(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`4 September 2008 (04.09.2008)
`
`PCT
`
`(51) International Patent Classification:
`A61K 33/00 (2006.01)
`A61P 11/00 (2006.01)
`A61K 35/74 (2006.01)
`A61P 3/00 (2006.01)
`A61K 36/185 (2006.01)
`A61P 9/00 (2006.01)
`
`(21) International Application Number:
`PCT/SE2008/050212
`
`(22) International Filing Date:
`26 February 2008 (26.02.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`0700520-0
`60/919,709
`
`26 February 2007 (26.02.2007)
`22 March 2007 (22.03.2007)
`
`SE
`US
`
`(71) Applicants and
`(72) Inventors: LUNDBERG, Jon [SE/SE]; Stenbocksvagen
`6, S-182 62 Djursholm (SE). WEITZBERG, Eddie
`[SE/SE]; Nybrogatan 24, S-1 14 39 Stockholm (SE).
`
`(10) International Publication Number
`WO 2008/105731 Al
`(74) Agent: BERGENSTRAHLE & LINDVALL AB; Box
`17704, S-1 18 93 Stockholm (SE).
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT,AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG, ES, FT, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC,
`LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN,
`MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH,
`PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV,
`SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA, ZM, ZW
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT,BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HR, HU, IE, IS, IT, LT,LU, LV,MC, MT, NL,
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`Published:
`— with international search report
`
`(54) Title: NEW USE OF NITRITES AND NITRATES AND COMPOSITIONS CONTAINING THESE
`
`Sodium nitrite infused iv
`
`nmoles/kg/min
`
`(57) Abstract: Inorganic anions nitrate and nitrite influence metabolic rate and glucose homeostasis. Infusion of nitrite iv caused
`an acute drop in resting energy expenditure (oxygen consumption) and nitrate, when given perorally, caused a drop in oxygen con-
`sumption during exercise and a depression of the increase in blood glucose observed after an oral glucose tolerance test. The doses
`of nitrate and nitrite did not cause any detectable change in methemoglobin levels of blood. Also, nitrate and nitrite did not alter
`lactate levels in blood. This discovery provides useful treatments to regulate the energy expenditure and glucose homeostasis of a
`mammal by administration of inorganic nitrite and/or nitrate.
`
`Human Power of N Company
`EX1007
`Page 1 of 65
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`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 (NO3 ) and nitrite (NO2 ) 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. 1974, 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, Jon
`
`O., et al. Nitrate, becteria and human health. Nat Rev Microbiol. 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.9-1 6) 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
`
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`dependent on commensal bacteria (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 Cosmet 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 O., et al. Inorganic nitrate is a possible source for systemic
`
`generation of nitric oxide. Free Radic 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.1232-1 240; 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 2005/004884 A (US GOVERNMENT ET AL.) 2005-01-20 and WO
`
`2005/007173 A (US GOVERNMENT ET AL.) 2005-01-27 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
`
`homeostasis 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 (locally in isolated
`
`tissues or organs or systemically in the whole body), by administering
`
`inorganic nitrite (NO2 ) and/or nitrate (NO3 ) to said mammal
`
`in an amount of
`
`nitrite and/or nitrate sufficient to decrease oxygen consumption. The oxygen
`
`consumption is decreased without causing significant hypotension and does
`
`not cause any significant increase of the methemoglobin level in said
`
`mammal.
`
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`[0007] The invention also makes available a method for lowering the metabolic rate
`
`in a mammal, wherein a pharmaceutical composition comprising inorganic
`
`nitrite (NO2 ) and nitrate (NO3 ) is administered to said mammal
`
`in an amount
`
`of nitrite and nitrate which is sufficient to decrease oxygen consumption. The
`
`oxygen consumption is decreased without causing significant hypotension.
`
`[0008]
`
`In particular, the invention makes available a method for lowering the
`
`metabolic rate in a mammal, wherein a pharmaceutical composition
`
`comprising inorganic nitrite (NO2 ) is administered to said mammal
`
`in an
`
`amount of nitrite which is sufficient to decrease oxygen consumption. The
`
`oxygen consumption is decreased without causing 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 10 000 nmoles/kg/min.
