US20160038533A1 - Nitrite formulations and their use as nitric oxide prodrugs - Google Patents
Nitrite formulations and their use as nitric oxide prodrugs Download PDFInfo
- Publication number
- US20160038533A1 US20160038533A1 US14/827,100 US201514827100A US2016038533A1 US 20160038533 A1 US20160038533 A1 US 20160038533A1 US 201514827100 A US201514827100 A US 201514827100A US 2016038533 A1 US2016038533 A1 US 2016038533A1
- Authority
- US
- United States
- Prior art keywords
- nitrite
- weight parts
- nitrate
- mixtures
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 386
- 239000000203 mixture Substances 0.000 title claims abstract description 145
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 title description 514
- 238000009472 formulation Methods 0.000 title description 38
- 239000000651 prodrug Substances 0.000 title description 2
- 229940002612 prodrug Drugs 0.000 title description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims abstract description 152
- 229910002651 NO3 Inorganic materials 0.000 claims abstract description 151
- 238000000034 method Methods 0.000 claims abstract description 40
- 150000002826 nitrites Chemical class 0.000 claims abstract description 30
- 230000010757 Reduction Activity Effects 0.000 claims abstract description 18
- 108010074051 C-Reactive Protein Proteins 0.000 claims abstract description 4
- 102100032752 C-reactive protein Human genes 0.000 claims abstract description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 109
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 claims description 62
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 58
- 229930064664 L-arginine Natural products 0.000 claims description 58
- 235000014852 L-arginine Nutrition 0.000 claims description 58
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 claims description 36
- 235000010323 ascorbic acid Nutrition 0.000 claims description 34
- 239000011668 ascorbic acid Substances 0.000 claims description 34
- 229960005070 ascorbic acid Drugs 0.000 claims description 33
- 235000010288 sodium nitrite Nutrition 0.000 claims description 31
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 30
- 235000021537 Beetroot Nutrition 0.000 claims description 20
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 claims description 20
- 229930003268 Vitamin C Natural products 0.000 claims description 20
- 235000019154 vitamin C Nutrition 0.000 claims description 20
- 239000011718 vitamin C Substances 0.000 claims description 20
- 229960002173 citrulline Drugs 0.000 claims description 19
- 150000002823 nitrates Chemical class 0.000 claims description 18
- RMRCNWBMXRMIRW-BYFNXCQMSA-M cyanocobalamin Chemical compound N#C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O RMRCNWBMXRMIRW-BYFNXCQMSA-M 0.000 claims description 16
- 229920002678 cellulose Polymers 0.000 claims description 15
- 239000004317 sodium nitrate Substances 0.000 claims description 15
- 235000010344 sodium nitrate Nutrition 0.000 claims description 15
- 244000269722 Thea sinensis Species 0.000 claims description 14
- 239000007937 lozenge Substances 0.000 claims description 13
- 240000006079 Schisandra chinensis Species 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 12
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 claims description 12
- 235000008422 Schisandra chinensis Nutrition 0.000 claims description 11
- 235000021028 berry Nutrition 0.000 claims description 11
- 239000000796 flavoring agent Substances 0.000 claims description 11
- 235000019634 flavors Nutrition 0.000 claims description 11
- 235000009569 green tea Nutrition 0.000 claims description 11
- 229940096428 hawthorn berry Drugs 0.000 claims description 11
- 229930003779 Vitamin B12 Natural products 0.000 claims description 10
- AZFNGPAYDKGCRB-XCPIVNJJSA-M [(1s,2s)-2-amino-1,2-diphenylethyl]-(4-methylphenyl)sulfonylazanide;chlororuthenium(1+);1-methyl-4-propan-2-ylbenzene Chemical compound [Ru+]Cl.CC(C)C1=CC=C(C)C=C1.C1=CC(C)=CC=C1S(=O)(=O)[N-][C@@H](C=1C=CC=CC=1)[C@@H](N)C1=CC=CC=C1 AZFNGPAYDKGCRB-XCPIVNJJSA-M 0.000 claims description 10
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- 239000004615 ingredient Substances 0.000 claims description 10
- 239000004304 potassium nitrite Substances 0.000 claims description 10
- 235000010289 potassium nitrite Nutrition 0.000 claims description 10
- 235000019163 vitamin B12 Nutrition 0.000 claims description 10
- 239000011715 vitamin B12 Substances 0.000 claims description 10
- 244000194101 Ginkgo biloba Species 0.000 claims description 9
- 235000004072 Ocimum sanctum Nutrition 0.000 claims description 9
- 235000010204 pine bark Nutrition 0.000 claims description 9
- 239000011666 cyanocobalamin Substances 0.000 claims description 8
- 235000000639 cyanocobalamin Nutrition 0.000 claims description 8
- 229960002104 cyanocobalamin Drugs 0.000 claims description 8
- 229940074358 magnesium ascorbate Drugs 0.000 claims description 8
- AIOKQVJVNPDJKA-ZZMNMWMASA-L magnesium;(2r)-2-[(1s)-1,2-dihydroxyethyl]-4-hydroxy-5-oxo-2h-furan-3-olate Chemical compound [Mg+2].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] AIOKQVJVNPDJKA-ZZMNMWMASA-L 0.000 claims description 8
- 239000011585 methylcobalamin Substances 0.000 claims description 8
- 235000007672 methylcobalamin Nutrition 0.000 claims description 8
- JEWJRMKHSMTXPP-BYFNXCQMSA-M methylcobalamin Chemical compound C[Co+]N([C@]1([H])[C@H](CC(N)=O)[C@]\2(CCC(=O)NC[C@H](C)OP(O)(=O)OC3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)C)C/2=C(C)\C([C@H](C/2(C)C)CCC(N)=O)=N\C\2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O JEWJRMKHSMTXPP-BYFNXCQMSA-M 0.000 claims description 8
- 244000019459 Cynara cardunculus Species 0.000 claims description 7
- 235000019106 Cynara scolymus Nutrition 0.000 claims description 7
- 235000016520 artichoke thistle Nutrition 0.000 claims description 7
- 239000011230 binding agent Substances 0.000 claims description 7
- 239000003086 colorant Substances 0.000 claims description 7
- 235000003599 food sweetener Nutrition 0.000 claims description 7
- 239000003765 sweetening agent Substances 0.000 claims description 7
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 6
- 229930195725 Mannitol Natural products 0.000 claims description 6
- 244000299461 Theobroma cacao Species 0.000 claims description 6
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 claims description 6
- 230000000181 anti-adherent effect Effects 0.000 claims description 6
- 239000003911 antiadherent Substances 0.000 claims description 6
- 239000001913 cellulose Substances 0.000 claims description 6
- 239000000314 lubricant Substances 0.000 claims description 6
- 235000019359 magnesium stearate Nutrition 0.000 claims description 6
- 239000000594 mannitol Substances 0.000 claims description 6
- 235000010355 mannitol Nutrition 0.000 claims description 6
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- 239000000377 silicon dioxide Substances 0.000 claims description 6
- 239000000811 xylitol Substances 0.000 claims description 6
- 235000010447 xylitol Nutrition 0.000 claims description 6
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 claims description 6
- 229960002675 xylitol Drugs 0.000 claims description 6
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 claims description 5
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 5
- 235000009337 Spinacia oleracea Nutrition 0.000 claims description 5
- 244000300264 Spinacia oleracea Species 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 229920003086 cellulose ether Polymers 0.000 claims description 5
- 150000002016 disaccharides Chemical class 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 150000004676 glycans Chemical class 0.000 claims description 5
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical class CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 5
- 229920001542 oligosaccharide Polymers 0.000 claims description 5
- 150000002482 oligosaccharides Chemical class 0.000 claims description 5
- 229920001223 polyethylene glycol Polymers 0.000 claims description 5
- 229920001282 polysaccharide Polymers 0.000 claims description 5
- 239000005017 polysaccharide Substances 0.000 claims description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 claims description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 5
- 239000000600 sorbitol Substances 0.000 claims description 5
- 235000010356 sorbitol Nutrition 0.000 claims description 5
- 235000019698 starch Nutrition 0.000 claims description 5
- 239000008107 starch Substances 0.000 claims description 5
- 150000005846 sugar alcohols Chemical class 0.000 claims description 5
- 241000219094 Vitaceae Species 0.000 claims description 4
- 235000021021 grapes Nutrition 0.000 claims description 4
- MUKYLHIZBOASDM-UHFFFAOYSA-N 2-[carbamimidoyl(methyl)amino]acetic acid 2,3,4,5,6-pentahydroxyhexanoic acid Chemical compound NC(=N)N(C)CC(O)=O.OCC(O)C(O)C(O)C(O)C(O)=O MUKYLHIZBOASDM-UHFFFAOYSA-N 0.000 claims description 3
- 244000105624 Arachis hypogaea Species 0.000 claims description 3
- 241000512259 Ascophyllum nodosum Species 0.000 claims description 3
- 239000009405 Ashwagandha Substances 0.000 claims description 3
- 241001061264 Astragalus Species 0.000 claims description 3
- 244000166075 Borojoa patinoi Species 0.000 claims description 3
- 235000003558 Borojoa patinoi Nutrition 0.000 claims description 3
- 235000011299 Brassica oleracea var botrytis Nutrition 0.000 claims description 3
- 235000017647 Brassica oleracea var italica Nutrition 0.000 claims description 3
- 240000003259 Brassica oleracea var. botrytis Species 0.000 claims description 3
- 240000007154 Coffea arabica Species 0.000 claims description 3
- 241000190633 Cordyceps Species 0.000 claims description 3
- 240000007371 Cuscuta campestris Species 0.000 claims description 3
- 241001632410 Eleutherococcus senticosus Species 0.000 claims description 3
- 241000893536 Epimedium Species 0.000 claims description 3
- 241000208688 Eucommia Species 0.000 claims description 3
- 241000208253 Gymnema sylvestre Species 0.000 claims description 3
- 235000017309 Hypericum perforatum Nutrition 0.000 claims description 3
- 244000188472 Ilex paraguariensis Species 0.000 claims description 3
- 235000003368 Ilex paraguariensis Nutrition 0.000 claims description 3
- 241000758791 Juglandaceae Species 0.000 claims description 3
- 240000004371 Panax ginseng Species 0.000 claims description 3
- 235000002789 Panax ginseng Nutrition 0.000 claims description 3
- 244000294611 Punica granatum Species 0.000 claims description 3
- 235000014360 Punica granatum Nutrition 0.000 claims description 3
- 241001165494 Rhodiola Species 0.000 claims description 3
- 235000017276 Salvia Nutrition 0.000 claims description 3
- 240000007164 Salvia officinalis Species 0.000 claims description 3
- 244000228451 Stevia rebaudiana Species 0.000 claims description 3
- 235000009470 Theobroma cacao Nutrition 0.000 claims description 3
- 241000819233 Tribulus <sea snail> Species 0.000 claims description 3
- 240000004482 Withania somnifera Species 0.000 claims description 3
- 235000001978 Withania somnifera Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
- 241000482268 Zea mays subsp. mays Species 0.000 claims description 3
- 235000006533 astragalus Nutrition 0.000 claims description 3
- 235000013405 beer Nutrition 0.000 claims description 3
- 235000019219 chocolate Nutrition 0.000 claims description 3
- 235000016213 coffee Nutrition 0.000 claims description 3
- 235000013353 coffee beverage Nutrition 0.000 claims description 3
- 235000018905 epimedium Nutrition 0.000 claims description 3
- 235000008434 ginseng Nutrition 0.000 claims description 3
- 235000008390 olive oil Nutrition 0.000 claims description 3
- 239000004006 olive oil Substances 0.000 claims description 3
- 235000020232 peanut Nutrition 0.000 claims description 3
- 239000004323 potassium nitrate Substances 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 claims description 3
- 210000004233 talus Anatomy 0.000 claims description 3
- 235000013616 tea Nutrition 0.000 claims description 3
- 229940047183 tribulus Drugs 0.000 claims description 3
- 235000020234 walnut Nutrition 0.000 claims description 3
- 235000014101 wine Nutrition 0.000 claims description 3
- DBRXOUCRJQVYJQ-CKNDUULBSA-N withaferin A Chemical compound C([C@@H]1[C@H]([C@@H]2[C@]3(CC[C@@H]4[C@@]5(C)C(=O)C=C[C@H](O)[C@@]65O[C@@H]6C[C@H]4[C@@H]3CC2)C)C)C(C)=C(CO)C(=O)O1 DBRXOUCRJQVYJQ-CKNDUULBSA-N 0.000 claims description 3
- 239000004260 Potassium ascorbate Substances 0.000 claims description 2
- AAJBNRZDTJPMTJ-UHFFFAOYSA-L magnesium;dinitrite Chemical compound [Mg+2].[O-]N=O.[O-]N=O AAJBNRZDTJPMTJ-UHFFFAOYSA-L 0.000 claims description 2
- RAFRTSDUWORDLA-UHFFFAOYSA-N phenyl 3-chloropropanoate Chemical compound ClCCC(=O)OC1=CC=CC=C1 RAFRTSDUWORDLA-UHFFFAOYSA-N 0.000 claims description 2
- 235000019275 potassium ascorbate Nutrition 0.000 claims description 2
- 229940017794 potassium ascorbate Drugs 0.000 claims description 2
- CONVKSGEGAVTMB-RXSVEWSESA-M potassium-L-ascorbate Chemical compound [K+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] CONVKSGEGAVTMB-RXSVEWSESA-M 0.000 claims description 2
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 2
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 2
- 229960005055 sodium ascorbate Drugs 0.000 claims description 2
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 2
- 244000136948 Ocimum sanctum Species 0.000 claims 3
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 claims 3
- 150000003626 triacylglycerols Chemical class 0.000 abstract description 5
- 235000005911 diet Nutrition 0.000 description 58
- 102000008299 Nitric Oxide Synthase Human genes 0.000 description 55
- 108010021487 Nitric Oxide Synthase Proteins 0.000 description 55
- 210000001519 tissue Anatomy 0.000 description 51
- 230000009467 reduction Effects 0.000 description 45
- 241000282414 Homo sapiens Species 0.000 description 40
- 230000015572 biosynthetic process Effects 0.000 description 40
- 241000699670 Mus sp. Species 0.000 description 37
- 238000004519 manufacturing process Methods 0.000 description 35
- 210000004369 blood Anatomy 0.000 description 32
- 239000008280 blood Substances 0.000 description 32
- 230000000694 effects Effects 0.000 description 32
- 230000037213 diet Effects 0.000 description 31
- 208000028867 ischemia Diseases 0.000 description 29
- 230000010410 reperfusion Effects 0.000 description 29
- 239000001301 oxygen Substances 0.000 description 28
- 229910052760 oxygen Inorganic materials 0.000 description 28
- JCXJVPUVTGWSNB-UHFFFAOYSA-N Nitrogen dioxide Chemical compound O=[N]=O JCXJVPUVTGWSNB-UHFFFAOYSA-N 0.000 description 27
- 230000000378 dietary effect Effects 0.000 description 27
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 26
- 229910002089 NOx Inorganic materials 0.000 description 25
- 210000002216 heart Anatomy 0.000 description 23
- 230000037361 pathway Effects 0.000 description 23
- 241000282412 Homo Species 0.000 description 22
- 208000031225 myocardial ischemia Diseases 0.000 description 22
- 230000009469 supplementation Effects 0.000 description 22
- 230000001965 increasing effect Effects 0.000 description 21
- 239000000758 substrate Substances 0.000 description 21
- 206010021143 Hypoxia Diseases 0.000 description 20
- 102100028452 Nitric oxide synthase, endothelial Human genes 0.000 description 20
- 125000000018 nitroso group Chemical group N(=O)* 0.000 description 19
- 101710090055 Nitric oxide synthase, endothelial Proteins 0.000 description 18
- 206010063837 Reperfusion injury Diseases 0.000 description 17
- 230000006378 damage Effects 0.000 description 17
- 208000024172 Cardiovascular disease Diseases 0.000 description 16
- 238000001727 in vivo Methods 0.000 description 16
- 208000027418 Wounds and injury Diseases 0.000 description 15
- 208000014674 injury Diseases 0.000 description 15
- 210000000214 mouth Anatomy 0.000 description 15
- 208000010125 myocardial infarction Diseases 0.000 description 15
- 239000000047 product Substances 0.000 description 15
- 230000002829 reductive effect Effects 0.000 description 15
- 210000002784 stomach Anatomy 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 14
- 108010025915 Nitrite Reductases Proteins 0.000 description 14
- 235000020188 drinking water Nutrition 0.000 description 14
- 239000003651 drinking water Substances 0.000 description 14
- 230000000302 ischemic effect Effects 0.000 description 14
- 230000004060 metabolic process Effects 0.000 description 14
- 210000003205 muscle Anatomy 0.000 description 14
- 230000003647 oxidation Effects 0.000 description 14
- 238000007254 oxidation reaction Methods 0.000 description 14
- 235000013311 vegetables Nutrition 0.000 description 14
- 241000124008 Mammalia Species 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 13
- 239000003814 drug Substances 0.000 description 13
- 230000002526 effect on cardiovascular system Effects 0.000 description 13
- 230000001146 hypoxic effect Effects 0.000 description 13
- 241000894006 Bacteria Species 0.000 description 12
- 206010028980 Neoplasm Diseases 0.000 description 12
- 235000013305 food Nutrition 0.000 description 12
- 239000013589 supplement Substances 0.000 description 12
- 230000001225 therapeutic effect Effects 0.000 description 12
- 238000002560 therapeutic procedure Methods 0.000 description 12
- 230000006870 function Effects 0.000 description 11
- 210000001035 gastrointestinal tract Anatomy 0.000 description 11
- 241000196324 Embryophyta Species 0.000 description 10
- 201000011510 cancer Diseases 0.000 description 10
- 229940079593 drug Drugs 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 201000001320 Atherosclerosis Diseases 0.000 description 9
- 230000009471 action Effects 0.000 description 9
- 235000010980 cellulose Nutrition 0.000 description 9
- 230000004087 circulation Effects 0.000 description 9
- 230000001419 dependent effect Effects 0.000 description 9
- 230000002496 gastric effect Effects 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 230000007246 mechanism Effects 0.000 description 9
- 230000001681 protective effect Effects 0.000 description 9
- 210000003296 saliva Anatomy 0.000 description 9
- 229940124549 vasodilator Drugs 0.000 description 9
- 239000003071 vasodilator agent Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 230000008901 benefit Effects 0.000 description 8
- 230000017531 blood circulation Effects 0.000 description 8
- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 description 8
- 230000003247 decreasing effect Effects 0.000 description 8
- 230000002255 enzymatic effect Effects 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 230000036541 health Effects 0.000 description 8
- 230000035479 physiological effects, processes and functions Effects 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000011664 signaling Effects 0.000 description 8
- 235000013175 Crataegus laevigata Nutrition 0.000 description 7
- 206010048554 Endothelial dysfunction Diseases 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 241000283984 Rodentia Species 0.000 description 7
- 108010093894 Xanthine oxidase Proteins 0.000 description 7
- 102100033220 Xanthine oxidase Human genes 0.000 description 7
- 230000007423 decrease Effects 0.000 description 7
- 230000008694 endothelial dysfunction Effects 0.000 description 7
- 150000003278 haem Chemical class 0.000 description 7
- 230000007954 hypoxia Effects 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 230000032424 nitric oxide homeostasis Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 102100022397 Nitric oxide synthase, brain Human genes 0.000 description 6
- 101710111444 Nitric oxide synthase, brain Proteins 0.000 description 6
- 240000002837 Ocimum tenuiflorum Species 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- 230000002378 acidificating effect Effects 0.000 description 6
- 230000001154 acute effect Effects 0.000 description 6
- LZDSILRDTDCIQT-UHFFFAOYSA-N dinitrogen trioxide Chemical compound [O-][N+](=O)N=O LZDSILRDTDCIQT-UHFFFAOYSA-N 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 235000008216 herbs Nutrition 0.000 description 6
- 235000009200 high fat diet Nutrition 0.000 description 6
- 235000013372 meat Nutrition 0.000 description 6
- 210000003470 mitochondria Anatomy 0.000 description 6
- 239000002840 nitric oxide donor Substances 0.000 description 6
- 150000004005 nitrosamines Chemical class 0.000 description 6
- 238000007034 nitrosation reaction Methods 0.000 description 6
- 230000000153 supplemental effect Effects 0.000 description 6
- 230000002792 vascular Effects 0.000 description 6
- 108010054147 Hemoglobins Proteins 0.000 description 5
- 102000001554 Hemoglobins Human genes 0.000 description 5
- 150000004008 N-nitroso compounds Chemical class 0.000 description 5
- 108010076864 Nitric Oxide Synthase Type II Proteins 0.000 description 5
- 230000000747 cardiac effect Effects 0.000 description 5
- 230000003293 cardioprotective effect Effects 0.000 description 5
- 230000007211 cardiovascular event Effects 0.000 description 5
- 230000037406 food intake Effects 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 230000002107 myocardial effect Effects 0.000 description 5
- UMFJAHHVKNCGLG-UHFFFAOYSA-N n-Nitrosodimethylamine Chemical compound CN(C)N=O UMFJAHHVKNCGLG-UHFFFAOYSA-N 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 230000003389 potentiating effect Effects 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- INGWEZCOABYORO-UHFFFAOYSA-N 2-(furan-2-yl)-7-methyl-1h-1,8-naphthyridin-4-one Chemical compound N=1C2=NC(C)=CC=C2C(O)=CC=1C1=CC=CO1 INGWEZCOABYORO-UHFFFAOYSA-N 0.000 description 4
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 4
- 206010061216 Infarction Diseases 0.000 description 4
- 229920000881 Modified starch Polymers 0.000 description 4
- 208000007201 Myocardial reperfusion injury Diseases 0.000 description 4
- 108090000913 Nitrate Reductases Proteins 0.000 description 4
- 102100029438 Nitric oxide synthase, inducible Human genes 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 229940024606 amino acid Drugs 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 230000000711 cancerogenic effect Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000001120 cytoprotective effect Effects 0.000 description 4
- 108010002255 deoxyhemoglobin Proteins 0.000 description 4
- 230000003292 diminished effect Effects 0.000 description 4
- 229910001882 dioxygen Inorganic materials 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001802 infusion Methods 0.000 description 4
- 210000000936 intestine Anatomy 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 235000019426 modified starch Nutrition 0.000 description 4
- 210000004165 myocardium Anatomy 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000002441 reversible effect Effects 0.000 description 4
- FNKQXYHWGSIFBK-RPDRRWSUSA-N sapropterin Chemical compound N1=C(N)NC(=O)C2=C1NC[C@H]([C@@H](O)[C@@H](O)C)N2 FNKQXYHWGSIFBK-RPDRRWSUSA-N 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- 150000003573 thiols Chemical class 0.000 description 4
- 238000012549 training Methods 0.000 description 4
- 238000011282 treatment Methods 0.000 description 4
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- 206010020772 Hypertension Diseases 0.000 description 3
- KEJOCWOXCDWNID-UHFFFAOYSA-N Nitrilooxonium Chemical compound [O+]#N KEJOCWOXCDWNID-UHFFFAOYSA-N 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 230000009102 absorption Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 206010000891 acute myocardial infarction Diseases 0.000 description 3
- 229960001456 adenosine triphosphate Drugs 0.000 description 3
- 230000002411 adverse Effects 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 235000009697 arginine Nutrition 0.000 description 3
- 229960003121 arginine Drugs 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000036996 cardiovascular health Effects 0.000 description 3
- 210000000748 cardiovascular system Anatomy 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 230000007123 defense Effects 0.000 description 3
- 210000003038 endothelium Anatomy 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 238000013265 extended release Methods 0.000 description 3
- FVTCRASFADXXNN-SCRDCRAPSA-N flavin mononucleotide Chemical compound OP(=O)(O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-SCRDCRAPSA-N 0.000 description 3
- FVTCRASFADXXNN-UHFFFAOYSA-N flavin mononucleotide Natural products OP(=O)(O)OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O FVTCRASFADXXNN-UHFFFAOYSA-N 0.000 description 3
- 239000011768 flavin mononucleotide Substances 0.000 description 3
- 235000012055 fruits and vegetables Nutrition 0.000 description 3
- 239000007789 gas Substances 0.000 description 3
- 235000021384 green leafy vegetables Nutrition 0.000 description 3
- 208000019622 heart disease Diseases 0.000 description 3
- 210000005003 heart tissue Anatomy 0.000 description 3
- 208000001286 intracranial vasospasm Diseases 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000000670 limiting effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 208000005135 methemoglobinemia Diseases 0.000 description 3
- 230000005787 mitochondrial ATP synthesis coupled electron transport Effects 0.000 description 3
- 230000002438 mitochondrial effect Effects 0.000 description 3
- 238000010172 mouse model Methods 0.000 description 3
- 230000010417 nitric oxide pathway Effects 0.000 description 3
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 3
- 230000009935 nitrosation Effects 0.000 description 3
- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- -1 oxygen radicals Chemical class 0.000 description 3
- 230000000144 pharmacologic effect Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000003449 preventive effect Effects 0.000 description 3
- 230000005588 protonation Effects 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 235000019231 riboflavin-5'-phosphate Nutrition 0.000 description 3
- 230000000391 smoking effect Effects 0.000 description 3
- 229940126680 traditional chinese medicines Drugs 0.000 description 3
- 230000024883 vasodilation Effects 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 235000016068 Berberis vulgaris Nutrition 0.000 description 2
