Attorney Docket No. 1255.101 CON2 BP
`
`COMPOSITIONS OF NITRATES AND METHODS OF USE THEREOF
`
`Related Applications
`
`[0001]
`
`This application is a continuation application of earlier U.S. Utility Patent Application
`
`to Jon Lundberg and Eddie Weitzberg, titled “Compositions of Nitrates and Methods of Use
`
`Thereof,” application serial number 15/966,508, filed April 30, 2018, now pending, whichis a
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`continuation application of earlier U.S. Utility Patent Application to Jon Lundberg and Eddie
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`Weitzberg, titled “Use of Nitrites and Nitrates and Compositions Containing These,” application
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`serial number 12/528,794,filed June 17, 2013, now pending, whichis a national stage application
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`of PCT application No. PCT/SE08/50212, filed February 26, 2008, which claims the benefit of the
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`filing date of U.S. Provisional Patent Application 60/919,709 to Jon Lundberg and Eddie
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`Weitzberg, filed on March 22, 2007, the disclosures of all of which being hereby incorporated
`
`entirely herein by reference.
`
`[0002]
`
`U.S. Patent Application No. 15/966,508 is also a continuation application of earlier
`
`U.S. Utility Patent Application to Jon Lundberg and Eddie Weitzberg,
`
`titled “Performance
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`Enhancing Compositions and Use Thereof,” U.S. Patent Application No. 14/830,937, filed August
`
`20, 2015, now pending, which is a continuation application of U.S. Patent Application No.
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`12/528,798, filed August 26, 2009, now issued as U.S. Patent No. 9,180,140, which wasa national
`
`stage application of PCT application No. PCT/SE 2008/050211, filed February 26, 2008, which
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`claims priority to Swedish Patent Application 0700520-0, which wasfiled February 26, 2007, and
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`Swedish Patent Application 0700729-0, which was filed March 22, 2007, the disclosures ofall of
`
`which being hereby incorporated entirely herein by reference.
`
`Field of the Invention
`
`[0003]
`
`The present invention relates to the field of performance enhancing nutritional foods
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`and food supplements, liquid and solid edible products such as sport drinks, energy drinks and
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`energy bars. The present invention also relates to the field of medicine and pharmaceuticals, in
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`particular pharmaceuticals and therapeutic methods for
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`lowering metabolic rate, oxygen
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`consumption and/or glucose homeostasis in a human patient or another mammal, based on the
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`administration of nitrates and/ornitrites to said patient or mammal.
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`1
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`Background of the Invention
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`[0004]
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`NOis involved in control of cellular respiration through interaction with enzymes of
`
`the mitochondrial respiratory chain (for review see MONCADA,§, et al. Does nitric oxide
`
`modulate mitochondrial energy generation and apoptosis?. Nat Rev Mol Cell Biol. 2002, vol. 3,
`
`no. 3, p. 214-20). The classical means by which NO production occurs is the L-arginine pathway,
`
`where NO is synthesized by specific enzymes, the NO-synthases. A fundamentally different
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`alternative way of generating NO has been described more recently (LUNDBERG,J O,etal.
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`Intragastric nitric oxide production in humans: measurements in expelled air. Gut. 1994, vol. 35,
`
`no. 11, p. 1543-6; BENJAMIN,N,et al. Stomach NO synthesis. Nature. 7 Apr. 1994, vol. 368, no.