`
`[0009] The invention also makes available a method for lowering the metabolic rate
`
`in a mammal, wherein a pharmaceutical composition comprising inorganic
`
`nitrate (NO3 ) 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 100
`
`mmol/kg/24h. When given perorally, nitrate can be seen as a precursor of
`
`nitrite.
`
`[0010] The invention also makes available a composition, use and method wherein
`
`inorganic nitrite and/or nitrate is/are combined with polyphenols.
`
`[001 1] 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 solid
`
`organ transplantation or patients having undergone an solid organ
`
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`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 (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.
`
`[0012] 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 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.
`
`[0013] 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.
`
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`[0014] 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/or 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.
`
`[0015] 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 the drawings
`
`[0016] 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:
`
`[0017] Figure 1 shows a graph illustrating numerous ways in which the combination
`
`of nitrate and polyphenols synergistically act to increase the bioavailability of
`
`nitric oxide and at the same time to reduce the formation of harmful
`
`compounds such as oxygen radicals and nitrosamines. For detailed
`
`explanation see text.
`
`[0018] Figure 2 is a graph showing changes in oxygen consumption (VO2) following
`
`iv infusion of sodium nitrite in increasing doses. Nitrite was infused over a 10
`
`min period in non-smoking healthy male volunteers (30-70 years).
`
`[0019] Figure 3 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.
`
`[0020] Figure 4 is a bar diagram showing the oxygen consumption (VO2) 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
`
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`cross-over
`
`design with a washout period of at least 10 days between the
`
`tests.
`
`* p<0.05,
`
`* * p<0.01 .
`
`[0021]
`
`during bicycle exercise at
`
`Figure 5 is a graph showing oxygen consumption
`80% of V q
`a 3-day dietary supplementation
`
`2peak in 9 healthy male volunteers. Measurements were made after
`
`with sodium nitrate (0.1 mmol/kg/day)
`
`o r a n
`
`equal amount of sodium chloride (placebo). The difference between nitrate
`
`and placebo periods was significant
`
`(p<0.01).
`
`[0022]
`
`Figure 6 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) o r a n equal amount of sodium chloride
`
`(placebo, empty bars).
`
`[0023]
`
`Figure 7 consists of three graphs, showing changes in blood glucose levels
`
`after a n oral challenge with glucose for three test subjects in a double-blind,
`
`placebo-controlled
`
`cross-over
`
`study (Fig. 7a, 7 b and 7c). 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) o r sodium nitrate (Nitrate) at a dose of 0.1 mmol/kg/day.
`
`[0024]
`
`Figure 8 is a graph showing changes in blood glucose levels after a n oral
`
`challenge with glucose in 8 additional
`
`subjects in a double-blind,
`
`placebo-
`
`controlled cross-over
`
`study. 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)
`
`o r sodium
`
`nitrate (NITRATE)
`
`at a dose of 0.1 mmol/kg/day. Data are presented as
`
`mean+SEM.
`
`[0025]
`
`Figure 9 is a graph showing the effect of a two-week
`
`intervention with
`
`beetroot
`
`juce (fresh juice 3-4 dl/day) o n systolic, diastolic and mean arterial
`
`(MAP) blood pressure in a 43 year old male with hypertension.
`
`[0026]
`
`Figure 10 shows the plasma nitrate and nitrite concentrations
`
`after
`
`intravenous
`
`infusion of nitrate a) shows plasma nitrate concentrations,
`
`b)
`
`shows plasma nitrite concentrations
`
`and c) shows plasma nitrite
`
`concentrations
`
`in wild type (C57BL/6),
`
`germ free and knockout
`
`(eNOS) mice.
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`[0027] Figure 11 is a graph showing enhanced post-ischemic blood flow after nitrate
`
`infusion.
`
`Detailed description of the invention
`
`Definitions
`
`[0028] Before the present method and compositions are described in the form of
`
`embodiments thereof, it is to be understood that this invention is not 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.
