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`S:—COMBE
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`UNITED STATES DEPARTM TOF COMMERCE
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`United States Patent anc! Trademark CHYVice
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`PatXML
`
`Description
`
`\
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`1/28
`
`MH57154
`
`New use of nitrites and nitrates and compositions containing these
`
`Technical Field
`
`[0001] This invention relates to the field of medicine and pharmaceuticals, in
`
`particular pharmaceuticals and therapeutic methods for lowering metabolic
`
`rate, oxygen consumption and/or glucose homeostasis in a human patient
`
`or another mammal, based on the administration of nitrates and/or nitrites
`
`to said patient or mammal.
`
`Background Art
`
`[0002] Nitrate (N03-) and nitrite (NO£) are generally viewed as unwanted
`residues in the food chain with potentially harmful effects ( Joint
`
`FAO/WHOExpert Committee on Food Additives (JECFA). Safety
`
`Evaluation of Certain Food Additives. WHO, 1970. ISBN 9241660503.;
`
`TANNENBAUM, S.R., et al. Nitrite in human saliva. Its possible
`
`relationship to nitrosamine formation .. J cancer Ins. 197 4, vol.53, p. 79-84.
`
`; BARTSCH, H., et al. Inhibitors of endogenous nitrosation: mechanisms
`
`and implications in human cancer prevention. Mutation Res. 1988, vol.202,
`
`p.307-324. ) Proposed harmful effects of these anions include promotion
`
`of gastric cancers and other malignancies and development of
`
`methemoglobinemia in infants. Because of this the levels of nitrate/nitrite
`
`are strictly regulated in food and drinking water.
`
`[0003] Recent studies indicate that nitrate and nitrite can have significant
`
`biological effects in the body and that these effects may be beneficial. (
`
`LUNDBERG, Jori 0., et al. Nitrate, becteria and human health. Nat Rev
`
`Microbial. 2004, no.2, p.593-602. ) For example the nitrite anion can cause
`
`vasodilatation at near physiological concentrations when tested in vitro (
`
`MODIN, A, et al. Nitrite-derived nitric oxide: a possible mediator of
`
`'acidic-metabolic' vasodilation. Acta Physiol Scand. 2001, vol.171, p.9-16.
`) or when infused intra-arterially to humans ( COSBY, K., et al. Nitrite
`reduction to nitric oxide by deoxyhemoglobin vasodilates the human
`
`circulation . Nat Med 2003, no.9, p.1498-505. ). Nitrate can be converted
`
`to nitrite in vivo in a process dependent on commensal bacteria. (
`
`Page 7 of 40
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`

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`PatxML
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`2/28
`
`MH57154
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`SPIEGELHALDER, B., et al. Influence of dietary nitrate on nitrite content
`
`of human saliva: possible relevance to in vivo formation of N-nitroso
`
`compounds. Food Cosme! Toxicol. 1976, no.14, p.545-548. ) When nitrate
`
`is ingested it is rapidly absorbed into blood and then accumulates in saliva.
`
`In the oral cavity bacteria reduce parts of the dietary nitrate to nitrite and
`
`nitrite can then enter the systemic circulation. ( LUNDBERG, Jon 0., et al.
`
`Inorganic nitrate is a possible source for systemic generation of nitric oxide
`
`. Free Radie Biol Med 2004, vol.37, p.395-400.)
`
`[0004] To date the focus among researchers has been on the cardiovascular
`
`effects of nitrite after its in vivo reduction to the vasodilator nitric oxide
`
`(NO). (COSBY et al. (supra); DURANSKI, M.R., et al. Cytoprotective
`
`efects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J
`
`Clin Invest 2005, vol.115, p.1232-1240. ; GLADWIN, M.T., et al. The
`
`emerging biology of the nitrite anion. Nat Chem Biol. 2005, no.1, p.308-14.
`
`; LARSEN, F.J., et al. Effects of dietary nitrate on blood pressure in halthy
`
`volunteers. N Engl J Med 2006, vol.355, p.2792-3. )
`
`[0005] WO 20051004884 A (US GOVERNMENT ET AL.) 20.01.2005 and WO
`
`2005/007173 A (US GOVERNMENT ET AL.) 27.01.2005 describe a
`
`method to administer a nitrite salt specifically to obtain vasodilatation in a
`
`subject. No effects of low-dose nitrate/nitrite on energy expenditure or
`
`glucose ho~eostasis have been described.