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`6471, p. 502; ZWEIER,J L, et al. Enzyme-independent formation of nitric oxide in biological
`
`tissues. Nat Med. 1995, vol. 1, no. 8, p. 804-9; and WEITZBERG,E,et al. Nonenzymatic nitric
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`oxide production in humans. NO Biol. Chem. 1998, no. 2, p. 1-7). In this NOS-independent
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`pathway the inorganic anions nitrate (NO3°) and nitrite (NO.sub.2--) are reduced in vivo to form
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`NO. Dietary nitrate (found mainly in green leafy vegetables) (MCKNIGHT, G M. Chemical
`
`synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut. 1997, no. 40, p. 211-
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`214; and Weitzberg, 1998, supra) is absorbed from the circulation by the salivary glands, secreted
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`in saliva and partly converted to nitrite in the oral cavity by nitrate reducing bacteria. Swallowed
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`nitrite can then enter the systemic circulation. Indeed, a recent study showsthat ingestion ofnitrate
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`results in a sustained increase in circulating nitrite levels (LUNDBERG,J O,et al. Inorganic nitrate
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`is a possible source for systemic generation of nitric oxide. Free Rad Bio Med. 2004, vol. 37, no.
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`3, p. 395-400). Further reduction of nitrite into bioactive NO can occur spontaneously in acidic or
`
`reducing environments (Benjaminet al. 1994, supra, Lundberget al. 1994, supra) butis also greatly
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`enhanced by various proteins and enzymesincluding deoxyhemoglobin in blood (COSBY,K,et
`
`al. Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat
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`Med. 2003, vol. 9, no. 12, p. 1498-505), deoxymyoglobin (SHIVA,S. et al. Deoxymyoglobin is a
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`Nitrite Reductase That Generates Nitric Oxide and Regulates Mitochondrial Respiration. Circ Res.
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`9 Feb. 2007), xanthine oxidase (MILLAR, T M,et al. Xanthine oxidoreductase catalyses the
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`reduction of nitrates and nitrite to nitric oxide under hypoxic conditions. FEBS Lett. 8 May 1998,
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`vol. 427, no. 2, p. 225-8) and possibly by enzymes of the mitochondrial respiratory chain (for
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`review see LUNDBERG,J O,et al. Nitrate, bacteria and human health. Nat Rev Microbiol. 2004,
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`vol. 2, no. 7, p. 593-602; LUNDBERG,J O,et al. NO generation from nitrite and its role in vascular
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`Attorney Docket No. 1255.101 CON2 BP
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`control. Arterioscler Thromb Vasc Biol. 2005, vol. 25, no. 5, p. 915-22; and GLADWIN,M T,et
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`al. The emerging biology ofthe nitrite anion. Nat Chem. Biol. 2005, vol. 1, no. 6, p. 308-14). NOS-
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`independent NO production seems to complement the endogenous NO production especially
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`during ischemia and acidosis when oxygen availability is low and the NO synthases operate poorly
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`(Zweier et al. 1995, supra; Weitzberg et al, 1998, supra; DURANSKI, M R,et al. Cytoprotective
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`effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest. 2005,
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`vol. 115, no. 5, p. 1232-40; Lundberget al, 2004, supra). Tissue acidosis and relative hypoxia is
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`present also during physical exercise and in this metabolic state, bioactivation of nitrite is likely
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`enhanced.
`
`[0005]
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`Recentstudies indicate that nitrate and nitrite can have significant biological effects in
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`the body and that these effects may be beneficial (LUNDBERG,Jon O., et al. Nitrate, becteria and
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`human health. Nat Rev Microbiol. 2004, no. 2, p. 593-602). For example, the nitrite anion can
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`cause vasodilatation at near physiological concentrations when tested in vitro (MODIN,A., etal.
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`Nitrite-derived nitric oxide: a possible mediator of ‘acidic-metabolic’ vasodilation. Acta Physiol
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`Scand. 2001, vol. 171, p. 9-16) or when infused intra-arterially to humans (COSBY,K., et al.
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`Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nat Med.
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`2003, no. 9, p. 1498-505). Nitrate can be converted to nitrite in vivo in a process dependent on
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`commensal bacteria (SPIEGELHALDER,B., et al. Influence of dietary nitrate on nitrite content
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`of humansaliva: possible relevance to in vivo formation of N-nitroso compounds. Food Cosmet
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`Toxicol. 1976, no. 14, p. 545-548). When nitrate is ingested it is rapidly absorbed into blood and
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`then accumulates in saliva. In the oral cavity bacteria reduce parts of the dietary nitrate to nitrite
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`and nitrite can then enter the systemic circulation. (LUNDBERG,Jon O., et al. Inorganic nitrate
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`is a possible source for systemic generation of nitric oxide. Free Radic Biol Med. 2004, vol. 37, p.