`
`[0029]
`
`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.
`
`[0030] 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 +/- 2 % of the given value, preferably +/- 5 % , and most
`preferably +/- 10 % of the numeric values, where applicable.
`[0031] 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.
`
`[0032] 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.
`
`[0033] The term "metabolic syndrome" is here defined as a combination of medical
`
`disorders that increase the risk for cardiovascular disease and diabetes in a
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`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.
`
`[0034] 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.
`
`[0035] 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.
`
`[0036] The term "oxygen consumption" is defined as the amount of oxygen (O2)
`
`consumed by a mammal and is usually expressed as ml of pure oxygen
`
`consumed/min. "Oxygen consumption" relates to the amount of oxygen
`
`consumed by a mammal as whole but also to oxygen consumption in an
`
`isolated tissue or organ, such as, but not limited to, heart, liver brain or other
`
`tissue exposed to ischemia.
`
`[0037] 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.
`
`[0038] 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.
`
`[0039] The term "functional food" relates to any fresh or processed food claimed to
`
`have a health-promoting and/or disease-preventing property beyond the basic
`
`nutritional function of supplying nutrients. Functional foods are sometimes
`
`called nutraceuticals. The general category includes processed food made
`
`from functional food ingredients, or fortified with health-promoting additives,
`
`like "vitamin-enriched" products, and also, fresh foods (e g vegetables) that
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`have specific claims attached. Fermented foods with live cultures are often
`
`also considered to be functional foods with probiotic benefits.
`
`[0040] The inventors have surprisingly shown that the metabolic rate and/or the
`
`oxygen consumption can be influenced in a mammal (locally or systemically),
`
`by administering inorganic nitrite (NO2 ) and/or nitrate (NO3 ) to said mammal
`
`in an amount of nitrite and/or nitrate sufficient to decrease oxygen
`
`consumption. The decreased oxygen consumption is achieved without
`
`causing significant hypotension and without causing any significant increase
`
`of the methemoglobin level in said mammal. In case of local reduction of the
`
`metabolic rate, the oxygen consumption is decreased in an isolated tissue or
`
`organ such as the heart, liver, brain or other tissue that is exposed to
`
`ischemia (a condition in which blood flow, and thus oxygen, is restricted to a
`
`part of the body). In such cases the interaction of reaction products (including
`
`NO) of nitrite and/or nitrate with enzymes of the mitochondrial respiratory
`
`chain and subsequent
`
`inhibition of respiration leads to lowering of oxygen
`
`demand which is beneficial for an ischemic tissue. This effect resembles
`
`hibernation. Because the generation of active nitrite and/or nitrate reaction
`
`products are maximized in ischemic tissues the effect of oxygen consumption
`
`will be most pronounced at these sites. In one particular embodiment,
`
`the
`
`oxygen consumption is lowered in the heart.
`
`[0041] 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,
`
`transcutaneous, by inhalation, rectally, vaginally, topical,
`
`intraperitoneally,
`
`intra muscular, subcutaneous, sublingual or any other way
`
`of parenteral administration). The nitrite and/or nitrate comprising composition
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`and possible further combinations described herein can be administered
`
`continuously or as single bolus doses.
`
`[0042]
`
`In principle, also other additional approaches can lead to increased systemic
`
`or local 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
`
`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 enhanced by
`
`interfering with the oral microflora in order to maximise the number of nitrate
`
`reducing species. This can be achieved through the delivery of "probiotic"
`
`nitrate reducing bacteria or selective treatment with an antibiotic to favour the
`
`nitrate reducing species.
`
`[0043]
`
`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 10 000nmoles/kg/min,
`
`preferably about 0.01 to about 1000 nmoles/kg/min, more preferably about
`
`0.1 to about 100 nmoles/kg/min, most preferably about 1 to about 20
`
`nmoles/kg/min.
