactions also result in the rapid inactivation of nitric oxide, thus eliminating any beneficial pharmacological effect. (Furchgott R. F. et al., I. Endothelium- Derived Relaxing Factors and Nitric Oxide; eds. Rubanyi G. M., pp. 8-21 (1990); Gryglewski, R. J. et al., Nature 320:454-456 (1986)).

Thus, a clinical need exists for pharmacological agents which can diagnose, treat or prevent lung disorders such as bronchopulmonary dysplasia and hyperoxia-induced inflammation without adverse effects.

SUMMARY OF THE INVENTION

This invention is based on the discovery that ethyl nitrite exerts a potent antiinflammatory effect. This concept led to the discovery that compositions comprising ethyl nitrite can be used as a prophylactic or therapeutic modality in disorders which involve inflammation. The present invention provides methods of treating or preventing hyperoxia-induced lung disease or hyperoxia-impaired alveolar development in a subject in need thereof by administering a therapeutically effective amount of a composition comprising a gas and ethyl nitrite, thereby treating or preventing the hyperoxia-induced lung disease. In some embodiments, the subject is a premature newborn and in more preferred embodiments, the subject is an extremely low birth weight premature newborn. In some embodiments, the lung disease is a chronic lung disease and in more preferred embodiments, the lung disease is bronchopulmonary dysplasia.

The present invention also provides methods of reducing hyperoxia-induced inflammation, hyperoxia-induced NF-κB activation, or hyperoxia-induced cytokine expression in the lung in the subject. The reduction in cytokine expression can be a reduction in cytokine protein expression or a reduction in cytokine RNA expression. In preferred embodiments, the cytokine can be Cytokine Induced Neutrophil Chemoattractant-1 (CINC- 1), Macrophage Inflammatory Protein-2 (MIP-2), Monocyte Chemotactic Protein-1 (MCP-I) or Tumor Necrosis Factor Alpha (TNF-α).

The present invention also provides methods for inducing lung organogenesis in a subject in need thereof comprising administering a therapeutically effective amount of a S composition comprising a gas and ethyl nitrite to said subject.

The compositions for use in the methods of the present invention can comprise a gas and ethyl nitrite in a therapeutically effective amount. The gas can be nitrogen or oxygen. Preferably, the gas is nitrogen. In preferred embodiments where the gas is nitrogen, the nitrogen gas is admixed with oxygen prior to administration. The therapeutically effect 0 amount can range from about 0.1 to about 20 ppm, from about 1 to about 10 ppm or more preferably from about 1 to about 5 ppm. Preferably, the gas comprises minimal impurities. More preferably, the gas comprises less than 5 ppm NO₂, less than 25 ppm NO and/or less than 32 ppmv ethanol. Preferably, the agent does not form NO2 or NO_x in the presence of oxygen or reactive oxygen species at body temperature. The compounds of the invention can 5 be administered by any means known in the art. Preferably, the administration is inhalation. In other embodiments, the administration can be intranasal or the compositions can be administered as an aerosol or in the form of an inhalant for pulmonary delivery. Administration can occur for as long as symptoms persist. Preferably, compositions are administered from about 7 to about 14 days. 0

DETAILED DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on body weight.

Figure 2A is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on 5 bronchoalveolar lavage leukocytes and neutrophils. Figure 2B is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on myeloperoxidase (MPO) activity.

Figure 3 A is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on whole lung CINC-I mRNA. Figure 3B is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on whole lung CINC-I protein. 0 Figure 4 is a graph showing the effect of air or 95% O2 ± inhaled ethyl nitrite on whole lung TNF-α mRNA. Figure 5A is a photograph of immunohistochemistry showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on VEGF. Figure 5B is a graph showing the effect on VEGF in whole lung homogenates.

Figure 6 A is a graph showing the amount of Nuclear Factor (NF)- KB activation in air-exposed adult rat lung (negative control), LPS-treated adult rat lung, or tissue plasminogen activator-stimulated jurkat cells (positive control group), 8-day old rat lung from air, 95% O₂-exposed, 95% O₂ + ENO, and 95% O₂ + NO treatment groups (mean + SD, n=3 per group). Figure 6B is a photograph of immunohistochemistry showing the localization of activated (nuclear localization signal) NF-κB in representative sections from each of the LPS- treated adult rat lung, 8-day old rat lung from air, 95% 0₂-exposed, 95% O₂ + ENO, and 95% O₂ + NO treatment groups (AE = alveolar epithelium, BE = bronchioloar epithelium).

Figure 7 is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite on 3- nitrotyrosine.

Figure 8A is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite followed by recovery in air on pressure-volume loops. Figure 8B is a graph showing the effect of air or 95% O₂ ± inhaled ethyl nitrite followed by recovery in air on lung resistance.

Figure 9A is a photograph of immunohistochemistry showing the effect of air followed by recovery in air on two alveolar crests identified by elastin staining. Figure 9B is a photograph of immunohistochemistry showing the effect of 95% O₂ followed by recovery in air on two alveolar crests identified by elastin staining. Figure 9C is a photograph of immunohistochemistry showing the effect of 95% O₂ + inhaled ethyl nitrite followed by recovery in air on two alveolar crests identified by elastin staining. Figure 9D is a photograph of immunohistochemistry showing alveolar development in representative sections stained with malachite green in air exposed pups. Figure 9E is a photograph of immunohistochemistry showing alveolar development in representative sections stained with malachite green in 95% O₂ exposed pups. Figure 9F is a photograph of immunohistochemistry showing alveolar development in representative sections stained with malachite green in 95% O₂ + ethyl nitrite exposed pups.

Figure 1OA is a graph showing the effect of air, 95% O₂ ± ethyl nitrite followed by recovery in air on alveolar volume density. Figure 1OB is a graph showing the effect of air, 95% O₂ ± ethyl nitrite followed by recovery in air on alveolar surface density.

Figure 11 is a graph showing the effect of air or 95%O₂ ± inhaled ethyl nitrite 0.2-20 ppm x 8 days on NO and SNO in bronchoalveolar lavage fluid. Figure 12A is a photograph of an immunoblot showing the effect of air or 95%O₂ , or 95%O2 + inhaled ethyl nitrite 10 ppm x 8 days on SODl, 2, and 3, catalase, and β-actin expression. Figure 12B is a graph showing quantified by image analysis. Figure 12C is a graph showing the effects on total lung SOD activity. Figure 12D is a graph showing the effects on total lung catalase activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating or preventing hyperoxia-induced lung disease in a subject in need thereof by administering a therapeutically effective amount of a composition comprising a gas and ethyl nitrite, thereby treating or preventing the hyperoxia-induced lung disease. Ethyl nitrite has the formula: CH₃CH₂ONO and is also referred to as O-nitrosoethanol. The terms "ethyl nitrite", "O-nitrosoethanol" and "ENO" are used interchangeably herein.

In some embodiments, the lung disease is chronic lung disease (CLD). Preferably, the invention is directed to the treatment or prevention of bronchopulmonary dysplasia (BPD) which encompasses chronic lung disease and long-term respiratory problems in premature babies.

In general, CLD results from lung injury to newborns who must use a mechanical ventilator and extra oxygen for breathing. The lungs of premature babies are fragile and are easily damaged. With injury, the tissues inside the lungs become inflamed and can break down causing scarring. This scarring can result in difficulty breathing and increased oxygen needs. Some of the causes of lung injury include prematurity (the lungs, especially the air sacs, are not fully developed); low amounts of surfactant (a substance in the lungs that helps keep the tiny air sacs open); oxygen use (high concentrations of oxygen can damage the cells of the lungs) and mechanical ventilation (the pressure of air from breathing machines, suctioning of the airways, use of an endotracheal tube), etc.

Chronic lung disease can develop in premature babies who have had mechanical ventilation. Risk factors for developing CLD include birth at less than 34 weeks gestation; birth weight less than 2,000 grams (4 pounds 6 1/2 ounces); hyaline membrane disease (lung disease of prematurity due to lack of surfactant that does not show the usual improvement by the third or fourth day); pulmonary interstitial emphysema (PIE) (a problem in which air leaks out of the airways into the spaces between the small air sacs of the lungs); patent ductus arteriosus (PDA) - (a connection between the blood vessels of the heart and lungs that does not close as it should after birth); being a Caucasian male; maternal womb infection (chorioamnionitis) and a family history of asthma, among others.