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`395-400).
`
`[0006]
`
`In vitro studies published in the 1990s show that NO is a modulator of mitochondrial
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`respiration via reversible inhibition of cytochrome c oxidase (CARR,GJ. et al. Nitric oxide formed
`
`by nitrite reductase of Paracoccus denitrificansis sufficiently stable to inhibit cytochrome oxidase
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`activity and is reduced by its reductase under aerobic conditions. Biochim Biophys Acta. 15 May
`
`1990, vol. 1017, no. 1, p. 57-62.; BOLANOS, J P, et al. Nitric oxide-mediated inhibition of the
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`mitochondrial respiratory chain in cultured astrocytes. J. Neurochem. 1994, vol. 63, no. 2, p. 910-
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`6; BROWN, GC,et al. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal
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`Attorney Docket No. 1255.101 CON2 BP
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`respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 19 Dec. 1994, vol. 356,
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`no. 2-3, p. 295-8; CLEETER, M W,et al. Reversible inhibition of cytochrome c oxidase, the
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`terminal enzyme of the mitochondrial
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`respiratory chain, by nitric oxide.
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`Implications for
`
`neurodegenerative diseases. FEBS Lett. 23 May 1994, vol. 345, no. 1, p. 50-4; and SCHWEIZER,
`
`M,et al. Nitric oxide potently and reversibly deenergizes mitochondria at low oxygen tension.
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`Biochem Biophys Res Comm. 1994, no. 204, p. 169-75). NO mayalsointeract at other sites of the
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`mitochondrial respiratory chain and in the Krebs cycle (for review see Moncada, supra). While
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`this important action of NO has been very well characterised in cell cultures, less is known about
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`its physiological relevance in vivo. To date the focus among researchers has been on the
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`cardiovascular effects of nitrite after its in vivo reduction to the vasodilator nitric oxide (NO)
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`(COSBYet al. (supra); DURANSKI, M. R., et al. Cytoprotective effects 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 healthy volunteers.
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`N Engl J Med. 2006, vol. 355, p. 2792-3). WO 2005/004884 A (US GOVERNMENTETAL.)
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`2005-01-20 and WO 2005/007173 A (US GOVERNMENT ET AL.) 2005-01-27 describe a
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`method to administer a nitrite salt specifically to obtain vasodilatation in a subject. No effects of
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`low-dose nitrate/nitrite on energy expenditure or glucose homeostasis or the effects of NO on
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`cellular respiration during physical exercise have been described.
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`[0007]
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`Physiological adaptation to exercise involves major cardiovascular and metabolic
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`changes. Oxygen consumption increases dramatically in the active muscles with a parallel increase
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`in muscle blood flow. In these processes, the endogenousgas nitric oxide (NO)plays an important
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`regulatory role. NO increases blood flow to the muscles and modulates muscular contraction and
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`glucose uptake (for review see STAMLER,J S. et al. Physiology ofnitric oxide in skeletal muscle.
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`Physiol Rev. 2001, vol. 81, no. 1, p. 209-37).
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`[0008]
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`The available information on the role of NO in healthy subjects and in particular in
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`athletes during work or exercise is both insufficient and contradictory. Shen and colleagues
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`showed that administration of NOS-inhibitors in vivo during submaximal exercise leads to
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`increased oxygen consumption in dogs (SHEN, W.et al. Role of NO in the regulation of oxygen
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`consumption in conscious dogs. Circulation Res. 1999, no. 84, p. 840-5) and Lacerda and
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`colleagues showed similarresults in rats (LACERDA, ACR,et al. Evidencethat brain nitric oxide
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`Attorney Docket No. 1255.101 CON2 BP
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`inhibition increases metabolic cost of exercise,
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`reducing running performance in rats.