- 241000335053 Beta vulgaris Species 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 206010007269 Carcinogenicity Diseases 0.000 description 2
- 241001340526 Chrysoclista linneella Species 0.000 description 2
- 241000252203 Clupea harengus Species 0.000 description 2
- 241001092040 Crataegus Species 0.000 description 2
- 235000014493 Crataegus Nutrition 0.000 description 2
- 244000265913 Crataegus laevigata Species 0.000 description 2
- 235000017156 Crataegus rhipidophylla Nutrition 0.000 description 2
- 206010011703 Cyanosis Diseases 0.000 description 2
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 2
- 102000015782 Electron Transport Complex III Human genes 0.000 description 2
- 108010024882 Electron Transport Complex III Proteins 0.000 description 2
- 206010014486 Elevated triglycerides Diseases 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N Fe2+ Chemical compound [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
- 206010056328 Hepatic ischaemia Diseases 0.000 description 2
- 208000031226 Hyperlipidaemia Diseases 0.000 description 2
- 235000003228 Lactuca sativa Nutrition 0.000 description 2
- 240000008415 Lactuca sativa Species 0.000 description 2
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 2
- 108010075520 Nitric Oxide Synthase Type III Proteins 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- VVGPECAOVDZTLZ-UHFFFAOYSA-N [N]NC(N)=N Chemical compound [N]NC(N)=N VVGPECAOVDZTLZ-UHFFFAOYSA-N 0.000 description 2
- XJLXINKUBYWONI-DQQFMEOOSA-N [[(2r,3r,4r,5r)-5-(6-aminopurin-9-yl)-3-hydroxy-4-phosphonooxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2s,3r,4s,5s)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl phosphate Chemical compound NC(=O)C1=CC=C[N+]([C@@H]2[C@H]([C@@H](O)[C@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-DQQFMEOOSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 210000000709 aorta Anatomy 0.000 description 2
- 210000000702 aorta abdominal Anatomy 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 230000036770 blood supply Effects 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000357 carcinogen Toxicity 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 231100000315 carcinogenic Toxicity 0.000 description 2
- 239000003183 carcinogenic agent Substances 0.000 description 2
- 231100000260 carcinogenicity Toxicity 0.000 description 2
- 230000007670 carcinogenicity Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 235000013477 citrulline Nutrition 0.000 description 2
- 235000020971 citrus fruits Nutrition 0.000 description 2
- 208000029039 cyanide poisoning Diseases 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 235000015872 dietary supplement Nutrition 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 230000009939 endogenous nitrosation Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 235000020774 essential nutrients Nutrition 0.000 description 2
- 235000019688 fish Nutrition 0.000 description 2
- 235000019162 flavin adenine dinucleotide Nutrition 0.000 description 2
- 239000011714 flavin adenine dinucleotide Substances 0.000 description 2
- 230000004907 flux Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000008821 health effect Effects 0.000 description 2
- 235000019514 herring Nutrition 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 230000007574 infarction Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 208000037906 ischaemic injury Diseases 0.000 description 2
- 208000012947 ischemia reperfusion injury Diseases 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 230000006540 mitochondrial respiration Effects 0.000 description 2
- 210000003097 mucus Anatomy 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- YBVAXJOZZAJCLA-UHFFFAOYSA-N nitric acid nitrous acid Chemical compound ON=O.O[N+]([O-])=O YBVAXJOZZAJCLA-UHFFFAOYSA-N 0.000 description 2
- 210000004789 organ system Anatomy 0.000 description 2
- 230000036542 oxidative stress Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 230000005180 public health Effects 0.000 description 2
- 238000010926 purge Methods 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000006965 reversible inhibition Effects 0.000 description 2
- 210000003079 salivary gland Anatomy 0.000 description 2
- 229960004617 sapropterin Drugs 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 210000002027 skeletal muscle Anatomy 0.000 description 2
- 235000000891 standard diet Nutrition 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 210000004243 sweat Anatomy 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 230000000304 vasodilatating effect Effects 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 235000020681 well water Nutrition 0.000 description 2
- 239000002349 well water Substances 0.000 description 2
- JWZZKOKVBUJMES-UHFFFAOYSA-N (+-)-Isoprenaline Chemical compound CC(C)NCC(O)C1=CC=C(O)C(O)=C1 JWZZKOKVBUJMES-UHFFFAOYSA-N 0.000 description 1
- ZONOMVGGONAQQA-QTBHSTJXSA-N (2s)-2-acetamidopropanoic acid;[3-[[(2r)-1-ethenoxy-3-sulfanylidenepropan-2-yl]amino]-2,2-dimethyl-3-oxopropyl] nitrate Chemical compound OC(=O)[C@H](C)NC(C)=O.[O-][N+](=O)OCC(C)(C)C(=O)N[C@H](COC=C)C=S ZONOMVGGONAQQA-QTBHSTJXSA-N 0.000 description 1
- UOKINRKWPYVMSZ-LADGPHEKSA-N (5r,11as)-2-benzyl-5-(3-hydroxyphenyl)-6h-1,2,3,5,11,11a-hexahydro-imidazo[1,5-b]-β-carboline-1,3-dione Chemical compound OC1=CC=CC([C@@H]2C3=C(C4=CC=CC=C4N3)C[C@@H]3N2C(N(CC=2C=CC=CC=2)C3=O)=O)=C1 UOKINRKWPYVMSZ-LADGPHEKSA-N 0.000 description 1
- UCTWMZQNUQWSLP-VIFPVBQESA-N (R)-adrenaline Chemical compound CNC[C@H](O)C1=CC=C(O)C(O)=C1 UCTWMZQNUQWSLP-VIFPVBQESA-N 0.000 description 1
- 229930182837 (R)-adrenaline Natural products 0.000 description 1
- ZOOGRGPOEVQQDX-UUOKFMHZSA-N 3',5'-cyclic GMP Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=C(NC2=O)N)=C2N=C1 ZOOGRGPOEVQQDX-UUOKFMHZSA-N 0.000 description 1
- MGWGWNFMUOTEHG-UHFFFAOYSA-N 4-(3,5-dimethylphenyl)-1,3-thiazol-2-amine Chemical compound CC1=CC(C)=CC(C=2N=C(N)SC=2)=C1 MGWGWNFMUOTEHG-UHFFFAOYSA-N 0.000 description 1
- 241000191291 Abies alba Species 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 description 1
- 102000011727 Caspases Human genes 0.000 description 1
- 108010076667 Caspases Proteins 0.000 description 1
- 108010075016 Ceruloplasmin Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000819038 Chichester Species 0.000 description 1
- 241000195654 Chlorella sorokiniana Species 0.000 description 1
- 241000207199 Citrus Species 0.000 description 1
- 235000019542 Cured Meats Nutrition 0.000 description 1
- 206010011705 Cyanosis neonatal Diseases 0.000 description 1
- 102100030497 Cytochrome c Human genes 0.000 description 1
- 108050008072 Cytochrome c oxidase subunit IV Proteins 0.000 description 1
- 108090000365 Cytochrome-c oxidases Proteins 0.000 description 1
- 108010075031 Cytochromes c Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 description 1
- 102000008015 Hemeproteins Human genes 0.000 description 1
- 108010089792 Hemeproteins Proteins 0.000 description 1
- 208000035150 Hypercholesterolemia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 206010022680 Intestinal ischaemia Diseases 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 108010044467 Isoenzymes Proteins 0.000 description 1
- 125000002059 L-arginyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C([H])([H])N([H])C(=N[H])N([H])[H] 0.000 description 1
- 235000001412 Mediterranean diet Nutrition 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- FQWRAVYMZULPNK-BYPYZUCNSA-N N(5)-[(hydroxyamino)(imino)methyl]-L-ornithine Chemical compound OC(=O)[C@@H](N)CCCNC(=N)NO FQWRAVYMZULPNK-BYPYZUCNSA-N 0.000 description 1
- YDGMGEXADBMOMJ-LURJTMIESA-N N(g)-dimethylarginine Chemical compound CN(C)C(\N)=N\CCC[C@H](N)C(O)=O YDGMGEXADBMOMJ-LURJTMIESA-N 0.000 description 1
- KCWZGJVSDFYRIX-YFKPBYRVSA-N N(gamma)-nitro-L-arginine methyl ester Chemical compound COC(=O)[C@@H](N)CCCN=C(N)N[N+]([O-])=O KCWZGJVSDFYRIX-YFKPBYRVSA-N 0.000 description 1
- 108010029138 N-nitratopivaloyl-S-(N'-acetylalanyl)-cysteine ethyl ester Proteins 0.000 description 1
- 150000001204 N-oxides Chemical class 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 102000011779 Nitric Oxide Synthase Type II Human genes 0.000 description 1
- SWINTVPHHIJZJR-UHFFFAOYSA-N O=[N+].[O-][N+]([O-])=O Chemical compound O=[N+].[O-][N+]([O-])=O SWINTVPHHIJZJR-UHFFFAOYSA-N 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108090000417 Oxygenases Proteins 0.000 description 1
- 102000004020 Oxygenases Human genes 0.000 description 1
- 241000590428 Panacea Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 208000010378 Pulmonary Embolism Diseases 0.000 description 1
- 241000558607 Schisandra rubriflora Species 0.000 description 1
- 206010040943 Skin Ulcer Diseases 0.000 description 1
- 208000032851 Subarachnoid Hemorrhage Diseases 0.000 description 1
- 239000004376 Sucralose Substances 0.000 description 1
- 206010049418 Sudden Cardiac Death Diseases 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- JLRGJRBPOGGCBT-UHFFFAOYSA-N Tolbutamide Chemical compound CCCCNC(=O)NS(=O)(=O)C1=CC=C(C)C=C1 JLRGJRBPOGGCBT-UHFFFAOYSA-N 0.000 description 1
- 235000021068 Western diet Nutrition 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000006538 anaerobic glycolysis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 150000001449 anionic compounds Chemical class 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 208000037849 arterial hypertension Diseases 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 229940072107 ascorbate Drugs 0.000 description 1
- 235000015173 baked goods and baking mixes Nutrition 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000008238 biochemical pathway Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940124630 bronchodilator Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 229940045200 cardioprotective agent Drugs 0.000 description 1
- 239000012659 cardioprotective agent Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000008370 chocolate flavor Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 235000017471 coenzyme Q10 Nutrition 0.000 description 1
- ACTIUHUUMQJHFO-UPTCCGCDSA-N coenzyme Q10 Chemical compound COC1=C(OC)C(=O)C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)=C(C)C1=O ACTIUHUUMQJHFO-UPTCCGCDSA-N 0.000 description 1
- 230000001447 compensatory effect Effects 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000009091 contractile dysfunction Effects 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 125000000664 diazo group Chemical group [N-]=[N+]=[*] 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 235000020805 dietary restrictions Nutrition 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 235000021271 drinking Nutrition 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 229920001971 elastomer Polymers 0.000 description 1
- 230000008753 endothelial function Effects 0.000 description 1
- 239000000066 endothelium dependent relaxing factor Substances 0.000 description 1
- 210000003989 endothelium vascular Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229960005139 epinephrine Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 235000020937 fasting conditions Nutrition 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- YAGKRVSRTSUGEY-UHFFFAOYSA-N ferricyanide Chemical compound [Fe+3].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] YAGKRVSRTSUGEY-UHFFFAOYSA-N 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- VWWQXMAJTJZDQX-UYBVJOGSSA-N flavin adenine dinucleotide Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]([C@H](O)[C@@H]1O)O[C@@H]1CO[P@](O)(=O)O[P@@](O)(=O)OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C2=NC(=O)NC(=O)C2=NC2=C1C=C(C)C(C)=C2 VWWQXMAJTJZDQX-UYBVJOGSSA-N 0.000 description 1
- 229940013640 flavin mononucleotide Drugs 0.000 description 1
- 229940093632 flavin-adenine dinucleotide Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 210000000245 forearm Anatomy 0.000 description 1
- 239000003574 free electron Substances 0.000 description 1
- 239000008369 fruit flavor Substances 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 235000021472 generally recognized as safe Nutrition 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 229940096427 hawthorn berry extract Drugs 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000001378 hepatocarcinogenic effect Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 231100000334 hepatotoxic Toxicity 0.000 description 1
- 230000003082 hepatotoxic effect Effects 0.000 description 1
- 239000012676 herbal extract Substances 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 230000009215 host defense mechanism Effects 0.000 description 1
- 235000006486 human diet Nutrition 0.000 description 1
- 238000007455 ileostomy Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229910001412 inorganic anion Inorganic materials 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 229910001959 inorganic nitrate Inorganic materials 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000037041 intracellular level Effects 0.000 description 1
- 230000010499 intrinsic vasodilatating effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229940124280 l-arginine Drugs 0.000 description 1
- 239000003041 laboratory chemical Substances 0.000 description 1
- 230000032820 leukocyte apoptotic process Effects 0.000 description 1
- 230000011268 leukocyte chemotaxis Effects 0.000 description 1
- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000029052 metamorphosis Effects 0.000 description 1
- 230000004089 microcirculation Effects 0.000 description 1
- 230000004065 mitochondrial dysfunction Effects 0.000 description 1
- 239000002324 mouth wash Substances 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 230000008881 mucosal defense Effects 0.000 description 1
- 230000004678 mucosal integrity Effects 0.000 description 1
- 230000008345 muscle blood flow Effects 0.000 description 1
- 208000037891 myocardial injury Diseases 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 229910017464 nitrogen compound Inorganic materials 0.000 description 1
- 150000002830 nitrogen compounds Chemical class 0.000 description 1
- 150000002832 nitroso derivatives Chemical class 0.000 description 1
- 235000021590 normal diet Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 239000007968 orange flavor Substances 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011809 primate model Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011321 prophylaxis Methods 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 230000004088 pulmonary circulation Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011867 re-evaluation Methods 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- NPCOQXAVBJJZBQ-UHFFFAOYSA-N reduced coenzyme Q9 Natural products COC1=C(O)C(C)=C(CC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)C(O)=C1OC NPCOQXAVBJJZBQ-UHFFFAOYSA-N 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 210000001581 salivary duct Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000021003 saturated fats Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000276 sedentary effect Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000007781 signaling event Effects 0.000 description 1
- 206010040872 skin infection Diseases 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 235000002639 sodium chloride Nutrition 0.000 description 1
- 235000021058 soft food Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- BAQAVOSOZGMPRM-QBMZZYIRSA-N sucralose Chemical compound O[C@@H]1[C@@H](O)[C@@H](Cl)[C@@H](CO)O[C@@H]1O[C@@]1(CCl)[C@@H](O)[C@H](O)[C@@H](CCl)O1 BAQAVOSOZGMPRM-QBMZZYIRSA-N 0.000 description 1
- 235000019408 sucralose Nutrition 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 230000008337 systemic blood flow Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-L thiosulfate(2-) Chemical compound [O-]S([S-])(=O)=O DHCDFWKWKRSZHF-UHFFFAOYSA-L 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000010245 tubular reabsorption Effects 0.000 description 1
- 229940040064 ubiquinol Drugs 0.000 description 1
- QNTNKSLOFHEFPK-UPTCCGCDSA-N ubiquinol-10 Chemical compound COC1=C(O)C(C)=C(C\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)C(O)=C1OC QNTNKSLOFHEFPK-UPTCCGCDSA-N 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000004143 urea cycle Effects 0.000 description 1
- 239000008371 vanilla flavor Substances 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 235000003563 vegetarian diet Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/15—Vitamins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/175—Amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
- A61K31/198—Alpha-aminoacids, e.g. alanine, edetic acids [EDTA]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
- A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7135—Compounds containing heavy metals
- A61K31/714—Cobalamins, e.g. cyanocobalamin, i.e. vitamin B12
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/21—Amaranthaceae (Amaranth family), e.g. pigweed, rockwort or globe amaranth
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/734—Crataegus (hawthorn)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Definitions
- the present invention relates generally to the field of cardiovascular health and performance. More particularly, several embodiments of the invention relate to nitrite formulations and their use as nitric oxide prodrugs.
- the present invention is directed to a composition, comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- the present invention is directed to a method of reducing a patient's triglyceride level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- the present invention is directed to a method of reducing a patient's C-reactive protein level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- FIG. 2 Steady-state plasma and heart NOx and nitros(yl)ation levels in mice following nitrite insufficiency and supplementation.
- Mice were fed a standard rodent chow or a low NOx chow ⁇ 50 mg/L nitrite supplementation for 7 days at which time steady-state levels of plasma and heart nitrite (A), nitrate (B), nitroso (C), and heart nitrosyl-heme (D) were measured.
- Mice fed a low NOx diet for 7 days exhibited an exacerbated injury following myocardial ischemia/reperfusion (I/R) (E) which was reversed in those animals supplemented with nitrite.
- I/R myocardial ischemia/reperfusion
- FIG. 3 Nitrite levels in heart tissue and plasma during 30 minutes of ischemia in a mouse model. These data demonstrate that nitrite is consumed during ischemia and restored during reperfusion.
- FIG. 4 Dietary nitrite insufficiency unmasks NO biochemistry (A) nitrite, (B) nitrate, (C) nitroso, and (D) NO-heme in eNOS knockout mice and is restored by supplementation in the drinking water. Methods: eNOS knockout mice on either standard diet or low NOx diet ⁇ 50 mg/L nitrite supplementation were compared to C57 control mice on standard diet to reveal dietary nitrite can restore NO biochemistry in mice unable to produce NO.
- FIG. 5 Mice were fed 50 mg/L nitrite supplement for 12 weeks, resulting in increased circulating nitrite and nitrate levels (A and B).
- the nitrite fed group had 20% less lesion formation on the abdominal aorta than the control group fed high fat diet with nitrite free water (C), demonstrating that nitrite supplementation can inhibit the progression of atherosclerosis in a mouse model of atherosclerosis.
- FIG. 6 Blood nitrite concentration after ingestion of an oral formulation containing sodium nitrite in a human model.
- FIG. 7 Blood nitrate concentration after ingestion of an oral formulation containing L-arginine in a human model.
- FIG. 8 Oxidation and reduction of nitrate, nitrite, and nitric oxide.
- FIG. 9 Effect of a composition according to the present invention on NO concentration and blood NO levels compared to blood NO levels from a nutritional supplement containing L-arginine and antioxidants.
- FIG. 10 A 30 day twice per day regimen of a composition according to the present invention significantly increase plasma levels of nitrite and nitrate to normal healthy levels;
- C Patients taking a composition according to the present invention daily for 30 days saw a 10%-55% decrease in fasting triglycerides.
- Nitrite (NO 2 —) is such a compound that is naturally occurring in nature and biology. Over the years, the pharmacological stance on nitrite has undergone a surprising metamorphosis, from a vilified substance that generates carcinogenic nitrosamines in the stomach, to a life-saving drug that liberates a protective agent (NO) during hypoxic events. Nitrite has been investigated as a vasodilator in mammals for over 125 years and is a known by-product of organic nitrate metabolism.
- Nitrite is emerging as an endogenous signaling molecule and regulator of gene expression that can not only serve as a diagnostic marker but also as potential therapy of cardiovascular disease. Up until recently nitrite was thought to be an inert oxidative breakdown product of endogenous nitric oxide synthesis.
- Certain embodiments of the present invention disclosed herein provide a formulation and a process to enhance and extend the therapeutic half-life of nitrite and therefore increase nitric oxide (NO) bioavailability.
- NO nitric oxide
- several embodiments provide the basis for new preventive or therapeutic strategies in diseases associated with NO insufficiency and new guidelines for optimal health as well as extend the therapeutic window in which one may intervene during a heart attack.
- Extension of nitrite half-life is desirable in the design of cardioprotective therapeutics or preventative medicines.
- several embodiments prevent the onset or progression of cardiovascular or heart disease and protect from myocardial infarction thru nitrite/nitrate supplementation.
- certain embodiments provide an extended half-life of nitrite, out to 1 hour, which is the “golden hour” in terms of recovery from heart attack and stroke.
- NO endogenous signaling pathway
- Ischemic heart disease including myocardial infarction, remains the leading cause of morbidity and mortality in all industrialized nations (Myerburg, R. J. (2001). “Sudden cardiac death: exploring the limits of our knowledge.” J Cardiovasc Electrophysiol 12(3): 369-81).
- ischemic injury and reperfusion injury.
- the myocardium is able to tolerate brief periods of ischemia (an absolute or relative shortage of the blood supply to an organ) as activation of inherent, adaptive mechanisms can preserve energy levels and prevent injury. These include switching metabolism to anaerobic glycolysis and fatty acid utilization, increasing glucose uptake, and decreasing contractility.
- Reperfusion is characterized by the formation of oxygen radicals upon reintroduction of molecular oxygen to ischemic tissues, resulting in widespread lipid and protein oxidative modifications, mitochondrial injury, as well as tissue apoptosis and necrosis (Nayler, W. G. (1981). “The role of calcium in the ischemic myocardium.” Am J Pathol 102(2): 262-70; McCord, J. M., R. S. Roy, et al. ( 1985 ). “Free radicals and myocardial ischemia. The role of xanthine oxidase.” Adv Myocardiol 5: 183-9).
- nitric oxide (NO) generation is a very likely cause of heart disease (Esper, R. J., R. A. Nordaby, et al. (2006). “Endothelial dysfunction: a comprehensive appraisal.” Cardiovasc Diabetol 5: 4).
- Continuous generation of NO is essential for the integrity of the cardiovascular system and a decreased production and/or bioavailability of NO is central to the development of cardiovascular disorders (Ignarro, L. J. (2002). “Nitric oxide as a unique signaling molecule in the vascular system: a historical overview.” J Physiol Pharmacol 53(4 Pt 1): 503-14; Herman, A. G. and S. Moncada (2005).
- NO is a highly reactive and diffusible gas formed by three NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS).
- NO has been extensively studied in the setting of ischemia-reperfusion (ischemia/reperfusion) injury. Previous studies clearly demonstrate that the deficiency of eNOS exacerbates myocardial ischemia/reperfusion injury (Jones, S. P., W. G. Girod, et al. (1999).
- NO synthesis is influenced by various cofactors such as tetrahydrobiopterin, flavin mononucleotide and flavin adenine dinucleotide, the presence of reduced thiols, the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and substrate and oxygen availability. Without an adequate delivery of substrate and co-factors (conditions that exist during ischemia), NOS no longer produces NO but instead transfers the free electrons to oxygen and thus produces free oxygen radicals (Becker, B. F., C. Kupatt, et al. (2000).
- cofactors such as tetrahydrobiopterin, flavin mononucleotide and flavin adenine dinucleotide
- ADMA asymmetric dimethylarginine
- Nitrite is an oxidative breakdown product of NO that has been shown to serve as an acute marker of NO flux/formation (Kleinbongard, P., A. Dejam, et al. (2003). “Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals.” Free Radic Biol Med 35(7): 790-6). Nitrite has recently moved to the forefront of NO biology (Gladwin, M. T., A. N. Schechter, et al. (2005). “The emerging biology of the nitrite anion.” Nat Chem Biol 1(6): 308-14), as it represents a major storage form of NO in blood and tissues (Bryan, N. S. (2006).
- nitrite in nitric oxide biology cause or consequence? A systems-based review.” Free Radic Biol Med 41(5): 691-701, hereby incorporated by reference herein).
- nitrite is also derived from reduction of salivary nitrate by commensal bacteria in the mouth and gastrointestinal tract (Tannenbaum, S. R., A. J. Sinskey, et al. (1974). “Nitrite in human saliva. Its possible relationship to nitrosamine formation.” J Natl Cancer Inst 53: 79-84; van Maanen, J. M., A. A. van Geel, et al. ( 1996 ).
- Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, C. L., R. J. Casselden, et al. (1967). “Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments.” Biochim Biophys Acta 143(2): 310-8; Reutov, V. P. and E. G. Sorokina (1998).
- a reduced NO availability is a hallmark of a number of cardiovascular disorders.
- Hyperlipidemia, arterial hypertension, diabetes, smoking and aging are major risk factors for the manifestation of cardiovascular events (Widlansky, M. E., N. Gokce, et al. ( 2003 ). “The clinical implications of endothelial dysfunction.” J. Am Coll Cardiol 42: 1149-1160).