`
`It is presently contemplated that the most preferred dose is
`
`less than about 15 nmoles/kg/min. For comparison,
`
`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. The nitrite can also
`
`be administered as one or more bolus doses, preferably 0.01 - 100 umol/kg
`
`body weight, more preferably 0.1 - 10 umol/kg body weight, and even more
`
`preferably 0.1 - 2 umol/kg body weight.
`
`[0044] For the use of a nitrate salt perorally, a dose of about 0.01-100 mmol/kg/24 h
`
`is currently preferred or more preferably a dose of about 0.01-10
`
`mmol/kg/24h, even more preferably 0.1 - 1 mmol/kg/24h.
`
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`[0045]
`
`Importantly,
`
`the administered dose of nitrate or nitrite should not induce
`
`production of more than about 10% methemoglobin, preferably not more than
`
`about 5 % methemoglobin, and more preferably not more than about 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.
`
`[0046] Combinations of nitrate and nitrite salts can also be used. According to one
`
`embodiment, nitrate and nitrite are given orally in a dose ratio interval of
`
`about 5:1 to about 100:1 (nitrate:nitrite), such as 5:1 , 10:1 , 30:1 , 50:1 , 70:1
`
`and 100:1 . Preferably the dose ratio is about 10:1 . 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.
`
`[0047]
`
`In one embodiment
`
`the composition comprising inorganic nitrite and/or nitrate
`
`is a pharmaceutical composition comprising inorganic nitrite and/or nitrate in
`
`an amount which is sufficient to decrease oxygen consumption, but which
`
`does not increase the methemoglobin level in a subject when administered to
`
`said subject in a prescribed dose. Optionally the composition comprises
`
`another pharmaceutically active compound.
`
`[0048] Alternatively,
`
`the composition comprising nitrite and/or nitrate is a nutritional
`
`supplement, an enteral nutritional solution, an infant formula, a snack product
`
`or a parenteral nutritional solution comprising inorganic nitrite and/or nitrate in
`
`an amount which is sufficient to decrease oxygen consumption, but which
`
`does not cause significant hypotension in a subject when ingested by said
`
`subject in a prescribed dose.
`
`[0049] 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 include, but are not limited to spinach,
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`lettuce, fennel, cabbage,
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`Chinese cabbage and beetroot. Concentrates, such as juices or dried
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`concentrates of these and other nitrate-rich vegetables or fruits are suitably
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`used for the manufacture of food products (including functional
`
`food products)
`
`or nutritional supplements according to the present
`
`invention.
`
`[0050] Polyphenols are a group of chemical substances found in plants,
`
`characterized by the presence of more than one phenol group per molecule.
`
`Polyphenols are generally further subdivided into hydrolyzable tannins, which
`
`are gallic acid esters of glucose and other sugars; and phenylpropanoids,
`
`such as lignins, flavonoids, and condensed tannins.
`
`In one embodiment of the
`
`present
`
`invention the inorganic nitrite and/or nitrate is/are mixed with a
`
`compound that contains high levels of polyphenols.
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`It is contemplated that
`
`this combination will have synergistic health promoting effects via potentiation
`
`of NO bioavailability. Polyphenols will enhance NO generation by several
`
`separate mechanisms highlighted in Fig 1. First, such agents can directly
`
`stimulate endogenous NO formation from NO synthase enzymes ( 1 in figure
`
`1). Second,
`
`it is contemplated that these compounds will enhance the
`
`reduction of nitrite to bioactive NO due to the presence of reductive -OH
`
`groups on the phenol ring (2 in figure 1). Third, by acting as scavengers of
`
`free radicals such as superoxide,
`
`they prevent these radicals from interacting
`
`with (and destroying) NO and thereby, NO becomes more long-lived (3 in
`
`figure 1).
`
`In addition to this, nitrite or its reaction products can interact with the
`
`polyphenol
`
`itself and modify it chemically via nitration or nitrosation reactions
`
`(4a in figure 1). The resulting compound can act as a long-lived NO donor (4b
`
`in figure 1). A n additional effect is that the presence of polyphenols will divert
`
`the chemical reactions away from formation of potentially carcinogenic
`
`nitrosamines (5 in figure 1). Nitrates reaction product nitrite can react with
`
`amines to form nitrosamines but polyphenols will
`
`inhibit this reaction by a dual
`
`mechanism. First they help to rapidly reduce HNO2 directly to NO thereby
`
`minimizing the formation of nitrosating species (N2O3, HNO2). Second,
`
`they
`
`can directly compete for nitrosation with the amines by being nitrosated
`
`themselves.