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`Neuroscience Letters. 2006, no. 393, p. 260-3). The majority of studies have been done using
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`NOS-inhibitors while the effects of administering exogenous NO on exercise are largely unknown.
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`In addition, studies in healthy humansare scarce.
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`[0009]
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`Interestingly, the marketing of some currently available food supplements for athletes
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`and bodybuilders refer to the vasodilatory effect of NO. One example is “NOX2” (Bodyonics,
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`Ltd., USA), a product said to contain arginine alpha-ketoglutarate (A-AKG) and arginine-
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`ketoisocaproate (A-KIC) and allegedly capable of boosting short term nitric oxide levels. Other
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`products contain L-arginine, from which NO is synthesized by the NOS enzymes, and the
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`beneficial effects of NO are often referred to, however without offering more detailed
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`explanations.
`
`[0010]
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`The relation between peak work rate and resting levels of nitrate in plasma and urine
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`from subjects with different levels of physical fitness has been studied (Jungersten et al., Both
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`physical fitness and acute exercise regulate nitric oxide formation in healthy humans. J Appl
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`Physiol 82:760-764, 1997). A positive relationship between physical fitness and formation of NO
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`at rest was found and it was hypothesised that this positive relationship helps to explain the
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`beneficial effects of physical exercise on cardiovascular health. In Jungersten’s study nitrate was
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`used solely as a marker of NO production and the authors state several times that nitrate is a stable
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`and inert end product of NO andthatit is biologically inactive.
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`[0011]
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`Nitrate (NO3°) and nitrite (NO2’) are generally viewed as unwantedresidues in the food
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`chain with potentially harmful effects (Joint FAO/WHO Expert Committee on Food Additives
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`(JECFA). Safety Evaluation of Certain Food Additives. WHO, 1970.
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`ISBN 9241660503;
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`TANNENBAUM,S. R., et al. Nitrite in human saliva. Its possible relationship to nitrosamine
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`formation. J cancer Ins. 1974, vol. 53, p. 79-84; BARTSCH,H., et al. Inhibitors of endogenous
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`nitrosation: mechanisms and implications in human cancer prevention. Mutation Res. 1988, vol.
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`202, p. 307-324). Proposed harmful effects of these anions include promotion of gastric cancers
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`and other malignancies and development of methemoglobinemia in infants. Because of this the
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`levels of nitrate/nitrite are strictly regulated in food and drinking water.
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`Attorney Docket No. 1255.101 CON2 BP
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`Summaryof the Invention
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`[0012]
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`The disclosure is directed to methods of promoting health in a human (healthy or non-
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`healthy) and methods of decreasing systolic blood pressure in an adult human subject. The methods
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`include administering inorganic nitrate (NO3) to a human or adult human subject. In some
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`implementations, administration of the inorganic nitrate (NO3°) to the human or adult human
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`subject reduces diastolic blood pressure of the human or adult human subject.
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`In some
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`implementations, the human is a healthy human, and in other implementations the human is an
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`unhealthy human suffering from one or more of the conditions exemplified in this disclosure for
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`example.
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`In some implementations, the inorganic nitrate (NO3°) is administered orally. In some
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`implementations, the inorganic nitrate (NO3°) is administered in a composition in the form of a
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`liquid, a paste, a bar, a cake, a powder, a granulate, an effervescent tablet, a chewing gum,a tablet,
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`a capsule, a lozenge, a fast melting tablet or wafer, a sublingual tablet, or a spray. In someaspects,
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`the composition is a nutritional supplementor functional food.