- Plasma nitrite reflects acute changes in endothelial NOS activity in various mammals (Kleinbongard, Dejam et al. 2003) and thus may provide an accurate measurement of patients at risk for cardiovascular events.
- Kleinbongard et al. Kleinbongard, P., A. Dejam, et al. (2006).
- nitrite concentrations reflect the degree of endothelial dysfunction in humans.” Free Radic Biol Med 40(2): 295-302), demonstrated that plasma nitrite levels progressively decrease with increasing cardiovascular risk load. Risk factors considered include age, hypertension, smoking, and hypercholesterolemia. Since nitrite acts as a protective molecule during ischemic events these data raise the intriguing possibility that the underlying problem with these patients is their diminished nitrite bioavailability. Since a substantial portion of steady state nitrite concentrations in blood and tissue are derived from dietary sources (Bryan, Fernandez et al. 2005), modulation of nitrite and/or nitrate intake may provide a first line of defense for ischemic heart disease.
- nitrite availability through diet or supplementation and provide an alternate route to increased NO availability as well as conferring protection from ischemia/reperfusion injury or other adverse cardiovascular event (e.g., heart attack, stroke, etc.). Since the half life of nitrite is on the order of seconds, it can only be used acutely and repeatedly for any therapeutic benefit. Dietary nitrite and nitrate supplementation for 7 days restores NO homeostasis and protects the heart from ischemia/reperfusion injury (Bryan, N. S., J. W. Calvert, et al. (2007).
- the method comprises providing a nitrite salt, wherein the nitrite salt is provided in an amount ranging from about 10 mg to about 100 mg; providing a nitrate salt, wherein the nitrate salt is provided in an amount ranging from about 50 mg to about 500 mg; and providing an ascorbic acid, wherein the ascorbic acid is provided in an amount ranging from about 100 mg to about 2000 mg.
- the nitrate salt and ascorbic acid are administered in combination with the nitrite salt.
- the nitrite salt and the nitrate salt convert to nitrite in vivo.
- the ascorbic acid reduces the conversion of nitrite into N-nitroso compounds in vivo, thereby extending the half-life of the nitrite. “Reduces” in this context encompasses, but is not limited to, “minimizes” or “prevents.”
- the ascorbic acid is particularly advantageous in some embodiments because it reduces carcinogens (e.g., N-nitroso compounds) while increasing the bioavailability of nitrite.
- L-arginine is added in some embodiments.
- L-arginine serves as a substrate for nitric oxide synthase, thereby increasing NO formation, which in turn can increase nitrate and/or nitrite formation.
- the nitrite salt, nitrate salt, ascorbic acid, and optionally L-arginine are provide to a mammal in a single dose.
- Nitrite has recently been implicated in hypoxic vasodilation in the circulation (Kim-Shapiro, D. B., M. T. Gladwin, et al. (2005). “The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation.” Journal of Inorganic Biochemistry 99: 237-246). As early as 1880, nitrite was described in terms of its vasodilatory abilities (Reichert, E. T. and S. W. Mitchell (1880).
- Nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action.” Proc Natl Acad Sci USA 98(22): 12814-12819). This discrepancy is likely due to kinetics and duration of infusion. Nitrite is found in high abundance throughout the mammalian organ system (Bryan, Rassaf et al. 2004). It is normally a short-lived, highly regulated ion in the circulation (200-600 nM) with a half life in whole blood of 110 seconds (Kelm, M. (1999). “Nitric oxide metabolism and breakdown.” Biochim Biophys Acta 1411: 273-289).
- nitrite-derived NO seems to play an important role in host defense (Duncan, C., H. Dougall, et al. (1995). “Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate.” Nat Med 1(6): 546-551; Dykhuizen, R. S., R. Frazer, et al. (1996). “Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.” Antimicrob Agents Chemother 40(6): 1422-1425) and in regulation of gastric mucosal integrity (Bjorne, H. H., J. Petersson, et al. (2004).
- Nitrite has clearly emerged as an important molecule in biology, but its effects on the endogenous NO pathway have been poorly investigated. Furthermore, its use as a potential therapy needs further safety consideration. Historically nitrite was considered a strong oxidant and potential carcinogen. It has been in widespread use for many years. It is used as a color fixative and preservation in meats and fish and is naturally occurring in the soil and in vegetables. It is also used in manufacturing diazo dyes, nitroso compounds, in the textile industry, in photography and in the manufacture of rubber chemicals. Nitrite is also a common clinical and laboratory chemical that is used as a vasodilator (Reichert and Mitchell 1880), bronchodilator (Hunter, Dejam et al.
- nitrite is in the range of 22-23 mg/kg body weight (from USFDA Generally Recognized as Safe Food Ingredient: Nitrates and Nitrites (Including Nitrosamines) 1972 by Battele-Columbus Laboratories and Department of Commence, Springfield Va.).
- Lower doses of either nitrite or nitrate have caused acute methemoglobinemia, particularly in infants.
- a high nitrite or nitrate intake has been associated with “blue baby syndrome” caused by methemoglobinemia (Comly, H. H. (1945). “Cyanosis in infants caused by nitrates in well water.” JAMA 129: 112-116; Donohoe, W. E. (1949).
- Nitrite and nitrate are excreted in the kidneys. Nitrate is excreted in the urine as such or after conversion to urea (Green, L. C., K. Ruiz de Luzuriaga, et al. (1981). “Nitrate biosynthesis in man.” Proc. Natl. Acad Sci. USA 78(12): 7764-7768). Clearance of nitrate from blood to urine approximates 20 ml/min in adults (Wennmalm, A., G. Benthin, et al. (1993). “Metabolism and excretion of nitric oxide in humans.
- NO 2 , N 2 O 3 and NO 2 ⁇ represent nitrogen dioxide, dinitrogen trioxide and nitrite, respectively.
- N 2 O 3 is a potent nitrosating agent by virtue of its ability to generate the nitrosonium ion (NO + ).
- NO and nitrite are rapidly oxidized to nitrate in whole blood.
- the half life of NO 2 ⁇ in human blood is about 110 seconds (Kelm 1999).
- Nitrate on the other hand has a circulating half life of 5-8 hours (Tannenbaum, S. R. (1994). “Nitrate and nitrite: origin in humans.” Science 205: 1333-1335, hereby incorporated by reference herein; Kelm, M. and K.
- Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, C. L., R. J. Casselden, and A. M. Taylor, Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments . Biochim Biophys Acta, 1967. 143: p. 310-318; Reutov, V. P. and E. G.
- Nohl, Nitrite reductase activity is a novel function of mammalian mitochondria .
- nitrite from a vilified substance that generates carcinogenic nitrosamines in the stomach, to a life-saving drug that liberates a protective agent (NO) during hypoxic events, as well as performs many actions independent of NO, warrants a re-evaluation of nitrite in biology.
- NO protective agent
- ascorbic acid reduces endogenous nitrosation reaction in the gastrointestinal tract, which enhances the half life of nitrite.
- the nitrate provides an additional source of nitrite, again extending the functional half life, e.g., by increasing stores, of nitrite.
- the human diet exerts important long-term effects on vital body functions and thereby makes an important contribution to health and disease. While high intake of cholesterol, saturated fat, salt, and sugar are associated with a greater risk for cardiovascular disease, conventional wisdom has it that the opposite is true for abundant consumption of fruits and vegetables. A diet rich in fruits and vegetables is associated with a lower risk of certain forms of cancer and cardiovascular disease. Recent epidemiological studies suggest a cardioprotective action afforded specifically by green leafy vegetables. Green leafy vegetables such as spinach and lettuce, in addition to being rich in antioxidants are especially rich in nitrite and nitrate as are berries, grapes, and a few other fruits.
- nitrite and nitrate are major factors contributing to the positive health effects of certain vegetables via bioconversion to NO which exerts protective effects on the cardiovascular system.
- a continuous intake of nitrite- and nitrate-containing food such as green leafy vegetables and berries may ensure that blood and tissue levels of NO are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis.
- Dietary source of NO metabolites could therefore improve circulation and oxygen delivery and lead to better health and increased energy. This dietary pathway may therefore not only provide essential nutrients for NO production but also provide a rescue pathway for people at risk for cardiovascular disease.
- Several embodiments provide the nutrition and protection of a high vegetable diet in the form of a daily supplement formulation which renders subjects protected from injury from heart attack or other cardiovascular events, i.e. stroke, pulmonary embolism.
- This strategy including nitrite/nitrate supplementation in combination with ascorbic acid may serve as an inexpensive cardioprotective regimen which may delay or reduce the onset or progression of cardiovascular or heart disease and protect from myocardial infarction.
- nitrite, nitrate and Vitamin C are particularly advantageous because they provide a supplement formulation of nitrite, nitrate and Vitamin C.
- such amounts may be found in a high vegetable diet, the time it would take to consume the required assortment of vegetables as well as the impact on the digestive system would adversely impact the absorption and/or bioavailability of the nitrite, nitrate and Vitamin C.
- the reaction of other compounds and nutrients in the naturally occurring vegetable assortment may also adversely impact the impact the absorption and/or bioavailability of the nitrite, nitrate and Vitamin C.
- the formulation comprises purified or isolated nitrite, nitrate and Vitamin C.
- the formulation consists essentially of purified or isolated nitrite, nitrate and Vitamin C.
- the formulation consists of purified or isolated nitrite, nitrate and Vitamin C.
- the formulation consists essentially of purified or isolated nitrite, nitrate, Vitamin C, and L-arginine. In yet other embodiments, the formulation consists of purified or isolated nitrite, nitrate, Vitamin C, and L -arginine
- composition may further contain one or more components selected from the group consisting of water and flavorants.
- the present invention can provide a novel therapy for patients experiencing myocardial infarction or stroke.
- Nitrite has been shown to be protective in animal models of stroke and both cardiac and hepatic ischemia-reperfusion injury. Conversely, nitrite insufficiency is associated with increased injury from ischemia-reperfusion insult.
- the therapeutic window for nitrite alone is very narrow. Therefore, several embodiments provide patients with an extended-release formulation comprising nitrite, among additional components, to be used upon onset of symptoms to provide at least some protection from injury until the patient can be provided with reperfusion therapy, such as in a hospital setting.
- the present invention relates to the use of supplemental nitrite in combination with nitrate and vitamin C (ascorbic acid) as a preventive agent in cardiovascular disease.
- nitrate acts as an extended release nitrite source that is absorbed and re-circulated through the enterosalivary pathway and is reduced to nitrite by commensal bacteria in the mouth.
- nitrite acts as a reservoir for nitric oxide activity. Reduced nitric oxide availability is a hallmark of a number of cardiovascular disorders and plasma nitrite levels progressively decrease with increasing cardiovascular risk load. Therefore, several embodiments provide a sufficient daily intake of nitrite, which is beneficial to optimal cardiovascular health.
- a typical Western diet is low in nitrite and nitrate compared to a vegetarian or Mediterranean diet and may therefore account for the increased incidence of cardiovascular disease in the United States, Europe, and other developed countries.
- a daily nitrite supplementation may provide the missing nutrient, analogously to a daily multivitamin.
- the Nobel Prize in Physiology or Medicine was awarded in 1998 for the discovery of nitric oxide in the cardiovascular system. Maintaining nitric oxide availability is essential for optimal health, particularly for those at risk for cardiovascular events, and therefore, in several embodiments, supplemental nitrite acts to increase the reservoir of nitric oxide which can be bio-activated upon need as a prevention rather than a treatment or therapy once disease has occurred.
- the present invention relates to a formulation for an alternate source of nitric oxide during cardiovascular exercise and/or muscle training.
- the formulation further comprises L-arginine.
- L-arginine is a natural amino acid substrate for nitric oxide synthase enzymes which produces L-citrulline and NO from L-arginine in a complex reaction requiring oxygen.
- L-arginine can be given as a pre-workout drink to saturate the NOS enzyme to produce sufficient NO and dilate vessels.
- oxygen availability is diminished and therefore NOS can no longer produce NO. Therefore an alternate substrate must be supplied to produce NO under anaerobic conditions. The substrate then becomes nitrite.
- nitrite supply blood and muscles with nitrite before a workout , which provides an additional source of NO during the workout and improves muscle blood flow during exercise, thereby enhancing performance and muscle building capacity.
- sodium nitrite is added to existing workout beverage formulations , thereby increasing NO and providing sufficient NO before during and/or after a workout. Since the L-aginine:NO pathway is not functional during workout, the addition of nitrite provides the substrate for anaerobic formation of NO, an alternate pathway for NO generation.
- the presence of nitrite in certain embodiments of the formulation will allow NO production from nitrite reduction during the workout, a time at which it is advantageous to increase blood flow and supply the muscles with essential nutrients and oxygen.
- the present invention relates to a composition
- a composition comprising a nitrite salt, a nitrate salt, and ascorbic acid.
- any positively-charged ion safe for use as a food additive or a component of a pharmaceutical formulation can be used as the counterion to nitrite in the nitrite salt or the counterion to nitrate in the nitrate salt.
- the positively-charged ion is an inorganic ion.
- the positively-charged ion is selected from the group consisting of sodium and potassium; e.g., the nitrite salt is sodium nitrite or potassium nitrite and the nitrate salt is sodium nitrate or potassium nitrate.
- the composition comprises from about 1 weight part to about 8 weight parts sodium nitrite, from about 5 weight parts to about 50 weight parts sodium nitrate, and from about 20 weight parts to about 200 weight parts ascorbic acid.
- the composition further comprises L-arginine. In a further embodiment, the composition comprises from about 20 weight parts to about 200 weight parts L -arginine.
- sodium nitrite is included in a range of about 0.01 mg/kg to about 15 mg/kg. In some embodiments, sodium nitrate is included in a range of about 1.0 mg/kg to about 50 mg/kg. In some embodiments, ascorbic acid is included in a range of about 1.0 mg/kg to about 25 mg/kg. In certain embodiments, L-arginine may also be included in a range of about 2.0 mg/kg to about 50 mg/kg.
- sodium nitrite is included in a range of about 30 mg to about 40 mg. In certain embodiments, sodium nitrate is included in a range of about 250 mg to about 300 mg. In certain such embodiments, ascorbic acid is included in an amount of about 1000 mg. In certain other embodiments, L-arginine may also be included in an amount of about 1000 mg.
- sodium nitrite is included in an amount of about 20 mg. In certain such embodiments, sodium nitrate is included in an amount of about 150 mg. In certain embodiments, ascorbic acid is included in an amount of about 500 mg. In certain other embodiments, L-arginine may also be included in an amount of about 500 mg.
- composition can further comprise other materials.
- the composition further comprises water.
- it can also further comprise other materials.
- the composition may comprise a flavorant, such as a citrus flavor, a non-citrus fruit flavor, an herbal flavor, a vanilla flavor, or a chocolate flavor, and other appropriate flavorings.
- the present invention relates to a method of enhancing cardiovascular performance in a mammal, comprising administering to the mammal a composition according to any of the embodiments described herein.
- the composition comprises a nitrite salt, a nitrate salt, and ascorbic acid.
- any mammal for which enhanced cardiovascular performance is desired can be the subject of the method.
- the mammal is Homo sapiens .
- Other mammals for which enhanced cardiovascular performance may be desired include, but are not limited to, draft animals, beasts of burden, animals useful in transportation (e.g., horses), racing animals (e.g., horses or greyhounds), meat animals, wool- or fur-bearing animals, milk animals, working dogs, and household pets, among others.
- Enhanced cardiovascular performance can be desired for a person or animal engaged in physical exertion.
- a composition as described herein may be used as a treatment or prophylaxis for a medical condition characterized by or associated with reduced blood flow to an organ of the body.
- Administering the composition can be by any route, such as oral, intravenous, or intraarterial, among others. In one embodiment, administering is by the oral route. In this embodiment, it is desirable that the components of the composition be dissolved in a neutral- or pleasant-tasting liquid, such as water, flavored water, milk, or fruit juice, among others. Additionally, the components of the composition may be in tablet or capsule form and in this form the composition may be dissolvable in liquid. In other embodiments, the composition is provided as a tablet that dissolves when placed in the mouth of a user. In some embodiments, a composition according to any of the embodiments described herein can be provided in powder, tablet, capsule, gel, aerosol or liquid form.
- administering is of a dosage from about 0.01 mg/kg/day to about 15 mg/kg/day sodium nitrite, from about 1 mg/kg/day to about 50 mg/kg/day sodium nitrate, and from about 1 mg/kg/day to about 25 mg/kg/day ascorbic acid.
- administering is of a dosage from about 2 mg/kg/day to about 50 mg/kg/day L -arginine.
- the present invention is directed to a composition, comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- a botanical nitrate source is any plant matter, extract of plant matter, or product of plant matter containing nitrate. Generally, it is desirable that the botanical nitrate source be generally regarded as safe for human or animal consumption. In one embodiment, the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia, St.
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof.
- a botanical source of nitrite reduction activity is any plant matter, extract of plant matter, or product of plant matter, containing nitrite reductase enzyme, a compound capable of reducing nitrite, or both. Generally, it is desirable that the botanical source of nitrite reduction activity be generally regarded as safe for human or animal consumption.
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra , green tea, beet root, pine bark, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia, St.
- Berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee, walnuts, peanuts, borojo, pomegranates, popcorn, and yerba mate are known to contain polyphenols, which are known to have antioxidant properties.
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra , green tea, beet root, pine bark, and mixtures thereof.
- Hawthorn berry herein refers to any portion of a plant of the genus Crataegus (for example, Crataegus oxyacantha ), such as the berry, leaf, or flower, among others, as well as extracts of any portion thereof. In a particular embodiment, it refers to the berry of a plant of the genus Crataegus (for example, Crataegus oxyacantha ).
- Schisandra refers to any portion of a plant of the genus Schisandra (for example, S. chinensis and S. rubiflora , among others), such as the fruit, leaf, or flower, among others, as well as extracts of any portion thereof.
- a nitrite salt comprising any counterion may be used. Generally, it is desirable that the nitrite salt be generally regarded as safe for human or animal consumption.
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, magnesium nitrite, calcium nitrite, and mixtures thereof.
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof.
- composition can further comprise one or more additional materials.
- the composition can further comprise from about 20 weight parts to about 500 weight parts L -citrulline.
- the composition can further comprise from about 20 weight parts to about 1000 weight parts L -arginine.
- L-Arginine supplementation may be detrimental in some populations.
- a clinical trial designed to enhance NO production in humans that have suffered a heart attack revealed that L-arginine, when added to standard postinfarction therapies, does not improve vascular stiffness measurements or ejection fraction and may be associated with higher postinfarction mortality.
- the composition can further comprise from about 0.2 weight parts to about 5 weight parts vitamin B12.
- the vitamin B12 can be in any form of cobalamin.
- the vitamin B12 is in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof.
- the composition can further comprise from about 20 weight parts to about 500 weight parts vitamin C.
- the vitamin C can be in any form of ascorbate or ascorbic acid.
- the vitamin C is in a form selected from the group consisting of magnesium ascorbate, sodium ascorbate, potassium ascorbate, ascorbic acid, and mixtures thereof.
- the composition can further comprise from about from about 20 weight parts to about 500 weight parts of a nitrate salt selected from the group consisting of sodium nitrate, potassium nitrate, and mixtures thereof.
- a nitrate salt selected from the group consisting of sodium nitrate, potassium nitrate, and mixtures thereof.
- the composition can further comprise one or more additional materials suitable for forming the composition into a vehicle deliverable for human or animal consumption.
- additional materials include, but are not limited to, binders, flavorants, colorants, sweeteners, adujvants, and excipients, among others.
- the composition can further comprise from about 50 weight parts to about 1500 weight parts of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- These one or more ingredients can act as one or more of binders, flavorants, colorants, sweeteners, antiadherents, or lubricants, among other functions.
- composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 weight parts;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra , green tea, beet root, pine bark, and mixtures thereof and is present at about 100 weight parts; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 weight parts;
- composition further comprises:
- vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof;
- mannitol from about 50 weight parts to about 1500 weight parts of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides
- the composition can be formulated, using techniques known in the art, into any vehicle suitable for human consumption.
- the composition can be formulated as a powder dissolvable or suspendable in a potable beverage, a soft food, or both; as an ingredient that can be baked into a baked cookie, cracker, or bar; a tablet or capsule that can be swallowed; or a lozenge dissolvable in the mouth; among others.
- the composition can be in a form of a lozenge dissolvable in the mouth.
- the lozenge can have a weight from about 600 mg to about 2000 mg.
- the present invention is directed to a method of reducing a patient's triglyceride level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- composition and its components can be as described above. Also, the formulation of the composition as a lozenge can be as described above.
- 1 weight part is 1 mg.
- the administered composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 mg;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra , green tea, beet root, pine bark, and mixtures thereof and is present at about 100 mg; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 mg;
- composition further comprises:
- vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof;
- mannitol from about 50 mg to about 1500 mg of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, ge
- composition can be administered according to any dosing regimen. Particular details of how administering is to be performed are within the ability of the person of ordinary skill in the art having the benefit of the present disclosure. In one embodiment, the administering is performed once or twice daily.
- the present invention is directed to a method of reducing a patient's C-reactive protein level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- composition and its components can be as described above. Also, the formulation of the composition as a lozenge can be as described above.
- 1 weight part is 1 mg.
- the administered composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 mg;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra , green tea, beet root, pine bark, and mixtures thereof and is present at about 100 mg; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 mg;
- composition further comprises:
- vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof;
- mannitol from about 50 mg to about 1500 mg of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, ge
- composition can be administered according to any dosing regimen. Particular details of how administering is to be performed are within the ability of the person of ordinary skill in the art having the benefit of the present disclosure. In one embodiment, the administering is performed once or twice daily.
- Nitric oxide is the body's most potent vasodilator. Nitric oxide is produced in the body by the enzyme nitric oxide synthase (NOS). NOS enzymes produce .NO by catalyzing a five electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing N ⁇ hydroxy L-arginine as an intermediate. Two moles of O 2 and 1.5 moles of NADPH are consumed per mole of .NO formed (Liu, Q. and G. S.
- NOS enzymes are the only enzymes known to simultaneously require five bound cofactors/prosthetic groups: FAD, FMN, heme, tetrahydrobiopterin (BH 4 ) and Ca 2+ -calmodulin (CaM). All NOS isozymes are catalytically self-sufficient provided all required substrates and co-factors are available. CaM binding to nNOS has been shown to regulate catalytic activity by triggering electron flux from FMN to heme, thereby coupling the oxygenase and reductase domains.
- CaM also facilitates NADPH dependent reduction of cytochrome c and ferricyanide in BH 4 and heme depleted nNOS. If any of the co-factors become limiting, then NO production from NOS shuts down, and in many cases NOS then produces superoxide instead. This is indeed a very complex and coordinated effort to enzymatically produce NO which normally proceeds very efficiently. However, in disease characterized by oxidative stress where cofactors become oxidized, NOS uncoupling, or conditions of hypoxia where oxygen is limiting, this process can no longer maintain NO production. Therefore there has to be an alternate route to NO production. It is highly unlikely that Nature devised such a sophisticated mechanism of NO production as a sole source of a critical molecule. Nitrite reduction then acts as a backup system to the NOS system. Part of this may occur through nitrite reduction during low oxygen availability. Nitrite supplementation can then support NO production during exercise when enzymatic NO production is shut down.
- Nitrite reduction to NO can occur via a simple mechanism.
- the 1-electron reduction of nitrite can occur by ferrous heme proteins (or any redox active metal) and an electron donor through the following reaction:
- Nitrite supplementation may therefore act as a protective measure to compensate for insufficient NOS activity under conditions of hypoxia such as during anaerobic metabolism during exercise or muscle training Nitrite contributes to whole body NO production and homeostasis.
- nitrite can act as a circulating NO donor (Dejam, A., et al., Emerging role of nitrite in human biology . Blood Cells Mol Dis, 2004. 32(3): p. 423-429) and nitrite can itself perform many actions previously attributable to NO (Gladwin, M. T., et al., The emerging biology of the nitrite anion . Nature Chemical Biology, 2005. 1(6): p. 308-314) without the intermediacy of NO (Bryan, N.
- Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues . Nature Chemical Biology, 2005. 1(5): p. 290-297). While L-arginine supplementation may provide moderate amounts of NO prior to workout, during a workout, this system becomes inefficient and very little NO from L-arginine can be produced due to lack of oxygen substrate. Therefore NO from nitrite provides an alternate mechanism to maintain NO production during exercise. Supplemental nitrite taken 15-20 minutes prior to workout can titrate up tissue and muscle nitrite concentrations in order to produce NO locally during exercise and therefore enhance blood flow and performance.
