This disclosure concerns formulations of nitrite containing an anti-caking agent, and uses thereof. This disclosure further concerns treatment of lung injuries resulting from chemical or smoke inhalation or viral infection by administration of nitrite.
Exposure to chlorine (Cl2) gas can cause extensive lung injury, including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (White et al., Proc Am Thorac Soc 7:257-263, 2010; Yadav et al., Proc Am Thorac Soc 7:278-283, 2010). Even after cessation of exposure, lung injury can continue to occur for hours or even days, resulting in inflammation, oxidative stress, ALI and ARDS (Samal et al., Proc Am Thorac Soc 7:290-293, 2010; Honavar et al., Am J Respir Cell Mol Biol 45: 419-425, 2010; Martin et al., Am J Respir Crit Care Med 168: 568-574, 2003; Leustik et al., Am J Physiol Lung Cell Mol Physiol 295: L733-L743, 2008; Tuck et al., Respir Res 9:61, 2008). Studies in animal models have demonstrated that administration of antioxidants or β2-agonists following chlorine gas exposure can provide some protection against lung injury (Leustik et al., Am J Physiol Lung Cell Mol Physiol 295: L733-L743, 2008; McGovern et al., Free Radic Biol Med 50:602-608, 2011; McGovern et al., 11: 138, 2010; Zarogiannis et al., Am J Respir Cell Mol Biol 45: 386-392, 2010). Intraperitoneal or intramuscular administration of nitrite has also been shown to reduce ALI in animals exposed to chlorine gas (Samal et al., Free Radic Biol Med 53(7): 1431-1439, 2012; Yadav et al., Am J Physiol Lung Cell Mol Physiol 300(3): L362-369, 2011). However, a needs remains for a safe and effective treatment for inhalational chemical and smoke injuries, as well as lung injury due to viral infection.
Treatment of lung injuries resulting from, for example, chemical inhalation, smoke inhalation, microbial infection, trauma or mechanical injury, by administration of nitrite is described. Also described are formulations of nitrite that include a nitrite salt and an anti-caking agent, and use of the formulation for the treatment of a variety of diseases and conditions.
Provided herein is a method of treating a lung injury in a subject by administering to the subject a therapeutically effective amount of a nitrite salt. In some embodiments, the lung injury is caused by inhalation of a chemical, inhalation of smoke, an infection (such as SARS-CoV-2 infection), trauma, or mechanical injury. In particular embodiments, the chemical is not chlorine. In some examples, the nitrite salt is administered as a formulation that includes the nitrite salt and an anti-caking agent, such as sodium bicarbonate.
Also provided is a formulation that includes a nitrite salt and an anti-caking agent, such as sodium bicarbonate. In some embodiments, the anti-caking agent is present at a concentration of at least 20 parts per million. In some embodiments, the nitrite salt is sodium nitrite, potassium nitrite or arginine nitrite.
Further provided are methods of treating a disease or condition in a subject by administering a therapeutically effective amount of the nitrite formulation disclosed herein. In some embodiments, the disease or condition is selected from a lung injury, pulmonary hypertension, heart failure, cardiogenic shock, hypertension, respiratory failure, a metabolic syndrome, diabetes, a lipid disorder, an endocrine disorder, a gastroenterological disorder, hypoperfusion, inflammation, cystic fibrosis and aging.
The foregoing and other objects and features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.
Chlorine gas exposure results in increased oxidative stress, inflammation and dysfunction in endogenous repair processes (Bessac and Jordt, Proc Am Thorac Soc 7:269-277, 2010; Chang et al., Toxicol Appl Pharmacol 263:251-258, 2012; Chen et al., Toxicol Appl Pharmacol 272(2): 408-413, 2013; Fanucchi et al., Am J Respir Cell Mol Biol 46:599-606, 2012; Gessner et al., Am J Physiol Lung Cell Mol Physiol 304:L765-773, 2013; Honavar et al., Am J Respir Cell Mol Biol 45(2): 419-425, 2010; Koohsari et al., Respir Res 8:21, 2007; Leustik et al., Am J Physiol Lung Cell Mol Physiol 295:L733-743, 2008; Martin et al., Am J Respir Crit Care Med 168:568-574, 2003; McGovern et al., Free Radic Biol Med 50:602-608, 2011; McGovern et al., Respir Res 11:138, 2010; Musah et al., Respir Res 13:107, 2012; O'Koren et al., Am J Respir Cell Mol Biol 49(5): 788-797, 2013; Song et al., Am J Respir Cell Mol Biol 45:88-94, 2011; Yadav et al., Am J Physiol Lung Cell Mol Physiol 300:L362-369, 2011; Zarogiannis et al., Am J Respir Cell Mol Biol 45:386-392, 2011). The endothelial dysfunction associated with chlorine injury is characterized by a loss of nitric oxide (NO) bioavailability, which predisposes tissues to inflammation and oxidative stress. Repleting NO reduces inflammation and oxidative stress and improves survival. Available data also suggests that antioxidant (vitamin C) and chelator (desferoxamine) therapies can improve survival and enhance lung epithelial repair (Fanucchi et al., Am J Respir Cell Mol Biol 46:599-606, 2012).
The present disclosure describes the administration of nitrite in intramuscular, intravenous, subcutaneous, oral or inhalational forms to treat post-exposure chemical lung injury due to chemical inhalation or smoke inhalation, as well lung injury resulting from infectious disease, such as SARS-CoV-2. Administration of nitrite as soon as possible post exposure to chemicals, smoke or infectious agents is proposed to improve lung injury and survival.
As demonstrated in the Examples herein, post-exposure treatment with intramuscular sodium nitrite resulted in significant decreases in acute lung injury and improved survival in mice, rats and rabbits after exposure to chlorine, bromine and/or phosgene gas (see also Honavar et al., Toxicol Lett 271:20-25, 2017; Honavar et al., Am J Physiol Lung Cell Mol Physiol 307(11):L888-L894, 2014; Samal et al., Free Radic Biol Med 53(7):1431-1439, 2012).
ALI acute lung injury
ARDS acute respiratory distress syndrome
BAL bronchoalveolar lavage
BALF bronchoalveolar lavage fluid
Br2 bromine
CFU colony forming unit
CHX chlorohexidine
Cl2 chlorine
COCl2 phosgene
CoV coronavirus
ELISA enzyme-linked immunosorbent assay
eNOS endothelial nitric oxide synthase
GI gastrointestinal
Hb hemoglobin
IM intramuscular
IP intraperitoneal
NO nitric oxide
NOS nitric oxide synthase
NR nitrite reductase
PMN polymorphonuclear
ppm parts per million
RBC red blood cell
SARS severe acute respiratory syndrome
Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes X, published by Jones & Bartlett Publishers, 2009; and Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, published by Wiley-VCH in 16 volumes, 2008; and other similar references.