`
`Summary of the invention
`
`[0006] The inventors have surprisingly shown that the metabolic rate and/or the
`
`oxygen consumption can be influenced in a mammal, by administering
`
`inorganic nitrite (N02·) or nitrate (N03·) to said mammal in an amount of
`nitrite or nitrate sufficient to decrease oxygen consumption, but without
`
`significant hypotension and which does not cause any significant increase
`
`of the methemoglobin level in said mammal.
`
`[0007]
`
`In particular, the invention makes available a method for lowering the
`
`metabolic rate in a mammal, wherein a pharmaceutical composition
`comprising inorganic nitrite (N02·) is administered to said mammal in an
`amount of nitrite which is sufficient to decrease oxygen consumption, but
`
`Page 8 of 40
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`

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`PatxML
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`3/28
`
`MH57154
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`without significant hypotension. The nitrite can be administered perorally
`
`or parenterally. When administered parenterally, said .nitrite can be
`
`administered intravenously at a dose of about 0.01 to about 1000
`
`nmoles/kg/min.
`
`[0008] The invention also makes available a method for lowering the metabolic
`
`rate in a mammal, wherein a pharmaceutical composition comprising
`
`inorganic nitrate (N03-) is administered to said mammal in an amount of
`nitrate which is sufficient to decrease oxygen consumption, but which does
`
`not increase methemoglobin levels in said mammal, wherein said nitrate is
`
`administered perorally in the form of a nitrate salt at a dose of about 0.01
`to about 10 mmol/kg/24h. When given perorally, nitrate can be seen as a
`
`precursor of nitrite.
`
`[0009] The influence on, or regulation of, metabolic rate and/or oxygen
`
`consumption can be used as a step in the treatment, prevention or
`
`amelioration of a pathological or physiological condition where metabolic
`
`stress is a distinctive feature. Such stress may be present in e.g. intensive
`
`care patients, patients undergoing surgery, patients with malnutrition,
`
`patients with cancer and anorexia, patients with burn injury, trauma
`
`patients, neonates and prematures, patients with anorexia nervosa,
`
`patients scheduled for organ transplantation or patients having undergone
`
`an organ transplantation. In addition, a decrease in oxygen consumption
`
`with a lower need for oxygen in the tissues, is desirable in any pathological
`
`situation characterized by low oxygen availability, including; chronic
`
`obstructive pulmonary disease (CQPD), inflammatory airway disease such
`
`as asthma, pulmonary hypertension, congestive heart disease, interstitial
`
`lung disease, pulmonary embolism, ischemic heart disease, peripheral
`
`artery disease; sleep apnea syndrome.
`
`[001 OJ The inventive findings can also be applied to the regulation of the
`
`metabolic rate as a step in treating, preventing or ameliorating a condition
`
`of disturbed glucose homeostasis (glucose control) including: diabetes
`
`mellitus type 1 and type 2, prediabetes, intensive care patients, surgical
`
`trauma, metabolic syndrome, obesity, burn injury, drug-induced diabetes.
`
`The term "glucose control" is used in its broadest sense, as it is known that
`
`Page 9 of 40
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`

`

`PatXML
`
`4/28
`
`MH57154
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`the stabilization of blood glucose levels is important for many reasons and
`
`concerns many patient groups, as well as healthy subjects. It is general
`
`knowledge that diabetic patients are susceptible to complications such as
`
`neuropathies, cardiomyopathy, vascular disease, poor wound healing and
`
`blindness. Even patients suffering with simple hypoglycaemia would
`
`benefit from glucose control. Further it is hypothesised that many common
`
`health conditions other than diabetes have a component of insufficient or
`
`disturbed glucose control. For example obesitas is closely associated with
`
`glucose and insulin. It is also suggested that emotional problems such as
`
`concentration difficulties and mood swings are associated with poor
`
`glucose control. Due to the well documented adverse effects of
`
`hyperglycaemia as well as hypoglycemia, it is important to maintain proper
`
`glucose control in both diabetic and non-diabetic patients/subjects.
`
`[0011] The invention also provides methods for treatment, alleviation and/or
`
`prevention of clinical conditions, comprising administering an effective
`
`amount of a nitrate and/or a nitrite to a patient in need thereof sufficient to
`
`treat, alleviate and/or prevent such condition.