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`[0013]
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`In some implementations, between 0.01 to 100 mmol
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`inorganic nitrate (NO3°) is
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`administered per kg body weight of the human or adult human subject, for example, between 0.01
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`mmol and 10 mmolinorganic nitrate (NO3°), between 0.1 mmol and 1 mmolinorganic nitrate, or
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`0.1 mmolinorganic nitrate (NO3°) is administered per kg body weight of the human or adult human
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`subject. Because the molecular weight of NO3° is 62.005 g/mol, the amount of inorganic nitrate
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`(NO3°) administered per kg body weight of the humanor adult human subject is between 0.62 mg
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`and 6.2 g, or for example, between 0.62 mg and 620 mg, between 6.2 mg and 62 mg, or 6.2 mg.
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`In some implementations, the inorganic nitrate (NO3°) is administered once a day.
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`[0014]
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`In some implementations, the inorganic nitrate (NO3°) is administered in the form of
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`beetroot juice. For example, the methods may include administering to the human or adult human
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`subject at least 3 deciliters fresh beetroot juice. In some aspects, the human or adult human subject
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`is administered the inorganic nitrate once a day. In some implementations, the human or adult
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`human subject is administered the inorganic nitrate for at least two days, for example three
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`consecutive days or two weeks.
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`[0015]
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`In some implementations,
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`the inorganic nitrate is administered in a composition
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`consisting of the inorganic nitrate and at least one additive. The additive can be a natural flavor,
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`an artificial flavor, a sweetener, a flavor enhancer, a color additive, an emulsifier, a stabilizer, a
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`fat, or a preservative.
`
`In other implementations,
`
`the inorganic nitrate is administered in a
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`Attorney Docket No. 1255.101 CON2 BP
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`composition comprising the inorganic nitrate and a polyphenol. Such a composition may be a
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`functional food. The polyphenol can be provided from at least one natural source selected from
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`the group consisting of: apple, pear, grapes, lemon, orange, lime, peach, pomegranate, grapefruit,
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`kiwi, ginger, pineapple, blackberries, black raspberries, blueberries, cranberries, red raspberries,
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`cherries, bog wortleberry,
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`lingonberries, black elderberry, black chokeberry, black currant,
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`cloudberries, strawberries, carrots, chili, rhubarb, onions, cacao products, green tea, black tea, nuts,
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`Yerba mate, and coffee. In these implementations, the amount of the inorganic nitrate in the
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`composition is at least 50%.
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`[0016]
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`In some implementations, the inorganic nitrate is provided from a naturalnitrate source.
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`For example, the natural inorganic nitrate source is juice or dried concentrate from at least one of
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`spinach, lettuce, fennel, cabbage, Chinese cabbage, and beetroot. In other implementations, the
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`source of the inorganic nitrate is in the form ofa nitrate salt. For example, the nitrate salt is sodium
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`nitrate, potassium nitrate, calcium nitrate, zinc nitrate, ammonium nitrate, or argininenitrate.
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`[0017]
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`Further implementations will becomeevident to the skilled person upon study of the
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`figures, description and examples, as well as the appended claims,
`
`incorporated herein by
`
`reference.
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`Description of the Figures
`
`[0018]
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`The invention will be described in closer detail in the following description, examples
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`and non-limiting claims, with reference to the attached drawings in which:
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`[0019]
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`FIG. 1 shows a graph illustrating numerous ways in which the combination of nitrate
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`and polyphenols synergistically act to increase the bioavailability of nitric oxide and at the same
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`time to reduce the formation of harmful compounds such as oxygen radicals and nitrosamines. For
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`detailed explanation see text.
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`[0020]
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`FIG. 2 is a graph showing changes in oxygen consumption (VOz) following iv infusion
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`of sodium nitrite in increasing doses. Nitrite was infused over a 10 min period in non-smoking
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`healthy male volunteers (30-70 years).
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`[0021]
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`FIG. 3 is a graph showingthe effects of a dietary supplementation with sodium nitrate
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`or sodium chloride (placebo) on plasma concentrations of nitrite measured at rest and immediately
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`after exercise in 9 healthy male volunteers.