As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a salivary gland” includes single or plural cells and can be considered equivalent to the phrase “at least one salivary gland.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various embodiments, the following explanations of terms are provided:
Administration: To provide or give a subject an agent, such as nitrite, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), intraglandular, oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
Anti-caking agent: An agent that inhibits or prevents caking of a drug substance, such as a nitrite salt. In the context the present disclosure, the formulation of nitrite salt generally contains an anti-caking agent in at least 20 parts per million. Examples of anti-caking agents include, but are not limited to, sodium bicarbonate, aluminum caprate, aluminum caprylate, aluminum laurate, aluminum myristate, aluminum oleate, aluminum palmitate, aluminum salts of fatty acids, aluminum stearate, calcium caprate, calcium caprylate, calcium carbonate, calcium chloride, calcium myristate, calcium oxide, calcium palmitate, calcium phosphate (dibasic or tribasic), calcium salts of fatty acids, calcium silicate, calcium stearate, calcium sulfate, carboxymethyl cellulose, carnauba wax, carrageenan, castor oil, microcrystalline cellulose, dextrin, dextrose, glycerin, glycerin monooleate, iron ammonium citrate, magnesium caprate, magnesium caprylate, magnesium carbonate, magnesium laurate, magnesium myristate, magnesium oleate, magnesium oxide, magnesium palmitate, magnesium salts of fatty acids, magnesium silicate, magnesium stearate, magnesium sulfate, maltodextrin, mannitol, potassium caprate, potassium caprylate, potassium laurate, potassium myristate, potassium palmitate, potassium permanganate, potassium salts of fatty acids, propylene glycol, silicon dioxide, sodium aluminum phosphate (acidic or basic), sodium aluminum silicate, sodium caprate, sodium caseinate, sodium chloride, sodium laurate, sodium mono- and dimethyl naphthalene sulfonates, sodium myristate, sodium oleate, sodium palmitate, sodium phosphate (monobasic, dibasic or tribasic), sodium pyrophosphate, sodium salts of fatty acids, sodium silicate, sodium stearate, sodium tripolyphosphate, tartaric acid, titanium dioxide, and xanthan gum.
Bromine (Br2): A naturally occurring element that is a liquid at room temperature. Bromine has a dark reddish-brown color with a pungent bleach-like odor. Bromine gas is toxic by inhalation, causing damage to mucous membranes and other tissues, such as the lung. Bromine liquid or gas can cause skin irritation and burns. Inhalation of bromine gas can cause long-term injury to the lungs, as well as kidney and brain damage.
Chemical lung injury: Lung injury as a result of exposure to a chemical, such as bromide, methyl bromide, mustard gas (sulfur mustard), nitrogen mustard (HN-1, HN-2, HN-3), phosgene, phosgene oxime, diphosgene, phosphine, ammonia, bromine, methyl isocyanate, hydrogen chloride, chlorine, osmium tetroxide, phosphorous (elemental, white or yellow), sulfuryl fluoride, lewisite, riot control agents (such as chloroacetophenone (CN), chlorobenzylidenemalononitrile, chloropicrin, bromobenzylcyanide (CA) and/or dibenzoxazepine (CR)).
Heart failure: A condition that occurs when the heart is no longer capable of pumping enough blood to other parts of the body. Types of heart failure include left-sided heart failure, right-sided heart failure and congestive heart failure. Left-sided heart failure is the most common type and includes heart failure with reduced ejection fraction (HFrEF) (also known as systolic failure) and heart failure with preserved ejection fraction (HFpEF) (also known as diastolic failure). With HFrEF, the left ventricles lose their ability to contract normally and the heart can't pump with enough force to circulate blood. Blood then builds up in the pulmonary veins, which causes shortness of breath. In subjects with HFpEF, the left ventricles lose their ability to relax, and the heart can't properly fill with blood between each heartbeat. Right-sided heart failure (also known as right ventricular heart failure) typically occurs as a result of left-sided heart failure. With right-sided heart failure, the heart is too weak to pump enough blood to the lungs so blood builds up in the veins. With congestive heart failure (CHF), as blood flow out of the heart slows, blood returning to the heart backs up, resulting in congestion in body tissues. CHF often results in swelling of the legs, ankles and other parts of the body.
Lung injury: In the context of the present disclosure, “lung injury” encompasses any disease, disorder, condition or injury that causes damage to the lung. Examples of lung injuries include, but are not limited to, inhalation chemical injuries, inhalation smoke injuries, injuries resulting from viral, bacterial or fungal infection, trauma or mechanical injuries. Lung injuries can result in acute respiratory distress syndrome or acute lung injury from any disease, disorder or condition, including sepsis, trauma, bacterial infection, viral infection, fungal infection and drug reaction. In some instances, lung injury is caused by pneumonia. In specific examples, the lung injury is caused by a viral infection, such as SARS-CoV-2 infection.
Nitrite: The inorganic anion−NO2 or a salt of nitrous acid (NO2−). Nitrites are often highly soluble, and can be oxidized to form nitrates or reduced to form nitric oxide or ammonia. Nitrite may form salts with alkali metals, such as sodium (NaNO2, also known as nitrous acid sodium salt), potassium and lithium, with alkali earth metals, such as calcium, magnesium and barium, with organic bases, such as amine bases, for example, dicyclohexylamine, pyridine, arginine, lysine and the like. Other nitrite salts may be formed from a variety of organic and inorganic bases. In particular embodiments, the nitrite is a salt of an anionic nitrite delivered with a cation, which cation is selected from sodium, potassium, and arginine. Many nitrite salts are commercially available, and/or readily produced using conventional techniques.
Parenteral: Administered outside of the intestine, for example, not via the alimentary tract. Generally, parenteral formulations are those that will be administered through any possible mode except ingestion. This term especially refers to injections, whether administered intravenously, intrathecally, intramuscularly, intraperitoneally, or subcutaneously, and various surface applications including intranasal, intradermal, and topical application, for instance.
Pharmaceutically acceptable carrier: The pharmaceutically acceptable carriers useful in this disclosure are conventional. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, Pa., 21st Edition (2005), describes compositions and formulations Suitable for pharmaceutical delivery of the compounds herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (for example, powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
Phosgene (carbonyl chloride, CoCl2): An industrial chemical used to make plastics and pesticides. At room temperature, phosgene is a gas. Phosgene gas can appear colorless or white to pale yellow, and has an odor of newly mown hay at low concentrations, and a stronger unpleasant odor at higher concentrations. Exposure to phosgene gas can result in damage to the skin, eyes, nose, throat and lungs. Signs of exposure include coughing, burning in the eyes and throat, watery eyes, blurred vision, difficulty breathing, nausea and vomiting. Delayed effects of phosgene gas exposure include low blood pressure, pulmonary edema and heart failure.
Preventing or treating a disease: “Preventing” a disease refers to inhibiting the full development of a disease. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
Pulmonary hypertension: Abnormally elevated blood pressure in the pulmonary circulation. Pulmonary hypertension affects the arteries in the lungs and right side of the heart. In this condition, the pulmonary arteries can become stiff, swollen and thick, which blocks or slows blood flow, leading to pulmonary hypertension.
Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified nitrite salt preparation is one in which the specified nitrite salt is more enriched than it is in its generative environment, for instance within a biochemical reaction chamber. Preferably, a preparation of a specified nitrite salt is purified such that the salt represents at least 50% of the total nitrite content of the preparation. In some embodiments, a purified preparation contains at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or more of the specified compound, such as a particular nitrite salt.
Pneumonia: An infection in one or both lungs caused by bacteria, viruses or fungi. Pneumonia causes inflammation of the alveoli of the lung, resulting in accumulation of fluid in the alveoli, which can cause difficulty in breathing.
Respiratory failure: A condition that occurs when not enough oxygen passes from the lungs into the blood. Respiratory failure can be either acute or chronic. Diseases and disorders that impair breathing can lead to respiratory failure. Examples includes chronic obstructive lung disease (COPD), pneumonia, acute respiratory syndrome, pulmonary embolism, cystic fibrosis, and acute lung injuries, such as inhalation of harmful chemicals or smoke.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): A betacoronavirus that causes coronavirus disease referred to as COVID-19 (coronavirus disease 2019). SARS-CoV-2 was first reported in Wuhan, China in late December 2019. Infection with SARS-CoV-2 can cause fever, chills, cough, pneumonia, difficulty breathing, respiratory failure, heart failure and in some cases, death.
Standard methods for detecting viral infection may be used to detect SARS-CoV-2 infection, including but not limited to, assessment of patient symptoms and background and genetic tests such as reverse transcription-polymerase chain reaction (rRT-PCR). The test can be done on patient samples such as respiratory or blood samples.