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`In one embodiment of the present invention inorganic nitrite and/or nitrate is
`
`administered or used in combination with a polyphenol rich compound or
`
`product. The ratio nitrite/nitrate comprising composition:polyphenol-rich
`
`compound should be chosen to obtain enough supply of nitrate. The
`
`nitrite/nitrate comprising composition should therefore be at least about 10%,
`
`preferably at least about 20 % , more preferably at least about 30%, even
`
`more preferably at least about 40 % and most preferably at least about 50%
`
`or even more. It is contemplated that the combination of nitrite/nitrate and
`
`polyphenol rich compound or product will act synergistically to enhance NO
`
`formation in the body at the expense of detrimental compounds such as
`
`nitrosamines. The beneficial effects of this includes a reduction in blood
`
`pressure and platelet aggregation, reduced atherosclerosis, reduced risk of
`
`myocardial
`
`infarction, stroke and other cardiovascular disorders, reduced risk
`
`of cancer in any form. Examples of polyphenol rich fruit or juices thereof
`
`include, but are not limited to, apple, pear, grapes, lemon, orange, lime,
`
`peach, pomegranate, grapefruit, kiwi, ginger and pineapple. Juice from
`
`berries are also usable including blackberries, black raspberries, blueberries,
`
`cranberries, red raspberries, cherries, bog wortleberry,
`
`lingonberries, black
`
`elderberry, black chokeberry, black currant, blueberry, cloudberries and
`
`strawberries. Other natural sources of polyphenols include vegetables such
`
`as carrots, chili, rhubarb, onions. In addition, cacao products (rich in
`
`flavanols), green or black tea, nuts, Yerba mate and coffee are all rich in
`
`polyphenols.
`
`In one preferred embodiment the nitrate in the inventive
`
`composition originates from beetroot (such as beetroot juice) which is
`
`blended with one or several polyphenol-rich compounds or products. The
`
`ratio beetroot juice:polyphenol-rich compound should be chosen to obtain
`
`enough supply of nitrate and therefore the beetroot juice part should be at
`
`least about 10%, preferably at least about 20 % , more preferably at least
`
`about 30%, even more preferably at least about 40 % and most preferably at
`
`least about 50%.
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`[0052] According to one embodiment of the present invention the dose of nitrite
`
`and/or nitrate or nitrite and/or nitrate together with polyphenols is
`
`administered or manufactured as a chewing gum, lozenge or pastille, wafer,
`
`cake, bar or the like which optionally also comprises live non-pathogenic
`
`bacteria. It can also be administered or manufactured as a functional food
`
`product or as a part of a functional food product.
`
`[0053] The use of nitrite and/or nitrate or a combination of nitrate and/or nitrite with
`
`polyphenols may also have beneficial effects on the blood pressure. Thus, in
`
`one embodiment the present invention relates to a method to reduce blood
`
`pressure, preferably to normal levels (about 140/80 mmHg). Examples of
`
`nitrite and/or nitrate sources are natural sources of nitrate (such as
`
`vegetables as mentioned above or juices thereof) or salts of nitrite and/or
`
`nitrate. In one particular embodiment beetroot or a juice thereof is
`
`administered in order to reduce the blood pressure in a mammal.
`
`[0054] The purpose with the combination with non-pathogenic live bacteria is to
`
`further enhance the generation of bioactive compounds such as NO, nitroso
`
`adducts or chemically related compounds. This enhancement will occur
`
`locally in the G l tract via bacteria-dependent reduction of nitrate and nitrite to
`
`NO and other bioactive nitrogen oxides. In particular, this combination will be
`
`effective in treating and preventing G l disorders such as ulcers in the
`
`sto