- Nitrite reduction to NO under aerobic and anaerobic conditions using chemiluminescent detection of free NO has been quantified and characterized. Under aerobic conditions, established by continuous sample purging with air, NO production by blood-free tissues and RBCs from nitrite was minimal and fleeting. However, switching the purge gas to N 2 (i.e., hypoxia) acutely enhanced tissue NO formation from NO 2 ⁇ ( FIG. 1 , left). Hypoxic tissue NO 2 ⁇ reduction exhibited compartment-specific properties (initial kinetics, amount, duration) and was most dramatic and sustained in liver homogenate.
- N 2 i.e., hypoxia
- Nitrite can Protect Tissues from Ischemia-Reperfusion Injury
- mice fed a purified amino acid diet the diet lowest in NOx (20.5 ⁇ 0.7 pmol/g nitrite and 503.1 ⁇ 17.9 pmol/g nitrate) but with the same L-arginine content.
- mice fed a low NOx diet for 1 week demonstrated a significant reduction in plasma and heart nitroso levels compared to mice fed standard chow, which could be replenished and increased with 50 mg/L nitrite in the drinking water for 1 week ( FIG. 2C ).
- Nitrosyl-heme products FIG. 2D ) were also reduced in the mice fed a low NOx diet and replenished by nitrite supplementation in the drinking water.
- mice fed a low NOx diet displayed a 59% increase in infarct relative to the area at risk (AAR) compared to mice fed a standard chow.
- AAR area at risk
- Nitrite supplementation in animals on the low NOx diet reversed the increased myocardial infarct size by 57%. Additionally, mice fed the low NOx diet displayed a higher mortality rate (57.7% survival) 24 hours post-myocardial infarction than mice on the standard rodent chow (70.6% survival). Likewise, survival improved in mice on the low NOx diet with nitrite-supplemented drinking water to 76.9%. Since nitrite is derived both from diet and oxidation of enzymatic NO production from NOS, potential compensatory changes in NOS expression following one week low NOx intake were investigated.
- Nitrite may provide a valuable nutrient to these athletes as a pre-workout or pre-marathon supplement to protect from ischemic injury during the event. Nitrite may then serve multiple purposes in the setting of myocardial ischemia/reperfusion. First, by titrating up tissue concentrations of nitrite when it is administered just prior to reperfusion, one can protect the heart from ischemia/reperfusion. Second, acute nitrite administration may initiate a signaling cascade that results in the upregulation of other protective proteins which afford protection hours later.
- Nitrite has been shown to be protective in both the heart and the liver following ischemia/reperfusion (Webb, A., et al., Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia - reperfusion damage . Proc Natl Acad Sci USA, 2004. 101(13683-13688); Duranski, M. R., et al., Cytoprotective effects of nitrite during in vivo ischemia - reperfusion of the heart and liver . J Clin Invest, 2005. 115(5): p. 1232-1240).
- nitrite is reduced to NO under ischemic conditions to provide an alternate source of NO when NOS is inactive due to decreased substrate delivery and decreased oxygen saturation.
- a time course of nitrite metabolism both after ischemia and during reperfusion was conducted. As shown in FIG. 3 , nitrite is consumed in the heart tissue during 30 minutes of ischemia but is unaffected in the plasma. The consumption of nitrite appears to lead to a concomitant increase in cardiac nitroso products ( FIG. 3B ).
- Nitrite can form nitrosothiols in a first order reaction requiring heme and thiols and can also be reduced to NO under anaerobic conditions (Bryan, N. S., et al., Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues . Nat Chem Biol, 2005. 1(5): p. 290-7).
- nitrite is gradually increased and restored whereby tissue nitroso decompose during the reperfusion phase ( FIG. 3 ).
- the inventor proposes that nitrite serves two functions in the setting of ischemia reperfusion.
- nitrite may react with critical thiols to form nitrosothiols. It is possible that this nitroso modification acts as a reversible protective shield which reduces irreversible oxidation during the oxidative burst of reperfusion.
- nitroso products can then release the NO moiety during the reperfusion phase and act an a redox sensitive NO donor (Hogg, N., Biological chemistry and clinical potential of S - nitrosothiols . Free Radic Biol Med, 2000. 28(10): p. 1478-86).
- Biochemical data support this notion by the increase in nitroso at the expense of nitrite followed by the decay of nitroso over time during reperfusion.
- nitrite insufficiency leads to increased injury because there is not enough stored in blood or tissue to perform these actions.
- Enzymatic NO insufficiency is a hallmark of a number of diseases including cardiovascular disease.
- eNOS ⁇ / ⁇ mice were fed standard rodent chow or low NOx diet.
- eNOS ⁇ / ⁇ mice revealed lower plasma nitrite concentrations consistent with earlier findings (Kleinbongard, P., et al., Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals . Free Radical Biology & Medicine, 2003. 35(7): p.
- Plasma nitrite could be further decreased by feeding eNOS ⁇ / ⁇ mice a low NOx diet demonstrating that plasma nitrite is a reflection of both NOS and diet. Feeding low NOx diet to eNOS ⁇ / ⁇ mice completely eliminated steady state concentrations of plasma nitroso without any significant effect on cardiac nitroso. Supplementation of 50 mg/L nitrite in the drinking water for 7 days restores plasma nitrite in eNOS ⁇ / ⁇ to control levels and increases both plasma and cardiac nitroso to above C57 control levels.
- mice deficient in eNOS have increased injury to ischemia/reperfusion insult (Jones, S. P., et al., Myocardial ischemia - reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase . Am J Physiol, 1999. 276(5 Pt 2): p. H1567-73) and data shown above reveal these mice also have reduced nitrite and nitroso compared to C57 wild type.
- eNOS ⁇ / ⁇ mice on low NOx diet ⁇ 50 mg/L nitrite in drinking water were subjected to 30 minutes ischemia and 24 hour reperfusion as above. These mice are also protected from myocardial ischemia/reperfusion injury suggesting that dietary nitrite supplementation can provide benefit under conditions of dysfunctional NOS.
- Nitrite can be used to Delay or Prevent the Onset and Development of Atherosclerosis
- a high fat diet was fed to 8 female LDb mice for 12 weeks.
- Four of the mice received nitrite free water and the other 4 mice were supplemented with 50 mg/L nitrite throughout the 12 weeks on high fat diet.
- the LDb mice spontaneously develop atherosclerosis on normal rodent chow, the addition of a high fat diet will accelerate the process from 8 months to 12 weeks.
- plasma was collected for nitrite, nitrate determination as well as lipid profile determination. As shown in FIG. 5A-B , there significantly more circulating nitrite and nitrate in the nitrite fed mice than the nitrite free water group.
- the nitrite fed group had 20% less lesion formation on the abdominal aorta than the control group fed high fat diet with nitrite free water ( FIG. 5C ). These data demonstrate that nitrite supplementation can inhibit the progression of atherosclerosis in the female LDb mice using a high fat diet.
- Aim To develop specific formulation that will extend the circulating half life of nitrite from 110 seconds to 45-60 minutes.
- nitrite is derived from NO oxidation, diet, and from the reduction of nitrate in the human body, to enhance nitrite bioavailability, substrates from all 3 pathways were included: L-arginine to enhance NO production from nitric oxide synthase which will subsequently produce nitrite; sodium nitrite to increase acute circulating nitrite concentrations; ascorbic acid to inhibit endogenous nitrosation reactions in the stomach; and sodium nitrate to provide a slow release source of nitrite.
- This specific formulation was compared to 3 g L-arginine that is marketed commercially to enhance NO production. An intravenous line was obtained by the inventor on himself with 21 gauge infusion set needle and blood collected. The first 5 ml of blood was discarded. Blood was then collected at baseline. Then the prescribed formulation was dissolved in 50 ml of water and taken orally. A timer was started and blood was sampled for analysis at 1 minute and every 2 minutes for 60 minutes.
- the specific formulation developed can increase plasma nitrite to therapeutic levels within 3 minutes of ingestion and can maintain therapeutic levels until 50 minutes after drinking This represents a novel formulation whereby this product can be given immediately upon patient presentation of ischemic episode that will maintain the protective nitrite levels for up to 1 hour. There is a golden hour in clinical medicine whereby the patient survival and outcome from ischemic episodes greatly declines.
- the safety of nitrite and nitrate at these doses is well established and therefore the formulations according to several embodiments discussed above represent a safe, novel use for oral nitrite as a cardioprotective agent.
- Disclosed herein is an immediate and extended release form of nitrite extending the biological half life from hundreds of seconds to minutes and hours.
- two possible applications for this technology may be as a revolutionary nitric oxide based supplement for the workout industry and a daily supplement to restore NO homeostasis in the aging population.
- a range of effective doses of each ingredient is a range of effective doses of each ingredient.
- Sodium nitrate (1.0 mg/kg-50 mg/kg; Poisoning in man may result from a total oral daily dose in excess of 4 g or from a single dose of more than 1 g. 8 g may be fatal and 13-15 g are generally fatal (Sollmann, 1957).
- concentrations are sufficiently low that the volume (or mass) that would need to be consumed to provide the same degree of supplementation as compared to several embodiments disclosed herein would be prohibitively large. For example, one liter of beetroot juice contains about 2.79 g of nitrate.
- a workout supplement formulation to enhance NO formation in working muscle consists, consists essentially of or comprises:
- a workout supplement formulation to enhance NO formation in working muscle consists, consists essentially of or comprises:
- a daily supplement formulation to restore NO homeostasis consists, consists essentially of or comprises:
- FIG. 6 shows blood nitrite and nitrate levels from 0 min to 60 min after ingestion of an oral formulation containing 30 mg sodium nitrite, 300 mg sodium nitrate, 1000 mg ascorbic acid, and 1000 mg L-arginine in a human volunteer.
- the formulation consists, consists essentially of or comprises 30 mg sodium nitrite, 300 mg sodium nitrate, 1000 mg ascorbic acid, and 1000 mg L-arginine.
- NO is formed by oxidation of the guanidino nitrogen of L-arginine with molecular oxygen as the electron acceptor (Moncada, S. and A. Higgs (1993). “The L-arginine-nitric oxide pathway.” N Engl J Med 329(27): 2002-12).
- This complex reaction is catalysed by specific heme-containing enzymes, the NO synthases, and the reaction requires several co-factors.
- the alternative pathway was fundamentally different; instead of L-arginine it used the simple inorganic anions nitrate (NO 3 ⁇ ) and nitrite (NO 2 ⁇ ) as substrates in a stepwise reduction process that did not require NO synthase or multiple co-factors.
- a Novel Nitric Oxide System This recognition now provides a new paradigm for restoring NO homeostasis that is vastly different than the NOS system.
- the stepwise reduction of nitrate to nitrite to nitric oxide is, by necessity, an inefficient process by which each step yields a 3-log lower concentration of product than substrate (Jansson, E. A., L. Huang, et al. (2008). “A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis.” Nat Chem Biol 4(7): 411-7).
- Steady state levels of nitrate and nitrite in healthy humans is 20-40 ⁇ M and ⁇ 0.5 ⁇ M respectively.
- TCM Traditional Chinese Medicines
- Hawthorn berry contains an active nitrite reductase that is 10 times more active than the Chinese herbs we tested.
- Neo40 was sourced and developed into a quick dissolving lozenge by a GMP-certified facility. We then tested the pharmacokinetics of Neo40 in humans. When allowed to dissolve in the mouth, Neo40 leads to a slow and steady rise in plasma nitrite of humans as shown in FIG. 9B .
- Neo40 To demonstrate the difference between Neo40 and compositions of L-arginine and anti-oxidants that have been used as nutritional supplements designed to enhance NO production, we compared Neo40 to one of the most popular products on the market. As shown in FIG. 9B , the L-arginine product did not lead to any appreciable formation of NO when consumed. This is in stark contrast to the NO formation resulting from administration of Neo40 .
- Neo40 could restore NO levels
- we conducted a small clinical trial in healthy volunteers (age 34-64; 6 male, 6 female; 6 smokers, 6 non-smokers) and found that after taking Neo40 (one lozenge, twice a day) for 30 days lead to a modest but significant increase in both plasma nitrite and nitrate ( FIG. 10A-B ).
- Utilizing beet root and Hawthorne berry along with nitrite and L-citrulline provides a novel system for generating NO whereby the metabolism of nitrite is specifically directed towards reduction to NO by the Hawthorne berry.
- an active reductase in the formulation is an important addition as it allows a more efficient means to produce NO from nitrite.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Abstract
Compositions comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt. Use of said composition in methods of reducing triglycerides or reducing C-reactive protein levels are also provided.
Description
- This application is a continuation of U.S. patent application Ser. No. 14/610,492 filed on Jan. 30, 2015, now U.S. Pat. No. 9,119,823 pending issuance on Sep. 1, 2015; which is a continuation of U.S. patent application Ser. No. 13/668,776 filed on Nov. 5, 2012, now U.S. Pat. No. 8,962,038 issued Feb. 24, 2015; which is a continuation of U.S. patent application Ser. No. 12/856,957 filed on Aug. 16, 2010, now U.S. Pat. No. 8,303,995 issued Nov. 6, 2012; which is a continuation-in-part of U.S. patent application Ser. No. 12/484,364 filed on Jun. 15, 2009, now U.S. Pat. No. 8,298,589 issued Oct. 30, 2012; which claims the benefit of priority to U.S. Provisional Application No. 61/061,251 filed on Jun. 13, 2008, all of which are incorporated herein by reference in their entity.
- The present invention relates generally to the field of cardiovascular health and performance. More particularly, several embodiments of the invention relate to nitrite formulations and their use as nitric oxide prodrugs.
- In one embodiment, the present invention is directed to a composition, comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- In one embodiment, the present invention is directed to a method of reducing a patient's triglyceride level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts
L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt. - In one embodiment, the present invention is directed to a method of reducing a patient's C-reactive protein level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts
L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt. - The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention according to several embodiments disclosed herein. The invention according to several embodiments disclosed herein may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.
-
FIG. 1 . (top) Original tracing from blood and tissue aerobic and anaerobic nitrite reduction to NO. (bottom) Quantification of NO generation from nitrite under aerobic and anaerobic conditions in blood and tissues after the addition of 200 μM nitrite. Data represent n=3-4 for each tissue and NO quantified over 4 minutes. Inset: Compartment-specific fold increase from aerobic to anaerobic NO formation from nitrite. -
FIG. 2 . Steady-state plasma and heart NOx and nitros(yl)ation levels in mice following nitrite insufficiency and supplementation. Mice were fed a standard rodent chow or a low NOx chow ±50 mg/L nitrite supplementation for 7 days at which time steady-state levels of plasma and heart nitrite (A), nitrate (B), nitroso (C), and heart nitrosyl-heme (D) were measured. Mice fed a low NOx diet for 7 days exhibited an exacerbated injury following myocardial ischemia/reperfusion (I/R) (E) which was reversed in those animals supplemented with nitrite. Representative immunoblots of eNOS, iNOS, and nNOS from myocardial tissue homogenates of mice on standard chow and low NOx diet for 7 days reveal no changes in NOS protein expression, (F). These data demonstrate that supplemental nitrite can protect the heart from damage following heart attack. -
FIG. 3 . Nitrite levels in heart tissue and plasma during 30 minutes of ischemia in a mouse model. These data demonstrate that nitrite is consumed during ischemia and restored during reperfusion. -
FIG. 4 . Dietary nitrite insufficiency unmasks NO biochemistry (A) nitrite, (B) nitrate, (C) nitroso, and (D) NO-heme in eNOS knockout mice and is restored by supplementation in the drinking water. Methods: eNOS knockout mice on either standard diet or low NOx diet ±50 mg/L nitrite supplementation were compared to C57 control mice on standard diet to reveal dietary nitrite can restore NO biochemistry in mice unable to produce NO. -
FIG. 5 . Mice were fed 50 mg/L nitrite supplement for 12 weeks, resulting in increased circulating nitrite and nitrate levels (A and B). The nitrite fed group had 20% less lesion formation on the abdominal aorta than the control group fed high fat diet with nitrite free water (C), demonstrating that nitrite supplementation can inhibit the progression of atherosclerosis in a mouse model of atherosclerosis. -
FIG. 6 . Blood nitrite concentration after ingestion of an oral formulation containing sodium nitrite in a human model. -
FIG. 7 . Blood nitrate concentration after ingestion of an oral formulation containing L-arginine in a human model. -
FIG. 8 . Oxidation and reduction of nitrate, nitrite, and nitric oxide. -
FIG. 9 . Effect of a composition according to the present invention on NO concentration and blood NO levels compared to blood NO levels from a nutritional supplement containing L-arginine and antioxidants. -
FIG. 10 . (A&B) A 30 day twice per day regimen of a composition according to the present invention significantly increase plasma levels of nitrite and nitrate to normal healthy levels; (C) Patients taking a composition according to the present invention daily for 30 days saw a 10%-55% decrease in fasting triglycerides. (D) Collectively all patients taking a composition according to the present invention daily experienced a statistically significant reduction in fasting triglycerides (data are ave±SEM of n=13 patients) - Significance
- All life requires nitrogen-compounds. Nitrite (NO2—) is such a compound that is naturally occurring in nature and biology. Over the years, the pharmacological stance on nitrite has undergone a surprising metamorphosis, from a vilified substance that generates carcinogenic nitrosamines in the stomach, to a life-saving drug that liberates a protective agent (NO) during hypoxic events. Nitrite has been investigated as a vasodilator in mammals for over 125 years and is a known by-product of organic nitrate metabolism. There has been a recent re-discovery of some of the vasodilator actions of nitrite in physiology along with novel discoveries which may render nitrite a fundamental molecule in biology. Nitrite is emerging as an endogenous signaling molecule and regulator of gene expression that can not only serve as a diagnostic marker but also as potential therapy of cardiovascular disease. Up until recently nitrite was thought to be an inert oxidative breakdown product of endogenous nitric oxide synthesis.
- Certain embodiments of the present invention disclosed herein provide a formulation and a process to enhance and extend the therapeutic half-life of nitrite and therefore increase nitric oxide (NO) bioavailability. Thus, several embodiments provide the basis for new preventive or therapeutic strategies in diseases associated with NO insufficiency and new guidelines for optimal health as well as extend the therapeutic window in which one may intervene during a heart attack. Extension of nitrite half-life is desirable in the design of cardioprotective therapeutics or preventative medicines. As such, several embodiments prevent the onset or progression of cardiovascular or heart disease and protect from myocardial infarction thru nitrite/nitrate supplementation. Furthermore, certain embodiments provide an extended half-life of nitrite, out to 1 hour, which is the “golden hour” in terms of recovery from heart attack and stroke. The possibility of modulating an endogenous signaling pathway (NO) known to be involved in many physiological and pathophysiological events through a molecule found in certain foods is revolutionary and intriguing.
- Nitrite, NO and Cardiovascular Disease
- Ischemic heart disease, including myocardial infarction, remains the leading cause of morbidity and mortality in all industrialized nations (Myerburg, R. J. (2001). “Sudden cardiac death: exploring the limits of our knowledge.” J Cardiovasc Electrophysiol 12(3): 369-81). There are two distinct components of damage to the heart in patients who experience acute myocardial infarction: ischemic injury and reperfusion injury. The myocardium is able to tolerate brief periods of ischemia (an absolute or relative shortage of the blood supply to an organ) as activation of inherent, adaptive mechanisms can preserve energy levels and prevent injury. These include switching metabolism to anaerobic glycolysis and fatty acid utilization, increasing glucose uptake, and decreasing contractility. If ischemia persists however, the myocardium will develop a severe adenosine tri-phosphate (ATP) deficit, resulting in irreversible injury and culminating in cell death. Although reperfusion of ischemic tissues provides oxygen and metabolic substrates necessary for the recovery and survival of reversibly injured cells, reperfusion itself paradoxically results in the acceleration of cellular necrosis (Braunwald, E. and R. A. Kloner (1985). “Myocardial reperfusion: a double-edged sword?” J Clin Invest 76(5): 1713-9). Reperfusion is characterized by the formation of oxygen radicals upon reintroduction of molecular oxygen to ischemic tissues, resulting in widespread lipid and protein oxidative modifications, mitochondrial injury, as well as tissue apoptosis and necrosis (Nayler, W. G. (1981). “The role of calcium in the ischemic myocardium.” Am J Pathol 102(2): 262-70; McCord, J. M., R. S. Roy, et al. (1985). “Free radicals and myocardial ischemia. The role of xanthine oxidase.” Adv Myocardiol 5: 183-9).
- The loss of nitric oxide (NO) generation as a result of a dysfunctional vascular endothelium is a very likely cause of heart disease (Esper, R. J., R. A. Nordaby, et al. (2006). “Endothelial dysfunction: a comprehensive appraisal.” Cardiovasc Diabetol 5: 4). Continuous generation of NO is essential for the integrity of the cardiovascular system and a decreased production and/or bioavailability of NO is central to the development of cardiovascular disorders (Ignarro, L. J. (2002). “Nitric oxide as a unique signaling molecule in the vascular system: a historical overview.” J Physiol Pharmacol 53(4 Pt 1): 503-14; Herman, A. G. and S. Moncada (2005). “Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis.” Eur Heart J 26(19): 1945-55). NO is a highly reactive and diffusible gas formed by three NO synthase (NOS) isoforms: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). NO has been extensively studied in the setting of ischemia-reperfusion (ischemia/reperfusion) injury. Previous studies clearly demonstrate that the deficiency of eNOS exacerbates myocardial ischemia/reperfusion injury (Jones, S. P., W. G. Girod, et al. (1999). “Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase.” Am J Physiol 276(5 Pt 2): H1567-73; Sharp, B. R., S. P. Jones, et al. (2002). “Differential response to myocardial reperfusion injury in eNOS-deficient mice.” Am J Physiol Heart Circ Physiol 282(6): H2422-6), whereas the overexpression of eNOS (Jones, S. P., J. J. Greer, et al. (2004). “Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury.” Am J Physiol Heart Circ Physiol 286(1): H276-82; Elrod, J. W., J. J. Greer, et al. (2006). “Cardiomyocyte-specific overexpression of NO synthase-3 protects against myocardial ischemia-reperfusion injury.” Arterioscler Thromb Vasc Biol 26(7): 1517-23), NO donor (Siegfried, M. R., C. Carey, et al. (1992). “Beneficial effects of SPM-5185, a cysteine-containing NO donor in myocardial ischemia-reperfusion.” Am J Physiol 263(3 Pt 2): H771-7; Pabla, R., A. J. Buda, et al. (1996). “Nitric oxide attenuates neutrophil-mediated myocardial contractile dysfunction after ischemia and reperfusion.” Circ Res 78(1): 65-72) or inhaled NO gas (Hataishi, R., A. C. Rodrigues, et al. (2006). “Inhaled nitric oxide decreases infarction size and improves left ventricular function in a murine model of myocardial ischemia-reperfusion injury.” Am J Physiol Heart Circ Physiol 291(1): H379-84) therapy significantly protect the myocardium (Bolli, R. (2001). “Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research.” J Mol Cell Cardiol 33(11): 1897-918). NO possesses a number of physiological properties that makes it a potent cardioprotective-signaling molecule. These include vasodilation and the inhibition of oxidative stress, platelet aggregation, leukocyte chemotaxis and apoptosis (Ignarro, L. J., G. M. Buga, et al. (1987). “Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide.” Proc Natl Acad Sci U S A 84(24): 9265-9;, X. L., A. S. Weyrich, et al. (1993). “Diminished basal nitric oxide release after myocardial ischemia and reperfusion promotes neutrophil adherence to coronary endothelium.” Circ Res 72(2): 403-12; Li, J., C. A. Bombeck, et al. (1999). “Nitric oxide suppresses apoptosis via interrupting caspase activation and mitochondrial dysfunction in cultured hepatocytes.” J Biol Chem 274(24): 17325-33). NO synthesis is influenced by various cofactors such as tetrahydrobiopterin, flavin mononucleotide and flavin adenine dinucleotide, the presence of reduced thiols, the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) and substrate and oxygen availability. Without an adequate delivery of substrate and co-factors (conditions that exist during ischemia), NOS no longer produces NO but instead transfers the free electrons to oxygen and thus produces free oxygen radicals (Becker, B. F., C. Kupatt, et al. (2000). “Reactive oxygen species and nitric oxide in myocardial ischemia and reperfusion.” Z Kardiol 89 Suppl 9: IX/88-91, hereby incorporated by reference herein). Thus, there is a need for additional NO production in ischemic tissues that may limit ischemia/reperfusion injury.