Sodium bicarbonate: A chemical compound with the formula NaHCO3. Sodium bicarbonate is also known as sodium hydrogen carbonate, baking soda and bicarbonate of soda. Sodium bicarbonate can be used as an anti-caking agent.
Subject: Living multi-cellular vertebrate organisms, a category that includes both human and veterinary subjects, including human and non-human mammals.
Therapeutically effective amount: A quantity of a compound, such as a nitrite salt, sufficient to achieve a desired effect in a subject being treated. For instance, this can be the amount necessary to treat or ameliorate a lung injury, or to measurably decrease hypertension in a subject.
An effective amount of a compound, such as a nitrite salt, can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount will be dependent on the particular compound applied, the subject being treated, the severity and type of the affliction, and the manner of administration of the compound. For example, a therapeutically effective amount of an active ingredient can be measured as the concentration (moles per liter or molar-M) of the active ingredient (such as a pharmaceutically-acceptable salt of nitrite) in blood (in vivo) or a buffer (in vitro) that produces an effect. Alternatively, the therapeutically effective amount can be measured by weight, such as in milligrams, grams or kilograms.
In some embodiments, the therapeutically effective amount is an amount sufficient to achieve about 0.5 to about 500 μM final concentration of nitrite in the circulating blood of a subject, which level can be determined empirically or through calculations. In specific examples, the concentration of nitrite in the circulation is about 1 to about 250 μM, about 2.5 to about 200 μM, about 5 to about 100 μM, or about 10 to about 50 μM. In other embodiments, the therapeutically effective amount of a nitrite salt is less than about 300 mg or less nitrite in a single dose, or a dose provided over a period of time (e.g., by infusion or inhalation). In some examples, the effective amount is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250 or about 300 mg. Specific example dosages of nitrite salts are provided herein, though the examples are not intended to be limiting. Exact dosage amounts will vary by the size of the subject being treated, the duration of the treatment, the mode of administration, and so forth.
Particularly beneficial therapeutically effective amounts of a nitrite salt (e.g., sodium nitrite), are those that are effective for treating lung injury (or another disease or condition), but not so high that a significant or toxic level of methemoglobin is produced in the subject to which the nitrite salt is administered. In specific embodiments, for instance, no more than about 25% methemoglobin is produced in the subject. In specific examples, no more than 20%, no more than 15%, no more than 10%, no more than 8% or less methemoglobin is produced, for instance as little as 5% or 3% or less, in response to treatment with the nitrite salt.
It is disclosed herein that in animal models of chlorine, bromine and/or phosgene gas poisoning, post-exposure treatment with nitrite results in significant decreases in acute lung injury and improved survival. In view of these findings, the present disclosure describes the administration of nitrite in intramuscular, intravenous, subcutaneous, oral or inhalational forms to treat post-exposure chemical lung injury due to chemical inhalation or smoke inhalation, as well lung injury resulting from an infectious disease, such as viral infection, for example SARS-CoV-2. Administration of nitrite as soon as possible post exposure to chemicals, smoke or infectious agents is proposed to improve lung injury and survival.
Provided herein are methods for the treatment of lung injury caused by chemical inhalation, smoke inhalation, viral infection, bacterial infection or fungal infection. In some embodiments, the methods include administering a therapeutically effective amount of nitrite, such as a nitrite salt, or a formulation thereof. In some examples, the chemical causing the lung injury is not chlorine (Cl2).
In some embodiments, the lung injury is caused by a chemical, and the chemical includes bromide, methyl bromide, mustard gas, nitrogen mustard, phosgene, phosgene oxime, diphosgene, phosphine, ammonia, bromine, methyl isocyanate, hydrogen chloride, osmium tetroxide, phosphorous, sulfuryl fluoride, lewisite, chloroacetophenone, chlorobenzylidenemalononitrile, chloropicrin, bromobenzylcyanide, dibenzoxazepine, or a combination or two or more, three or more or four or more thereof. In some examples, the chemical comprises bromine or phosgene gas.
In some embodiments, the lung injury is caused by smoke, and the smoke is from a fire, exhaust fumes or an explosion.
In other embodiments, the lung injury is caused by a viral infection, such as an infection by a coronavirus, for example SARS-CoV-2. In some examples, the subject being treated has pneumonia or respiratory failure resulting from SARS-CoV-2 infection. The lung injury associated with SARS-CoV-2 can cause pneumonia alterations of pulmonary hemodynamics, shock and hypoxia.
In some embodiments, the nitrite salt is sodium nitrite, potassium nitrite or arginine nitrite.
In some embodiments, the therapeutically effective amount of nitrite salt is about 5 mg to about 600 mg, such as about 10 mg to about 300 mg, about 20 mg to about 200 mg, about 30 mg to about 150 mg, about 40 mg to about 100 mg or about 50 to about 75 mg of nitrite salt. In some examples, the therapeutically effective amount is 600 mg or less, 500 mg or less, 400 mg or less, 300 mg or less, 275 mg or less, 250 mg or less, 225 mg or less, 200 mg or less, 175 mg or less, 150 mg or less, 125 mg or less, 100 mg or less, 75 mg or less, or 50 mg or less of nitrite salt.
In some examples, the therapeutically effective amount of nitrite salt is about 0.5 to about 10 mg/kg, such as about 2 to about 8 mg/kg, about 2 to about 6 mg/kg or about to about 8 mg/kg. In specific examples, the therapeutically effective amount of nitrite salt is about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kb or about 10 mg/kg. In specific non-limiting embodiments, the therapeutically effective amount of nitrite salt does not exceed 150 mg, 300 mg, 450 mg or 600 mg.
In some embodiments, the nitrite salt is administered as a formulation that includes the nitrite salt and an anti-caking agent. In some examples, the anti-caking agent is selected from any one of sodium bicarbonate, aluminum caprate, aluminum caprylate, aluminum laurate, aluminum myristate, aluminum oleate, aluminum palmitate, aluminum salts of fatty acids, aluminum stearate, calcium caprate, calcium caprylate, calcium carbonate, calcium chloride, calcium myristate, calcium oxide, calcium palmitate, calcium phosphate (dibasic or tribasic), calcium salts of fatty acids, calcium silicate, calcium stearate, calcium sulfate, carboxymethyl cellulose, carnauba wax, carrageenan, castor oil, microcrystalline cellulose, dextrin, dextrose, glycerin, glycerin monooleate, iron ammonium citrate, magnesium caprate, magnesium caprylate, magnesium carbonate, magnesium laurate, magnesium myristate, magnesium oleate, magnesium oxide, magnesium palmitate, magnesium salts of fatty acids, magnesium silicate, magnesium stearate, magnesium sulfate, maltodextrin, mannitol, potassium caprate, potassium caprylate, potassium laurate, potassium myristate, potassium palmitate, potassium permanganate, potassium salts of fatty acids, propylene glycol, silicon dioxide, sodium aluminum phosphate (acidic or basic), sodium aluminum silicate, sodium caprate, sodium caseinate, sodium chloride, sodium laurate, sodium mono- and dimethyl naphthalene sulfonates, sodium myristate, sodium oleate, sodium palmitate, sodium phosphate (monobasic, dibasic or tribasic), sodium pyrophosphate, sodium salts of fatty acids, sodium silicate, sodium stearate, sodium tripolyphosphate, tartaric acid, titanium dioxide, and xanthan gum. In specific examples, the anti-caking agent is sodium bicarbonate.
In some examples, the concentration of the anti-caking agent in the nitrite formulation is at least 20 parts per million (ppm), such as at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, or at least 50 ppm. In specific non-limiting examples, the anti-caking agent is sodium bicarbonate and the nitrite formulation includes at least 20 ppm of sodium bicarbonate.
In some embodiments, the nitrite salt is administered by an intramuscular, intravenous, subcutaneous, oral or inhalation route.
In some embodiments, the method further includes selecting a subject with a lung injury prior to administering the nitrite salt.