The following are the most common symptoms of CLD. However, each baby may experience different symptoms of the condition. Symptoms may include respiratory distress S (rapid breathing, flaring of the nostrils, chest retractions) or continued need for mechanical ventilation or oxygen after a premature baby reaches 36 weeks gestation. Symptoms of CLD may resemble other conditions or medical problems.

Diagnosis for CLD varies. Because CLD is a chronic disease and appears gradually, physicians must look at several factors. It is often diagnosed when a premature baby with 0 respiratory problems continues to need additional oxygen after reaching 36 weeks gestational age. Chest x-rays compared with previous x-rays may show changes in the appearance of the lungs. The x-rays of lungs with CLD often have a bubbly, sponge-like appearance. X-rays are diagnostic tests which use invisible electromagnetic energy beams to produce images of internal tissues, bones, and organs onto film. The pathology of CLD shows impaired alveolar S formation and often shows, thick, disorganized elastin deposition. In normal lung development, elastin is found at the tips of the alveolar septae.

CLD can be a long-term condition. Some babies with CLD require mechanical ventilators for several months. Some babies will continue to require oxygen when they go home from the hospital, but most can be weaned from oxygen by the end of their first year. 0 Babies with CLD may be at increased risk for respiratory infection and may have to be re- hospitalized.

Current treatment methodologies also vary, with limited success. Specific treatment for CLD in general is determined on a patient by patient basis taking the following factors into consideration on: the baby's gestational age, overall health, and medical history; extent of 5 the disease; the baby's tolerance for specific medications, procedures, or therapies; expectations for the course of the disease

Current treatment of CLD can include extra oxygen (to make up for the decreased breathing ability of the damaged lungs); mechanical ventilation with gradual weaning as the baby's lungs grow and can do more of the work of breathing; limiting fluids and giving a 0 diuretic medication to help reduce excess fluid which can worsen breathing ability; nutrition (to help the baby and the lungs grow); immunization against lung infection by respiratory syncytial virus (RSV) and influenza and medications, such as, bronchodilators (to help open the airways) or steroids (to help reduce inflammation). Although there are current treatment methodologies for CLD, none, with the exception of complete lung transplant, completely ameliorate the condition; and most, such as extra oxygen, have adverse side effects, such as hyperoxia-induced protein nitration. More recent studies have indicated that nitric oxide can be used to treat lung inflammation in rat and lamb models and that lung inflammation may be an underling cause of CLD and impaired alveolar development in premature newborns. However, as described nitric oxide is unstable under physiologic conditions and therefore must be stabilized in vivo in a form that preserves its biological activity. It is preferred that when the compositions of the invention are administered to a subject for therapeutic or diagnostic purposes, the gas comprising ethyl nitrite does not form NO_∑ or NO_x in the presence ofjoxygen or reactive oxygen species at body temperature or exert systemic blood pressure compromising effect.

The compositions of the present invention overcome the limitations of current CLD treatment. Advantages of the methods described herein include: administration of ethyl nitrite reduces the toxicity caused by Nθ₂/NO_x formation when NO is administered; the option of administering the compound comprising ethyl nitrite together with oxygen, without NO_∑/NO_* production; some patients respond to administration of ethyl nitrite who do not respond to administration of NO.

As used herein the term "NO_x" means NO, N₂O₃, N₂O₄, OONO^", OONO^" and any products of their interaction or their reaction with NO or NO₂. As used herein the term "reactive oxygen species" is singlet oxygen, superoxide, hydrogen peroxide or hydroxyl radical.

When the ethyl nitrite is administered as part of a gas, it must be formulated so as to produce a homogenous gas blend for administration. The ethyl nitrite is not expected to harm the lungs or respiratory tract or condense in the lungs or respiratory tract. Ethyl nitrite is available commercially, e.g., diluted in ethanol; however, this mixture is not suitable for preparation of homogenous gas blends as described herein. The ethyl nitrite can be admixed with an inert gas to provide the gas for administration, e.g., by conventional gas blending methods or more preferably by the methods described herein. The inert gas can be nitrogen or oxygen, or a mixture thereof. Preferably, the composition of the present invention comprises ethyl nitrite in nitrogen.

Preferably, the ethyl nitrite is produced as a homogenous gas blend in nitrogen as described herein. Thus, the present invention also provides methods for producing ethyl nitrite comprising the steps of (a) mixing sodium nitrite and ethanol to form a first solution; and Qo) adding a second solution comprising sulfuric acid and ethanol to the first solution, wherein the rate of addition of the second solution provides a reaction temperature which enhances conversion. Preferably, the ethyl nitrite is produced in at least 92% yield. Preferably, the reaction temperature is between 0-30° C. The first solution and/or second solution can be homogenous. As used herein, the term "homogenous" means that all of the solids are dissolved. The mixing of the first solution and/or the addition of the second solution can further include a nitrogen purge. The rate of addition can be dropwise. Preferably, the addition is at a rate such that the reaction temperature remains below 30°C. The ethyl nitrite is collected into an ice chilled receiving flask and can be stored under an inert atmosphere. The present invention also provides methods for producing ethyl nitrite in nitrogen comprising the steps of (a) introducing ethyl nitrite which comprises less than 3% (by weight) ethanol to an evacuated cylinder, (b) adding nitrogen gas to the cylinder; and, (c) homogenizing the ethyl nitrite and nitrogen gas in the cylinder, such that the ethyl nitrite in nitrogen comprises less than 5 ppm nitrogen dioxide. The ethyl nitrite in nitrogen further comprises less than 25 ppm nitric oxide. Preferably, the ethyl nitrite in nitrogen is at about 1000 ppm. The ethyl nitrite in nitrogen can be stable for at least two years. Preferably, the cylinder is evacuated to at least 10 microns prior to addition of ethyl nitrite. Preferably, the ethyl nitrite in step (a) is introduced at about 5.625 G. Preferably, the ethyl nitrite in step (a) is introduced to the cylinder by gas-tight syringe. Preferably, the nitrogen gas in step (b) is added at about 2.1 kG. Preferably, the nitrogen gas in step (b) is added by means of a high purity stainless steel manifold. Preferably, the homogenization in step (c) occurs by rolling the cylinder on a four position cylinder roller for at least 30 minutes.

Where the ethyl nitrite is administered as part of a gas, it is administered in a therapeutically effective amount ranging from 0.1 to 1000 ppm. The therapeutically effective amount can range from 0.1 to 100 ppm. The therapeutically effective amount can also range from 0.1 to 10 ppm. As an example, if the therapeutically effective amount is from 0.1 to 10 ppm, the therapeutically effective amount can be any specific amount within the range of 0.1 to 10 ppm (e.g., 0.1, 0.2 ...1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ppm, or any fraction thereof). The therapeutically effective amount can also be any range within 0.1 to 10 ppm (e.g., 0.1 to 2, 0.1 to 3 ... 0.1 to 10, 0.2 to 3, 0.2 to 4 ... 0.2 to 10, etc. or any fraction thereof). Preferably, the gas comprises minimal impurities. More preferably, the gas comprises less than 5 ppm NO₂, less than 25 ppm NO and/or less than 32 ppmv ethanol.

These compounds may be used to diagnose, treat or prevent those pathophysiologic conditions which result from, or involve, lung disease, lung inflammation, activation of NF- KB in the lung, increased cytokine expression in the lung, alveolar destruction or those which necessitate therapeutic intervention to achieve correction of lung disorders. The present invention is also directed to methods of treating or preventing alveolar destruction or promoting the formation of alveoli in a subject in need thereof by administering the compounds of the invention. BPD is characterized mainly by a failure of the infant to form a sufficient number of appropriately-sized alveoli.

According to the present invention, a "therapeutically effective amount" of a pharmaceutical composition is an amount which is sufficient to achieve the desired pharmacological effect. Preferably, the desired pharmacological effect is the treatment or prevention of lung disease or alveolar destruction or promoting the formation of alveoli. In other embodiments, the desired pharmacological effect is the reduction of hyperoxia-induced inflammation, hyperoxia-induced NF-κB activation, or hyperoxia-induced cytokine expression in the lung in the subject. Generally, the dosage required to provide an effective mount of the composition, and which can be adjusted by one of ordinary skill in the art, will vary, depending upon the age, health, physical condition, sex, weight and extent of disease, of the recipient. Additionally, the dosage may be determined by the frequency of treatment and the nature and scope of the desired effect

Administration can be carried out for as long as symptoms ameliorate. In some embodiments, duration of treatment can range from about 7 to about 14 days. The duration of treatment can be any specific amount within the about 7 to about 14 days (e.g., 7, 8, 9, 10, 11, 12, 13, 14 days). The duration of treatment can also be any range within about 7 to about 14 days (e.g., 7 to 8, 7 to 9 ... 7 to 14, 8 to 8, 8 to 10 ... 8 to 14, etc). Preferably, the duration of treatment is 8 or 14 days. The dosage will vary from patient to patient. Upon administration, results are noted with variation in dosage and then the dosage is preferably used where the best results are achieved. The most effective dosage can be lower than some of the dosages tried; thus, if after increases in dosage are tried, an increased dosage provides less improvement, then return to the more effective lower dose is indicated.