- Nitrite is an oxidative breakdown product of NO that has been shown to serve as an acute marker of NO flux/formation (Kleinbongard, P., A. Dejam, et al. (2003). “Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals.” Free Radic Biol Med 35(7): 790-6). Nitrite has recently moved to the forefront of NO biology (Gladwin, M. T., A. N. Schechter, et al. (2005). “The emerging biology of the nitrite anion.” Nat Chem Biol 1(6): 308-14), as it represents a major storage form of NO in blood and tissues (Bryan, N. S. (2006). “Nitrite in nitric oxide biology: cause or consequence? A systems-based review.” Free Radic Biol Med 41(5): 691-701, hereby incorporated by reference herein). In addition to the oxidation of NO, nitrite is also derived from reduction of salivary nitrate by commensal bacteria in the mouth and gastrointestinal tract (Tannenbaum, S. R., A. J. Sinskey, et al. (1974). “Nitrite in human saliva. Its possible relationship to nitrosamine formation.” J Natl Cancer Inst 53: 79-84; van Maanen, J. M., A. A. van Geel, et al. (1996). “Modulation of nitrate-nitrite conversion in the oral cavity.” Cancer Detect Prey 20(6): 590-6) as well as from dietary sources such as meat, vegetables and drinking water. Much of the recent focus on nitrite physiology is due to its ability to be reduced to NO during ischemic or hypoxic events (Zweier, J. L., P. Wang, et al. (1995). “Enzyme-independent formation of nitric oxide in biological tissues.” Nat Med 1(8): 804-9; Bryan, N. S., T. Rassaf, et al. (2004). “Cellular Targets and Mechanisms of Nitros(yl)ation: An Insight into Their Nature and Kinetics in vivo.” Proc. Natl. Acad Sci. USA 101(12): 4308-4313, hereby incorporated by reference herein; Lundberg, J. O. and E. Weitzberg (2005). “NO generation from nitrite and its role in vascular control.” Arterioscler Thromb Vasc Biol 25(5): 915-22; Bryan 2006). Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, C. L., R. J. Casselden, et al. (1967). “Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments.” Biochim Biophys Acta 143(2): 310-8; Reutov, V. P. and E. G. Sorokina (1998). “NO-synthase and nitrite-reductase components of nitric oxide cycle.” Biochemistry (Mosc) 63(7): 874-84; Kozlov, A. V., K. Staniek, et al. (1999). “Nitrite reductase activity is a novel function of mammalian mitochondria.” FEBS Lett 454(1-2): 127-30), protonation (Zweier, Wang et al. 1995), deoxyhemoglobin (Cosby, K., K. S. Partovi, et al. (2003). “Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation.” Nature Medicine 9: 1498-1505, hereby incorporated by reference herein), and xanthine oxidase (Alikulov, Z. A., P. L'Vov N, et al. (1980). “[Nitrate and nitrite reductase activity of milk xanthine oxidase].” Biokhimiia 45(9): 1714-8; Li, H., A. Samouilov, et al. (2004). “Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation.” J Biol Chem 279(17): 16939-46; Webb, A., R. Bond, et al. (2004). “Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage.” Proc Natl Acad Sci U S A 101(37): 13683-8). Nitrite can also transiently form nitrosothiols (RSNOs) under both normoxic and hypoxic conditions (Bryan, Rassaf et al. 2004) and a recent study by Bryan et al demonstrates that steady state concentrations of tissue nitrite and nitroso are affected by changes in dietary NOx (nitrite and nitrate) intake (Bryan, N. S., B. O. Fernandez, et al. (2005). “Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues.” Nat Chem Biol 1(5): 290-7, hereby incorporated by reference herein). Previous studies have shown that nitrite therapy prior to reperfusion protects against hepatic and myocardial ischemia/reperfusion injury (Webb, Bond et al. 2004; Duranski, M. R., J. J. Greer, et al. (2005). “Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver.” J Clin Invest 115(5): 1232-1240). Additionally, experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm (Pluta, R. M., A. Dejam, et al. (2005). “Nitrite infusions to prevent delayed cerebral vasospasm in a primate model of subarachnoid hemorrhage.” Jama 293(12): 1477-84). Oral nitrite has also been shown to reverse L-NAME induced hypertension and serve as an alternate source of NO in vivo (Tsuchiya, K., Y. Kanematsu, et al. (2005). “Nitrite is an alternative source of NO in vivo.” Am J Physiol Heart Circ Physiol 288(5): H2163-70).
- A reduced NO availability is a hallmark of a number of cardiovascular disorders. Hyperlipidemia, arterial hypertension, diabetes, smoking and aging are major risk factors for the manifestation of cardiovascular events (Widlansky, M. E., N. Gokce, et al. (2003). “The clinical implications of endothelial dysfunction.” J. Am Coll Cardiol 42: 1149-1160). Plasma nitrite reflects acute changes in endothelial NOS activity in various mammals (Kleinbongard, Dejam et al. 2003) and thus may provide an accurate measurement of patients at risk for cardiovascular events. A recent report by Kleinbongard et al. (Kleinbongard, P., A. Dejam, et al. (2006). “Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans.” Free Radic Biol Med 40(2): 295-302), demonstrated that plasma nitrite levels progressively decrease with increasing cardiovascular risk load. Risk factors considered include age, hypertension, smoking, and hypercholesterolemia. Since nitrite acts as a protective molecule during ischemic events these data raise the intriguing possibility that the underlying problem with these patients is their diminished nitrite bioavailability. Since a substantial portion of steady state nitrite concentrations in blood and tissue are derived from dietary sources (Bryan, Fernandez et al. 2005), modulation of nitrite and/or nitrate intake may provide a first line of defense for ischemic heart disease.
- Therefore, several embodiments increase nitrite availability through diet or supplementation and provide an alternate route to increased NO availability as well as conferring protection from ischemia/reperfusion injury or other adverse cardiovascular event (e.g., heart attack, stroke, etc.). Since the half life of nitrite is on the order of seconds, it can only be used acutely and repeatedly for any therapeutic benefit. Dietary nitrite and nitrate supplementation for 7 days restores NO homeostasis and protects the heart from ischemia/reperfusion injury (Bryan, N. S., J. W. Calvert, et al. (2007). “Dietary nitrite supplementation protects against myocardial ischemia-reperfusion injury.” Proc Natl Acad Sci U S A, vol. 104 no. 48 19144-19149, hereby incorporated by reference herein) providing the first proof of concept that nitrite can be chronically administered and have a profound effect on outcome from heart attack.
- Thus, several embodiments of the present invention provide a method for prolonging the half life of nitrate and extending therapeutic benefit. In one embodiment, the method comprises providing a nitrite salt, wherein the nitrite salt is provided in an amount ranging from about 10 mg to about 100 mg; providing a nitrate salt, wherein the nitrate salt is provided in an amount ranging from about 50 mg to about 500 mg; and providing an ascorbic acid, wherein the ascorbic acid is provided in an amount ranging from about 100 mg to about 2000 mg. The nitrate salt and ascorbic acid are administered in combination with the nitrite salt. The nitrite salt and the nitrate salt convert to nitrite in vivo. The ascorbic acid reduces the conversion of nitrite into N-nitroso compounds in vivo, thereby extending the half-life of the nitrite. “Reduces” in this context encompasses, but is not limited to, “minimizes” or “prevents.” The ascorbic acid is particularly advantageous in some embodiments because it reduces carcinogens (e.g., N-nitroso compounds) while increasing the bioavailability of nitrite. L-arginine is added in some embodiments. L-arginine, in some embodiments, serves as a substrate for nitric oxide synthase, thereby increasing NO formation, which in turn can increase nitrate and/or nitrite formation. In one embodiment, the nitrite salt, nitrate salt, ascorbic acid, and optionally L-arginine are provide to a mammal in a single dose.
- Nitrite in NO Biology
- Nitrite has recently been implicated in hypoxic vasodilation in the circulation (Kim-Shapiro, D. B., M. T. Gladwin, et al. (2005). “The reaction between nitrite and hemoglobin: the role of nitrite in hemoglobin-mediated hypoxic vasodilation.” Journal of Inorganic Biochemistry 99: 237-246). As early as 1880, nitrite was described in terms of its vasodilatory abilities (Reichert, E. T. and S. W. Mitchell (1880). “On the physiological action of potassium nitrite, with a note on the physiological action on man.” Am J Med Sci 159: 158-180) and much later, Furchgott used acidified sodium nitrite to relax precontracted aortic strips in 1953 (Furchgott, R. F. and S. Bhadrakom (1953). “Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs.” J Pharcol Exp Ther 108(2): 129-143). Both studies used supra-physiological concentrations of nitrite. However, recent studies have rediscovered the vasodilatory effect of nitrite on forearm and systemic blood flow after nitrite infusion. Cosby et al. (Cosby, Partovi et al. 2003) suggested that nitrite is a large intravascular storage pool for NO and that nitrite bioactivation to NO could dilate regions with tissue oxygen debt in the human circulation. However, earlier a study by Lauer et al. reported that nitrite lacked intrinsic vasodilatory properties (Lauer, T., M. Preik, et al. (2001). “Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action.” Proc Natl Acad Sci USA 98(22): 12814-12819). This discrepancy is likely due to kinetics and duration of infusion. Nitrite is found in high abundance throughout the mammalian organ system (Bryan, Rassaf et al. 2004). It is normally a short-lived, highly regulated ion in the circulation (200-600 nM) with a half life in whole blood of 110 seconds (Kelm, M. (1999). “Nitric oxide metabolism and breakdown.” Biochim Biophys Acta 1411: 273-289). Two independent groups have recently demonstrated the cytoprotective effects of nitrite in ischemia-reperfusion injury (Webb, Bond et al. 2004; Duranski, Greer et al. 2005). Duranski et al attribute nitrite's protective effects to the reduction of nitrite to NO by the reductase activity of hemoglobin. The study by Webb et al using an isolated heart setup was in the absence of blood, clearly demonstrating that the myocardial tissue itself can metabolize nitrite without the need for hemoglobin. Moreover, inhalation of nitrite selectively dilates the pulmonary circulation under hypoxic conditions in vivo in sheep (Hunter, C. J., A. Dejam, et al. (2004). “Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator.” Nat Med 10: 1122-1127). Experiments in primates revealed a beneficial effect of long-term application of nitrite on cerebral vasospasm (Pluta, Dejam et al. 2005). Topical application of nitrite improves skin infections and ulcerations (Hardwick, J. B., A. T. Tucker, et al. (2001). “A novel method for the delivery of nitric oxide therapy to the skin of human subjects using a semi-permeable membrane.” Clin Sci (Loud) 100(4): 395-400). Furthermore, in the stomach, nitrite-derived NO seems to play an important role in host defense (Duncan, C., H. Dougall, et al. (1995). “Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate.” Nat Med 1(6): 546-551; Dykhuizen, R. S., R. Frazer, et al. (1996). “Antimicrobial effect of acidified nitrite on gut pathogens: importance of dietary nitrate in host defense.” Antimicrob Agents Chemother 40(6): 1422-1425) and in regulation of gastric mucosal integrity (Bjorne, H. H., J. Petersson, et al. (2004). “Nitrite in saliva increases gastric mucosal blood flow and mucus thickness.” J Clin Invest 113(1): 106-114). All of these studies together along with the observation that nitrite can act as a marker of NOS activity (Kleinbongard, Dejam et al. 2003) opened a new avenue for the diagnostic and therapeutic application of nitrite, especially in cardiovascular diseases, using nitrite as marker as well as an active agent. However, it is still not known how and to what extent nitrite reduction to NO occurs or how the NO-independent effects of nitrite contribute to the cytoprotection of ischemia/reperfusion insult.
- The History of Nitrite and Nitrate
- Nitrite has clearly emerged as an important molecule in biology, but its effects on the endogenous NO pathway have been poorly investigated. Furthermore, its use as a potential therapy needs further safety consideration. Historically nitrite was considered a strong oxidant and potential carcinogen. It has been in widespread use for many years. It is used as a color fixative and preservation in meats and fish and is naturally occurring in the soil and in vegetables. It is also used in manufacturing diazo dyes, nitroso compounds, in the textile industry, in photography and in the manufacture of rubber chemicals. Nitrite is also a common clinical and laboratory chemical that is used as a vasodilator (Reichert and Mitchell 1880), bronchodilator (Hunter, Dejam et al. 2004), intestinal relaxant (Kozlov, A. V., B. Sobhian, et al. (2001). “Organ specific formation of nitrosyl complexes under intestinal ischemia-reperfusion in rats involves NOS-independent mechanism(s).” Shock 15: 366-371) and used as an antidote for cyanide poisoning (Chen, K. K. and C. L. Rose (1952). “Nitrite and thiosulfate therapy in cyanide poisoning.” J Am Med Assoc 149(2): 113-119). Considering its widespread use there have been many toxicological studies on acute and chronic exposure to nitrite. The fatal dose of nitrite is in the range of 22-23 mg/kg body weight (from USFDA Generally Recognized as Safe Food Ingredient: Nitrates and Nitrites (Including Nitrosamines) 1972 by Battele-Columbus Laboratories and Department of Commence, Springfield Va.). Lower doses of either nitrite or nitrate have caused acute methemoglobinemia, particularly in infants. In infants, a high nitrite or nitrate intake has been associated with “blue baby syndrome” caused by methemoglobinemia (Comly, H. H. (1945). “Cyanosis in infants caused by nitrates in well water.” JAMA 129: 112-116; Donohoe, W. E. (1949). “Cyanosis in infants with drinking water as a cause.” Paediatrics 3: 308-311; Lecks, H. I. (1950). “Methemoglobinemia in infancy.” Am J Dis Child 79: 117-123). The major public health concern, particularly in the 1970s, was the endogenous formation of N-nitrosamines from nitrite and nitrate and its relevance to human cancer. The first report in the 1950s on the hepatocarcinogenic effects of N-nitrosodimethylamine (NDMA) (Magee, P. H. and J. M. Barnes (1956). “The production of malignant primary heptic tumors in the rat by feeding dimethylnitrosamine.” Br. J. Cancer 10: 114-122), and the suggestion that low molecular weight N-nitrosamines (RNNO) can be formed following nitrosation of various amines (Druckrey, H. and R. Preussmann (1962b). “Die Bilding carcinogener Nitrosamine am Beispiel des Tabakrauchs.” Naturewissenschaften 49: 498-499) ignited an enormous interest in N-nitrosamines and their association with cancer. Direct proof that such nitrosation reactions can occur was provided by Ender et al. (Ender, F., C. Havre, et al. (1964). “Isolation and identification of a hepatotoxic factor in herring meat produced from sodium nitrite preserved herring.” Naturwissenschaften 51: 637-638) who identified NDMA in nitrite preserved fish, and by Sander and Sief (Sander, J. and F. Seif (1969). “Bakterielle Reduction von nitrat im magen des menschen als ursache einer Nitrosamin-Bildung.” Arzneimittel-Forsch 19: 1091-1093) who demonstrated the in vivo formation of a nitrosamine in the acidic conditions of the human stomach. Because of the potent carcinogenicity, wide environmental occurrence and ease of formation of nitrosamines, considerable effort has been made to determine the levels of nitrite and nitrate in the external and internal human environment, and to assess exposure in order to correlate it with human cancer at specific sites (Bartsch, H. and R. Montėsano (1984). “Relevance of nitrosamines to human cancer.” Carcinogenesis 5(11): 1381-1393). Since the early 1980s there have been numerous reports on the association of N-nitrosamines and human cancers (Craddock, V. M. (1983). “Nitrosamines and human cancer: proof of an association?” Nature 306: 638; Bartsch and Montesano 1984) but a causative link between nitrite exposure and cancer is still missing (Ward, M. H., T. M. deKok, et al. (2005). “Workgroup report: Drinking-water nitrate and health-recent findings and research needs.” Environ Health Perspect 113(11): 1607-14). Furthermore, a two year study on the carcinogenicity of nitrite by NIH has conclusively found that there was no evidence of carcinogenic activity by sodium nitrite in male or female rats or mice (Program, N. T. (2001). On The Toxicology and Carcinogenesis Studues of Sodium Nitrite. U. S. D. o. H. a. H. Services, National Institute of Health. NTP TR 495: 1-276). These negative connotations of nitrite and nitrate have led the United States government to regulate and restrict the levels in food and drinking water. Early studies on nitrogen balance in humans and analyses of fecal and ileostomy samples indicated that nitrite and nitrate are formed de novo in the intestine. It was these early findings by Tannenbaum et al. (Tannenbaum, S. R., D. Fett, et al. (1978). “Nitrite and nitrate are formed by endogenous synthesis in the human intestine.” Science 200: 1487-1488) that significantly altered conceptions of human exposure to exogenous nitrite and nitrates and represented the original observations that would eventually lead to the discovery of the L-arginine: NO pathway. Prior to these studies it was thought that steady-state levels of nitrite and nitrate originated solely from the diet and from nitrogen-fixing enteric bacteria. Endogenous sources of nitrite in mammals are derived from: 1. oxidation of endogenous nitric oxide, 2. nutritional sources such as meat, vegetable and drinking water, 3. reduction of salivary nitrate by commensal bacteria in the mouth and gastrointestinal tract. The discovery of the NO pathway and the emerging biomedical applications of nitrite and nitrate necessitate a paradigm shift on the role of nitrite and nitrate in physiology.
- Nitrate/Nitrite Reduction to NO
- Humans, unlike prokaryotes, are thought to lack the enzymatic machinery to reduce nitrate back to nitrite. However, due to the commensal bacteria that reside within the human body it has been demonstrated that these bacteria can reduce nitrate thereby supplying an alternative source of nitrite (Goaz, P. W. and H. A. Biswell (1961). “Nitrite reduction in whole saliva.” J Dent Res 40: 355-365; Tannenbaum, Sinskey et al. 1974; Ishiwata, H., A. Tanimura, et al. (1975). “Nitrite and nitrate concentrations in human saliva collected from salivary ducts.” J Food Hyg Soc Jpn 16: 89-92; van Maanen, van Geel et al. 1996). Therefore dietary and enzymatic sources of nitrate are now a potentially large source of nitrite in the human body. Nitrate is rapidly absorbed in the small intestines and readily distributed throughout the body (Walker, R. (1996). “The metabolism of dietary nitrites and nitrates.” Biochem Soc Trans 24(3): 780-785). As much as 25% of the ingested nitrate is actively taken up by the salivary glands to be excreted in the saliva (Spiegelhalder, B., G. Eisenbrand, et al. (1976). “Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds.” Food Cosmet Toxicol 14: 545-548). Approximately 20% of the salivary nitrate is then reduced to nitrite by bacteria in the mouth (Spiegelhalder, Eisenbrand et al. 1976) and then disproportionates with formation of NO after entering the acidic environment of the stomach. This nitrate pathway to NO has been shown to help reduce gastrointestinal tract infection, increase mucous barrier thickness and gastric blood flow (Pique, J. M., B. J. Whittle, et al. (1989). “The vasodilator role of endogenous nitric oxide in the rat gastric microcirculation.” Eur. J. Pharmacol 174(2-3): 293-296; Brown, J. F., P. J. Hanson, et al. (1992). “Nitric oxide donors increase mucus gel thickness in rat stomach.” Eur. J. Pharmacol 223(1): 103-104; McKnight, G. M., L. M. Smith, et al. (1994). “Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans.” Gut 40(2): 211-214; Walker 1996). The concentrations of nitrate in drinking water are usually <10 mg/L in the absence of bacterial contamination (Kross, B. C., G. R. Hallberg, et al. (1993). “The nitrate concentration of private well water in Iowa.” Am J Public Health 83(2): 270-272). Vegetables, especially beets, celery, and leafy vegetables like lettuce and spinach are rich in nitrates (Meah, M. N., N. Harrison, et al. (1994). “Nitrate and nitrite in foods and the diet.” Food Addit Contam 11(4): 519-532; Walker 1996; Vallance, P. (1997). “Dietary nitrate: poison or panacea?” Gut 40(2): 211-214). Other vegetables contain nitrate at lower concentrations, but because they are consumed in greater quantity, they may contribute more nitrate and thus nitrite from the diet. For the average population, most nitrate exposure (86%) comes from vegetables, whereas the primary contributors to nitrite intake are cured meats (39%), baked goods and cereals (34%), and vegetables (16%). The National Research Council report The Health Effects of Nitrate, Nitrite, and N-Nitroso Compounds (NRC 1981) reported estimates of nitrite and nitrate intake based on food consumption tables. They report that the average total nitrite and nitrate intake in the U.S. was 0.77 mg and 76 mg, respectively per day. Nitrite and nitrate are excreted in the kidneys. Nitrate is excreted in the urine as such or after conversion to urea (Green, L. C., K. Ruiz de Luzuriaga, et al. (1981). “Nitrate biosynthesis in man.” Proc. Natl. Acad Sci. USA 78(12): 7764-7768). Clearance of nitrate from blood to urine approximates 20 ml/min in adults (Wennmalm, A., G. Benthin, et al. (1993). “Metabolism and excretion of nitric oxide in humans. An experimental and clinical study.” Circ Res 73(6): 1121-1127), indicating considerable renal tubular reabsorption of this ion. There is little detectable nitrite or nitrate in feces (Bednar, C. and C. Kies (1994). “Nitrate and Vitamin C from fruits and vegetables: impact of intake variations on nitrate and nitrite excretions in humans.” Plant Foods Hum Nutr 45(1): 71-80). There is some loss of nitrate and nitrite in sweat, but is not a major route of excretion (Weller, R., S. Pattullo, et al. (1996). “Nitric oxide is generated on the skin surface by reduction of sweat nitrate.” J Invest Dermatol 107(3): 327-331). Assuming the human body (70 kg) produces 1.68 mmole NO per day (based on 1 μmole/kg/hr NO production), an average daily intake of 0.77 mg of nitrite would equate to 11.1 μmoles per day and 76 mg nitrate would equate to 894 μmoles per day or roughly 1 mmole NOx per day from diet. This almost matches what the human body makes from NO, assuming most of the NO goes to stepwise oxidation to nitrite and nitrate.
- Nitrite Physiology
- The endogenous production of NO by NOS has been established as playing an important role in vascular homeostasis, neurotransmission, and host defense mechanisms (Moncada, S., R. M. J. Palmer, et al. (1991). “Nitric oxide: physiology, pathophysiology and pharmacology.” Pharmacol Rev 43(2): 109-142). The major pathway for NO metabolism is the stepwise oxidation to nitrite and nitrate (Yoshida, K., K. Kasama, et al. (1983). “Biotransformation of nitric oxide, nitrite and nitrate.” Int Arch Occup Environ Health 52: 103-115). In plasma or other physiological fluids or buffers, NO is oxidized almost completely to nitrite, where it remains stable for several hours (Kelm, M., M. Feelisch, et al. (1992). The Biology of nitric oxide. Physiological and Clinical Aspects. S. Moncada, M. A. Marletta, J. B. Hibbs and E. A. Higgs. London, Portland Press. 1: 319-322, hereby incorporated by reference herein; Grube, R., M. Kelm, et al. (1994). The Biology of Nitric Oxide. Enzymology, Biochemistry, and Immunology. S. Moncada, M. Feelisch, R. Busse and E. A. Higgs. London, Portland Press. 4: 201-204, hereby incorporated by reference herein); however, the half life of NO2 − in human whole blood is about 110 seconds (Kelm 1999).
- The oxidation of NO by molecular oxygen is second order with respect to NO:
-
2NO+O2→2NO2 (1) -
2NO+2NO2→2N2O3 (2) -
2N2O3+2H2O→4NO2 −+4H+ (3) - whereby NO2, N2O3 and NO2 − represent nitrogen dioxide, dinitrogen trioxide and nitrite, respectively. It should be noted that N2O3 is a potent nitrosating agent by virtue of its ability to generate the nitrosonium ion (NO+). NO and nitrite are rapidly oxidized to nitrate in whole blood. As stated above, the half life of NO2 − in human blood is about 110 seconds (Kelm 1999). Nitrate on the other hand has a circulating half life of 5-8 hours (Tannenbaum, S. R. (1994). “Nitrate and nitrite: origin in humans.” Science 205: 1333-1335, hereby incorporated by reference herein; Kelm, M. and K. Yoshida (1996). Metabolic Fate of Nitric Oxide and Related N-oxides. Methods in Nitric Oxide Research. M. Feelisch and J. S. Stamler. Chichester, John Wiley and Sons: 47-58, hereby incorporated by reference herein). Although the mechanisms by which NO and NO2 − are converted to NO3 − in vivo are not entirely clear, there are several possibilities. During fasting conditions with low intake of nitrite/nitrate, enzymatic NO formation from NOS accounts for the majority of nitrite (Rhodes, P., A. M. Leone, et al. (1995). “The L-arginine: nitric oxide pathway is the major source of plasma nitrite in fasted humans.” Biochem Biophys Res Commun 209: 590-596).