Also provided herein are formulations of nitrite. In some embodiments, the formulation includes a nitrite salt and an anti-caking agent. In some examples, the anti-caking agent is present at a concentration of at least 20 parts per million, such as at least 25 ppm, at least 30 ppm, at least 35 ppm, at least 40 ppm, at least 45 ppm, or at least 50 ppm.
In some embodiments of the nitrite formulation, the anti-caking agent is selected from any one of sodium bicarbonate, aluminum caprate, aluminum caprylate, aluminum laurate, aluminum myristate, aluminum oleate, aluminum palmitate, aluminum salts of fatty acids, aluminum stearate, calcium caprate, calcium caprylate, calcium carbonate, calcium chloride, calcium myristate, calcium oxide, calcium palmitate, calcium phosphate (dibasic or tribasic), calcium salts of fatty acids, calcium silicate, calcium stearate, calcium sulfate, carboxymethyl cellulose, carnauba wax, carrageenan, castor oil, microcrystalline cellulose, dextrin, dextrose, glycerin, glycerin monooleate, iron ammonium citrate, magnesium caprate, magnesium caprylate, magnesium carbonate, magnesium laurate, magnesium myristate, magnesium oleate, magnesium oxide, magnesium palmitate, magnesium salts of fatty acids, magnesium silicate, magnesium stearate, magnesium sulfate, maltodextrin, mannitol, potassium caprate, potassium caprylate, potassium laurate, potassium myristate, potassium palmitate, potassium permanganate, potassium salts of fatty acids, propylene glycol, silicon dioxide, sodium aluminum phosphate (acidic or basic), sodium aluminum silicate, sodium caprate, sodium caseinate, sodium chloride, sodium laurate, sodium mono- and dimethyl naphthalene sulfonates, sodium myristate, sodium oleate, sodium palmitate, sodium phosphate (monobasic, dibasic or tribasic), sodium pyrophosphate, sodium salts of fatty acids, sodium silicate, sodium stearate, sodium tripolyphosphate, tartaric acid, titanium dioxide, and xanthan gum. In specific examples, the anti-caking agent is sodium bicarbonate. In specific non-limiting examples, the anti-caking agent is sodium bicarbonate and the nitrite formulation includes at least 20 ppm of sodium bicarbonate.
In some embodiments, the formulation includes a phosphate buffered solution, a lactated Ringer's solution or physiological saline. In some examples, the phosphate buffered solution, lactated Ringer's solution or physiological saline is sterile. In some examples, the phosphate buffer is comprised of sodium hydroxide, phosphoric acid, sodium phosphate (monobasic; dibasic or a combination thereof), or any combination thereof.
In some embodiments, the formulation includes water.
In some embodiments, the nitrite salt of the formulation is sodium nitrite, potassium nitrite or arginine nitrite.
In some embodiments, the concentration of nitrite salt in the formulation is about 30 mg/ml, about 40 mg/ml, about 60 mg/ml or about 80 mg/ml. In some embodiments, the formulation is provided in unit dose form. In some examples, such as when the formulation is administered intramuscularly, the total volume of the unit dose is about 0.5 ml, about 1 ml, about 3 ml, about 5 ml, about 8 ml, about 10 ml or about 20 ml.
Further provided are methods of treating a disease or condition in a subject by administering to the subject a therapeutically effective amount of a nitrite formulation disclosed herein. In some embodiments, the disease or condition is selected from a lung injury, pulmonary hypertension, heart failure, cardiogenic shock, hypertension, respiratory failure, a metabolic syndrome, diabetes, a lipid disorder, an endocrine disorder, a gastroenterological disorder, hypoperfusion, inflammation, cystic fibrosis and aging.
In some embodiments, the disease or condition is a lung injury and the lung injury is caused by inhalation of a chemical, inhalation of smoke, SARS-CoV-2 infection, trauma or mechanical injury. In some examples, the lung injury is caused by a chemical, and the chemical includes bromide, methyl bromide, mustard gas, nitrogen mustard, phosgene, phosgene oxime, diphosgene, phosphine, ammonia, bromine, methyl isocyanate, hydrogen chloride, osmium tetroxide, phosphorous, sulfuryl fluoride, lewisite, chloroacetophenone, chlorobenzylidenemalononitrile, chloropicrin, bromobenzylcyanide, dibenzoxazepine, or a combination or two or more, three or more or four or more thereof. In some examples, the chemical comprises bromine or phosgene gas.
In some examples, the lung injury is caused by smoke, and the smoke is from a fire, exhaust fumes or an explosion. In other examples, the lung injury is caused by a viral infection, such as an infection by SARS-CoV-2. In particular non-limiting examples, the subject being treated has pneumonia or respiratory failure resulting from SARS-CoV-2 infection.
In some embodiments of the method, the disease or condition comprises pulmonary hypertension and heart failure with a preserved ejection fraction (HFpEF).
In other embodiments, the disease or condition is heart failure, and the heart failure includes right-sided heart failure, left-sided heart failure or congestive heart failure.
In other embodiments, the disease or condition is hypertension, and the hypertension includes chronic hypertension, acute hypertension, urgency hypertension, emergency hypertension, prehypertension, or a combination thereof.
In other embodiments, the disease or condition is respiratory failure and the respiratory failure comprises acute respiratory distress syndrome, respiratory failure from trauma or mechanical injury, respiratory failure due to pneumonia, respiratory failure due to trauma, respiratory failure due to lung transplantation, respiratory failure due to lung transplantation rejection (acute and chronic), respiratory failure resulting from an infectious disease, respiratory failure resulting from pulmonary edema, respiratory failure resulting from pulmonary embolism, respiratory failure due to infant respiratory distress syndrome (IRDS; also known as neonatal respiratory distress syndrome (NRDS), respiratory distress syndrome of newborn, persistent pulmonary hypertension of the newborn (PPHN) and surfactant deficiency disorder (SDD), respiratory failure due to interstitial lung disease, or respiratory failure resulting from an autoimmune disease.
In some embodiments, the disease or condition is a lipid disorder and the lipid disorder includes hypercholesterolemia, hypertriglyceridemia, or both.
In some embodiments, the method further includes selecting a subject with a lung injury, pulmonary hypertension, heart failure, cardiogenic shock, hypertension, respiratory failure, a metabolic syndrome, diabetes, a lipid disorder, an endocrine disorder, a gastroenterological disorder, hypoperfusion, inflammation, cystic fibrosis or aging prior to administering the formulation.
Also provided herein is a method of enhancing cardiovascular performance in a subject by administering to the subject a therapeutically effective amount of the nitrite formulation disclosed herein.
Further provided is a method of providing a nutritional supplement to a subject, by administering to the subject the nitrite formulation disclosed herein.
The present disclosure further contemplates intramuscular, intravenous, subcutaneous, oral or inhalational administration of the disclosed nitrite formulations during the perioperative period. Also contemplated are use of the disclosed nitrite formulations as an antidote (such as an antidote for cyanide poisoning); use of the disclosed nitrite formulations as dietary supplements; use of the disclosed nitrite formulations to enhance cardiovascular performance; use of the disclosed nitrite formulations to improve oxygen delivery; use of the disclosed nitrite formulations to treat microbial infections and/or to inhibit microbial (such as bacterial) activity; and use of the disclosed nitrite formulations for alteration of the microbiome.
In some embodiments, the subject is administered an effective amount of the nitrite formulation, wherein the effective amount contains about 5 mg to about 600 mg, such as about 10 mg to about 300 mg, about 20 mg to about 200 mg, about 30 mg to about 150 mg, about 40 mg to about 100 mg or about 50 to about 75 mg of nitrite salt. In some examples, the effective amount contains 600 mg or less, 500 mg or less, 400 mg or less, 300 mg or less, 275 mg or less, 250 mg or less, 225 mg or less, 200 mg or less, 175 mg or less, 150 mg or less, 125 mg or less, 100 mg or less, 75 mg or less, or 50 mg or less of nitrite salt. In some examples, the effective amount contains about 0.5 to about 10 mg/kg, such as about 2 to about 8 mg/kg, about 2 to about 6 mg/kg or about to about 8 mg/kg of nitrite salt. In specific examples, the effective amount contains about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kb or about 10 mg/kg of nitrite salt. In specific non-limiting embodiments, the effective amount does not exceed 150 mg, 300 mg, 450 mg or 600 mg of nitrite salt.
In some embodiments, the concentration of nitrite salt in the nitrite formulation is about 30 mg/ml, about 40 mg/ml, about 60 mg/ml or about 80 mg/ml. In some embodiments, the formulation is provided in unit dose form. In some examples, such as when the formulation is administered intramuscularly, the total volume of the unit dose is about 0.5 ml, about 1 ml, about 3 ml, about 5 ml, about 8 ml, about 10 ml or about 20 ml.
Common features of injury caused by exposure to reactive gases include acute injury to point of contact areas (airways, skin) that is followed by acute and chronic inflammatory injury to the cardiopulmonary system. Exposure to chlorine (Cl2), bromine (Br2), or phosgene (COCl2) forms intermediates that cause red blood cell (RBC) hemolysis (Aggarwal et al., Toxicol Lett 312: 204-213, 2019; Aggarwal et al., JCI Insight 3(21), doi: 10.1172/jci.insight.120694, 2018; Aggarwal et al., Antioxid Redox Signal 24(2): 99-112, 2015). Released free hemoglobin and heme collectively decrease nitric oxide (NO) bioavailability, and promote mitochondrial damage, inflammation and oxidative stress. NO is an endogenous anti-inflammatory agent. While NO-production is regulated by NO-synthase enzymes, data indicate non-NO synthase sources of NO are also significant in mammals, specifically from dietary nitrate present in “healthy diets” comprising green leafy and root vegetables. Upon ingestion, nitrate is concentrated 10-20 fold into the saliva, where it is reduced to nitrite by commensal lingual nitrate-reductase (NR) expressing bacteria. Nitrite is swallowed, increases in the plasma and then mediates NO-signaling in all major tissues by a further 1-electron reduction process. Thus, it is believed that oral nitrate-reducing bacteria are significant mediators of physiologic mammalian NO-homeostasis.
A. Halogen and Phosgene Toxicity Mechanisms
Chlorine (Cl2) and bromine (Br2) are halogens used in various industrial processes and consequently stored and transported in large amounts. Phosgene (COCl2) is a high irritant gas currently used in the production of dyes, pesticides, and plastics, and is a breakdown product of chloroform. Exposure to COCl2 causes lung injury and inflammation (Aggarwal et al., Toxicol Lett 312: 204-213, 2019).8-10 Cl2, Br2 and COCl2 are all inhaled chemical threat agents. Accidental exposure in these settings, as well as intentional exposure in the military arena, have been documented, with facilities producing these gases considered by the Department of Homeland Security at high risk for terrorist attacks. The acute and chronic health effects of Cl2, Br2 or COCl2 exposure remain under investigation. During exposure to these gases, injury to the airways occurs and is then followed, over hours to days, by further airway/alveolar epithelial injury and pulmonary and systemic endothelial injury.11-13 Collectively, this manifests as progressively worsening acute lung injury (ALI), development of reactive airways, pulmonary and systemic vascular dysfunction (blood flow) and impaired cardiac contractility. While chemically distinct, the post-exposure injury phenotype is similar for all three gases. Moreover, the molecular and biochemical mechanisms underlying injury are believed to be similar. The present disclosure describes the finding that Cl2, Br2 or COCl2 exposure results in hemolysis; the released hemoglobin/heme then promotes post-exposure toxicity by promoting oxidative stress and inflammation in the lung, inhibiting nitric oxide (NO) bioavailability and causing mitochondrial DNA damage and cellular bioenergetic dysfunction.
B. NO-Homeostasis Mechanisms: Role of Diet and the Oral Microbiome
One contributing mechanism to post-Cl2 induced toxicity is dysregulation of endogenous nitric oxide (NO)-formation6,12, but mechanisms remain to be elucidated. Nitric oxide mediates vasodilation, limits inflammation and coagulation, and regulates cell death, proliferation and mitochondrial function14,15. Consequently, inhibition of this pathway predisposes to and/or leads to numerous acute and chronic inflammatory diseases. While NO-formation from nitric oxide synthases is well-accepted, it is now evident that mammalian NO-signaling is also regulated by non-enzymatic sources of NO. Typically, NO is thought to be inactivated by its oxidation to nitrite and nitrate. However, nitrite is not inert, and can be reduced (by 1-electron) back to NO or other NO-containing intermediates (e.g. S-nitrosothiols) during inflammation and hypoxia16-20. In this model, endogenous or therapeutic nitrite is a source of NO-signaling equivalents, especially during stress when endogenous NOS-dependent NO-formation is inhibited (e.g. after halogen exposure).
Furthermore, it is also clear that in mammals, nitrate is not an inert NO-oxidation product. Specifically, nitrate, which is formed endogenously and ingested in foods, is concentrated 10-20 fold (resulting in mM concentrations) in saliva21-26. Here, nitrate is reduced to nitrite by oral bacteria expressing nitrate-reductases27-34. Produced nitrite is swallowed, where it can elicit NO-signaling in the acidic stomach, but also enters the plasma and provides substrate for NO-formation via nitrite-reduction in all tissues as outlined above24,35-37. Thus, there exists a symbiotic relationship between oral microbes that use nitrate as a respiratory source, and the mammalian host, that is critical to NO-homeostasis.
C. Toxic Gas Exposure and Use of Nitrite
Cl2, Br2 and COCl2 cause hemolysis in mice, rats, and guinea pigs5,7,38,39. Moreover, transient drops in RBC count, likely due to hemolysis, can occur following human exposure to COCl240. The present disclosure investigates the hypothesis that NR containing bacteria present on the posterior tongue mediate NO-bioavailability in vivo (
The present disclosure further describes the use of nitrite as a treatment for inhalation chemical and smoke injuries. Low-dose nitrite therapy protects against injury caused by ischemia and inflammation (reviewed in18,60-64) Underlying this therapeutic efficacy is nitrite-dependent repletion of NO-signaling62-63,65-75. Developing a targeted efficacious therapy that is amenable to rapid administration in mass casualty scenarios is a focus of the CounterAct network. Importantly, nitrite can be stockpiled, is stable, is amenable to IM administration, and is an active ingredient in FDA-approved cyanide antidote kits. Nitrite has also undergone successful phase I/II studies for indications related to systemic/pulmonary hypertension and ischemia reperfusion injury46,76-80.
Embodiment 1. A method of treating a lung injury in a subject, wherein the lung injury is caused by inhalation of a chemical, inhalation of smoke, or SARS-CoV-2 infection, comprising administering to the subject a therapeutically effective amount of a nitrite salt, wherein the chemical is not chlorine, thereby treating the lung injury.
Embodiment 2. The method of embodiment 1, wherein the chemical comprises bromide, methyl bromide, mustard gas, nitrogen mustard, phosgene, phosgene oxime, diphosgene, phosphine, ammonia, bromine, methyl isocyanate, hydrogen chloride, osmium tetroxide, phosphorous, sulfuryl fluoride, lewisite, chloroacetophenone, chlorobenzylidenemalononitrile, chloropicrin, bromobenzylcyanide, dibenzoxazepine, or a combination or two or more thereof.
Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the chemical comprises bromine or phosgene gas.
Embodiment 4. The method of embodiment 1, wherein the smoke is from a fire, exhaust fumes or an explosion.
Embodiment 5. The method of embodiment 1, wherein the subject has pneumonia resulting from SARS-CoV-2 infection.
Embodiment 6. The method of any one of embodiments 1-5, wherein the nitrite salt comprises sodium nitrite, potassium nitrite or arginine nitrite.
Embodiment 7. The method of any one of embodiments 1-6, wherein the therapeutically effective amount of nitrite salt is about 5 mg to about 600 mg.
Embodiment 8. The method of any one of embodiments 1-7, wherein the nitrite salt is administered as a formulation comprising the nitrite salt and an anti-caking agent.
Embodiment 9. The method of embodiment 8, wherein the concentration of the anti-caking agent in the formulation is at least 20 parts per million.
Embodiment 10. The method of embodiment 8 or embodiment 9, wherein the anti-caking agent comprises sodium bicarbonate.
Embodiment 11. The method of any one of embodiments 1-10, wherein the nitrite salt is administered by an intramuscular, intravenous, subcutaneous, oral or inhalation route.
Embodiment 12. The method of any one of embodiments 1-11, further comprising selecting a subject with a lung injury prior to administering the nitrite salt.
Embodiment 13. A formulation comprising a nitrite salt and an anti-caking agent, wherein the anti-caking agent is present at a concentration of at least 20 parts per million.
Embodiment 14. The formulation of embodiment 13, wherein the nitrite salt is sodium nitrite, potassium nitrite or arginine nitrite.
Embodiment 15. The formulation of embodiment 13 or embodiment 14, wherein the anti-caking agent comprises sodium bicarbonate.
Embodiment 16. A method of treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the formulation of any one of embodiments 13-15, wherein the disease or condition is selected from a lung injury, pulmonary hypertension, heart failure, cardiogenic shock, hypertension, respiratory failure, a metabolic syndrome, diabetes, a lipid disorder, an endocrine disorder, a gastroenterological disorder, hypoperfusion, inflammation, cystic fibrosis and aging.
Embodiment 17. The method of embodiment 16, wherein the lung injury is caused by inhalation of a chemical, inhalation of smoke, or SARS-CoV-2 infection.
Embodiment 18. The method of embodiment 16, wherein the disease or condition comprises pulmonary hypertension and heart failure with a preserved ejection fraction (HFpEF).
Embodiment 19. The method of embodiment 16, wherein the heart failure comprises right-sided heart failure, left-sided heart failure or congestive heart failure.
Embodiment 20. The method of embodiment 16, wherein the hypertension comprises chronic hypertension, acute hypertension, urgency hypertension, emergency hypertension, prehypertension or a combination thereof.
Embodiment 21. The method of embodiment 16, wherein the respiratory failure comprises acute respiratory distress syndrome, respiratory failure from trauma or mechanical injury, respiratory failure due to pneumonia, respiratory failure resulting from an infectious disease, respiratory failure resulting from pulmonary edema, respiratory failure resulting from pulmonary embolism, respiratory failure due to infant respiratory distress syndrome, respiratory failure due to interstitial lung disease, or respiratory failure resulting from an autoimmune disease.
Embodiment 22. The method of embodiment 16, wherein the lipid disorder comprises hypercholesterolemia, hypertriglyceridemia, or both.
Embodiment 23. The method of any one of embodiments 16-22, further comprising selecting a subject with a lung injury, pulmonary hypertension, heart failure, cardiogenic shock, hypertension, respiratory failure, a metabolic syndrome, diabetes, a lipid disorder, an endocrine disorder, a gastroenterological disorder, hypoperfusion, inflammation, cystic fibrosis or aging prior to administering the formulation.
Embodiment 24. A method of enhancing cardiovascular performance in a subject, comprising administering to the subject a therapeutically effective amount of the formulation of any one of embodiments 13-15.
Embodiment 25. A method of providing a nutritional supplement to a subject, comprising administering to the subject the formulation of any one of embodiments 13-15.
The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the disclosure to the particular features or embodiments described.
This example describes studies to evaluate the use of nitrite for treating chemical lung injury.
Cl2/Br2/CoCl2 Exposure: All exposures (whole body) were performed as previously described (Honavar et al., Am J Physiol Lung Cell Mol Physiol 307: L888-894, 2014). After exposure, mice were housed singly to limit microbiome impacting variables.
Nitrite and nitrite measurement: Nitrite and nitrate were measured by HPLC based Griess reaction (Eicom) as described6,12. For plasma and saliva, nitrite and nitrate were extracted by methanol as described103.
Bacterial colony growth: were assessed as described4,29. Swab samples were serially diluted in tryptic soy broth, plated on agar plates and incubated for 12-24 h at 37° C. Defined colonies were counted by microscopy. Also, samples were diluted into nitrate medium (BD Gifco) to selectively assess denitrifying bacterial growth.
Sequencing: DNA was extracted from bacterial culture with DNA Isolation Kit (Zymo Research, cat. no. D6010). Amplicon library from individual samples was prepared by PCR using unique barcoded primers to amplify the hypervariable region V4 of the 16S rRNA. The PCR products from individual samples were run into and excised from agarose gel followed by purification using a commercial gel extraction kit. Purified PCR products were sequenced using the NextGen sequencing Illumina MiSeq platform. The microbiome analysis package, “Quantitative Insights Into Microbial Ecology”, was used for microbiome analysis as described104.
Nitrate-dependent modulation of NO-bioavailability, via the pathway described in
To test whether halogen gas exposure inhibits the enterosalivary nitrate-nitrite-NO pathway, mice were exposed to air or Br2 gas (400 ppm, 30 minutes) and then brought back to room air.
The following study is carried out to determine if Cl2, Br2 or COCl2 gas inhibit oral nitrate reductase activity ex vivo. Eight-week old male and female C57Bl/6 mice are exposed to room air, C12 (400 ppm for 30 minutes or 600 ppm for 30-45 minutes), Br2 (400 or 600 ppm, 30-45 minutes) or COCl2 (10 ppm or 20 ppm, 10 minutes), and then brought back to room air. Exposure conditions are based on prior publications14-7 and
The following study is carried out to determine if Cl2, Br2 or COCl2 inhibits oral NR activity in vivo. To assess whether inhibition of oral NR activity results in a loss of NO-bioavailability, the pathway outlined in
To further isolate the enterosalivary system, the study includes a group in which mice are treated with L-NAME to inhibit all nitric oxide synthases 1 hour prior to testing of nitrate-dependent effects. In addition, and to complement these studies, effects of Cl2, Br2 or COCl2 exposure on lung and aortic NOS activity are determined using enzyme activity assays, functional assays (aortic ring dilation) and expression (western blotting), as described previously6,12 Finally, a positive control group is included to ensure loss of oral NR activity. Specifically, the posterior tongues of air exposed mice are treated with water or chlorhexidine as described in Example 2 below (see
The following studies are carried out to determine how Cl2, Br2 or COCl2 inhibits oral NR activity. These studies will determine whether decreased NR activity post Cl2, Br2 or COCl2 exposure is due to lower bacterial number, and/or due to inhibition of NR activity without bacterial killing (i.e. lower specific activity). The studies will also profile the oral nitrate-reducing microbiota and assess if this is reprogramed by toxic gas exposure.
Mice are exposed to air or Cl2, Br2 or COCl2 using the dose that each resulted in maximal inhibition of NR activity. Mice are brought back to air and tongues are collected at various times post-exposure (6 hours, 24 hours, 48 hours, 72 hours, 4 days, 7 days) to assess acute and chronic changes in microbiome diversity. A pre-collection (1 day prior to Cl2 exposure) time point is also included. Tongues are scraped to collect resident bacteria. Since qPCR measures both live and dead cell derived DNA, bacterial number is determined using colony growth assays. NR activity is also determined and normalized to bacterial number to assess specific activity.
To profile the oral nitrate-reducing microbiota, total DNA is isolated, followed by PCR amplification of the V4 region of the rRNA gene and NextGen sequencing of the PCR product to assess microbial diversity and taxonomic identification. The goal is to determine how toxic gas exposure changes the oral microbial community and evaluate whether it is reprogrammed with specific attention to communities where oral nitrate-reducing activity resides. To this end, the distal dorsal surface of the tongue where NR bacteria reside are sampled (see Fanucchi et al., Am J Respir Cell Mol Biol 46:599-606, 2012). To compare, the proximal (front) of the tongue is also sampled to determine whether the effects of each gas on nitrate-reducing bacteria is dependent on lingual location.
Additionally, little is known on how oral microbial diversity is affected by diet, housing and other factors including mice being coprophagic. Therefore, mice are housed one per cage after air or toxic gas exposure to limit possible variability associated with these factors. Finally, samples of lung tissue, upper GI and lower GI tracts, and fecal matter are samples and microbiome sequencing is performed to compare with the composition of oral microbes. This will determine whether toxic gas exposure effects on the microbiome are limited to the oral cavity. Data shown in
Finally, due to differences between mouse and human microbiomes, and to assess whether the findings are translatable to humans, swabs from the back of the tongue are collected from ten male and ten female healthy age-matched human volunteers, cultured as described in methods below for 18 hours and then exposed in vitro to air, Cl2, Br2 (each at 0, 200, 400, 600 ppm) for 0-30 minutes, or COCl2 (10 or 20 ppm, 0-15 minutes). At various times post-exposure, bacterial number, diversity and NR activity are determined.
The data described in this example demonstrate that factors negatively affecting the enterosalivary NR system, namely low nitrate in the diet and/or lower levels of nitrate-reducing bacteria, pre-dispose mice to the injurious effects of hemolysis resulting in greater lung injury and toxicity after exposure to Cl2, Br2 or COCl2.
First, the effects of selective depletion of the oral microbiome on Cl2, Br2 or COCl2 toxicity was tested. For these studies, a method was developed for inhibiting NR activity in the oral cavity. C57Bl/6 mice were briefly anesthetized using ketamine/xylazine and a small volume (10 μl) of chlorhexidine (0.2%) or water (control) applied topically to the back of the tongue twice per day for 7 days. This protocol was optimized to ensure only the posterior tongue was exposed to chlorhexidine, and showed that compared to vehicle control, NR activity was inhibited by ˜70% (
In the next series of experiments, after chlorhexidine treatment, mice were exposed to Cl2, Br2 or COCl2 and acute lung injury (ALI) was assessed.
Next, mice were placed on control diet (that contains nitrite+nitrate), an isocaloric low nitrate diet (50% lower nitrite+nitrate content); low nitrate diets were formulated to ensure similar nutritional content between groups as described59.
Determine if the Oral Microbiome Limits Cl2, Br2 or COCl2 Gas Toxicity
Chlorhexidine (10 μl, 0.2% in water) or vehicle control is applied topically on the posterior tongues of C57Bl/6 male and female mice 2x per day, for 7 days, to inhibit oral NR activity. Effectiveness of chlorhexidine treatment is assessed by measuring oral NR activity, microbial viability and diversity, and plasma nitrite and nitrate. Then mice are exposed to air, Cl2 (400 ppm, 30 minutes) Br2 (400 ppm, 30 minutes) or COCl2 (10 ppm, 10 minutes) and the following variables are measured at 6 hours, 24 hours, and 48 hours post-exposure (i) ALI, (ii) airway reactivity to methacholine, (iii) eNOS-dependent dilation in aortas (which is inhibited in Cl2 exposed mice12), and (iv) susceptibility to P. Aeruginosa infection. Separately, mice are exposed to C12 (600 ppm, 45 minutes), Br2 (600 ppm, 45 minutes), or COCl2 (20 ppm, 10 minutes) and 24 hour survival is determined. Previous studies with COCl2 have established ˜50% mortality for these gases using these exposure conditions1,4-7. It is expected that mice in which their oral NR activity has been depleted will display a greater severity of post-toxic gas exposure toxicities.
Determine if Low NOx Diet Predisposes to Greater Cl2, Br2 or COCl2 Induced Toxicity
Eight week old C57bl/6 male and female mice are fed regular chow, an isocaloric low nitrate diet, or an isocaloric low nitrate diet with nitrate supplemented drinking water, for 14 days prior to exposure to Cl2, Br2 or COCl2 using conditions that will allow testing of acute lung injury and 24 hour survival end points, as described above. Nitrate in the drinking water is titrated to restore circulating levels observed in the normal chow group. Consumption of food, water, and animal weight, oral NR activity and plasma nitrate and nitrite levels are measured every 3 days. It is expected that low NOx diet groups will show greater lung and systemic vascular injuries compared to normal diet, and that nitrate in the drinking water will prevent this. It is further expected that these results will be highly significant due to the relatively low nitrate, and hence lower plasma nitrite levels in individuals consuming typical western and nutrient poor diets.
Determine the Effects of Low NOx Diet and Oral-Microbial Disruption on Cl2, Br2 or COCl2 Induced Toxicity
The studies above test whether selective depletion of oral cavity dorsal lingual bacteria, or nitrate from the diet exacerbate toxic gas injury. In this study, it will be determined if the combination of these interventions leads to further increases in injury. C57Bl/6 male and female mice are placed on normal chow, low nitrate diet or low nitrate diet+nitrate in the drinking water for 2 weeks. Over the last 7 days, mice are further split into groups to receive vehicle or oral chlorhexidine and then Cl2, Br2, COCl2 lung injury (using one end-point showing greatest sensitivity) and mortality determined. It is expected that injury will be most severe in combined low NOx and chlorhexidine groups, and that protective effects of nitrate in the drinking water will be lost in mice treated with chlorhexidine due to an inability to reduce nitrate to nitrite.
Determine the Mechanism(s) by which Inhibition of the Enterosalivary Nitrate-Reduction System Increases Cl2, Br2, COCl2 Toxicity
NO is an antioxidant, and an anti-inflammatory agent. It is hypothesized that exacerbated injury in mice with an inhibited enterosalivary NR system is due to a greater magnitude of damage along the pathway of Cl2, Br2, COCl2 derived oxidized or halogenated lipids, hemolysis, and free hemoglobin/free heme-dependent oxidative and inflammatory injury to the lung. To test where along this pathway NO exerts protective actions, conditions will be used that are expected to show maximal exacerbation of injury caused by each gas and the following will be tested:
i) Measure Cl-lipids, Br-lipids or oxidized lipids in the lung and plasma immediately following, and 6 hours and 24 hours post-exposure.
ii) Measure plasma and BAL free hemoglobin, heme, non-transferrin bound iron (NTBI), hemopoexin and haptoglobin. A spectral deconvolution assay is used for Hb and heme107. In addition, it is tested whether RBC fragility to osmotic and mechanical stress is enhanced in mice with inhibited NR activities; prior studies have shown NO improves RBC deformability108.
iii) Measure indices of oxidative and inflammatory injury including F2-isoprostanes, ferrylHb and protein oxidation; these parameters report on hemolysis-derived products and lipid peroxidation and are measured using protocols described in published studies109,110. Heme is also a TLR4 ligand and downstream inflammatory pathways are inhibited by NO99. To test this, NF-κB signaling and candidate target genes including ICAM-1 and VCAM1 are tested by Western blotting. It is hypothesized that heme-dependent TLR4 activation will be greater in low nitrate diet and chlorhexidine treated mice. Data suggest heme-mediated cellular injury via inducing mitochondrial DNA damage. This is assessed by measuring circulating mitochondrial DNA fragments.
iv) Test whether low nitrate diet and chlorhexidine treatment sensitizes mice to exogenous hemoglobin and heme-dependent ALI, and increased P. Aeruginosa infection independent of toxic gas exposure. Specifically, oxyhemoglobin (0-50 μM), methemoglobin (0-50 μM) or heme (0-50 μM) are administered intratracheally as previously described” and ALI measured (see below) at 6 hours, and compared to vehicle controls. End points related to oxidative and inflammatory stress are also measured.