The compositions of the present invention can be administered in any therapeutically effective manner or form, and in conjunction with any pharmacologically effective vehicle. For example, in a particularly preferred aspect, the compositions of the invention may be administered in the form of an inhalant as a powdered or liquid aerosol. Aerosolized forms may be administered to optimize delivery such as including droplets of aerosol in a size range which allows for deposition in the respiratory tract. Such a formulation may comprise the active agent solubilized in a micronized hydrophobic/hydrophilic emulsion. Such compositions are well known to those of skill in the art.

The compositions utilized in this invention can be administered by any means known in the art, for example, inhalation, intranasal, topical or local means. Preferably, the compositions of the present invention are administered or utilized via inhalation or intranasal.

Preferably, the compositions are delivered to the lung or respiratory system as part of a gas.

The compositions may be administered by any medical instrumentation including, but not limited to, inhalers or ventilators.

The compositions of the present invention comprising ethyl nitrite can further comprise a pharmaceutically acceptable carrier, to achieve the physiological effects described herein. The compounds of this invention can be employed in combination with conventional excipients; e.g., pharmaceutically acceptable organic or inorganic carrier substance suitable for the proposed method of application which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohol, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, tale, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. Such carriers are familiar to those skilled in the art.

It will be appreciated that the actually preferred amounts of active compounds used will vary according to the specific compound being utilized, the particular compositions formulated, the mode of application and the particular site of administration. Optimal administration rates for a given protocol of administration can be readily ascertained by those skilled in the art, using conventional dosage determination tests conducted with regard to the foregoing guidelines. Ampules are convenient unit dosages.

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The entire text of all publications herein are hereby incorporated by reference. EXAMPLES

Animals

Time-mated pregnant Sprague Dawley rats (Worthington) were allowed to deliver spontaneously, and litters were reassigned to achieve balance among 4 litters, with average liter sizes of 10 pups/litter. Dams and pups were housed in polystyrene rat cages with custom adapted sealed lids with gas fittings. Gases were supplied at 2.5 liters per minute, in the following mixtures: air, 95% O₂ + 5% N₂, or 95% O₂ + ethyl nitrite 0.2, 1.0, 10, and 20 ppm + N₂ for the balance gas. Gas flows were regulated by microprocessor mass flow controllers (Aalborg, Orangeburg, NY). Food and water were provided ad libitum and cages were changed daily. Nursing dams were switched between air and 95% O₂ (or O₂ + ethyl nitrite) exposures daily to avoid oxygen toxicity, as previously described in detail (Auten et al., Am J Physiol Lung Cell MoI Physiol 281 (2):L336-44 (2001 )). Air and 95% O₂ ± ethyl nitrite (0.2 — 20 ppm) exposures were continued for 8 days, and pups were euthanized with sodium pentobarbital 150 mg/kg i.p. Methemoglobin was measured in 3 pups/treatment group in blood samples obtained by cardiac puncture at day 8 (model 482 co-oximeter,

Instrumentation Laboratories, Lexington, MA). Additional litters were exposed to air or 95% O₂ ± ethyl nitrite 10 ppm x 8 days then recovered in an air environment for 6 more days (14 total postnatal days).

Antibodies:

Anti-CINC-1 antibodies were provided by John Zagorski and R&D Systems (Minneapolis, MN) as previously described in detail (Deng et al., Am J Respir Crit Care Med 162(6):2316-23 (2000)). Antibody coated beads for fluorescent VEGF antibody (Luminex, BioRad) analysis were from Linco Research, St. Charles, MO. Anti-vascular endothelial growth factor for immunohistochemistry (anti- VEGF) was from Santa Cruz Biotechnology, Santa Cruz, CA. Anti-β-actin was from Abeam. Secondary antibodies, avidin-biotin detection reagents, and substrates were from Vector Laboratories, Portland, OR. Other reagents were provided by Sigma-Aldrich, St. Louis, MO.

Data Analysis

Data are expressed as the mean ± S.E.M. Significant differences between treatment groups were determined by ANOVA. Post hoc differences were identified by Tukey- Kramer, and paired comparisons were performed using Kruskall-Wallis tests. Significance was accepted for comparisons at/7 < 0.05, and calculations were made using statistical analysis software (SPSS version 14, Chicago, IL).

Example 1: Ethyl Nitrite Ethyl nitrite is readily delivered to a subject in gaseous form. As known to those ordinarily skilled in the art, gaseous ethyl nitrite for delivery is produced by bubbling nitrogen or oxygen through a Fisher-Milligan ("FM") gas diffuser containing ethyl nitrite diluted in ethanol (e.g., from 0.00125 to 0.5% ethyl nitrite in ethanol (v/v), preferably from 0.0025 to 0.125% ethyl nitrite in ethanol (v/v)), e.g., at a flow rate of 0.5 liters/min to 1.5 liters/min, to produce nitrogen or oxygen containing ethyl nitrite. The actual composition of the gas resulting from this type of exposure has been assessed by Fourier Transform Infrared Spectroscopy (FTIR). As is apparent from the following study and resulting data, however, the EtOH content provided to patients by methods of the prior art was significantly higher (e.g., 30X more ethanol was delivered than ethyl nitrite) than in the present invention, wherein the constant concentration of EtOH is less than 32 ppmv in the ENO/EtOH composition.

200ml of solution of ethyl nitrite in ethanol (final concentrations as given below) was loaded into the FM gas diffuser and 1 LPM flow of air was delivered for 1 hour. Effluent gas from the FM was combined with an additional 9 LPM of air post-FM and the resulting gas was analyzed by FTIR for concentration of ethyl nitrite and ethanol. FTIR data was collected at 0.5 wavenumber resolution with 128 scans over the range of 650-4000 wavenumbers. FTIR gas cell length is 6.5 meters, cell pressure was ~5 psig (controlled), and temperature was 150° F (controlled). Sample flow was controlled at 3 LPM through the FTIR gas cell. A water vapor spectrum was taken and subtracted from the data prior to quantitation. The ethyl nitrite quantitation was performed using the region of 1722- 1572 wavenumbers. Data was collected approximately every ten minutes and, in the low and mid concentration ranges, data was taken 2 minutes into the run. FTIR analyses took 2.5 minutes to run and represent an average value over that time.

For ethyl nitrite measurement, a calibration curve was prepared using a 250 ppm ENO/N₂ standard and a 10 point capillary gas divider. For ethanol, a single ethanol standard prepared at 2000 ppm in nitrogen was used to estimate the quantity of ethanol in the delivered gas stream. ENO/EtOH Concentrations Used in FM Experiments:

High Cone - 1.4 mL of ENO/ETOH solutions from Aldrich (17.8% cone) diluted to 200 mL solution (i.e., 0.125%)

Mid Cone - 0.28 mL of ENO/ETOH solutions from Aldrich (17.8% cone) diluted to 200 mL solution (i.e., 0.025%)

Low Cone - 0.028 mL of ENO/ETOH solutions from Aldrich (17.8% cone) diluted to 200 mL solution (i.e., 0.0025%)

Results of ENO Analyses

The results of the ethyl nitrite analyses are provided in Table 1 below. As is apparent from the results, the ethyl nitrite content provided to patients by methods of the prior art was likely significantly more variable over time than the content provided by the methods of the current invention:

Table 1

Results of Ethanol Analyses

The ethanol amounts varied with time over the course of the vaporization but less than 10% over the hour. Variance was probably due to the cooling of the solution as the gas vaporized. The ethanol was quantified over the region of 3762-3551 wavenumbers. The results were scaled to the standard as a simple linear relationship. The quantitation results represent a lower limit of the actual values. As can be seen, the ethanol concentrations delivered in the prior art (represented in Table 2) were significantly higher than the concentrations delivered by the methods in the current invention (e.g., less than 32 ppmv). Table 2

Liquid ethyl nitrite is synthesized in one step from ethanol by nitrosylation under acidic conditions as described (Org. Syn. Coll. Vol. 2, A H Blatt ed, 1943, p 204-205). The product distills from the reaction mixture and is isolated by collection of the condensate. The isolated product can be used with or without further purification for blending of the pharmaceutical composition.

The synthesis of ethyl nitrite is shown below and the appropriate reagents and solvents used for the reaction are shown in Table 3. Preferably, the yield of the reaction is greater than 92%.

H₂SO₄

CH₃CH₂OH + NaNO₂ CH₃CH₂ONO

N₂, 0 - 15C

Table 3

The following description of the synthesis represents typical charge quantities and yields. Reactions of larger or smaller scale may be performed with adjustment of reagent charges.

1. Solution 1: Approximately 90 mL of ethanol, 725 mL of water, and 210 grams of sodium nitrite are combined in an appropriate vessel with stirring. The vessel is purged with nitrogen and the material stirred until all solids dissolve resulting in a pale yellow solution. 2. Solution 2: Approximately 90 mL of sulfuric acid, 90 mL of ethanol, and 632 mL of water are combined and mixed to assure homogeneity.

3. Solution 2 is added to Solution 1 under nitrogen in a dropwise manner at a rate such that the reaction temperature remains below 30°C while the temperature of the condenser is maintained between 0 - 10⁰C.

4. The ethyl nitrite product distills into the ice water chilled receiving flask over the course of the reaction and visibly stops collecting near the end of the addition of Solution 2.

5. The product is transferred to an aluminum or glass bottle under an inert atmosphere and stored under refrigeration.

The specifications preferred for ethyl nitrite liquid prepared from the foregoing procedure are shown in Table 4. Table 4

The ethyl nitrite synthesis process was optimized by conducting a series of experiments in which parameters of reaction temperature and rate of addition of solution 2 were varied.

Ethyl nitrite in nitrogen can be prepared by blending ethyl nitrite liquid in nitrogen to form a homogeneous gas blend at a preferred concentration of 1000 ppm and preferred pressure of approximately 900 psi. The blending is performed gravimetrically in accordance with standard compressed gas manufacturing procedures familiar to those skilled in the art. Manufacturing information is provided below for the preferred concentration of ethyl nitrite

¹ calculated at 1000:1 dilution

² calculated at 1000:1 dilution in nitrogen, 1000 ppm. Other final blend concentrations can be prepared as required according to these instructions with the appropriate modifications to ethyl nitrite and nitrogen gas charge quantities. The material specifications are shown in Table 5. Table 5

The following is a stepwise description of a preferred manufacturing process:

1. Empty aluminum cylinders are placed in a cylinder oven and connected to a stainless steel manifold with purge and evacuation capabilities. The manifold is opened to a vacuum pump and the integrity of the system is tested (vacuum must be better than 10 microns). The oven heater/blower is activated and set to 150⁰F. The vacuum is shut off and the cylinder valves are opened to the manifold. High-pressure nitrogen is allowed to flow into the cylinders to a final pressure of ~ 100 psig and then vented to atmosphere. This procedure is repeated a total of three times. After the final purge cycle, the manifold is isolated from the purge gas supply and vent and placed under vacuum. The cylinders are allowed to evacuate under heated conditions until the vacuum achieved is better than 10 microns (usually ~2 hours time). The cylinder valves are closed and the cylinders are removed from the oven under vacuum and placed in the quarantine area for prepared cylinders.

2. Ethyl nitrite liquid as prepared above, ~5.625 G, is withdrawn into a gas-tight syringe and is added to an evacuated cylinder. 3. Nitrogen, approximately 2.1 kG, is added with the resulting pressure typically being 850-1000 psi.

4. The gaseous mixture is homogenized by rolling for no less than 1/2 hour.

5. The cylinders are then tested and stored at ambient warehouse conditions.

Ethyl nitrite in nitrogen, 1000 ppm, is stable for at least two years after manufacture when stored under standard compressed gas cylinder conditions (typical warehouse conditions). The specifications preferred for ethyl nitrite in nitrogen, 1000 ppm. prepared from the foregoing procedure are shown in Table 6.

Example 2: Survival, Weight Gain and Methemoglobin.

Methemoglobin was measured in 3 pups/treatment group by co-oximetry (Instrumentation Laboratories, model 482 cooximeter) in blood samples obtained by cardiac puncture into sodium heparin coated 1 ml syringes. Survival was slightly less in hyperoxia-exposed groups (95%) compared with air exposed groups (97%), but was not statistically different between hyperoxia and hyperoxia + ethyl nitrite exposed groups at day 8 and 14. Figure 1 (Left Panel) shows the effect of air or 95% O₂ ± inhaled ethyl nitrite at 0.2,

1, 10, or 20 ppm * 8 days on body weight. N=10/group, mean ± S.E.M. *p<.0l v. 8day O₂ control. Figure 1 (Right Panel) shows the effect of air x 14 days or 95% O₂ ± inhaled ethyl nitrite at 10 ppm x 8 days, followed by recovery in air x 6 days on body weight. +p<.005 v. 14 day O₂ control. As expected, hyperoxia exposure impaired weight gain, measured as body weight at day 8 and 14, as shown in Figure 1, but this effect was prevented in pups treated with hyperoxia + ethyl nitrite 20 ppm at 8 days, and in pups treated with 10 ppm at 14 days. Methemoglobin was slightly increased in the ethyl nitrite 10 ppm group (N=3/group), but in no case exceeded 5%.

Example 3: Leukocyte Influx

Earlier studies (Yi et al., Am J Respir Crit Care Med 170(1 1): 1188-96 (2004); Auten et al., Am J Physiol Lung. Cell MoI Physiol 281(2):L336-44 (2001); Deng et al., Am J Respir Crit Care Med 162(6):2316-23 (2000)) strongly implicate leukocyte influx as a key mechanism that impairs alveolar development in hyperoxia-exposed newborns, in part through DNA (Auten et al., Am J Respir Cell MoI Biol 26(4):391 -7 (2002)) and protein (Vozzelli et al., Am J Physiol Lung Cell MoI Physiol 286(3):L488-93 (2004)) oxidation. Since neutrophils are a major source of in vivo superoxide generation, it may be that blocking neutrophil influx reduces tissue superoxide accumulation, thereby preserving nitric oxide bioavailability. Co-administration of superoxide dismutase and inhaled nitric oxide potentiates the effects of nitric oxide on pulmonary vascular resistance (Van Meurs et al., N EnglJ Med 353(l):13-22 (2005)), so it is plausible that preventing inflammation could preserve adaptive, physiologic nitric oxide bioavailability. To analyze bronchoalveolar lavage fluid (BALF), animals were treated for 8 days then euthanized. A blunt Luer-stub 18 G catheter was used to cannulate the trachea, secured with suture. The lungs were perfused free of blood with 150 mM NaCl ImM EDTA pH 7.0 through the pulmonary artery after clipping the left atrial appendage. Lungs were then lavaged with four 0.5 ml aliquots of buffer, and pooled aliquots were centrifuged to collect cells for cytology and differential as previously described (Auten et al., Am J Physiol Lung Cell MoI Physiol 281(2):L336-44 (2001)). To assess whole lung myeloperoxidase, lungs from day 8 pups were snap frozen in liquid nitrogen following perfusion and lavage. Pulverized lungs were extracted in buffer and reacted with σ-dianisidine to detect myeloperoxidase activity using a microplate method previously described (Auten et al., Am J Physiol Lung Cell MoI Physiol 281(2):L336-44 (2001)). Figure 2 shows the effect of air or 95% O₂ ± inhaled ethyl nitrite at 0.2, 1, 10, or 20 ppm * 8 days, on bronchoalveolar lavage leukocytes and neutrophils (Panel A) and myeloperoxidase (MPO) activity (Panel B) in perfused, lavaged whole lung. N=8/group, mean ± S.E.M. */y<.05 v. O₂ control. As shown in Figure 2, treatment with ethyl nitrite for 8 days dose-dependently decreased 95% 0₂-induced BALF leukocyte and neutrophil accumulation, and tissue myeloperoxidase activity, N=8/group.

Example 4: Whole lung CINC-I Expression

Lungs from pups exposed to air or 95% O₂ ± ethyl nitrite 10 ppm for 8 days were extracted in lysis buffer, and supernatants analyzed in duplicate by ELISA for cytokine- induced neutrophil chemoattractant-1 (CINC-I), as previously described (Auten et al., Am J Physiol Lung Cell MoI Physiol 281(2):L336-44 (2001)). Total lung RNA was extracted with TriZol (Invitrogen). Total RNA was reverse transcribed (0.5 μg) using Superscript IIF^M (Invitrogen) and oligo dT primers in duplicate from each animal (N = 4-5/group) at 55°C according to the manufacturer's directions and as previously described. cDNA was then amplified by real-time PCR (Applied Biosystems 7300) using primers targeting CINC-I and L32 cyclin (control) mRNA and a master PCR mix containing Sybr Green (Applied Biosystems), according to the manufacturer's directions and as previously described, (Auten et al., Am J Physiol Lung Cell MoI Physiol 281(2):L336-44 (2001)). Samples were heated at 50⁰C x 2 min, then 95°C * 10 min to activate the Taq polymerase. PCR was continued for 40 cycles: 95°C * 15 sec → 58°C * 60 sec.

The crossing point absorbance for CINC-I RT-PCR products were compared after X cycles, and for L32 cyclin, after Y cycles. The values for CINC-I were normalized to the values for L32 (loading control) in each sample. CINC- 1/L32 ratios from 95% O₂ ± ethyl nitrite 10 ppm-exposed rats were expressed a proportion of the mean CINC- 1/L32 ratio in the air exposed treatment group.

Figure 3 shows the effect of air or 95% O₂ ± inhaled ethyl nitrite 10 ppm * 8 days on whole lung cytokine induced neutrophil chemoattractant-1 (CINC-I) mRNA, N = 4/group (Panel A) and protein (Panel B), N=8/group, mean S.E.M., *p<.05 v. O₂ control. As shown in Figure 3, ethyl nitrite treatment 10 ppm prevented hyperoxia-induced CINC- 1 expression in whole lung measured at day 8. CINC-I mRNA measured by real-time reverse transcriptase PCR was induced by hyperoxia at day 8, but this was prevented in pups treated with ethyl nitrite 10 ppm. The effects on CINC-I protein measured by ELISA were parallel. As shown in Figure 4, similar results were found with TNF-α following the same experimental procedure outlined for CINC-I above.

Like studies conducted with nitric oxide, (Marshall and Stamler, Biochemistry 40(6): 1688-93 (2001)) it may be that ethyl nitrite treatment can prevent NF-κB activation, which could account for the decreased CINC-I mRNA, a transcriptional target of NF-κB. To determine the expression of NF-κB, lung sections were blocked in 1% BSA in PBS and incubated with anti-p65 (activated subunit of NF-κB, Santa Cruz Biotechnology, Santa Cruz CA), and detected with horse anti-goat-biotin (Vector, Burlingame CA), followed by avidin-AlexaFluor 485™(Invitrogen, Eugene OR). Nuclei were counterstained with propidium iodide. The results of these in vitro studies shows that ethyl nitrite prevents superoxide-induced NF-κB activation and chemokine expression at day 8. The mechanisms by which ethyl nitrite might regulate NF-KB activation are unknown, but may include S-nitrosylation of multiple vulnerable cysteine residues of the NF-κB complex, by which nitric oxide has been shown to contribute to NF-κB inactivation in vitro (Marshall and Stamler, Biochemistry 40(6): 1688-93 (2001);

Marshall et al., Pr oc Natl Acad Sd USA 101(24):8841-2 (2004)). Example 5: VEGF Expression

Whole lung homogenates were analyzed by fluorescent antibody bead adsorption (Luminex), 75 μg protein/sample in duplicate from lungs of 4 pups/treatment group. Random sections from 4-5 animals/treatment group were detected with polyclonal rabbit anti-VEGF-1 1:1 ,000 followed by goat-anti-rabbit-biotin 1 :2,000 and detection with ABC Elite(Vector) according to the manufacturer's directions.

Figure 5 shows the effect of air or 95% O₂ ± inhaled ethyl nitrite 10 ppm * 8 days on VEGF immunohistochemistry, bar = 50 μm. The graph in Figure 5 shows the effect on VEGF in whole lung homogenates. In contrast with other reports of effects of hyperoxia in newborns, it was found that there was no statistically significant effect of hyperoxia on whole lung VEGF, measured by antibody-coated fluorescent bead assay, and ethyl nitrite treatment had no effect on whole lung VEGF at day 8 (Figure 5). There was significant variability among individuals, as previously reported in newborn rats using similar hyperoxia exposures (Lin et al., Pediatr Res 58(l):22-9 (2005)). Since the biological effect of VEGF on alveolar development is likely mediated via paracrine effects on alveolar capillary development, VEGF immunohistochemistry was performed to determine if there were effects on local expression. It was routinely found, as shown in Figure S, that hyperoxia depressed VEGF expression in bronchiolar epithelium, but it was also found that effects in alveolar parenchyma were more variable. VEGF signal was observed as intense staining in cells around alveoli, as well as diffuse staining within the alveolar septae. A consistent pattern of peripheral v. central alveolar immunolabeling in alveolar epithelium was not observed at day 8. Intense alveolar labeling was more typically observed in the ethyl nitrite treated pups, but this was not systematically quantified. The immunostaining pattern at day 14 was similar among all groups, with prominent VEGF signal in bronchiolar epithelium, but seldom in alveolar epithelium for all groups.

The results indicate that hyperoxia exposure uniformly depressed VEGF expression in distal bronchiolar epithelium, which was unaffected by ethyl nitrite treatment. These results are consistent with the findings of Lin and colleagues (Lin et al., Pediatr Res 58(l):22-9 (2005)). In contrast, hyperoxia-exposure did not significantly decreased whole lung VEGF. There was greater heterogeneity of alveolar epithelial expression in both hyperoxia treated groups than in air-treated groups. No obvious differences in alveolar epithelial expression were found among any of the groups, although this was not rigorously quantified. These data indicate that the beneficial effects of ethyl nitrite do not appear to be mediated through effects on total or cell-specific VEGF expression. The expression of VEGFRl or VEGFR2, which may be downregulated in clinical BPD (Bhatt et al., Am J Respir Crit Care Med 164(10 Pt 1): 1971-80 (2001); Maniscalco et al., Am J Physiol Lung Cell MoI Physiol 282(4):L81 1-23 (2002)) remains to be determined.

Example 6: NF-κB activation

Nuclear proteins were extracted from Day 8 pups, according to ordinary methods well known in the field of art. Nuclear protein from tumor necrosis factor-alpha (TNFα) activated Jurkat cells was used as a positive control, along with nuclear protein extracted from lungs from lipopolysaccharide (E. coli serotype 055, 6 mg/kg intraperitoneally)-treated adult rats, with vehicle-treated adult rat as additional negative controls. Activated NF-κB was detected using a commercial kit (TransAM NFKB, Active Motif, Carlsbad CA), binding nuclear protein extracts to NF-κB binding DNA sequence covalently bound to an ELISA. Samples (10 μg/pup) were assayed in duplicate from 3 pups/group.

Activated NF-κB was detected in histologic sections from each treatment group by immunohistochemistry using an antibody directed against the nuclear localization epitope of the p65 subunit, which is exposed during activation.

The results are depicted in Figure 6. Figure 6A shows a comparison of the amount of Nuclear Factor (NF)- KB activation in air-exposed adult rat lung (negative control), LPS- treated adult rat lung, or tissue plasminogen activator-stimulated jurkat cells (positive control group), 8-day old rat lung from air, 95% O₂-exposed, 95% O₂ + ENO, and 95% O₂ + NO treatment groups (mean + SD, n=3 per group). Figure 6B shows the immunohistochemical localization of activated NF-κB in representative sections from each of the LPS-treated adult rat lung, 8-day old rat lung from air, 95% O₂-exposed, 95% O₂ + ENO, and 95% O₂ + NO treatment groups. As shown in Figure 6B, hyperoxia activated NF-κB in a subpopulation of cells, mainly in bronchiolar epithelial cells (BE), but also in alveolar epithelium (AE), as detected by immunohistochemistry. This was in marked contrast to the widespread activation observed in lungs from LPS-treated adult rat. Treatment with ENO, but not NO decreased bronchiolar epithelial NF-κB activation. Effects on alveolar epithelium were modest and heterogeneous in all hyperoxia-exposed groups at 8 days. Hyperoxia did not significantly increase NF-κB activation in whole lung compared with air-exposed newborn pups, and this was unaffected by ENO or NO. The overall activation in lungs from each of the neonatal exposure groups was small compared with the activation in LPS-treated adults (or in stimulated Jurkat cells), in accord with the immunohistochemical studies (Figure 6B).

It was therefore hypothesized that ENO might be superior to NO in its ability to inactivate NF-κB, an important regulator of lung inflammation via induction of inflammatory cytokine transcription factors. Previous studies have shown that S-nitroso modifications inactivate I-κ kinase-kinaseβ in vitro, which de-represses NF-κB activation. S-nitrosylation inactivates the p50 subunit of NF-κB, preventing transcriptional activation. Preliminary studies show that ENO can block superoxide-induced NF-κB activation in vitro. To determine if hyperoxia induced NF-κB activation in newborn rat lung, or if inhaled ENO or NO blocked hyperoxia-induced pulmonary NF-κB activation in vivo, NF-κB activation was measured in nuclear protein extracts from whole lung in each treatment group at day 8. It was found that the overall magnitude of NF-κB activation in whole lung nuclear extracts was small compared with the more robust stimulus of LPS-injection in adult rat lung. It was also found that hyperoxia exposure increased activation in a subset of cells identified by immunohistochemistry, most prominently the bronchiolar epithelium, which was prevented in ENO but not in NO treated pups. In contrast with the LPS-treated adult rats, alveolar epithelial NF-κB activation was qualitatively decreased and heterogeneously expressed.

NF-κB abundance is reportedly induced by hyperoxia in neonatal rats, but activation and nuclear localization were not directly examined in the previous report. Hyperoxia- exposed transgenic newborn mice expressing NF-κB-driven luciferase demonstrate NF-κB activation which persisted until 72 hours of hyperoxia-exposure (later time points were not examined) but not in all cells, in agreement with the findings in newborn rats described herein. While not intending to be bound by any theory, these results suggest that NF-κB activation may protect some cells against oxidative-stress induced apoptosis, and may serve as an adaptive response under certain circumstances. Therefore, conducting similar studies using the NF-κB luciferase transgenic mouse could help to clarify timing and location of inhaled ENO or NO effects on pulmonary NF-κB activation.

Example 7: 3-Nitrotyrosine Levels Lung homogenates, 0.4 mg total protein/lung from pups exposed to air or 95% O₂ ± ethyl nitrite 10 ppm for 8 days (N = 6-7/group) were analyzed in duplicate for 3-nitrotyrosine by ELISA using a commercial kit, according to the manufacturer's directions (Northwest Life Science Specialists, Vancouver WA). Results below the limit of detection for the assay were assigned a value of one half of the detection limit for the purposes of statistical comparisons.

Figure 7 shows the effect of air or 95% O₂ ± inhaled ethyl nitrite at 8 days on 3- nitrotyrosine detected by ELISA, mean ± S.E.M., N = 6-7/group. */K.O5 v. air. As shown in Figure 7, hyperoxia induced a modest, but statistically significant increase in 3-nitrotyrosine, compared with air exposed animals at day 8. In contrast, hyperoxia did not significantly increase 3-nitrotyrosine in animals treated with ethyl nitrite 10 ppm.

Example 8: Static Lung Compliance Pups exposed to air or 95% O₂ ± ethyl nitrite 10 ppm for 8 days, then recovered in air for 6 days (total 14 postnatal days) were anesthetized with xylazine and ketamine, tracheae cannulated, and connected to a small animal ventilator (flexiVent, Scireq, Montreal, Canada) and ventilated at 7.5 ml/kg with positive end expiratory pressure at 4 cmH2O. Pressures and flows at specified tidal volumes were measured using forced oscillatory with a small animal ventilator (FlexiVent, SCIREQ, Montreal, Canada) and used to generate pressure-volume loops, and to calculate compliance as described in detail (Liu et al., Essential roles of S- nitrosothiols in vascular homeostasis and endotoxic shock. Cell 116(4): 617-28 (2004)).

In order to determine longer term effects of ethyl nitrite on hyperoxia-impaired postnatal lung function and development, pups were allowed to recover in air for 6 days after exposure to air, or 95% O2 ± ethyl nitrite 10 ppm for 8 days. Figure 8A shows the effect of air or 95% O₂ ± inhaled ethyl nitrite at 10 ppm x 8 days followed by recovery in air 6 days on pressure-volume loops. N=5-6/group, mean ± S.E.M. Figure 8B shows the effect of air or 95% O₂ ± inhaled ethyl nitrite at 10 ppm * 8 days followed by recovery in air 6 days on lung resistance. N=5-6/group, mean ± S.E.M. As shown in Figure 8A and B, hyperoxia-exposed pups showed decreased lung compliance, with a decreased pressure-volume loop hysteresis, compared with air-exposed pups. Treatment with ethyl nitrite significantly prevented hyperoxia-impaired static lung compliance and the effect on pressure-volume loop hysteresis.

Example 9: Alveolar Development Lungs from pups exposed to air or 95% O₂ ± ethyl nitrite 10 ppm for 8 days, then recovered in air, as above, N = 6 /group, were inflation fixed with 10% phosphate-buffered formalin pH 7.0 at 25 cm H₂O, paraffin embedded, and sectioned at 4-6 μm. Random sections were stained with malachite green to achieve optimum contrast or with hematoxylin and eosin as described (Yi et al., Am J Respir Crit Care Med 170(1 1): 1 188-96 (2004)). Three random sections per animal (N=5-6/treatment group) were stained with malachite green solution and three random, non-overlapping images were acquired per section at 200^χ magnification to measure alveolar volume and surface density as previously described (Auten et al., Am J Physiol Lung Cell MoI Physiol 281 (2):L336-44 (2001)). An 11 * 1 1 array of points was overlain atop the digitally thresholded image and instances of concurrence between these test points and alveolar tissue in a binary thresholded image (air, white, tissue, black) were automatically counted and divided by the total number of points overlying parenchyma to yield alveolar volume density, an estimate of alveolar number. Alveolar surface density was obtained by similarly superimposing 15 parallel lines of equal known length atop the digitally thresholded binary images. A computer algorithm calculated the number of times the black test line was intersected by the black septal tissue, which was divided by the total test line length using a calibration image obtained using a stage micrometer.

Figure 9 shows the effect of air (Panel A) , 95% O₂ (Panel B), or 95% O₂ + inhaled ethyl nitrite at 10 ppm (Panel C) ^χ 8 days followed by recovery in air 6 days on 2 alveolar crests identified by elastin staining, arrowheads, bar = 100 μm. Alveolar development is illustrated in representative sections stained with malachite green in air (Panel D), 95% O₂ (Panel E), or 95% O₂ + ethyl nitrite exposed pups (Panel F). As shown in Figure 9, alveolar sizes and elastin-stained secondary crest abundance in 95% O₂ +ethyl nitrite 10 ppm-exposed pups were quantitatively similar to those observed in air exposed pups. Quantitative measurements of alveolar volume density and alveolar surface density showed effects parallel to those on lung compliance. Figure 10 shows the effect of air, 95% O₂ ± ethyl nitrite 10 ppm 8 days followed by recovery in air for 6 days on alveolar volume density (Panel A) and alveolar surface density (Panel B), N=5-6/group, mean ± S.E.M, *p<.05 v. O₂ control. Parallel to the effects on lung compliance, Figure 10 shows that both alveolar volume density and alveolar surface density were significantly impaired in 95% O₂-exposed pups but not in 95% O₂ + ethyl nitrite 10 ppm-exposed pups.

Consistent with the results of improved alveolar development described above, Lin and colleagues (Lin et al., PediatrRes 58(l):22-9 (2005)) showed that exposure to 100% O₂ for 6 days and subsequent treatment with nitric oxide at 10 ppm from day 6-14 preserved alveolar development and reduced pulmonary vascular remodeling. In the present studies, ethyl nitrite was co-administered with 95% oxygen, making it potentially vulnerable to oxidation, yet there was no evidence of increased nitrosative stress estimated by 3-nitrotyrosine measurements in whole lung. The protection of alveolar development by ethyl nitrite administration was durable, as evidenced by significant protective effects on lung compliance and estimates of alveolar number and surface area six days after discontinuing hyperoxia and ethyl nitrite treatment. In studies using nitric oxide to prevent BPD in animal models that mimic clinical BPD, extremely premature baboons treated with inhaled nitric oxide co-administered with normoxic mechanical ventilation showed partial protection of alveolar development (Ballard et al., PediatrRes. 59(1): 157-62 (2006), as did similarly treated premature lambs (Bland, Biol Neonate 88(3): 181-91 (2005)), consistent with the instant studies using ethyl nitrite.

Example 10: S-Nitrosothiol (SNO) Accumulation

NO was measured in lavage by chemiluminescense before and after reduction of SNO as previously described (Jia et al., Nature 380(6571):221-6 (1996). Equal volumes of bronchoalveolar lavage fluid were analyzed.