- NO production from nitrite has been described in infarcted heart tissue (Zweier, J. L., et al., Enzyme-independent formation of nitric oxide in biological tissues. Nature Medicine, 1995. 1(8): p. 804-809). Nitrite reductase activity in mammalian tissues has been linked to the mitochondrial electron transport system (Walters, C. L., R. J. Casselden, and A. M. Taylor, Nitrite metabolism by skeletal muscle mitochondria in relation to haem pigments. Biochim Biophys Acta, 1967. 143: p. 310-318; Reutov, V. P. and E. G. Sorokina, NO-synthase and nitrite-reductase components of nitric oxide cycle. Biochemistry (Mosc), 1998. 63(7): p. 874-884; Kozlov, A.V., K. Staniek, and H. Nohl, Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett, 1999. 454: p. 127-130; Nohl, H., et al., Mitochondria recycle nitrite back to the bioregulator nitric monoxide. Acta Biochim Pol, 2000. 47: p. 913-921; Tischner, R., E. Planchet, and W. M. Kaiser, Mitochondrial electron transport as a source for nitric oxide in the unicellular green algae Chlorella sorokiniana. FEBS Lett, 2004. 576: p. 151-155), protonation (Zweier, J. L., et al., Enzyme-independent formation of nitric oxide in biological tissues. Nature Medicine, 1995. 1(8): p. 804-809; Hunter, C. J., et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med, 2004. 10: p. 1122-1127), deoxyhemoglobin (Hunter, C. J., et al., Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator. Nat Med, 2004. 10: p. 1122-1127; Cosby, K., et al., Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human circulation. Nature Medicine, 2003. 9: p. 1498-1505), and xanthine oxidase (Li, H., et al., Characterization of the effects of oxygen on xanthine oxidase-mediated nitric oxide formation. J. Biol Chem, 2004. 279: p. 16939-16946; Alikulov, Z. A., N. P. L'vov, and V. L. Kretovich, Nitrate and nitrite reductase activity of milk xanthine oxidase. Biokhimiia, 1980. 45(9): p. 1714-1718; Webb, A., et al., Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci USA, 2004. 101(13683-13688)). Mitochondrial nitrite reduction has been shown to occur by ubiquinol (Kozlov, A. V., K. Staniek, and H. Nohl, Nitrite reductase activity is a novel function of mammalian mitochondria. FEBS Lett, 1999. 454: p. 127-130; Nohl, H., et al., The multiple functions of coenzyme Q. Bioorg Chem, 2001. 29(1): p. 1-13) and cytochrome c oxidase (Castello, P. R., et al., Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes. Cell Metab, 2006. 3(4): p. 277-87) with subsequent binding of the NO produced to cytochrome bc1 site of complex III or complex IV resulting in oxygen-dependent reversible inhibition of mitochondrial respiration (Takehara, Y., et al., Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide. Cell Struct Funct, 1996. 21(4): p. 251-8). The acidic reduction of nitrite requires protonation and a one-electron reduction. The relatively low pKa of nitrite (3.34) (Principles of Modern Chemistry. Third ed, ed. D. W. Oxtoby and N. H. Nachtrieb. 1996, Fort Worth: Harcourt Brace College Publishers. 848) limits this activity in physiology but it can occur in the stomach or during ischemic events when tissue pH falls. Since many different pathways have been shown to be able to reduce nitrite but require different conditions and substrates for optimal nitrite reduction, it is likely that all pathways may become relevant but at different oxygen tension, substrate availability, and perhaps even compartment specific needs.
- The evolution of nitrite from a vilified substance that generates carcinogenic nitrosamines in the stomach, to a life-saving drug that liberates a protective agent (NO) during hypoxic events, as well as performs many actions independent of NO, warrants a re-evaluation of nitrite in biology. With nitrite acting as both an end product of NO synthesis and a reservoir for NO, it is therefore a critical homeostatic molecule in NO biology.
- The collective evidence reviewed in this section strongly supports the notion that there is a fundamental and physiological basis for developing nitrite-based therapeutics. It is not understood how orally ingested nitrite (pKa 3.8) can survive the acidic environment of the stomach (pH 1-2). Furthermore, once nitrite is absorbed into the bloodstream it is known to be quickly oxidized to nitrate with a half life of 110 seconds. Surprisingly, several embodiments of the present invention demonstrate that orally administered nitrite in specific combination with nitrate and ascorbic acid can extend the therapeutic range of nitrite from seconds to tens of minutes providing a novel approach to treat or reduce injury from heart attack with nitrite. In some embodiments ascorbic acid reduces endogenous nitrosation reaction in the gastrointestinal tract, which enhances the half life of nitrite. In some embodiments, the nitrate provides an additional source of nitrite, again extending the functional half life, e.g., by increasing stores, of nitrite.
- The human diet exerts important long-term effects on vital body functions and thereby makes an important contribution to health and disease. While high intake of cholesterol, saturated fat, salt, and sugar are associated with a greater risk for cardiovascular disease, conventional wisdom has it that the opposite is true for abundant consumption of fruits and vegetables. A diet rich in fruits and vegetables is associated with a lower risk of certain forms of cancer and cardiovascular disease. Recent epidemiological studies suggest a cardioprotective action afforded specifically by green leafy vegetables. Green leafy vegetables such as spinach and lettuce, in addition to being rich in antioxidants are especially rich in nitrite and nitrate as are berries, grapes, and a few other fruits. The high content of nitrite and nitrate is a major factor contributing to the positive health effects of certain vegetables via bioconversion to NO which exerts protective effects on the cardiovascular system. A continuous intake of nitrite- and nitrate-containing food such as green leafy vegetables and berries may ensure that blood and tissue levels of NO are maintained at a level sufficient to compensate for any disturbances in endogenous NO synthesis. Dietary source of NO metabolites could therefore improve circulation and oxygen delivery and lead to better health and increased energy. This dietary pathway may therefore not only provide essential nutrients for NO production but also provide a rescue pathway for people at risk for cardiovascular disease. Several embodiments provide the nutrition and protection of a high vegetable diet in the form of a daily supplement formulation which renders subjects protected from injury from heart attack or other cardiovascular events, i.e. stroke, pulmonary embolism. This strategy including nitrite/nitrate supplementation in combination with ascorbic acid may serve as an inexpensive cardioprotective regimen which may delay or reduce the onset or progression of cardiovascular or heart disease and protect from myocardial infarction.
- Several embodiments of the invention are particularly advantageous because they provide a supplement formulation of nitrite, nitrate and Vitamin C. Although, in some embodiments, such amounts may be found in a high vegetable diet, the time it would take to consume the required assortment of vegetables as well as the impact on the digestive system would adversely impact the absorption and/or bioavailability of the nitrite, nitrate and Vitamin C. Moreover, the reaction of other compounds and nutrients in the naturally occurring vegetable assortment may also adversely impact the impact the absorption and/or bioavailability of the nitrite, nitrate and Vitamin C. Thus, a supplement of nitrite, nitrate and Vitamin C in e.g., daily dose formulations are advantageous in several embodiments because it increases the absorption and/or bioavailability of the formulation. In some embodiments, the formulation comprises purified or isolated nitrite, nitrate and Vitamin C. In other embodiments, the formulation consists essentially of purified or isolated nitrite, nitrate and Vitamin C. In yet other embodiments, the formulation consists of purified or isolated nitrite, nitrate and Vitamin C.
- In other embodiments, the formulation consists essentially of purified or isolated nitrite, nitrate, Vitamin C, and L-arginine. In yet other embodiments, the formulation consists of purified or isolated nitrite, nitrate, Vitamin C, and
L -arginine - In one embodiment, “consists essentially of” means the composition may further contain one or more components selected from the group consisting of water and flavorants.
- With 1 in every 3 men and 1 in every 10 women in the U.S. expected to develop some major cardiovascular disease before reaching
age 60 is it desirable to take preventive measures now to enhance cardiovascular health. - In one embodiment, the present invention can provide a novel therapy for patients experiencing myocardial infarction or stroke. Nitrite has been shown to be protective in animal models of stroke and both cardiac and hepatic ischemia-reperfusion injury. Conversely, nitrite insufficiency is associated with increased injury from ischemia-reperfusion insult. However, because of the short half-life of nitrite in the circulation (110 sec), the therapeutic window for nitrite alone is very narrow. Therefore, several embodiments provide patients with an extended-release formulation comprising nitrite, among additional components, to be used upon onset of symptoms to provide at least some protection from injury until the patient can be provided with reperfusion therapy, such as in a hospital setting.
- In several embodiments, the present invention relates to the use of supplemental nitrite in combination with nitrate and vitamin C (ascorbic acid) as a preventive agent in cardiovascular disease. In some embodiments nitrate acts as an extended release nitrite source that is absorbed and re-circulated through the enterosalivary pathway and is reduced to nitrite by commensal bacteria in the mouth. In some embodiments nitrite acts as a reservoir for nitric oxide activity. Reduced nitric oxide availability is a hallmark of a number of cardiovascular disorders and plasma nitrite levels progressively decrease with increasing cardiovascular risk load. Therefore, several embodiments provide a sufficient daily intake of nitrite, which is beneficial to optimal cardiovascular health. A typical Western diet is low in nitrite and nitrate compared to a vegetarian or Mediterranean diet and may therefore account for the increased incidence of cardiovascular disease in the United States, Europe, and other developed countries. A daily nitrite supplementation may provide the missing nutrient, analogously to a daily multivitamin. The Nobel Prize in Physiology or Medicine was awarded in 1998 for the discovery of nitric oxide in the cardiovascular system. Maintaining nitric oxide availability is essential for optimal health, particularly for those at risk for cardiovascular events, and therefore, in several embodiments, supplemental nitrite acts to increase the reservoir of nitric oxide which can be bio-activated upon need as a prevention rather than a treatment or therapy once disease has occurred.
- In one embodiment, the present invention relates to a formulation for an alternate source of nitric oxide during cardiovascular exercise and/or muscle training. In a further embodiment, the formulation further comprises L-arginine. L-arginine is a natural amino acid substrate for nitric oxide synthase enzymes which produces L-citrulline and NO from L-arginine in a complex reaction requiring oxygen. L-arginine can be given as a pre-workout drink to saturate the NOS enzyme to produce sufficient NO and dilate vessels. However, under conditions where muscles are working during anaerobic metabolism, oxygen availability is diminished and therefore NOS can no longer produce NO. Therefore an alternate substrate must be supplied to produce NO under anaerobic conditions. The substrate then becomes nitrite. Several embodiments supply blood and muscles with nitrite before a workout , which provides an additional source of NO during the workout and improves muscle blood flow during exercise, thereby enhancing performance and muscle building capacity. In one embodiment, sodium nitrite is added to existing workout beverage formulations , thereby increasing NO and providing sufficient NO before during and/or after a workout. Since the L-aginine:NO pathway is not functional during workout, the addition of nitrite provides the substrate for anaerobic formation of NO, an alternate pathway for NO generation. So instead of increasing NO production before and after a workout through the L-arginine: NO pathway, the presence of nitrite in certain embodiments of the formulation will allow NO production from nitrite reduction during the workout, a time at which it is advantageous to increase blood flow and supply the muscles with essential nutrients and oxygen.
- In one embodiment, the present invention relates to a composition comprising a nitrite salt, a nitrate salt, and ascorbic acid.
- Any positively-charged ion safe for use as a food additive or a component of a pharmaceutical formulation can be used as the counterion to nitrite in the nitrite salt or the counterion to nitrate in the nitrate salt. In one embodiment, the positively-charged ion is an inorganic ion. In a further embodiment, the positively-charged ion is selected from the group consisting of sodium and potassium; e.g., the nitrite salt is sodium nitrite or potassium nitrite and the nitrate salt is sodium nitrate or potassium nitrate.
- Any proportions of the components of the composition can be used. In one embodiment, the composition comprises from about 1 weight part to about 8 weight parts sodium nitrite, from about 5 weight parts to about 50 weight parts sodium nitrate, and from about 20 weight parts to about 200 weight parts ascorbic acid.
- In one embodiment, the composition further comprises L-arginine. In a further embodiment, the composition comprises from about 20 weight parts to about 200 weight parts
L -arginine. - In some embodiments, sodium nitrite is included in a range of about 0.01 mg/kg to about 15 mg/kg. In some embodiments, sodium nitrate is included in a range of about 1.0 mg/kg to about 50 mg/kg. In some embodiments, ascorbic acid is included in a range of about 1.0 mg/kg to about 25 mg/kg. In certain embodiments, L-arginine may also be included in a range of about 2.0 mg/kg to about 50 mg/kg.
- In certain embodiments that enhance NO formation in working muscle, sodium nitrite is included in a range of about 30 mg to about 40 mg. In certain embodiments, sodium nitrate is included in a range of about 250 mg to about 300 mg. In certain such embodiments, ascorbic acid is included in an amount of about 1000 mg. In certain other embodiments, L-arginine may also be included in an amount of about 1000 mg.
- In certain embodiments that function to restore NO homeostasis in the user, sodium nitrite is included in an amount of about 20 mg. In certain such embodiments, sodium nitrate is included in an amount of about 150 mg. In certain embodiments, ascorbic acid is included in an amount of about 500 mg. In certain other embodiments, L-arginine may also be included in an amount of about 500 mg.
- The composition can further comprise other materials. In one embodiment, the composition further comprises water. Alternatively or in addition, it can also further comprise other materials. For example, the composition may comprise a flavorant, such as a citrus flavor, a non-citrus fruit flavor, an herbal flavor, a vanilla flavor, or a chocolate flavor, and other appropriate flavorings.
- In other embodiments, the present invention relates to a method of enhancing cardiovascular performance in a mammal, comprising administering to the mammal a composition according to any of the embodiments described herein. In one embodiment, the composition comprises a nitrite salt, a nitrate salt, and ascorbic acid.
- Any mammal for which enhanced cardiovascular performance is desired can be the subject of the method. In one embodiment, the mammal is Homo sapiens. Other mammals for which enhanced cardiovascular performance may be desired include, but are not limited to, draft animals, beasts of burden, animals useful in transportation (e.g., horses), racing animals (e.g., horses or greyhounds), meat animals, wool- or fur-bearing animals, milk animals, working dogs, and household pets, among others. Enhanced cardiovascular performance can be desired for a person or animal engaged in physical exertion. In other embodiments, a composition as described herein may be used as a treatment or prophylaxis for a medical condition characterized by or associated with reduced blood flow to an organ of the body.
- Administering the composition can be by any route, such as oral, intravenous, or intraarterial, among others. In one embodiment, administering is by the oral route. In this embodiment, it is desirable that the components of the composition be dissolved in a neutral- or pleasant-tasting liquid, such as water, flavored water, milk, or fruit juice, among others. Additionally, the components of the composition may be in tablet or capsule form and in this form the composition may be dissolvable in liquid. In other embodiments, the composition is provided as a tablet that dissolves when placed in the mouth of a user. In some embodiments, a composition according to any of the embodiments described herein can be provided in powder, tablet, capsule, gel, aerosol or liquid form.
- Any dosage of the components of the composition can be used, provided such dosage is safe for the mammal In one embodiment, administering is of a dosage from about 0.01 mg/kg/day to about 15 mg/kg/day sodium nitrite, from about 1 mg/kg/day to about 50 mg/kg/day sodium nitrate, and from about 1 mg/kg/day to about 25 mg/kg/day ascorbic acid. If the composition comprises L-arginine, administering is of a dosage from about 2 mg/kg/day to about 50 mg/kg/day
L -arginine. - In one embodiment, the present invention is directed to a composition, comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; and from about 4 weight parts to about 100 weight parts of a nitrite salt.
- A botanical nitrate source is any plant matter, extract of plant matter, or product of plant matter containing nitrate. Generally, it is desirable that the botanical nitrate source be generally regarded as safe for human or animal consumption. In one embodiment, the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia, St. John wort, broccoli, stevia, spinach, gingko, kelp, tribulus, eleuthero, epimedium, eucommia, hawthorn berry, rhodiola, green tea, codonopsys, panax ginseng, astragalus, pine bark, dodder seed, Schisandra, cordyceps, and mixtures thereof. In one particular embodiment, the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof.
- A botanical source of nitrite reduction activity is any plant matter, extract of plant matter, or product of plant matter, containing nitrite reductase enzyme, a compound capable of reducing nitrite, or both. Generally, it is desirable that the botanical source of nitrite reduction activity be generally regarded as safe for human or animal consumption. In one embodiment, the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia, St. John wort, broccoli, stevia, spinach, gingko, kelp, tribulus, eleuthero, epimedium, eucommia, rhodiola, green tea, codonopsys, panax ginseng, astragalus, dodder seed, cordyceps, berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee, walnuts, peanuts, borojo, pomegranates, popcorn, yerba mate, and mixtures thereof. Berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee, walnuts, peanuts, borojo, pomegranates, popcorn, and yerba mate are known to contain polyphenols, which are known to have antioxidant properties.
- In a particular embodiment, the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof.
- “Hawthorn berry” herein refers to any portion of a plant of the genus Crataegus (for example, Crataegus oxyacantha), such as the berry, leaf, or flower, among others, as well as extracts of any portion thereof. In a particular embodiment, it refers to the berry of a plant of the genus Crataegus (for example, Crataegus oxyacantha).
- “Schisandra” refers to any portion of a plant of the genus Schisandra (for example, S. chinensis and S. rubiflora, among others), such as the fruit, leaf, or flower, among others, as well as extracts of any portion thereof.
- A nitrite salt comprising any counterion may be used. Generally, it is desirable that the nitrite salt be generally regarded as safe for human or animal consumption. In one embodiment, the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, magnesium nitrite, calcium nitrite, and mixtures thereof. In a particular embodiment, the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof.
- In addition to the materials described above, the composition can further comprise one or more additional materials.
- In one embodiment, the composition can further comprise from about 20 weight parts to about 500 weight parts
L -citrulline. - In one embodiment, the composition can further comprise from about 20 weight parts to about 1000 weight parts
L -arginine. - The production of NO from L-arginine is a critical cellular function performed by nitric oxide synthase (NOS) in most, if not all, organ systems throughout the body. For years, physicians and scientists assumed simply feeding more substrate L-arginine would be sufficient to enhance NO production. It is becoming increasingly clear that this may not be the most effective strategy, especially in patients that are insufficient in NO due to endothelial dysfunction. The effect of L-arginine on endothelial function in humans is inconsistent. By definition endothelial dysfunction is the inability to produce NO from L-arginine. This pathway is dysfunctional and inoperative. Providing the enzyme NOS with substrate because of lowered availability of L-arginine does not appear to be rate limiting since the intracellular levels of the amino acid are in the millimolar range (Gold, M. E., P. A. Bush, and L. J. Ignarro, Depletion of arterial L-arginine causes reversible tolerance to endothelium-dependent relaxation. Biochem Biophys Res Commun, 1989. 164(2): p. 714-21), and the enzyme's Michaelis constanst (KM) for substrate is in the micromolar range (2.9 μmol/L) (Bredt, D. S. and S. H. Snyder, Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A, 1990. 87(2): p. 682-5). In contrast, circulating L-arginine measured in plasma of healthy humans as well as in plasma of patients with vascular disorders is in the range of 45-100 μmol/L (Boger, R. H. and S. M. Bode-Boger, The clinical pharmacology of L-arginine. Annu Rev Pharmacol Toxicol, 2001. 41: p. 79-99). This is up to 15 to 30-fold higher than the concentration required to saturate the NOS enzyme. This biochemical discrepancy is termed as the “arginine paradox.”
- L-Arginine supplementation may be detrimental in some populations. A clinical trial designed to enhance NO production in humans that have suffered a heart attack revealed that L-arginine, when added to standard postinfarction therapies, does not improve vascular stiffness measurements or ejection fraction and may be associated with higher postinfarction mortality. The study concluded that L-arginine should not be recommended following acute myocardial infarction (Schulman, S. P., et al., L-arginine therapy in acute myocardial infarction: the Vascular Interaction With Age in Myocardial Infarction (VINTAGE MI) randomized clinical trial. Jama, 2006. 295(1): p. 58-64).
- The vast majority of endogenous arginine synthesis in adult mammals (60%) occurs in the kidney, where citrulline produced by the intestine is extracted from the blood and converted to arginine by the action of ASS and ASL (Windmueller, H. G. and A. E. Spaeth, Source and fate of circulating citrulline. Am J Physiol, 1981. 241(6): p. E473-80). Therefore supplying more L-citrulline to the urea cycle will produce more L-arginine to be specifically directed to the nitric oxide pathway; in other words, supplying more L-citrulline provides precursors to the L-arginine nitric oxide pathway while avoiding the negative side effects of supplementing L-arginine directly.
- In one embodiment, the composition can further comprise from about 0.2 weight parts to about 5 weight parts vitamin B12. The vitamin B12 can be in any form of cobalamin. In one embodiment, the vitamin B12 is in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof.
- In one embodiment, the composition can further comprise from about 20 weight parts to about 500 weight parts vitamin C. The vitamin C can be in any form of ascorbate or ascorbic acid. In one embodiment, the vitamin C is in a form selected from the group consisting of magnesium ascorbate, sodium ascorbate, potassium ascorbate, ascorbic acid, and mixtures thereof.
- In one embodiment, the composition can further comprise from about from about 20 weight parts to about 500 weight parts of a nitrate salt selected from the group consisting of sodium nitrate, potassium nitrate, and mixtures thereof.
- In addition to the materials described above, the composition can further comprise one or more additional materials suitable for forming the composition into a vehicle deliverable for human or animal consumption. Such one or more additional materials include, but are not limited to, binders, flavorants, colorants, sweeteners, adujvants, and excipients, among others.
- In one embodiment, the composition can further comprise from about 50 weight parts to about 1500 weight parts of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants. These one or more ingredients can act as one or more of binders, flavorants, colorants, sweeteners, antiadherents, or lubricants, among other functions.
- In one particular embodiment, the composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 weight parts;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof and is present at about 100 weight parts; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 weight parts;
- and the composition further comprises:
- about 100 weight parts L-citrulline;
- about 1 weight part vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- about 100 weight parts vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof; and
- from about 50 weight parts to about 1500 weight parts of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- The composition can be formulated, using techniques known in the art, into any vehicle suitable for human consumption. For example, the composition can be formulated as a powder dissolvable or suspendable in a potable beverage, a soft food, or both; as an ingredient that can be baked into a baked cookie, cracker, or bar; a tablet or capsule that can be swallowed; or a lozenge dissolvable in the mouth; among others. In one embodiment, the composition can be in a form of a lozenge dissolvable in the mouth. The lozenge can have a weight from about 600 mg to about 2000 mg.
- In one embodiment, the present invention is directed to a method of reducing a patient's triglyceride level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts
L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt. - The composition and its components can be as described above. Also, the formulation of the composition as a lozenge can be as described above.
- In one embodiment, 1 weight part is 1 mg.
- In a particular embodiment, the administered composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 mg;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof and is present at about 100 mg; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 mg;
- and the composition further comprises:
- about 100 mg
L -citrulline; - about 1000 μg vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- about 100 mg vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof; and
- from about 50 mg to about 1500 mg of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- The composition can be administered according to any dosing regimen. Particular details of how administering is to be performed are within the ability of the person of ordinary skill in the art having the benefit of the present disclosure. In one embodiment, the administering is performed once or twice daily.
- In one embodiment, the present invention is directed to a method of reducing a patient's C-reactive protein level, comprising administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising from about 40 weight parts to about 1000 weight parts of a botanical nitrate source; from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity; from about 20 weight parts to about 500 weight parts
L -citrulline; and from about 4 weight parts to about 100 weight parts of a nitrite salt. - The composition and its components can be as described above. Also, the formulation of the composition as a lozenge can be as described above.
- In one embodiment, 1 weight part is 1 mg.
- In a particular embodiment, the administered composition can be as follows:
- the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 mg;
- the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof and is present at about 100 mg; and
- the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 mg;
- and the composition further comprises:
- about 100 mg
L -citrulline; - about 1000 μg vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
- about 100 mg vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof; and
- from about 50 mg to about 1500 mg of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, other modified celluloses, starch, modified starches, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
- The composition can be administered according to any dosing regimen. Particular details of how administering is to be performed are within the ability of the person of ordinary skill in the art having the benefit of the present disclosure. In one embodiment, the administering is performed once or twice daily.