`
`[0012] As a consequence of the present findings and the conclusions reached by
`
`the inventors, the present invention also makes available new uses of
`
`nitrites and nitrates, for the manufacture of pharmaceutical products,
`
`enteral or parenteral nutritional solutions, or nutritional supplements, for
`
`administration to both healthy persons, such as athletes, or to patients,
`
`suffering from one or more of the conditions exemplified in the description.
`
`[0013) Further embodiments will become evident to the skilled person upon study
`
`of the figures, description and examples, as well as the appended claims,
`
`incorporated herein by reference.
`
`Brief Description of Drawings
`
`[0014] The invention will be described in closer detail in the following description,
`
`non-limiting examples and claims, with reference to the attached drawings
`
`in which
`[0015] Figure 1 is a graph showing changes in oxygen consumption (V02)
`following iv infusion of sodium nitrite in increasing doses. Nitrite was
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`Page 10 of 40
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`PatXML
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`5/28
`
`MH57154
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`infused over a 10 min period in non-smoking healthy male volunteers
`
`(30-70 years).
`
`[0016] Figure 2 is a graph showing the effects of a dietary supplementation with
`'
`sodium nitrate or sodium chloride (placebo) on plasma concentrations of
`
`nitrite measured at rest and immediately after exercise in 9 healthy male
`
`volunteers.
`
`[0017] Figure 3 is a bar diagram showing the oxygen consumption (V02) and
`heart rate (HR) measured at 6 different work rates after a 3-day dietary
`
`supplementation with sodium nitrate (0.1 mmol/kg/min, NIT} or an equal
`
`amount of sodium chloride (CON). The study had a randomized
`
`double-blind cross-over design with a washout period of at least 10 days
`
`between the tests.* p<0.05, ** p<0.01.
`
`[0018] Figure 4 is a graph showing oxygen consumption during bicycle exercise
`
`at 80% of V02peak in 9 healthy male volunteers. Measurements were made
`after a 3-day dietary supplementation with sodium nitrate (0.1
`
`mmol/kg/day) or an equal amount of sodium chloride (placebo). The
`
`difference between nitrate and placebo periods was significant (p<0.01 ).
`
`[0019] Figure 5 is a bar diagram showing plasma lactate concentration measured
`
`at 6 different work rates after dietary supplementation with sodium nitrate
`
`(0.1 mmol/kg/day for 3 days, filled bars) or an equal amount of sodium
`
`chloride (placebo, empty bars).
`
`[0020] Figure 6 consists of three graphs, showing changes in blood glucose
`
`levels after an oral challenge with glucose forthree test subjects in a
`
`double-blind, placebo-controlled cross-over study (Fig. 6a, 6b and 6c). A
`
`standard oral glucose tolerance test was performed. The subjects (healthy
`
`non-smoking volunteers) had their diet supplemented for 3 days with either
`
`sodium chloride (placebo) or sodium nitrate (Nitrate) at a dose of 0.1
`
`mmol/kg/day.
`
`Detailed description of the invention
`
`Definitions
`
`[0021] Before the present method and compositions are described in the form of
`
`embodiments thereof, it is to be understood that this invention is not
`
`Page 11 of 40
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`

`PatXML
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`6/28
`
`MH57154
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`limited to the particular configurations, method steps, and materials
`
`disclosed herein as such configurations, steps and materials may vary
`
`somewhat. It is also to be understood that the terminology employed
`
`herein is used for the purpose of describing particular embodiments only
`
`and is not intended to be limiting since the scope of the present invention
`
`will be limited only by the appended claims and equivalents thereof.
`
`[0022]
`
`It must also be noted that, as used in this specification and the appended
`
`claims, the singular forms "a", "an", and "the" include plural referents
`
`unless the context clearly dictates otherwise.
`
`[0023] The term "about" when used in the context of numeric values denotes an
`
`interval of accuracy, familiar and acceptable to a person skilled in the art.
`Said interval can be ± 10 % or preferably± 5 %.
`In describing and claiming the present invention, the following terminology
`
`[0024]
`
`will be used in accordance with the definitions set out herein.
`
`[0025] The term "mammal" is intended to encompass all mammals, and in
`
`particular humans, pets and agriculturally significant animals, as well as
`
`animals used in competitions, such as horses and dogs.
`
`[0026] The term "significant hypotension" means in this context an acute
`
`reduction of systolic and/or diastolic blood pressure, accompanied by
`
`clinical symptoms of hypotension such as dizziness, nausea, pallor, loss of
`
`consciousness, etc. Said symptoms may occur in various degrees, and it
`
`is preferred that they are entirely avoided, minimized or eliminated as far
`
`as possible, or at least to an extent that they are clinically insignificant.
`
`[0027] The term "metabolic syndrome" is here defined as a combination of
`
`medical disorders that increase the risk for cardiovascular disease and
`
`diabetes in a human. Symptoms and feature include fasting
`
`hyperglycaemia, diabetes mellitus type 2 or impaired fasting glucose,
`
`impaired glucose tolerance or insulin resistance, high blood pressure,
`
`central obesity, decreased HDL cholesterol, elevated triglycerides and
`
`elevated uric acid levels.
`
`[0028] The term "insulin resistance" is here defined as a condition in which
`
`normal amounts of insulin are inadequate to produce a normal insulin
`
`response from fat, muscle and liver cells.
`
`Page 12 of 40
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`PatXML
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`7/28
`
`MH57154
`
`[0029] The term "metabolism" is used to define the complete set of chemical
`
`reactions that occur in living cells and "metabolic rate" is defined as the
`
`speed of metabolism of a mammal. The term "energy expenditure" is here
`
`defined as the amount of energy expended for a certain metabolic rate.
`
`[0030] The term "oxygen consumption" is defined as the amount of oxygen (02)
`consumed by a mammal and is usually expressed as ml of pure oxygen
`
`consumed/min.
`
`[0031] Methemoglobin is a form of hemoglobin in which the iron in the heme
`group is in the Fe3+ state, not the Fe2+ of normal hemoglobin.
`
`Methemoglobin is unable to carry oxygen. Methemoglobinemia is defined
`
`as a blood disorder characterized by the presence of a higher than normal
`
`level of methemoglobin in the blood.
`
`[0032] The term "catabolism" is defined as the metabolic process that breaks
`
`down molecules into smaller units. It is made up of degradative chemical
`
`reactions in the living cell.
`
`[0033] The surprising finding that nitrite and its precursor nitrate affects such vital
`
`physiological processes as metabolic rate and/or oxygen consumption can
`
`be used therapeutically, e.g. in prophylaxis, alleviation or treatment of
`
`several conditions. In an attempt to increase systemic nitrite levels, nitrite
`
`and/or nitrate can be given by enteral administration (orally, in the form of
`
`a liquid, semi-solid or solid preparation, such as a chewing gum, tablet,
`
`lozenge, wafer, cake, bar or the like) or by parenteral administration
`
`(intravenous, transdermal, by inhalation, topical, or any other way of
`
`parenteral administration).
`
`[0034]
`
`In principle, also other additional approaches can lead to increased
`
`systemic nitrite levels. The most obvious is to administer nitrite or its·
`
`precursor nitrate as such, but this may be supplemented by or enhanced
`
`by increasing the gastric pH (e.g. with an acid suppressive drug such as a
`proton pump inhibitor or H2 receptor antagonist or antacida) to maximise
`nitrite survival in the stomach and thereby the systemic delivery of nitrite.
`
`Alternatively, the administration of nitrite or nitrate can be supplemented
`
`by or enhance by interfering with the oral microflora in order to maximise
`
`the number of nitrate reducing species. This can be achieved through the
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`8/28
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`MH57154
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`delivery of "probiotic" nitrate reducing bacteria or selective treatment with
`
`an antibiotic to favour the nitrate reducing species.
`
`[0035]
`
`It is likely that an optimal dose-interval exists, meaning that below a certain
`
`plasma level of nitrite the effects are insufficient and correspondingly, that
`
`over a certain level the effect is lower, and possibly accompanied by side
`
`effects. Using intravenous administration, the nitrite is preferably
`
`administered in a dose within the interval of about 0.01 to about 1000
`
`nmoles/kg/min, more preferably about 0.1 to about 100 nmoles/kg/min,
`
`most preferably about 1to about 20 nmoles/kg/min. It is presently
`
`contemplated that the most preferred dose is less than about 15
`
`nmoles/kg/min. For c.omparison, it should be noted that the nitrite dose
`
`used in the treatment of cyanide poisoning is about 100 000 nmoles/kg, or
`
`about 300 - 400 mg given as a single dose.
`
`[0036] For the use of a nitrate salt perorally, a dose of 0.01-100 mmol/kg/day is
`
`currently preferred or more preferably a dose of 0.1-10 mmol/kg/day.
`
`[0037]
`
`Importantly, the administered dose of nitrate or nitrite should not induce
`
`production of more than 10% methemoglobin, preferably not more than
`
`about 5 % methemoglobin, more preferably not more than about 2%
`
`methemoglobin or less than 2% methemoglobin. Most preferred is that the
`
`dose of nitrate or nitrite does not induce any measurable change in
`
`methemoglobin in the subject when administered or ingested according to
`
`·the prescribed dose.
`
`[0038] Combinations of nitrate and nitrite salts can also be used. According to
`
`one embodiment, nitrate and nitrite are given orally in a dose ratio 1O:1
`
`(nitrate:nitrite). This will ensure acute effects of the nitrite as soon as it is
`
`absorbed, and then provide a sustained effect of the nitrate following its
`
`bioconversion into nitrite.
`
`[0039] Pharmaceutically acceptable salts of nitrate and nitrite include but are not
`
`limited to sodium, potassium, calcium, zinc, arginine and ammonium.
`
`Sodium and potassium salts are presently most preferred. The nitrite and
`
`nitrate salts may be of synthetic origin, but may also be isolated from
`
`natural sources, such as naturally nitrate containing plants, e.g. green
`
`leafy vegetables, examples including but not limited to spinach, lettuce,
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`MH57154
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`fennel, cabbage, and beetroot. Concentrates, such as juices or dried
`
`concentrates of these and other nitrate-rich vegetables or fruits are
`
`suitably used for the manufacture of food products or nutritional
`
`supplements according to the present invention.
`
`[0040] According to one preferred embodiment the dose of nitrate and/or nitrite is
`
`administered as a chewing gum or lozenge or pastille which optionally also
`
`comprises live bacteria. Such bacteria may include suitable probiotic
`
`bacteria (including lactobacilli or bifidobacteria) or suitable non-pathogenic
`
`bacteria that enhance nitrate reduction or nitrite reduction including e.g.
`
`VeH/onella species; Staphylococcus species, Actinomyces species or
`
`Rothia species.
`
`[0041] According to an embodiment of the invention, the nitrate and nitrite salts
`
`are combined with other pharmaceuticals including but not limited to:
`
`anti-diabetic drugs (insulin and oral anti-diabetics), cardiovascular drugs
`
`(statins, ACE inhibitors, beta-receptor antagonists, diuretics, angiotensin 2
`
`receptor antagonists, organic nitrates, calcium channel blockers) acid
`
`secretion-inhibitors (proton pump inhibitors, Histamine-2 receptor
`
`blockers), oral anti-diabetics including biguanides, sulphonureides,
`
`alpha-glucosidase inhibitors, thiazolidinediones, glinides; drugs for
`
`treatment of pulmonary hypertension including prostacyclin analogues,
`
`endothelin receptor antagonists and sildenafil.
`
`[0042] According to one embodiment the nitrite is delivered systemically via
`
`peroral treatment with an organic nitrate including nitroglycerine or
`
`isosorbide mono/di-nitrate. Nitroglycerine is used clinically to treat angina
`
`pectoris and it acts by releasing vasodilatory nitric oxide systemically.
`
`However, this drug must be given parenterally because the ~rst passage
`
`metabolism in the liver is considerable. Interestingly, it has been found that
`
`the liver metabolism of nitroglycerine yields predominantly nitrite.
`
`Considering the novel biological effects of nitrite described here,
`
`nitroglycerine may therefore be used as a "prodrug" of nitrite. Preferably
`
`the drug should then be given by the enteral route (with liver metabolism)
`
`to maximize nitrite generation while at the same time avoiding the acute
`
`vasodilatation and drop in systemic blood pressure associated With iv or
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`10/28
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`MH57154
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`sublingual administration of nitroglycerine. A suitable dose range when
`
`giving nitroglycerine perorally is about 0.001 to 1 mol/kg/24 hours, more
`
`preferably 0,01 to about 0.1 mmol/kg/24 hours. The tablets should
`
`preferably be coated to avoid absorption in the oral cavity.
`
`[0043] According to one embodiment nitrite and/or nitrate is added to parenteral
`
`and enteral feeding/nutrition solutions to be used in adults, children,
`
`neonates and prematures. Today such solutions are generally extremely
`
`low in nitrate and nitrite as noted in measurements performed by the
`
`present inventors (not shown). An intubated mechanically ventilated
`
`patient with parenteral nutrition is particularly deprived of nitrate/nitrite.
`
`First, these patients do not produce and swallow saliva properly and thus
`
`one great nitrate/nitrite source is disrupted. Secondly as stated above, the
`
`feeding they receive contains almost no nitrate/nitrite. Many intensive care
`
`patients suffer from metabolic disturbances, in particular catabolism due to
`
`stress and trauma and their metabolic rate is often increased. Moreover,
`
`insulin resistance is common and glucose homeostasis is disturbed. Tight
`
`control of plasma glucose by administering insulin is advocated in these
`
`patients. Also healthy subjects, such as infants or subjects taking enteral
`
`solutions for other reasons can benefit from the compositions and methods
`
`of the present invention. Enteral nutrition is thus meant to include also
`
`infant formulas and other enteral products.
`
`[0044]
`
`In one embodiment, provided herein are methods for the treatment,
`
`alleviation and/or prevention of clinical conditions, comprising
`
`administering an effective amount of a nitrate and/or a nitrite to a patient in
`
`need thereof sufficient to treat, alleviate and/or prevent such condition.
`
`[0045] There are many possible clinical conditions where such treatment,
`
`alleviation and/or prevention is performed using nitrate and/or nitrite
`
`according to the present invention:
`
`- glucose control in diabetes/prediabetes
`
`- metabolic syndrome
`
`[0046] The method and composition according to the invention also has general
`
`therapeutic, alleviating and/or prophylactic effects in patients under
`
`metabolic stress. Examples include but are not limited to:
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`MH57154
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`- intensive care patients
`
`- patients undergoing surgery
`
`- patients suffering from malnutrition of different genesis
`
`- cancer with anorexia
`
`- burn injury
`
`- trauma
`
`- neonates/prematures
`
`- anorexia nervosa
`
`- thyreoidea disorders (e.g. hyperthyreosis)
`
`- myocardial infarction, cardiac arrest
`
`- major systemic disease with a catabolic state
`
`- stress ulcers (gastric)
`
`- surgical gut anastomosis insufficiency
`
`- fever
`
`- myocardial infarction and cardiac arrest
`
`".' ischemia-reperfusion injury (Ml, stroke, arterial insufficiency or any other
`
`organ ischemia)
`
`- sleep apnoea syndrome
`
`- septic chock
`
`- insufficient perfusion of the intestines
`
`[0047] The method and composition according to the invention has general
`
`therapeutic and/or prophylactic effects also in patients with pathological
`
`conditions characterized by low oxygen availability, including but not
`
`limited to;
`
`- chronic obstructive pulmonary disease (COPD)
`
`- inflammatory airway disease such as asthma
`
`- pulmonary hypertension
`
`- congestive heart disease
`
`- interstitial lung disease
`
`- pulmonary embolism
`
`- ischemic heart disease
`
`- peripheral artery disease
`
`- sleep apnea syndrome
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`12/28
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`MH57154
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`- myocardial infarction, cardiac arrest
`
`- ischemia/reperfusion injury (Ml, stroke, arterial insufficiency or any other
`
`organ ischemia)
`
`- systemic inflammatory disorder
`
`[0048]
`
`In one preferred embodiment nitrate and/or nitrite is given to patients with
`
`a pathological condition characterized by low oxygen availability. In such
`
`situations it is desirable to reduce the tissues need for oxygen to prevent
`
`sequele and symptoms associated with the hypoxia. Examples include
`
`patients with COPD whose pulmonary oxygen uptake may be severely
`
`compromised and patients with peripheral artery disease where oxygen
`
`delivery to the tissues is reduced.
`
`[0049] The inventive method and composition is also useful for healthy subjects,
`
`e.g. athletes. The inventive method and composition provides for less
`
`oxygen demand at a certain workload and improves anabolism. The
`
`method and composition is also useful for oxygen sparing at high altitude,
`
`for example in work and sports performed in a low oxygen environment,
`
`such as but not limited to rescue activities, fire fighting, military operations,
`
`diving, mountain climbing, high-altitude flying, and the exploration of
`
`space.
`
`[0050] The present method and composition is also useful in organ or tiss