Figure 11 shows the effect of air or 95%O₂ ± inhaled ethyl nitrite 0.2-20 ppm x 8 days on NO and SNO in bronchoalveolar lavage fluid, N=4-5/gτoup. SNO was measured in lavage from pups treated with air, 95%O2 or 95%O₂ + ethyl nitrite 10 or 20 ppm x 8 days. As shown in Figure 11, measurable SNO was detected only in BALF from pups treated with ethyl nitrite.

Example 11 : Anti-Oxidant Enzyme Expression

Whole lung homogenates from pups at 8 days (N=4/group) were analyzed in triplicate for total superoxide dismutase (WST-I, Dojindo, Japan) and catalase (AmplexRed, Invitrogen, Carlsbad CA) according to the manufacturer's directions. SOD 1, 2, and 3 isoforms and catalase expression were analyzed by immunoblot, and quantified by densitometry normalized to β-actin signal as previously described (Auten et al., Am J Physiol Lung Cell MoI Physiol 290(l):L32-40 (2006)).

Figure 12 shows the effect of air or 95%O₂ , or 95%O2 + inhaled ethyl nitrite 10 ppm x 8 days on SODl, 2, and 3, catalase, and β-actin expression by immunoblot (Panel A) quantified by image analysis (Panel B), N=4/group, mean + SEM, *p<.05 v. air, ** /7<.05 v. 95%θ2 control. Figure 12 also shows the effects on total lung superoxide dismutase (SOD) activity (Panel C) and catalase activity (Panel D), N=4/group, mean + SEM. As shown in Figure 12, whole lung SOD activity was unaffected by hyperoxia or hyperoxia + ethyl nitrite 10 ppm. Catalase activity was decreased in hyperoxia-exposed pups, but this was not statistically significant (p=.O8). There was no significant effect of hyperoxia + ethyl nitrite on catalase activity. Whole lung SODl and SOD2 abundance in immunoblots normalized to β-actin were unaffected by 95% O_∑ ± ethyl nitrite at 8 days. SOD3 was increased in both hyperoxia-exposed groups, compared with air exposed pups, but with no difference between 95% O₂ and 95% O₂ + ethyl nitrite groups. Parallel to the effects on activity, lung catalase abundance was decreased in 95% O₂ exposed pups but not in 95% O₂ + ethyl nitrite exposed pups compared with air exposed pups.

Example 12: Ethyl Nitrite and Hyperoxia-Induced Lung Disease The present invention shows that co-administration of ethyl nitrite with hyperoxia reduced pulmonary neutrophil chemokine expression and pulmonary leukocyte and neutrophil influx at day 8, preceding the observation of improved lung compliance and alveolar development at day 14. Inhaled ethyl nitrite treated and prevented hyperoxia- induced lung inflammation and treated and prevented neutrophil chemokine expression, without increasing nitrosative stress. Inhaled ethyl nitrite also treated and prevented the adverse effects of hyperoxia, such as the induction of protein nitration, on newborn alveolar development and lung compliance.

Unlike nitric oxide, the propensity of ethyl nitrite to nitrosylate thiol groups is relatively independent of concentrations of oxygen and reactive oxygen species that disrupt bioactivity, (Moya et al., Proc Natl Acad Sd USA 98(10):5792-7 (2001)) and S-nitrosylation has been proposed as the major route through which bioactivity is conveyed (Hess et al., Nat Rev MoI Cell Biol 6(2): 150-66 (2005)). S-nitrosylation has been shown to inhibit nuclear factor kappa B (NF-κB) activity through nitrosylation of target cysteine residues in both the regulatory IKK kinase and the DNA binding domain of the p50 subunit of NF-κB (Marshall and Stamler, Biochemistry 40(6): 1688-93 (2001 )), accounting in part for the antiinflammatory effects of nitric oxide. Since ethyl nitrite does not release NO, (Moya et al., Proc Natl Acad Sd USA 98(10):5792-7 (2001)), it is less susceptible to forming deleterious higher order nitrogen oxides that accompany inhaled nitric oxide (Janssen-Heininger et al., Am JRespir Crit Care Med 166(12 Pt 2):S9-S16 (2002); van der Vliet et al., Respir Res l(2):67-72 (2000)).

To determine if inhaled ethyl nitrite could prevent inflammation and protect against hyperoxia-impaired alveolar development in vivo, hyperoxia-exposed newborn rat pups were treated with inhaled ethyl nitrite at 0.2 - 20 ppm and the effects on SNO accumulation in bronchoalveolar lavage, and on pulmonary leukocyte influx, inflammatory chemokine expression, antioxidant enzymes, lung compliance, and alveolar development were measured. Inhaled ethyl nitrite increased SNOs in bronchoalveolar lavage at the highest doses. Inhaled ethyl nitrite dose-dependently prevented pulmonary leukocyte accumulation. At the optimum anti-inflammatory dose, ethyl nitrite prevented hyperoxia-impaired lung compliance and alveolar development. The present invention provides that inhaled ethyl nitrite can serve as lung-targeted therapy to replete SNOs in the alveolar lining fluid, prevent maladaptive inflammation that follows severe oxidative stress in newborns, and prevent the development ofBPD. The anti-inflammatory effects of ethyl nitrite may be partly attributable to increased

S-nitrosoglutathione (GSNO) accumulation in ethyl nitrite treated pups. SNO was detected in the airway lining fluid in pups treated with high doses of inhaled ethyl nitrite, and this result most likely reflects GSNO accumulation (Que et al., Science 308(5728): 1618-21 (2005); Gaston et al., Proc Natl Acad Sci USA 90(23): 10957-61 (1993)). Treatment with ethyl nitrite has previously been shown to achieve significant increases in GSNO in vitro and in vivo (Moya et al., Proc Natl Acad Sci USA 9%(\ 0): 5792-7 (2001 ); Que et al., Science 1308(5728): 1618-21 (2005)). GSNO itself has been shown to possess anti-inflammatory properties when administered in vivo during cerebral ischemia reperfusion injury Khan et al., Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke. Nitric Oxide (2006); Khan et al., J Cereb Blood Flow Metab 25(2): 177-92 (2005)), and to decrease pulmonary 5-lipoxygenase activity (Zaman et al, Am JRespir Cell MoI Biol 34(4):387-93 (2006)).

Although not wanting to be bound by any particular theory, ethyl nitrite treatment may be inhibiting pulmonary nuclear factor kappa-B (NF-κB) through S-nitrosylation, which could account for the decreased CINC-I mRNA and protein. CINC-I is a rat analog to human IL-8, targets the CXCR2 neutrophil chemokine receptor, (Auten et al., J Pharmacol Exp Ther 299(l):90-5 (2001)) and is also a transcriptional target of NF-κB (Blackwell et al., Am JRespir Cell MoI Biol 11(4):464-72 (1994)). There are at least two cysteine residues of the NF-κB apparatus, by which S-nitrosylation has been shown to inactivate NF-κB in vitro (Marshall and Stamler, Biochemistry 40(6): 1688-93 (2001 ); Marshall et al., Proc Natl Acad Sci USA 101(24):8841-2 (2004)). Preliminary studies show that ethyl nitrite inhibits nuclear p65 localization in rat alveolar epithelium in vitro (Wratney et al., Pediatric Research 55(4):510A (2004)), recapitulating similar in vitro effects of low molecular weight SNOs (Marshall and Stamler, J Biol Chem 277(37):34223-8 (2002)). Inhaled ethyl nitrite may transnitrosylate airway glutathione, which in turn is converted into smaller weight S- nitrosothiols (e.g., L-Cys-Gly-SNO or L-Cys-NO) that are imported intracellularly, and are capable of transnitrosylating intracellular targets (Li and Whorton, J Biol Chem 280(20):20102-10 (2005)).

On the basis of the fact that ethyl nitrite is a nitrosylating agent, the results described herein support the concept that S-nitrosylation may serve as a braking mechanism that attenuates inflammation. Conditions with decreased endogenous NO production or increased NO oxidation such as the baboon model of BPD (Afshar et al., Am J Physiol Lung Cell MoI Physiol 284(5):L749-58 (2003)) or hyperoxia-exposed newborn rats (Belik et al., JAppl Physiol 96(2):725-30 (2004)) may lack this braking mechanism and be more vulnerable to maladaptive inflammation. Since NO reacts directly with superoxide to form peroxynitrite and other higher order nitrogen oxides, NO itself may promote inflammation under certain circumstances. Peroxynitrite formation is thought to contribute to deleterious effects of nitrogen oxides, and may account for some of the pro-inflammatory effects of nitric oxide (Janssen-Heininger et al., Am J Respir Crit Care Med 166(12 Pt 2):S9-S16 (2002); van der Vliet et al., Respir Res l(2):67-72 (2000)).

In contrast, ethyl nitrite does not react directly with superoxide but selectively targets thiols to form SNOs. The effect of inhaled ethyl nitrite on peroxynitrite accumulation in hyperoxia-exposed lung was tested by measuring 3-nitrotyrosine levels by ELISA, since nitrotyrosine is formed by reaction of peroxynitrite with tyrosine residues. It was found that 95% O₂ x 8 days increased 3-nitrotyrosine, as expected (Haddad et al., J Clin Invest 94(6):2407-13 (1994)), but that ethyl nitrite prevented this increase. Increased plasma nitrotyrosine parallels disease severity in clinical BPD, probably reflecting the effects of combined oxidative and nitrosative stress (Lorch et ali, Free Radio Biol Med 34(9): 1146-52 (2003)). Peroxynitrite is formed by reaction of nitric oxide with superoxide, which accumulates during hyperoxic exposure (Ho, Am J Respir Crit Care Med 166(12 Pt 2):S51-6 (2002)). Myeloperoxidase, which is induced during hyperoxia exposure, contributes to protein nitration as well (Narasaraju et al., Am J Physiol Lung Cell MoI Physiol 285(5):L1037-45 (2003)). Ethyl nitrite can prevent nitrotyrosine accumulation by preventing pulmonary superoxide production or myeloperoxidase accumulation through effects on cellular injury or neutrophil accumulation.

There are a wide variety of signaling pathways potentially modified by S-nitrosylation

(Hess et al., Nat Rev MoI Cell Biol 6(2): 150-66 (2005); Gaston et al., S-Nitrosothiol Signaling in Respiratory Biology. Am J Respir Crit Care Med. (2006)) that can protect lung alveolar development against oxidative stress, injury, and maladaptive apoptosis. Regulation of antioxidant enzyme activity by S-nitrosylation could be a mechanism by which ethyl nitrite confers protection. Catalase has been shown to be modified by S-nitrosylation (Foster et al., J. Biol. Chem. 279(24):25891 -25897 (2004)) and can be upregulated by NO-based signals (Iwai et al., J Biol Chem 278(11):9813-22. (2003)). The SpI transcription factor, linked to extracellular superoxide dismutase promoter activity (Zelko and FoIz, Free Radio Biol Med 37(8): 1256-71 (2004)), is regulated by S-nitrosylation (Zaman et al., Am. J. Respir. Crit. Care Med 165:A279 (2002)). The effects of hyperoxia + ethyl nitrite were measured on whole lung superoxide dismutase and catalase activity, and on superoxide dismutase isoform and catalase abundance by immunoblot. Catalase was modestly decreased in the 95%O2 exposed group (p =0.08), but not in the 95%O2 + ethyl nitrite exposed group. Although no global effects on antioxidant activity in whole lung were found, it is possible that there are local effects on antioxidant enzyme in the airway lining fluid or other relevant compartments. Preventing a decline in catalase activity might account for some of the beneficial effects on lung development (Walther et al., Exp Lung Res 16(3):177-89 (1990)). SOD3 expression was induced by hyperoxia as previously reported (Mamo et al., Am J Respir Crit Care Med 170(3):313-8 (2004)), but there was no additional increase in the ethyl nitrite treated group. The most striking protective effects of ethyl nitrite were on lung mechanics and alveolar development, which were measured six days after discontinuing both hyperoxia and ethyl nitrite. Effects on mechanics, particularly compliance, could have been mediated in part through effects on the surfactant system, either by directly protecting surfactant synthesis, or by preventing surfactant inactivation, as has been suggested in baboons treated with inhaled NO (Ballard et al., Pediatr Res 59(1): 157-62 (2006)). The protective effects of inhaled ethyl nitrite on alveolar development, alveolar number (estimated by alveolar volume density) and surface area (estimated by alveolar surface density), would contribute to the observed differences in compliance. ^* The beneficial effects of ethyl nitrite on qualitatively assessed secondary crest abundance, marked by elastin staining, are consistent with this interpretation. The present invention provides that inhaled ethyl nitrite compositions increased SNOs in lavage fluid, prevented neutrophil chemokine expression, and inhibited hyperoxia-induced inflammation, without increasing nitrosative stress. Inhaled ethyl nitrite compositions prevented the adverse effects of hyperoxia on newborn rat lung compliance and alveolar development. Based on the foregoing, compositions comprising ethyl nitrite can serve as a lung-targeted therapy to restore airway lining fluid SNO and prevent or treat maladaptive lung inflammation that contributes to BPD.

Claims

What is claimed:

1. A method of treating or preventing hyperoxia-induced lung disease in a subject in need thereof comprising administering a therapeutically effective amount of a composition comprising a gas and ethyl nitrite to said subject, wherein the therapeutically effect amount ranges from 1 to 5 ppm.

2. The method of claim 1 , wherein the subject is a extremely low birth weight premature newborn.

3. The method of claim 1 , wherein the lung disease is chronic.

4. The method of claim 3, wherein the chronic lung disease is bronchopulmonary dysplasia.

5. The method of claim 1, wherein the treatment or prevention of the lung disease reduces hyperoxia-induced lung inflammation in the subject.

6. The method of claim 1 , wherein the treatment or prevention of the lung disease reduces hyperoxia-induced leukocyte and neutrophil accumulation in the lung in the subject.

7. The method of claim 1 , wherein the treatment or prevention of the lung disease reduces hyperoxia-induced peroxynitrite or NO_x accumulation in the lung in the subject.

8. The method of claim 1 , wherein the treatment or prevention of the lung disease reduces hyperoxia-induced NF-tcB activation in the lung in the subject.

9. The method of claim 1 , wherein the treatment or prevention of the lung disease reduces hyperoxia-induced lung compliance and alveolar development in the lung in the subject.

10. The method of claim 1, wherein the administration of a therapeutically effective amount of a composition comprising a gas and ethyl nitrite does not result in an increase in nitrosative stress in the lung in the subject.

11. The method of claim 1 , wherein the administration of a therapeutically effective amount of a composition comprising a gas and ethyl nitrite results in an increase in S- nitrosothiol levels in the lung in the subject.

12. The method of claim 11, wherein the S-nitrosothiol is S-nitrosoglutathione.

13. The method of claim 1 , wherein the treatment or prevention of the lung disease reduces hyperoxia-induced cytokine expression in the lung in the subject.

14. The method of claim 13, wherein the cytokine is CINC-l/MIP-2.

15. The method of claim 13 , wherein the cytokine is MCP- 1.

16. The method of claim 13, wherein the cytokine is TNF-α.

17. The method of claim 13, wherein there is a reduction in cytokine protein expression.

18. The method of claim 13, wherein there is a reduction in cytokine RNA expression.

19. The method of claim 1 , wherein said gas is nitrogen.

20. The method of claim 19, wherein the nitrogen gas is admixed with oxygen prior to administration.

21. The method of claim 1 , wherein said gas comprises less than 5 ppm NCh.

22. The method of claim 1, wherein said gas comprises less than 25 ppm NO.

23. The method of claim 1, wherein said gas comprises less than 32 ppmv ethanol.

24. The method of claim 1 , wherein the composition does not form NO2 or NO_x in the presence of oxygen or reactive oxygen species at body temperature.

25. The method of claim 1, wherein said administration is inhalation.

26. The method of claim 1, wherein said administration is intranasal.

27. The method of claim 1, wherein the composition is administered as an aerosol.

28. The method of claim 1, wherein the composition is administered from about 7 to about 14 days.

29. A method of inducing lung organogenesis in a subject in need thereof comprising administering a therapeutically effective amount of a composition comprising a gas and ethyl nitrite to said subject.

30. The method of claim 29, wherein the subject is a extremely low birth weight premature newborn.

31. The method of claim 29, wherein the subject is subject is suffering from a chronic lung disease.

32. The method of claim 31, wherein the chronic lung disease is bronchopulmonary dysplasia.

33. The method of claim 29, wherein the therapeutically effect amount ranges from 1 to 20 ppm.

34. The method of claim 29, wherein the therapeutically effect amount ranges from 1 to 10 ppm.

35. The method of claim 29, wherein the therapeutically effect amount ranges from 1 to 5 ppm