- The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
- Exercising muscle demands increased blood flow in order to maintain sufficient nutrients and oxygen for metabolism. Nitric oxide is the body's most potent vasodilator. Nitric oxide is produced in the body by the enzyme nitric oxide synthase (NOS). NOS enzymes produce .NO by catalyzing a five electron oxidation of a guanidino nitrogen of L-arginine (L-Arg). Oxidation of L-Arg to L-citrulline occurs via two successive monooxygenation reactions producing Nω hydroxy L-arginine as an intermediate. Two moles of O2 and 1.5 moles of NADPH are consumed per mole of .NO formed (Liu, Q. and G. S. S., Binding sites of nitric oxide synthases. Methods Enzymol, 1996. 268: p. 311-324). NOS enzymes are the only enzymes known to simultaneously require five bound cofactors/prosthetic groups: FAD, FMN, heme, tetrahydrobiopterin (BH4) and Ca2+-calmodulin (CaM). All NOS isozymes are catalytically self-sufficient provided all required substrates and co-factors are available. CaM binding to nNOS has been shown to regulate catalytic activity by triggering electron flux from FMN to heme, thereby coupling the oxygenase and reductase domains. CaM also facilitates NADPH dependent reduction of cytochrome c and ferricyanide in BH4 and heme depleted nNOS. If any of the co-factors become limiting, then NO production from NOS shuts down, and in many cases NOS then produces superoxide instead. This is indeed a very complex and coordinated effort to enzymatically produce NO which normally proceeds very efficiently. However, in disease characterized by oxidative stress where cofactors become oxidized, NOS uncoupling, or conditions of hypoxia where oxygen is limiting, this process can no longer maintain NO production. Therefore there has to be an alternate route to NO production. It is highly unlikely that Nature devised such a sophisticated mechanism of NO production as a sole source of a critical molecule. Nitrite reduction then acts as a backup system to the NOS system. Part of this may occur through nitrite reduction during low oxygen availability. Nitrite supplementation can then support NO production during exercise when enzymatic NO production is shut down.
- Nitrite reduction to NO can occur via a simple mechanism. The 1-electron reduction of nitrite can occur by ferrous heme proteins (or any redox active metal) and an electron donor through the following reaction:
- This is the same biologically active NO as that produced by NOS just instead of using L-arg as the substrate, nitrite is used. Therefore for this to occur, the tissues or biological compartment must have a sufficient pool of nitrite stored. Nitrite supplementation may therefore act as a protective measure to compensate for insufficient NOS activity under conditions of hypoxia such as during anaerobic metabolism during exercise or muscle training Nitrite contributes to whole body NO production and homeostasis. Considerable published support for this theory derives from the following facts: NO produced from nitrite in the upper intestine is up to 10,000 times the concentrations that occur in tissues from enzymatic synthesis (McKnight, G. M., et al., Chemical synthesis of nitric oxide in the stomach from dietary nitrate in humans. Gut, 1997. 40(2): p. 211-214), nitrite can act as a circulating NO donor (Dejam, A., et al., Emerging role of nitrite in human biology. Blood Cells Mol Dis, 2004. 32(3): p. 423-429) and nitrite can itself perform many actions previously attributable to NO (Gladwin, M. T., et al., The emerging biology of the nitrite anion. Nature Chemical Biology, 2005. 1(6): p. 308-314) without the intermediacy of NO (Bryan, N. S., et al., Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nature Chemical Biology, 2005. 1(5): p. 290-297). While L-arginine supplementation may provide moderate amounts of NO prior to workout, during a workout, this system becomes inefficient and very little NO from L-arginine can be produced due to lack of oxygen substrate. Therefore NO from nitrite provides an alternate mechanism to maintain NO production during exercise. Supplemental nitrite taken 15-20 minutes prior to workout can titrate up tissue and muscle nitrite concentrations in order to produce NO locally during exercise and therefore enhance blood flow and performance.
- Nitrite Reduction to NO is an Oxygen Sensitive Process
- Much of the recent focus on nitrite physiology is due to its ability to be reduced to NO during ischemic or hypoxic events (Lundberg, J. O. and E. Weitzberg, NO generation from nitrite and its role in vascular control. Arterioscler Thromb Vasc Biol, 2005. 25(5): p. 915-22; Bryan, N. S., Nitrite in nitric oxide biology: Cause or consequence? A systems-based review. Free Radic Biol Med, 2006. 41(5): p. 691-701; Bryan, N. S., et al., Cellular Targets and Mechanisms of Nitros(yl)ation: An Insight into Their Nature and Kinetics in vivo. Proc. Natl. Acad Sci. USA, 2004. 101(12): p. 4308-4313). Nitrite reduction to NO under aerobic and anaerobic conditions using chemiluminescent detection of free NO has been quantified and characterized. Under aerobic conditions, established by continuous sample purging with air, NO production by blood-free tissues and RBCs from nitrite was minimal and fleeting. However, switching the purge gas to N2 (i.e., hypoxia) acutely enhanced tissue NO formation from NO2 − (
FIG. 1 , left). Hypoxic tissue NO2 − reduction exhibited compartment-specific properties (initial kinetics, amount, duration) and was most dramatic and sustained in liver homogenate. In addition to liver, all tissues sampled were capable of detecting an imposed decrease in O2 tension and transducing this information into a potentiation of tissue NO formation from NO2 −, as demonstrated by the marked, tissue-selective increases in NO production from NO2 − observed under aerobic (21% O2) vs. hypoxic (N2) conditions (FIG. 1 , right). Heart, liver, skeletal (gastrocnemius) muscle, and aorta exhibited the greatest capacity for NO2 − reduction to NO during O2 deprivation (FIG. 1 , right inset). These data demonstrate an intrinsic ability of tissues to sense the graded diminution of ambient O2 and transduce this information into the subsequent production of NO from NO2 −. This finding suggests that various tissues might auto-regulate their blood flow in disease states with a pathological component of O2 insufficiency through their capacity for hypoxic NO2 − reduction to NO provided sufficient nitrite exist. - There is a growing appreciation that nitrite therapy may provide benefit from I/R injury (Dezfulian, Raat et al. 2007). However, there are no data on the effects of nitrite insufficiency in the setting of I/R injury. Analysis of several different standard rodent chows revealed that Purina 5001 contains the highest concentrations of NOx (104.3±4.7 pmol/g nitrite and 6275±50.7 pmol/g nitrate) and nitroso as compared to any other standard rodent chow analyzed. Therefore this diet was used in comparison to mice fed a purified amino acid diet, the diet lowest in NOx (20.5±0.7 pmol/g nitrite and 503.1±17.9 pmol/g nitrate) but with the same L-arginine content. In order to reveal the biochemical and physiological effects of dietary nitrite insufficiency, mice were fed a standard rodent chow (Purina 5001) for 9 weeks and then switched to a purified amino acid diet low in nitrite and nitrate (Harlan TD99366) for 7 days. Control mice were fed Purina 5001 for 10 weeks. Consistent with an earlier report (Bryan, Fernandez et al. 2005), the low NOx diet significantly decreased plasma and heart steady-state nitrite and nitrate concentrations which could be restored by the addition of 50 mg/L nitrite in the drinking water for 1 week (
FIG. 2A-B ). Blood and tissue nitroso products have been shown to preserve NO bioactivity (Stamler, Simon et al. 1992) and protein nitrosation modification confer cGMP-independent NO signalling events (Stamler, Lamas et al. 2001). Changes in dietary nitrite consumption affect cellular signaling events (Bryan, Fernandez et al. 2005). Mice fed a low NOx diet for 1 week demonstrated a significant reduction in plasma and heart nitroso levels compared to mice fed standard chow, which could be replenished and increased with 50 mg/L nitrite in the drinking water for 1 week (FIG. 2C ). Nitrosyl-heme products (FIG. 2D ) were also reduced in the mice fed a low NOx diet and replenished by nitrite supplementation in the drinking water. These data reveal that changes in dietary nitrite and/or nitrate consumption can affect steady state concentrations of blood and tissue NO products/metabolites commonly used to assess NO production. - Whether dietary restriction of nitrite affected the severity of cardiac ischemia-reperfusion (I/R) injury was determined The decrease in steady state nitrite concentrations in blood and heart was found to significantly exacerbate myocardial injury (
FIG. 2E ). The mice fed a low NOx diet displayed a 59% increase in infarct relative to the area at risk (AAR) compared to mice fed a standard chow. To ensure the observed effect was dependent upon NOx intake, and not due to an alteration in the nutritional value of the low NOx diet, a subset of mice on the low NOx diet were given 50 mg/L sodium nitrite ad libitum in the drinking water to restore steady state concentrations of blood and tissue nitrite. Nitrite supplementation in animals on the low NOx diet reversed the increased myocardial infarct size by 57%. Additionally, mice fed the low NOx diet displayed a higher mortality rate (57.7% survival) 24 hours post-myocardial infarction than mice on the standard rodent chow (70.6% survival). Likewise, survival improved in mice on the low NOx diet with nitrite-supplemented drinking water to 76.9%. Since nitrite is derived both from diet and oxidation of enzymatic NO production from NOS, potential compensatory changes in NOS expression following one week low NOx intake were investigated. Western blot analysis of myocardial tissue lysate revealed no significant alterations in NOS expression (eNOS, nNOS, and iNOS) (FIG. 2F ). These data clearly suggest that the increased injury is due specifically to changes in steady state concentrations of plasma and heart nitrite as a result of decreased dietary NOx consumption and not from changes in enzymatic NO production. - Duranski et al. recently demonstrated that bolus addition of nitrite prior to reperfusion significantly protects the heart and liver from ischemia/reperfusion damage in an in vivo model (Duranski, M. R., et al., Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest, 2005. 115(5): p. 1232-1240). During the ischemic event, NOS is inactive since oxygen, a necessary co-factor, has been depleted. It is believed that nitrite is reduced to NO to compensate for the insufficient NOS derived NO. These data are very important because they helped us to recognize that the application of exogenous nitrite has profound effects. Endurance training is known to cause ischemic organ damage in such organs as the gut and kidneys due to the diversion of blood flow from these organs to supply working muscles. This is a significant problem in marathon runners. Nitrite may provide a valuable nutrient to these athletes as a pre-workout or pre-marathon supplement to protect from ischemic injury during the event. Nitrite may then serve multiple purposes in the setting of myocardial ischemia/reperfusion. First, by titrating up tissue concentrations of nitrite when it is administered just prior to reperfusion, one can protect the heart from ischemia/reperfusion. Second, acute nitrite administration may initiate a signaling cascade that results in the upregulation of other protective proteins which afford protection hours later.
- Nitrite has been shown to be protective in both the heart and the liver following ischemia/reperfusion (Webb, A., et al., Reduction of nitrite to nitric oxide during ischemia protects against myocardial ischemia-reperfusion damage. Proc Natl Acad Sci USA, 2004. 101(13683-13688); Duranski, M. R., et al., Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest, 2005. 115(5): p. 1232-1240). It is speculated that nitrite is reduced to NO under ischemic conditions to provide an alternate source of NO when NOS is inactive due to decreased substrate delivery and decreased oxygen saturation. To better understand the fate of nitrite during ischemia and reperfusion, a time course of nitrite metabolism both after ischemia and during reperfusion was conducted. As shown in
FIG. 3 , nitrite is consumed in the heart tissue during 30 minutes of ischemia but is unaffected in the plasma. The consumption of nitrite appears to lead to a concomitant increase in cardiac nitroso products (FIG. 3B ). Nitrite can form nitrosothiols in a first order reaction requiring heme and thiols and can also be reduced to NO under anaerobic conditions (Bryan, N. S., et al., Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues. Nat Chem Biol, 2005. 1(5): p. 290-7). During reperfusion, nitrite is gradually increased and restored whereby tissue nitroso decompose during the reperfusion phase (FIG. 3 ). Without being bound by a particular theory, the inventor proposes that nitrite serves two functions in the setting of ischemia reperfusion. It may first serve as a NOS-independent source of NO by which nitrite is reduced to NO under ischemic conditions when NOS is inactive. Secondly, nitrite may react with critical thiols to form nitrosothiols. It is possible that this nitroso modification acts as a reversible protective shield which reduces irreversible oxidation during the oxidative burst of reperfusion. Aside from “capping” critical thiols from oxidation, and without being bound by a particular theory, the inventor proposes that the nitroso products can then release the NO moiety during the reperfusion phase and act an a redox sensitive NO donor (Hogg, N., Biological chemistry and clinical potential of S-nitrosothiols. Free Radic Biol Med, 2000. 28(10): p. 1478-86). Biochemical data support this notion by the increase in nitroso at the expense of nitrite followed by the decay of nitroso over time during reperfusion. Therefore adding supplemental nitrite can increase plasma and tissue nitrite but also lead to an increase in steady state levels of nitroso and thereby afford protection during ischemia/reperfusion. On the contrary, nitrite insufficiency leads to increased injury because there is not enough stored in blood or tissue to perform these actions. - Enzymatic NO insufficiency is a hallmark of a number of diseases including cardiovascular disease. To test the hypothesis that dietary nitrite can compensate for NOS dysfunction, the above experiments were repeated in eNOS −/− mice. The mice were fed standard rodent chow or low NOx diet. As shown in
FIG. 4 , eNOS −/− mice revealed lower plasma nitrite concentrations consistent with earlier findings (Kleinbongard, P., et al., Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radical Biology & Medicine, 2003. 35(7): p. 790-796) but there is no significant difference in cardiac nitrite revealing that blood markers do not accurately reflect tissue status (Bryan, N. S., Nitrite in nitric oxide biology: Cause or consequence? A systems-based review. Free Radic Biol Med, 2006. 41(5): p. 691-701). Plasma nitrite could be further decreased by feeding eNOS −/− mice a low NOx diet demonstrating that plasma nitrite is a reflection of both NOS and diet. Feeding low NOx diet to eNOS −/− mice completely eliminated steady state concentrations of plasma nitroso without any significant effect on cardiac nitroso. Supplementation of 50 mg/L nitrite in the drinking water for 7 days restores plasma nitrite in eNOS −/− to control levels and increases both plasma and cardiac nitroso to above C57 control levels. - Mice deficient in eNOS have increased injury to ischemia/reperfusion insult (Jones, S. P., et al., Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol, 1999. 276(5 Pt 2): p. H1567-73) and data shown above reveal these mice also have reduced nitrite and nitroso compared to C57 wild type. To investigate if dietary nitrite can benefit eNOS −/− mice, eNOS −/− mice on low NOx diet ±50 mg/L nitrite in drinking water were subjected to 30 minutes ischemia and 24 hour reperfusion as above. These mice are also protected from myocardial ischemia/reperfusion injury suggesting that dietary nitrite supplementation can provide benefit under conditions of dysfunctional NOS.
- Initial studies in the characterization of the LDb mouse reveal that they have diminished blood and tissue nitrite and nitroso levels with no difference in nitrate at 11 months when atherosclerosis is well developed. These data indicate that there is deficiency in bioavailable NO and nitrite. Steady state tissue nitroso levels are also decreased suggesting a dysregulation of protein nitrosation and therefore provide the rationale and justification for early intervention of dietary nitrite supplementation on restoring NO-nitroso redox and on the progression of atherosclerosis.
- In order to demonstrate the utility of supplemental nitrite in affecting the progression of atherosclerosis, a high fat diet was fed to 8 female LDb mice for 12 weeks. Four of the mice received nitrite free water and the other 4 mice were supplemented with 50 mg/L nitrite throughout the 12 weeks on high fat diet. Although the LDb mice spontaneously develop atherosclerosis on normal rodent chow, the addition of a high fat diet will accelerate the process from 8 months to 12 weeks. At the time of sacrifice, plasma was collected for nitrite, nitrate determination as well as lipid profile determination. As shown in
FIG. 5A-B , there significantly more circulating nitrite and nitrate in the nitrite fed mice than the nitrite free water group. The nitrite fed group had 20% less lesion formation on the abdominal aorta than the control group fed high fat diet with nitrite free water (FIG. 5C ). These data demonstrate that nitrite supplementation can inhibit the progression of atherosclerosis in the female LDb mice using a high fat diet. - Aim: To develop specific formulation that will extend the circulating half life of nitrite from 110 seconds to 45-60 minutes.
- Methods: Since nitrite is derived from NO oxidation, diet, and from the reduction of nitrate in the human body, to enhance nitrite bioavailability, substrates from all 3 pathways were included: L-arginine to enhance NO production from nitric oxide synthase which will subsequently produce nitrite; sodium nitrite to increase acute circulating nitrite concentrations; ascorbic acid to inhibit endogenous nitrosation reactions in the stomach; and sodium nitrate to provide a slow release source of nitrite. This specific formulation was compared to 3 g L-arginine that is marketed commercially to enhance NO production. An intravenous line was obtained by the inventor on himself with 21 gauge infusion set needle and blood collected. The first 5 ml of blood was discarded. Blood was then collected at baseline. Then the prescribed formulation was dissolved in 50 ml of water and taken orally. A timer was started and blood was sampled for analysis at 1 minute and every 2 minutes for 60 minutes.
- Results: Direct analysis of the volunteer's blood revealed that the oral formulation increases blood nitrite within 3 minutes and reaches a maximum 9 minutes (
FIG. 6 ). This represents a 20 fold increase in plasma nitrite that lasts for 50 minutes. Plasma nitrate continues to rise throughout the course of the experiment. L-arginine in combination with NAD and ascorbic acid did not significantly affect plasma nitrite or nitrate concentrations (FIG. 7 ). - Conclusions: The specific formulation developed can increase plasma nitrite to therapeutic levels within 3 minutes of ingestion and can maintain therapeutic levels until 50 minutes after drinking This represents a novel formulation whereby this product can be given immediately upon patient presentation of ischemic episode that will maintain the protective nitrite levels for up to 1 hour. There is a golden hour in clinical medicine whereby the patient survival and outcome from ischemic episodes greatly declines. The safety of nitrite and nitrate at these doses is well established and therefore the formulations according to several embodiments discussed above represent a safe, novel use for oral nitrite as a cardioprotective agent.
- Disclosed herein is an immediate and extended release form of nitrite extending the biological half life from hundreds of seconds to minutes and hours. Among others, two possible applications for this technology may be as a revolutionary nitric oxide based supplement for the workout industry and a daily supplement to restore NO homeostasis in the aging population. Below is a range of effective doses of each ingredient.
- Workout Supplement and Daily Supplement
- Sodium nitrite (0.01 mg/kg-15 mg/kg: fatal dose is 22-23 mg/kg in humans)
- Sodium nitrate (1.0 mg/kg-50 mg/kg; Poisoning in man may result from a total oral daily dose in excess of 4 g or from a single dose of more than 1 g. 8 g may be fatal and 13-15 g are generally fatal (Sollmann, 1957). Although natural sources of nitrate are available, the concentrations are sufficiently low that the volume (or mass) that would need to be consumed to provide the same degree of supplementation as compared to several embodiments disclosed herein would be prohibitively large. For example, one liter of beetroot juice contains about 2.79 g of nitrate.
- Ascorbic acid (1 mg/kg-25 mg/kg)
- L-arginine (2 mg/kg-50 mg/kg)
- In one embodiment, a workout supplement formulation to enhance NO formation in working muscle consists, consists essentially of or comprises:
- 40 mg sodium nitrite
- 250 mg sodium nitrate
- 1000 mg ascorbic acid
- 1000 mg L-arginine
- In another embodiment, a workout supplement formulation to enhance NO formation in working muscle consists, consists essentially of or comprises:
- 30 mg sodium nitrite
- 300 mg sodium nitrate
- 1000 mg ascorbic acid
- 1000 mg L-arginine
- In one embodiment, a daily supplement formulation to restore NO homeostasis consists, consists essentially of or comprises:
- 20 mg sodium nitrite
- 150 mg sodium nitrate
- 500 mg ascorbic acid
- 500 mg L-arginine
-
FIG. 6 shows blood nitrite and nitrate levels from 0 min to 60 min after ingestion of an oral formulation containing 30 mg sodium nitrite, 300 mg sodium nitrate, 1000 mg ascorbic acid, and 1000 mg L-arginine in a human volunteer. In one embodiment, the formulation consists, consists essentially of or comprises 30 mg sodium nitrite, 300 mg sodium nitrate, 1000 mg ascorbic acid, and 1000 mg L-arginine. - In 1994 two groups independently presented evidence for generation of NO in the stomach resulting from the acidic reduction of inorganic nitrite (Benjamin, N., F. O'Driscoll, et al. (1994). “Stomach NO synthesis.” Nature 368(6471): 502; Lundberg, J. O., E. Weitzberg, et al. (1994). “Intragastric nitric oxide production in humans: measurements in expelled air.” Gut 35(11): 1543-6). Benjamin and colleagues demonstrated that the antibacterial effects of acid alone was markedly enhanced by addition of nitrite which is present in saliva whereas Lundberg and colleagues could measure high levels of NO in expelled air from the stomach in humans. These levels were abolished after pretreatment with a proton pump inhibitor and markedly increased after ingestion of nitrate, showing the importance of both luminal pH and the conversion of nitrate to nitrite for stomach NO generation. These were the first reports of NO synthase-independent formation of NO in vivo.
- In the classical NO synthase pathway, NO is formed by oxidation of the guanidino nitrogen of L-arginine with molecular oxygen as the electron acceptor (Moncada, S. and A. Higgs (1993). “The L-arginine-nitric oxide pathway.” N Engl J Med 329(27): 2002-12). This complex reaction is catalysed by specific heme-containing enzymes, the NO synthases, and the reaction requires several co-factors. The alternative pathway was fundamentally different; instead of L-arginine it used the simple inorganic anions nitrate (NO3 −) and nitrite (NO2 −) as substrates in a stepwise reduction process that did not require NO synthase or multiple co-factors. The biochemical pathway and biological effects of nitrate reduction to nitrite and further on to NO in the gastrointestinal tract have now been further characterized (Lundberg, J. O., E. Weitzberg, et al. (2004). “Nitrate, bacteria and human health.” Nat Rev Microbiol 2(7): 593-602). Oral commensal bacteria are essential for the first step in the nitrate-nitrite-NO pathway since they are responsible for the reduction of the higher nitrogen oxide nitrate to form nitrite. It was known from the literature that the salivary glands extract nitrate from plasma but the reason for this active process was not explained. This active process leads to levels of salivary nitrate that are 10-20 fold higher than in plasma. Oral facultative anaerobic bacteria residing mainly in the crypts of the tongue, then reduce nitrate to nitrite by the action of nitrate reductase enzymes (Spiegelhalder, B., G. Eisenbrand, et al. (1976). “Influence of dietary nitrate on nitrite content of human saliva: possible relevance to in vivo formation of N-nitroso compounds.” Food Cosmet Toxicol 14: 545-548; Duncan, C., H. Dougall, et al. (1995). “Chemical generation of nitric oxide in the mouth from the enterosalivary circulation of dietary nitrate [see comments].” Nat Med 1(6): 546-51). These bacteria use nitrate as an alternative electron acceptor to gain ATP in the absence of oxygen. This highly effective bacterial nitrate reduction results in salivary levels of nitrite that are 1000-fold higher than those found in plasma (Lundberg, J. O. and M. Govoni (2004). “Inorganic nitrate is a possible source for systemic generation of nitric oxide.” Free Radic Biol Med 37(3): 395-400). When nitrite-rich saliva meets the acidic gastric juice, nitrite is protonated to form nitrous acid (HNO2) which then decomposes to NO and a variety of other nitrogen oxides (Benjamin, O'Driscoll et al. 1994; Lundberg, Weitzberg et al. 1994). It is now established that oral commensal bacteria are pivotal in gastric NO formation, and gastric NO levels are consistently low in animals reared under complete germ-free conditions (Sobko, T., C. Reinders, et al. (2004). “Gastrointestinal nitric oxide generation in germ-free and conventional rats.” Am J Physiol Gastrointest Liver Physiol 287(5): G993-7). If the oral flora is selectively removed by topical treatment with an antibacterial mouthwash, the gastric NO levels decrease drastically (Petersson, J. (2008). Nitrate, nitrite and nitric oxide in gastric mucosal defense. Uppsala University. Uppsala, Sweden). This pathway is now known to contribute to at least half of the endogenous NO metabolism in humans.