Acute lung injury (ALI) was assessed by measuring: (i) BAL changes in protein, inflammatory cells and cytokines112,113. Mice were euthanized with IP ketamine/xylazine (100 and 10 mg/kg body weight) and a 3 mm endotracheal cannula was inserted in their tracheas. Lungs were lavaged with 2 ml of 0.9% NaCl three times. Recovered lavage fluid was centrifuged immediately at 300 g, 10 minutes to pellet cells. (ii) Wet:dry weight ratios: bloodless lung wet/dry weight ratios were determined as previously described113. (iii) Arterial blood gases, oxygen saturation and pH were measured as previously described. (iv) Airway/alveolar morphological evaluation of injury to the alveolar and mid-upper airways was made as recently described112. Specific measurements included assessment of apoptosis and necrosis, and pathology scoring, all performed in a blinded manner
Airway reactivity and chronic lung injury was measured under basal and methacholine challenge by Flexivent112.
Systemic endothelial function was assessed using ex vivo vasodilation studies and eNOS expression/activity measurements in isolated aorta as described12,66. Each experiment determined vessel contractility to increasing doses of phenylephrine followed by assessment of eNOS-dependent vasodilation using acetylcholine, and then endothelium independent vasodilation determined using Mahma nonoate.
Cl-/Br-lipids and oxidized lipids were measured as previously described3,105,114. Sample processing protocols that avoid artefactual oxidation were used including using metal chelators, BHT and limiting light exposure.
Hemolysis-derived mediators: Free hemoglobin, heme, non-transferrin bound iron were measured according to standard procedures. Haptoglobin and hemopexin were measured by ELISA as described96,107,115.
IM injection of nitrite, post-Cl2 gas exposure, decreases ALI and reactive airways in mice and rats1-3. Nitrite is chemically stable, can be stockpiled, and is amenable to administration in mass-casualty scenarios. Since hemolysis is a common injury causing pathway for Cl2, Br2 and COCl2, it was hypothesized that nitrite may be protective against Br2 and COCl2 as well. The studies in this example test whether a single IM injection of nitrite can afford protection against Br2 and COCl2 toxicity, and whether the efficacy of nitrite-cytoprotection is improved by combining with other therapeutics, specifically hemopexin, that also protect against hemolysis-dependent injury.
Additional studies in this example will determine if nitrite-therapy is effective in susceptible populations (e.g., mice with decreased NO bioavailability), and test whether efficacy is similar in males vs. females. The latter is an important consideration as studies show nitrite is a more potent anti-platelet effector in male subjects44. Consistent with this concept, it was observed that higher doses of nitrite are required to improve post-Cl2 gas survival in female mice compared to male mice.
Determine if Post-Exposure IM Injection of Nitrite Protects Against Cl2, Br2 or COCl2 Induced Toxicity
This study focuses on two areas: (i) Optimizing nitrite dosing and timing of nitrite administration post-toxic gas exposure, and (ii) optimizing these parameters in males vs. females exposed to Br2 or COCl2. Testing of nitrite against Cl2 has been completed (see1-3 and
Finally, using conditions defined in Examples 1-2 that show the most severe ALI (e.g., in low NOx diet and chlorhexidine treated mice), nitrite therapy dosing studies are performed to assess whether this will be different in populations more susceptible to injury due to decreased basal NO-bioavailability.
Determine Mechanisms by which Nitrite Protects Against Br2 or COCl2 Toxicity
This study aims to define the mechanism by which nitrite therapy is protective. Understanding the mechanism(s) will aid development of nitrite-therapy and provide insights into why nitrite is more potent in males compared to females. In Example 1, it was hypothesized that loss of NO exacerbates inflammatory and oxidative stress. In this study, it is expected that nitrite-reduction to NO prevents this 16,17,19,23,116
Using the nitrite dose that conferred maximal protection against Br2, and COCl2, studies will assess (i) whether nitrite-mediates protection via anti-inflammatory mechanisms and measure expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1 and P-selectin) and pro-inflammatory cytokines (IL1β, IL-6, IFN-γ, MCP-1 and TNFα) in the lungs by ELISA and/or Western blotting. Also, PMN depletion prevents protection mediated by nitrite towards Cl2 gas toxicity1. Thus, PMN is depleted using IP injection with 200 μg of anti Ly-6G (clone 1A8) (Bxcel: cat# BE0075-1) and compared to IgG2a Isotype control (Bxcel: cat# BE0089) 24 hr prior to Br2 or COCl2 gas exposure. (ii) Whether nitrite-mediated protection occurs via anti-oxidant effects: This is tested by measuring markers of reactive oxygen, nitrogen and bromine species in the lung and plasma including F2-isoprostanes, protein carbonyl, 3-nitrotyrosine and bromotyrosine adducts114. (iii) If nitrite-mediated protection occurs via preventing cell death. Apoptosis is determined in the lung by TUNEL staining. Anti-apoptotic effect is verified by measuring caspase-3 activity in the lung sections by immunofluorescence using anti-active caspase-3 antibodies. (iv) Determine NO-metabolite profile and pharmacokinetics after nitrite administration. Lungs, aorta and blood are collected for assessment of NO-metabolites (nitrite, nitrate, S-nitrosothiols, C—N-nitroso and heme nitrosyl). the pattern of NO-metabolites provides insights into nitrite-reactivity associated with cytoprotection and therefore insights into mechanism. Tissue is collected and immediately processed in ‘stabilization solutions’, that are required to stabilize different NO-metabolites and prevent artefactual inter-conversion between them103 (v) Test if the protective effects of nitrite are meditated by NO formation. Mice are treated with the NO-scavenger C-PTIO (1 mg/Kg) or saline vehicle immediately (1-2 minutes) before nitrite administration. Control studies include addition of C-PTIO alone to air, Br2 or COCl2 gas exposed mice. (vi) Determine effects of nitrite on halogenated lipids. It is possible that nitrite, via stimulation of blood flow and perfusion facilitates halogenated lipid clearance and thereby decreases the effects of these species as post-exposure injury mediators. Br- and oxidized lipids are measured by GC-MS. (vii) Determine effects of nitrite on heme and iron. Hemopexin protects against increased infection risk post Br2 exposure, implicating a role for free heme. Free heme and non-transferrin bound iron levels, hemopexin and haptoglobin are measured to specifically assess effects of nitrite on hemolysis.
Assess the Therapeutic Efficacy of Nitrite in Combination with Hemopexin
Data show that individually, nitrite or hemopexin administered post-Cl2, Br2, or COCl2 exposure protects against ALI and P. aeruginosa infection (
In view of the many possible embodiments to which the principles of the disclosed subject matter may be applied, it should be recognized that the illustrated embodiments are only examples of the disclosure and should not be taken as limiting the scope of the disclosure. Rather, the scope of the disclosure is defined by the following claims. We therefore claim all that comes within the scope and spirit of these claims.
This application claims the benefit of U.S. Provisional Application No. 63/014,902, filed Apr. 24, 2020 and U.S. Provisional Application No. 62/896,419, filed Sep. 5, 2019, both of which are herein incorporated by reference in their entirety.
This invention was made with government support under grant numbers ES023759 and ES02645808 awarded by the National Institutes of Health. The government has certain rights in the invention.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/US2020/049551 | 9/4/2020 | WO |
Number | Date | Country | |
---|---|---|---|
63014902 | Apr 2020 | US | |
62896419 | Sep 2019 | US |