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`Attorney Docket No. 1255.101 CON2 BP
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`[0022]
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`FIG. 4 is a bar diagram showing the oxygen consumption (VO2) and heart rate (HR)
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`measured at 6 different work rates after a 3-day dietary supplementation with sodium nitrate (0.1
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`mmol/kg body weight/min, NIT) or an equal amount of sodium chloride (CON). The study had a
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`randomized double-blind cross-over design with a washout period of at least 10 days between the
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`tests. * p < 0.05, ** p< 0.01.
`
`[0023]
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`FIG. 5 is a graph showing oxygen consumption during bicycle exercise at 80% of
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`VOzpeak in 9 healthy male volunteers. Measurements were made after a 3-day dietary
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`supplementation with sodium nitrate (0.1 mmol/kg body weight/day) or an equal amount of
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`sodium chloride (placebo). The difference between nitrate and placebo periods wassignificant (p
`
`< 0.01).
`
`[0024]
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`FIG. 6 is a bar diagram showing plasmalactate concentration measured at 6 different
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`workrates after dietary supplementation with sodium nitrate (0.1 mmol/kg body weight/day for 3
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`days, filled bars) or an equal amount of sodium chloride (placebo, empty bars).
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`[0025]
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`FIGS. 7a-7c consist of three graphs, showing changes in blood glucose levels after an
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`oral challenge with glucose for three test subjects in a double-blind, placebo-controlled cross-over
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`study (FIGS. 7a, 7b and 7c). A standard oral glucose tolerance test was performed. The subjects
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`(healthy non-smoking volunteers) had their diet supplemented for 3 days with either sodium
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`chloride (placebo) or sodium nitrate (Nitrate) at a dose of 0.1 mmol/kg body weight/day.
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`[0026]
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`FIG. 8 is a graph showing changesin blood glucoselevels after an oral challenge with
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`glucose in 8 additional subjects in a double-blind, placebo-controlled cross-over study. A standard
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`oral glucose tolerance test was performed. The subjects (healthy non-smoking volunteers) had
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`their diet supplemented for 3 days with either sodium chloride (PLACEBO) or sodium nitrate
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`(NITRATE)at a dose of 0.1 mmol/kg/day. Data are presented as mean + SEM.
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`[0027]
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`FIG. 9 is a graph showing the effect of a two-week intervention with beetroot juice
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`(fresh juice 3-4 dl/day) on systolic, diastolic and mean arterial (MAP) blood pressure in a 43-year-
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`old male with hypertension.
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`[0028]
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`FIG. 10 showsthe plasma nitrate and nitrite concentrations after intravenous infusion
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`of nitrate. Panel a)
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`shows plasma nitrate concentration; panel b)
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`shows plasma nitrite
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`concentrations; and panel c) shows plasmanitrite concentrations in wild type (C57BL/6), germ
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`free and knockout (eNOS) mice.
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`[0029]
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`FIG. 11 is a graph showing enhancedpost-ischemic blood flow after nitrate infusion.
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`Detailed Description
`
`[0030]
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`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 andis not intended to be limiting since the scope of the
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`present invention will be limited only by the appended claims and equivalents thereof.
`
`[0031]
`
`It must also be noted that, as used in this specification and the appendedclaims, the
`
`singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates
`
`otherwise.
`
`[0032]
`
`The term “about” when used in the context of numeric values denotes an interval of
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`accuracy, familiar and acceptable to a person skilled in the art. Said interval can be +/-2% of the
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`given value, preferably +/-5%, and most preferably +/-10% of the numeric values, where
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`applicable.
`
`[0033]
`
`The term “indirect calorimetry” is here defined as a method for calculating heat that
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`living organisms produce from their production of carbon dioxide and nitrogen waste and from
`
`their consumption of oxygen, well known to personsskilled in the relevantart.
`
`[0034]
`
`The term “catabolism”is defined as the metabolic process that breaks down molecules
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`into smaller units. It is made up of degradative chemical reactionsin theliving cell.
`
`[0035]
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`The term “edible” in this context means non-toxic and possible to ingest, however not
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`limited to particular modes of ingesting, such as drinking, chewing, applying to the oral cavity in
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`various forms, such as, for example a spray or aerosol.
`
`[0036]
`
`The term “energy expenditure”is here defined as the amount of energy expendedfor a
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`certain metabolic rate.
`
`[0037]
`
`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 madefrom functional food ingredients, or fortified with health-promoting
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`additives,
`
`like “vitamin-enriched” products, and also, fresh foods (e.g. vegetables) that have
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`specific claims attached. Fermented foods with live cultures are often also considered to be
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`functional foods with probiotic benefits.
`
`[0038]
`
`The term “insulin resistance” is here defined as a condition in which normal amounts
`
`of insulin are inadequate to produce a normalinsulin response from fat, muscle andliver cells.
`
`[0039]
`
`The term “mammal”is intended to encompassall mammals, and in particular humans,
`
`pets and agriculturally significant animals, as well as animals used in competitions, such as horses
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`and dogs.
`
`[0040]
`
`The term “metabolic syndrome”is here defined as a combination of medical disorders
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`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
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`glucose tolerance or insulin resistance, high blood pressure, central obesity, decreased HDL
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`cholesterol, elevated triglycerides and elevated uric acid levels.
`
`[0041]
`
`The term “metabolism” is used to define the complete set of chemical reactions that
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`occurin living cells and “metabolic rate” is defined as the speed of metabolism of a mammal.
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`[0042]
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`Methemoglobin is a form of hemoglobin in which the iron in the heme groupis in the
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`Fe3” state, not the Fe” of normal hemoglobin. Methemoglobin is unable to carry oxygen.
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`Methemoglobinemia is defined as a blood disorder characterized by the presence of a higher than
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`normallevel of methemoglobin in the blood.
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`[0043]
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`The term “oxygen consumption”is defined as the amount of oxygen (Ox) consumed by
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`a mammal andis usually expressed as ml of pure oxygen consumed/min. “Oxygen consumption”
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`relates to the amount of oxygen consumed by a mammalas whole but also to oxygen consumption
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`in an isolated tissue or organ, such as, but not limited to, heart, liver brain or other tissue exposed
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`to ischemia.
`
`[0044]
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`The term “performance enhancing food or food supplement” includes sport drinks and
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`energy drinks, as well as other liquid, semi-solid or solid forms, such as energy bars andtablets.
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`Nodistinction is intended between sports drinks and energy drinks. Sports drinks tend to be more
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`isotonic while energy drinks tend to contain more sugar and frequently also contain caffeine.
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`Usually, sports drinks are non-carbonated and frequently contain fructose or other sugars, and
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`complex carbohydrates, which are easily absorbed by the body, and are designed to promote the
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`availability of energy and/or prevent or treat mild dehydration. Sport drinks also contain
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`electrolytes (mainly sodium and potassium salts) and nutrients (proteins and aminoacids). Sport
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`Attorney Docket No. 1255.101 CON2 BP
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`drinks, energy drinks and other liquid, semi-solid and solid products, while marketed for athletes,
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`are also consumedby non-athletes, as a snack, in situations where extra energy and enduranceis
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`desired. It is currently believed that improved sports performance can be attained by the intake of
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`so-called sport drinks.
`
`[0045]
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`The term “significant hypotension” meansin this context an acute reduction of systolic
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`and/or diastolic blood pressure, accompanied by clinical symptoms of hypotension such as
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`dizziness, nausea, pallor, loss of consciousness, etc. Said symptoms may occurin various degrees,
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`andit is preferred that they are entirely avoided, minimized or eliminated as far as possible, or at
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`least to an extentthat they are clinically insignificant.
`
`[0046]
`
`The inventors have surprisingly shown that the metabolic rate and/or the oxygen
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`consumption can be influenced in a mammal(locally or systemically), by administering inorganic
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`nitrite (NO’) and/or nitrate (NO3°) to said mammalin an amountofnitrite and/or nitrate sufficient
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`to decrease oxygen consumption. The decreased oxygen consumptionis achieved without causing
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`significant hypotension and without causing any significant increase of the methemoglobin level
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`in said mammal. In case of local reduction of the metabolic rate, the oxygen consumption is
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`decreased in an isolated tissue or organ suchasthe heart, liver, brain or other tissue that is exposed
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`to ischemia (a condition in which blood flow, and thus oxygen,is restricted to a part of the body).
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`In such cases the interaction of reaction products (including NO) ofnitrite and/or nitrate with
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`enzymes of the mitochondrial respiratory chain and subsequent inhibition of respiration leads to
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`lowering of oxygen demand, which is beneficial for an ischemic tissue. This effect resembles
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`hibernation. Because the generation of active nitrite and/or nitrate reaction products is maximized
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`in ischemic tissues the effect of oxygen consumption will be most pronounced at these sites. In
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`one particular embodiment, the oxygen consumption is lowered in the heart.
`
`[0047]
`
`The inventors also showedthat dietary supplementation with inorganic nitrate results
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`in areduced VO» during physical exercise and a significant increase in muscularefficiency. These
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`effects occurred without any increase in plasmalactate.
`
`[0048]
`
`In principle, also other additional approaches can lead to increased systemic or local
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`nitrite levels. The most obviousis to administernitrite or its precursor nitrate as such, but this may
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`be supplemented by or enhanced by increasing the gastric pH (e.g. with an acid suppressive drug
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`such as a proton pumpinhibitor or H2 receptor antagonist or antacids) to maximizenitrite survival
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`Attorney Docket No. 1255.101 CON2 BP
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`in the stomach and thereby the systemic delivery of nitrite. Alternatively, the administration of
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`nitrite or nitrate can be supplemented by or enhanced by interfering with the oral microflora in
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`order to maximize the number of nitrate reducing species. This can be achieved through the
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`delivery of “probiotic” nitrate reducing bacteria or selective treatment with an antibiotic to favor
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`the nitrate reducing species.
`
`[0049]
`
`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,
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`e.g.
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`in prophylaxis, alleviation or treatment of several conditions. In an attempt to increase
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`systemic nitrite levels, nitrite and/or nitrate can be given by enteral administration (orally, in the
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`form of a liquid, semi-solid or solid preparation, such as a chewing gum,tablet, lozenge, wafer,
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`cake, bar or the like) or by parenteral administration (intravenous, transdermal, transcutaneous, by
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`inhalation, rectally, vaginally, topical, intraperitoneally, intra muscular, subcutaneous, sublingual
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`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
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`bolus doses. In some aspects, the inventors make available a composition, preferably an edible
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`composition, capable of enhancing performance manifested as a reduced oxygen uptake (VO2)
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`during physical exercise when ingested by a mammal, wherein said composition comprises
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`inorganic nitrate and/or nitrite, and in particular a composition wherein the effect of enhanced
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`performance is manifested as both a reduced oxygen uptake (VO) during physical work and a
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`significant increase in muscularefficiency.
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`Composition
`
`[0050]
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`It is likely that an optimal dose-interval exists, meaning that below a certain plasma
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`level of nitrite the effects are insufficient and, correspondingly, that over a certain level the effect
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`is lower and possibly accompanied by side effects.
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`In one embodiment,
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`the composition
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`comprising inorganic nitrite and/or nitrate is a pharmaceutical composition comprising inorganic
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`nitrite and/or nitrate in an amount whichis sufficient to decrease oxygen consumption, but which
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`does not increase the methemoglobin level in a subject when administered to said subject in a
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`prescribed dose. Optionally the composition comprises another pharmaceutically active
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`compound.
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`Attorney Docket No. 1255.101 CON2 BP
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`[0051]
`
`According to a particular embodiment, said composition comprises nitrite alone,
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`without the presence of nitrate. According to another embodiment, said composition in addition to
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`nitrate and/or nitrite, also comprises arginine.
`
`[0052]
`
`According to one embodiment, nitrate and nitrite are