- A Novel Nitric Oxide System: This recognition now provides a new paradigm for restoring NO homeostasis that is vastly different than the NOS system. The stepwise reduction of nitrate to nitrite to nitric oxide is, by necessity, an inefficient process by which each step yields a 3-log lower concentration of product than substrate (Jansson, E. A., L. Huang, et al. (2008). “A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis.” Nat Chem Biol 4(7): 411-7). Steady state levels of nitrate and nitrite in healthy humans is 20-40 μM and ˜0.5 μM respectively. The relative ratio of nitrate to nitrite in blood corroborates this 3-log reduction efficacy of nitrate to nitrite. Given this same efficiency for nitrite, one might expect ˜5 nM NO production from reduction of physiological concentrations of nitrite, which would certainly elicit a physiological response. However, we have found that oxygen is a potent inhibitor of nitrite reduction with 80% inhibition at 0.5% oxygen (Feelisch, M., B. O. Fernandez, et al. (2008). “Tissue Processing of Nitrite in Hypoxia: An Intricate Interplay of Nitric Oxide-Generating and -Scavenging Systems.” J Biol Chem 283(49): 33927-34). In vitro measurements indicate only 0.01% of nitrite is converted to NO in completely anaerobic and anoxic tissue homogenates and actually much less at physiological concentrations of oxygen (Bryan, N. S., B. O. Fernandez, et al. (2005). “Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues.” Nat Chem Biol 1(5): 290-7). Since humans with known cardiovascular risk factors and endothelial dysfunction have reduced plasma and presumably tissue levels of nitrite (Kleinbongard, P., A. Dejam, et al. (2006). “Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans.” Free Radic Biol Med 40(2): 295-302), this reservoir of NO activity is further compromised and therefore unable to be utilized for NO production by inherent biological systems. As a result, in order to get an appreciable amount of NO from this pathway, pharmacological doses of nitrate and nitrite are needed when relying simply on inherent biological systems for the reduction.
- Although both nitrite and nitrate are naturally occurring molecules and are part of our food supply (Hord, N. G., Y. Tang, et al. (2009). “Food sources of nitrates and nitrites: the physiologic context for potential health benefits.” Am J Clin Nutr 90(1): 1-10), they are not without toxicity. We have created a system whereby we can enhance the nitrate-nitrite-NO reduction efficacy at each step which justifies the use of physiological concentrations of nitrate and nitrite as a provision of NO which can overcome the body's inability to generate NO from L-arginine.
- Remarkably, we have identified natural products that are part of our normal diet which can enhance this pathway. We recently completed a nationwide survey of over 400 food products including meats and vegetables, both organically and conventionally grown and screened over 100 herbs that have been used for many years in traditional medical practices in Europe and Asia. The results of our study have revealed products that are enriched in nitrate (e.g., beet root) that provide the substrate nitrite in the pathway. We have also uncovered a select few herbs with very robust nitrite reductase activity that is unaffected by oxygen (e.g., Hawthorne berry).
- As illustrated in
FIG. 8 . the endogenous production of nitric oxide by NOS leads to the rapid oxidation to nitrite by molecular oxygen and plasma proteins such as ceruloplasmin. Alternatively, NO reacts directly with oxyheme-proteins such as hemoglobin and is oxidized to nitrate. We now have a system to reverse this process and regenerate NO from these oxidative end-products, nitrite and nitrate. The introduction of beet and Hawthorne provide a very robust system for generating NO that is novel and innovative and allows us to use much lower physiological concentrations of nitrite rather than pharmacological therapeutic doses. - We recently published our initial results from these investigations using Traditional Chinese Medicines (TCM) (Tang, Y., H. Garg, et al. (2009). “Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications.” Free Radic Biol Med 47(6): 835-40). Since many of these TCMs are used primarily for cardiovascular indications characterized by NO insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. The results from our study reveal that the herbs tested contained varying levels of nitrite and nitrate and varying nitrite reductase activity. Several of the herbal extracts contain a nitrite reductase activity greater by 1000 times than that of biological tissues (Feelisch, Fernandez et al. 2008). We have discovered that Hawthorn berry contains an active nitrite reductase that is 10 times more active than the Chinese herbs we tested.
- We concluded that repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs (Tang, Garg et al. 2009). Since all of these herbs and TCMs contain relatively high levels of nitrate and some residual nitrite, there is in essence over 5000 years of phase I safety trials in humans regarding nitrite and nitrate at these levels.
- Human data: Based on the pre-clinical data obtained from above we created a rationally designed formulation based on the ingredients with the greatest capacity for generating NO through the system in
FIG. 8 . Over two years of screening and testing a number of combinations of different herbs, we recognized a combination of beet root and hawthorne berry which gave us the most predictable and prolonged NO release profile. The NO release kinetics are shown inFIG. 9A . In this in vitro system, the NO product has a half life of roughly 1 hour with respect to NO release. - The following formulation (Neo40) was prepared:
-
- beet root powder, 200 mg;
- hawthorn berry extract, 100 mg;
- sodium nitrite, 20 mg;
-
L -citrulline, 100 mg; - vitamin B12, 1000 μg (as methylcobalamin and cyanocobalamin);
- vitamin C, 100 mg (as magnesium ascorbate and ascorbic acid); and
- about 250 mg to about 500 mg other ingredients (mannitol, modified cellulose, xylitol, natural orange flavor, magnesium stearate, sucralose, and silica).
- Neo40 was sourced and developed into a quick dissolving lozenge by a GMP-certified facility. We then tested the pharmacokinetics of Neo40 in humans. When allowed to dissolve in the mouth, Neo40 leads to a slow and steady rise in plasma nitrite of humans as shown in
FIG. 9B . To demonstrate the difference between Neo40 and compositions of L-arginine and anti-oxidants that have been used as nutritional supplements designed to enhance NO production, we compared Neo40 to one of the most popular products on the market. As shown inFIG. 9B , the L-arginine product did not lead to any appreciable formation of NO when consumed. This is in stark contrast to the NO formation resulting from administration of Neo40 . - In order to demonstrate that Neo40 could restore NO levels, we conducted a small clinical trial in healthy volunteers (age 34-64; 6 male, 6 female; 6 smokers, 6 non-smokers) and found that after taking Neo40 (one lozenge, twice a day) for 30 days lead to a modest but significant increase in both plasma nitrite and nitrate (
FIG. 10A-B ). - Based on this data, we began a clinical trial. Our inclusion criteria for this double-blinded, placebo-controlled study were patients over 40 with three or more of the following cardiovascular risk factors: hypertension, obesity, hyperlipidemia, smoking, sedentary lifestyle, family history of cardiovascular disease, and diabetes. As shown in
FIG. 10C , there was a modest to dramatic decease in triglycerides in the patients with elevated triglycerides >149 mg/dL). As shown inFIG. 10D , 100% of the patients with elevated triglycerides (>149 mg/dL) at baseline had a reduction in their triglycerides with the twice-daily regimen. Utilizing beet root and Hawthorne berry along with nitrite and L-citrulline provides a novel system for generating NO whereby the metabolism of nitrite is specifically directed towards reduction to NO by the Hawthorne berry. Though not to be bound by theory, we believe the presence of an active reductase in the formulation is an important addition as it allows a more efficient means to produce NO from nitrite. - All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Claims (11)
1. A method of reducing a patient's C-reactive protein level, comprising:
administering to the patient a composition in a form of a lozenge dissolvable in the mouth, the composition comprising:
from about 40 weight parts to about 1000 weight parts of a botanical nitrate source;
from about 20 weight parts to about 500 weight parts of a botanical source of nitrite reduction activity;
from about 20 weight parts to about 500 weight parts L-citrulline; and
from about 4 weight parts to about 100 weight parts of a nitrite salt.
2. The method of claim 1 , wherein the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 weight parts;
the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof and is present at about 100 weight parts;
the L-citrulline is present at about 100 weight parts; and
the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 weight parts;
and the composition further comprises:
about 1 weight part vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof;
about 100 weight parts vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof; and
from about 50 weight parts to about 1500 weight parts of one or more other ingredients selected from the group consisting of mannitol, xylitol, sorbitol, other sugar alcohols, cellulose, cellulose esters, cellulose ethers, starch, other polysaccharides, oligosaccharides, disaccharides, saccharides, gelatin, polyvinylpyrrolidone, polyethylene glycol, other binders, flavorants, colorants, magnesium stearate, other antiadherent agents, other stearate salts, sweeteners, silica, and other lubricants.
3. The method of claim 1 , wherein the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, magnesium nitrite, calcium nitrite, and mixtures thereof.
4. The method of claim 1 , wherein the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, holy basil, gymnema sylvestre, L9H, ashwagandha root, salvia, St. John wort, broccoli, stevia, spinach, gingko, kelp, tribulus, eleuthero, epimedium, eucommia, rhodiola, green tea, codonopsys, panax ginseng, astragalus, dodder seed, cordyceps, berries, tea, beer, grapes, wine, olive oil, chocolate, cocoa, coffee, walnuts, peanuts, borojo, pomegranates, popcorn, yerba mate, and mixtures thereof.
5. The method of claim 1 , wherein: the botanical nitrate source is selected from the group consisting of beet root, artichoke, holy basil, gingko, and mixtures thereof and is present at about 200 weight parts; the botanical source of nitrite reduction activity is selected from the group consisting of hawthorn berry, Schisandra, green tea, beet root, pine bark, and mixtures thereof and is present at about 100 weight parts; and the nitrite salt is selected from the group consisting of sodium nitrite, potassium nitrite, and mixtures thereof and is present at about 20 weight parts; and the composition further comprises: about 100 weight parts L-citrulline; about 1 weight part vitamin B12 in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof; and about 100 weight parts vitamin C in a form selected from the group consisting of magnesium ascorbate, ascorbic acid, and mixtures thereof.
6. The method of claim 2 , wherein the vitamin B12 is in a form selected from the group consisting of methylcobalamin, cyanocobalamin, and mixtures thereof.
7. The method of claim 2 , wherein the vitamin C is in a form selected from the group consisting of magnesium ascorbate, sodium ascorbate, potassium ascorbate, ascorbic acid, and mixtures thereof.
8. The method of claim 1 , further comprising from about 20 weight parts to about 1000 weight parts L-arginine.
9. The method of claim 1 , further comprising from about from about 20 weight parts to about 500 weight parts of a nitrate salt selected from the group consisting of sodium nitrate, potassium nitrate, and mixtures thereof.
10. The method of claim 1 , wherein the lozenge weighs from about 600 mg to about 2000 mg.
11. The method of claim 1 , wherein the administering is performed once or twice daily.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/827,100 US20160038533A1 (en) | 2008-06-13 | 2015-08-14 | Nitrite formulations and their use as nitric oxide prodrugs |
US16/150,155 US20190083528A1 (en) | 2008-06-13 | 2018-10-02 | Nitrite formulations and their use as nitric oxide prodrugs |
US16/842,550 US20200237808A1 (en) | 2008-06-13 | 2020-04-07 | Nitrite formulations and their use as nitric oxide prodrugs |
US17/303,510 US20210290663A1 (en) | 2008-06-13 | 2021-06-01 | Nitrite formulations and their use as nitric oxide prodrugs |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6125108P | 2008-06-13 | 2008-06-13 | |
US12/484,364 US8298589B1 (en) | 2008-06-13 | 2009-06-15 | Nitrite formulations and their use as nitric oxide prodrugs |
US12/856,957 US8303995B1 (en) | 2008-06-13 | 2010-08-16 | Nitrite formulations and their use as nitric oxide prodrugs |
US13/668,776 US8962038B2 (en) | 2008-06-13 | 2012-11-05 | Nitrite formulations and their use as nitric oxide prodrugs |
US14/610,492 US9119823B2 (en) | 2008-06-13 | 2015-01-30 | Nitrite formulations and their use as nitric oxide prodrugs |
US14/827,100 US20160038533A1 (en) | 2008-06-13 | 2015-08-14 | Nitrite formulations and their use as nitric oxide prodrugs |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/610,492 Continuation US9119823B2 (en) | 2008-06-13 | 2015-01-30 | Nitrite formulations and their use as nitric oxide prodrugs |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/150,155 Continuation US20190083528A1 (en) | 2008-06-13 | 2018-10-02 | Nitrite formulations and their use as nitric oxide prodrugs |
Publications (1)
Publication Number | Publication Date |
---|---|
US20160038533A1 true US20160038533A1 (en) | 2016-02-11 |
Family
ID=47075413
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/856,957 Active US8303995B1 (en) | 2008-06-13 | 2010-08-16 | Nitrite formulations and their use as nitric oxide prodrugs |
US13/668,776 Active 2029-09-07 US8962038B2 (en) | 2008-06-13 | 2012-11-05 | Nitrite formulations and their use as nitric oxide prodrugs |
US14/610,492 Active US9119823B2 (en) | 2008-06-13 | 2015-01-30 | Nitrite formulations and their use as nitric oxide prodrugs |
US14/827,100 Abandoned US20160038533A1 (en) | 2008-06-13 | 2015-08-14 | Nitrite formulations and their use as nitric oxide prodrugs |
US16/150,155 Abandoned US20190083528A1 (en) | 2008-06-13 | 2018-10-02 | Nitrite formulations and their use as nitric oxide prodrugs |
US16/842,550 Abandoned US20200237808A1 (en) | 2008-06-13 | 2020-04-07 | Nitrite formulations and their use as nitric oxide prodrugs |
US17/303,510 Abandoned US20210290663A1 (en) | 2008-06-13 | 2021-06-01 | Nitrite formulations and their use as nitric oxide prodrugs |
Family Applications Before (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/856,957 Active US8303995B1 (en) | 2008-06-13 | 2010-08-16 | Nitrite formulations and their use as nitric oxide prodrugs |
US13/668,776 Active 2029-09-07 US8962038B2 (en) | 2008-06-13 | 2012-11-05 | Nitrite formulations and their use as nitric oxide prodrugs |
US14/610,492 Active US9119823B2 (en) | 2008-06-13 | 2015-01-30 | Nitrite formulations and their use as nitric oxide prodrugs |
Family Applications After (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/150,155 Abandoned US20190083528A1 (en) | 2008-06-13 | 2018-10-02 | Nitrite formulations and their use as nitric oxide prodrugs |
US16/842,550 Abandoned US20200237808A1 (en) | 2008-06-13 | 2020-04-07 | Nitrite formulations and their use as nitric oxide prodrugs |
US17/303,510 Abandoned US20210290663A1 (en) | 2008-06-13 | 2021-06-01 | Nitrite formulations and their use as nitric oxide prodrugs |
Country Status (1)
Country | Link |
---|---|
US (7) | US8303995B1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190076755A1 (en) * | 2017-09-13 | 2019-03-14 | Thermolife International, Llc | Enriched Root Powder Products and Methods of Producing Thereof |
CN109549951A (en) * | 2019-01-25 | 2019-04-02 | 大连海洋大学 | Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer |
CN110343038A (en) * | 2018-04-02 | 2019-10-18 | 暨南大学 | A kind of treasured Rogor monomer, extract and its preparation method and application |
US11464816B2 (en) | 2019-04-16 | 2022-10-11 | The Procter & Gamble Company | Supplement for menopause |
Families Citing this family (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PL2124638T3 (en) * | 2007-02-26 | 2017-07-31 | Heartbeet Ltd. | Performance enhancing composition and use thereof |
US10821132B2 (en) | 2007-02-26 | 2020-11-03 | Heartbeet Ltd | Compositions of nitrates and methods of use thereof |
US10842813B2 (en) | 2007-02-26 | 2020-11-24 | Heartbeet Ltd | Compositions of nitrates and methods of use thereof |
US11759477B2 (en) | 2007-02-26 | 2023-09-19 | Heartbeet Ltd. | Compositions of nitrates and methods of use thereof |
US8303995B1 (en) * | 2008-06-13 | 2012-11-06 | Board Of Regents, The University Of Texas System | Nitrite formulations and their use as nitric oxide prodrugs |
EP2991660B1 (en) * | 2013-05-04 | 2021-03-31 | Board of Regents, The University of Texas System | Compositions and methods for promoting nitric oxide production through an oral delivery system |
CA2915396A1 (en) | 2013-06-13 | 2014-12-18 | The Hershey Company | Food compositions that enhance nitric oxide mediated signalling |
AU2014300493B2 (en) | 2013-06-28 | 2020-01-16 | Arjuna Natural Private Limited | A medicinal composition of Amaranth extract origin having enriched nitrate content and a method of preparing the same |
US11338005B2 (en) | 2013-06-28 | 2022-05-24 | Arjuna Natural Private Limited | Medicinal composition of amaranth origin for cardiovascular treatment |
US20160361353A1 (en) * | 2015-06-12 | 2016-12-15 | AusTech Pharmaceutical | Nitric oxide generator combined with pde5 inhibitors |
US10624921B2 (en) * | 2016-11-15 | 2020-04-21 | Berkeley Nox Limited | Dietary supplements |
EP3479703A1 (en) * | 2017-11-07 | 2019-05-08 | PM-International AG | Food supplement preparation |
CN108142881A (en) * | 2018-03-20 | 2018-06-12 | 爱可道生物科技有限公司 | A kind of arithoke alabastrum extract blood pressure lowering capsule product and its production technology |
US11844811B2 (en) | 2018-05-20 | 2023-12-19 | Myfitstrip Llc | Methods and compositions for alleviating respiratory dysfunction |
IT201800007747A1 (en) * | 2018-08-01 | 2020-02-01 | Pharmanutrition R&D Srl | Nutraceutical formulation for the control of mild to moderate arterial hypertension |
US11865139B2 (en) | 2020-11-12 | 2024-01-09 | Thermolife International, Llc | Method of treating migraines and headaches |
EP4240351A1 (en) | 2020-11-20 | 2023-09-13 | Institute of Life Sciences (ILS) | Composition for enhancing intra-cellular nitric oxide generation |
WO2022182968A1 (en) * | 2021-02-25 | 2022-09-01 | Calroy Health Sciences, Llc | Synergistic compositions and methods to increase vascular nitric oxide to treat endothelial dysfunction and related conditions |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050226907A1 (en) * | 2004-04-08 | 2005-10-13 | Moneymaker Ricky D | Pharmanutrient composition(s) and system(s) for individualized, responsive dosing regimens |
US7553817B2 (en) * | 1999-04-12 | 2009-06-30 | Bioenergy, Inc. | Methods for improving cardiac function |
US8114442B2 (en) * | 2000-08-30 | 2012-02-14 | Queen Mary & Westfield College | Transdermal pharmaceutical delivery compositions |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0724723B1 (en) | 1993-07-06 | 2000-04-05 | Aerocrine Ab | A system to be used for the determination of no levels in exhaled air and diagnostic methods for disorders related to abnormal no levels |
US6103275A (en) * | 1998-06-10 | 2000-08-15 | Nitric Oxide Solutions | Systems and methods for topical treatment with nitric oxide |
GB9905425D0 (en) * | 1999-03-09 | 1999-05-05 | Queen Mary & Westfield College | Pharmaceutical composition |
US6436366B2 (en) | 1999-03-19 | 2002-08-20 | Joseph V. Boykin, Jr. | Prediction of wound healing by urinary nitrate assay |
GB0119011D0 (en) * | 2001-08-03 | 2001-09-26 | Univ Aberdeen | Treatment of nail infections |
US20050226906A1 (en) * | 2004-04-08 | 2005-10-13 | Micro Nutrient, Llc | Nutrient system for individualized responsive dosing regimens |
US8298589B1 (en) * | 2008-06-13 | 2012-10-30 | Board Of Regents, The University Of Texas System | Nitrite formulations and their use as nitric oxide prodrugs |
US8303995B1 (en) * | 2008-06-13 | 2012-11-06 | Board Of Regents, The University Of Texas System | Nitrite formulations and their use as nitric oxide prodrugs |
WO2010141719A2 (en) | 2009-06-03 | 2010-12-09 | University Of Florida Research Foundation Inc. | Materials and methods for measuring nitric oxide levels in a biological fluid |
MX354702B (en) * | 2011-08-17 | 2018-03-16 | Univ Texas | Method of producing physiological and therapeutic levels of nitric oxide through an oral delivery system. |
-
2010
- 2010-08-16 US US12/856,957 patent/US8303995B1/en active Active
-
2012
- 2012-11-05 US US13/668,776 patent/US8962038B2/en active Active
-
2015
- 2015-01-30 US US14/610,492 patent/US9119823B2/en active Active
- 2015-08-14 US US14/827,100 patent/US20160038533A1/en not_active Abandoned
-
2018
- 2018-10-02 US US16/150,155 patent/US20190083528A1/en not_active Abandoned
-
2020
- 2020-04-07 US US16/842,550 patent/US20200237808A1/en not_active Abandoned
-
2021
- 2021-06-01 US US17/303,510 patent/US20210290663A1/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7553817B2 (en) * | 1999-04-12 | 2009-06-30 | Bioenergy, Inc. | Methods for improving cardiac function |
US8114442B2 (en) * | 2000-08-30 | 2012-02-14 | Queen Mary & Westfield College | Transdermal pharmaceutical delivery compositions |
US20050226907A1 (en) * | 2004-04-08 | 2005-10-13 | Moneymaker Ricky D | Pharmanutrient composition(s) and system(s) for individualized, responsive dosing regimens |
Non-Patent Citations (4)
Title |
---|
Bjarnadottir, A. (Beetroot 101: Nutrition Facts and Health Benefits [online] retrieved on 2/14/17 from: https://authoritynutrition.com/foods/beetroot/; 8 pages) * |
Croitoru, M.D. (Journal of Chromatography B, 2012;911:154-161). * |
Hord et al. (Am J Clin Nutr 2009;90:1-10). * |
Webb et al. (Hypertension 2008; (51):784-790) * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20190076755A1 (en) * | 2017-09-13 | 2019-03-14 | Thermolife International, Llc | Enriched Root Powder Products and Methods of Producing Thereof |
US10702796B2 (en) * | 2017-09-13 | 2020-07-07 | Thermolife International, Llc | Enriched root powder products and methods of producing thereof |
US10967294B1 (en) | 2017-09-13 | 2021-04-06 | Thermolife International, Llc | Enriched root powder products and methods of producing thereof |
CN110343038A (en) * | 2018-04-02 | 2019-10-18 | 暨南大学 | A kind of treasured Rogor monomer, extract and its preparation method and application |
CN109549951A (en) * | 2019-01-25 | 2019-04-02 | 大连海洋大学 | Cage mesh kelp fucoidan sulfuric ester compound immunoenhancer |
US11464816B2 (en) | 2019-04-16 | 2022-10-11 | The Procter & Gamble Company | Supplement for menopause |
Also Published As
Publication number | Publication date |
---|---|
US20200237808A1 (en) | 2020-07-30 |
US20190083528A1 (en) | 2019-03-21 |
US20150164839A1 (en) | 2015-06-18 |
US20210290663A1 (en) | 2021-09-23 |
US8962038B2 (en) | 2015-02-24 |
US8303995B1 (en) | 2012-11-06 |
US9119823B2 (en) | 2015-09-01 |
US20130071494A1 (en) | 2013-03-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210290663A1 (en) | Nitrite formulations and their use as nitric oxide prodrugs | |
US8435570B1 (en) | Nitrite formulations and their use as nitric oxide prodrugs | |
Bryan | Nitrite in nitric oxide biology: Cause or consequence?: A systems-based review | |
AU2016204931B2 (en) | Method of producing physiological and therapeutic levels of nitric oxide through an oral delivery system | |
Khatri et al. | It is rocket science–why dietary nitrate is hard to ‘beet’! Part I: twists and turns in the realization of the nitrate–nitrite–NO pathway | |
Omar et al. | A comparison of organic and inorganic nitrates/nitrites | |
Kevil et al. | Inorganic nitrite therapy: historical perspective and future directions | |
Bailey et al. | Pharmacology and therapeutic role of inorganic nitrite and nitrate in vasodilatation | |
Butler et al. | Therapeutic uses of inorganic nitrite and nitrate: from the past to the future | |
Van Faassen et al. | Nitrite as regulator of hypoxic signaling in mammalian physiology | |
US6936283B2 (en) | Composition for stimulation of specific metallo-enzymes | |
Bryan | Cardioprotective actions of nitrite therapy and dietary considerations | |
B Blood | The medicinal chemistry of nitrite as a source of nitric oxide signaling | |
Sirirat et al. | Pharmacokinetics and pharmacodynamics of single dose of inhaled nebulized sodium nitrite in healthy and hemoglobin E/β-thalassemia subjects | |
Lbban et al. | Is vitamin C a booster of the effects of dietary nitrate on endothelial function? Physiologic rationale and implications for research | |
ES2959184T3 (en) | Compositions and methods to increase athletic performance | |
US20210023126A1 (en) | Method of producing physiological and therapeutic levels of nitric oxide through an oral delivery system | |
Bryan | An Overview of Nitrite and Nitrate: New Paradigm of Nitric Oxide | |
US20230270711A1 (en) | Improved Anti-Hangover Composition, Its Preparation and Uses | |
Kapil | Nitrite and Nitrate as a Treatment for Hypertension | |
Luedike et al. | Bypassing the endothelial L-arginine-nitric oxide pathway: effects of dietary nitrite and nitrate on cardiovascular function. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: BOARD OF REGENTS, THE UNIVERSITY OF TEXAS SYSTEM, Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BRYAN, NATHAN SCOTT;REEL/FRAME:036332/0873 Effective date: 20090824 Owner name: NEOGENIS LABS, INC., TEXAS Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ZAND, JANET;REEL/FRAME:036333/0015 Effective date: 20120716 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |