WO2007113368A1 - Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products - Google Patents
Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products Download PDFInfo
- Publication number
- WO2007113368A1 WO2007113368A1 PCT/ES2007/070071 ES2007070071W WO2007113368A1 WO 2007113368 A1 WO2007113368 A1 WO 2007113368A1 ES 2007070071 W ES2007070071 W ES 2007070071W WO 2007113368 A1 WO2007113368 A1 WO 2007113368A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- angiotensin
- seq
- peptides
- converting enzyme
- vitro
- Prior art date
Links
- 102400000344 Angiotensin-1 Human genes 0.000 title abstract description 22
- 101800000734 Angiotensin-1 Proteins 0.000 title abstract description 22
- 102000004190 Enzymes Human genes 0.000 title description 16
- 108090000790 Enzymes Proteins 0.000 title description 16
- 229940126601 medicinal product Drugs 0.000 title 1
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 79
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 claims abstract description 27
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 claims abstract description 27
- 239000003112 inhibitor Substances 0.000 claims abstract description 27
- 150000001413 amino acids Chemical class 0.000 claims abstract description 22
- 238000000338 in vitro Methods 0.000 claims abstract description 17
- 230000000975 bioactive effect Effects 0.000 claims abstract description 14
- 230000000694 effects Effects 0.000 claims description 31
- 230000001419 dependent effect Effects 0.000 claims description 12
- 206010020772 Hypertension Diseases 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 7
- 235000013376 functional food Nutrition 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 206010047139 Vasoconstriction Diseases 0.000 claims 7
- 230000025033 vasoconstriction Effects 0.000 claims 7
- 239000000047 product Substances 0.000 claims 5
- 239000000654 additive Substances 0.000 claims 2
- 230000000996 additive effect Effects 0.000 claims 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 230000002349 favourable effect Effects 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000013589 supplement Substances 0.000 claims 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 58
- 230000002401 inhibitory effect Effects 0.000 abstract description 25
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 14
- 230000008602 contraction Effects 0.000 abstract description 12
- 235000013305 food Nutrition 0.000 abstract description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 abstract description 6
- 210000001715 carotid artery Anatomy 0.000 abstract description 5
- 230000009467 reduction Effects 0.000 abstract description 5
- 125000000539 amino acid group Chemical group 0.000 abstract description 2
- 239000002417 nutraceutical Substances 0.000 abstract 1
- 235000021436 nutraceutical agent Nutrition 0.000 abstract 1
- ORWYRWWVDCYOMK-HBZPZAIKSA-N angiotensin I Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 ORWYRWWVDCYOMK-HBZPZAIKSA-N 0.000 description 20
- 230000005764 inhibitory process Effects 0.000 description 19
- 238000012360 testing method Methods 0.000 description 16
- 239000005541 ACE inhibitor Substances 0.000 description 15
- 108010064733 Angiotensins Proteins 0.000 description 15
- 102000015427 Angiotensins Human genes 0.000 description 15
- 229940088598 enzyme Drugs 0.000 description 15
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 14
- 239000000758 substrate Substances 0.000 description 13
- 230000009471 action Effects 0.000 description 7
- 210000001367 artery Anatomy 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 239000006225 natural substrate Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 230000036772 blood pressure Effects 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 235000021245 dietary protein Nutrition 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 230000000144 pharmacologic effect Effects 0.000 description 5
- 101800004538 Bradykinin Proteins 0.000 description 4
- 102400000967 Bradykinin Human genes 0.000 description 4
- QXZGBUJJYSLZLT-UHFFFAOYSA-N H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH Natural products NC(N)=NCCCC(N)C(=O)N1CCCC1C(=O)N1C(C(=O)NCC(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CO)C(=O)N2C(CCC2)C(=O)NC(CC=2C=CC=CC=2)C(=O)NC(CCCN=C(N)N)C(O)=O)CCC1 QXZGBUJJYSLZLT-UHFFFAOYSA-N 0.000 description 4
- QIAFMBKCNZACKA-UHFFFAOYSA-N N-benzoylglycine Chemical compound OC(=O)CNC(=O)C1=CC=CC=C1 QIAFMBKCNZACKA-UHFFFAOYSA-N 0.000 description 4
- 108090000783 Renin Proteins 0.000 description 4
- 102100028255 Renin Human genes 0.000 description 4
- 210000001841 basilar artery Anatomy 0.000 description 4
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 3
- 102400000345 Angiotensin-2 Human genes 0.000 description 3
- 101800000733 Angiotensin-2 Proteins 0.000 description 3
- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 description 3
- 208000006011 Stroke Diseases 0.000 description 3
- 229950006323 angiotensin ii Drugs 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000000099 in vitro assay Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000005526 vasoconstrictor agent Substances 0.000 description 3
- -1 9- fluorenylmethoxycarbonyl amino Chemical class 0.000 description 2
- 240000002234 Allium sativum Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 108010016626 Dipeptides Proteins 0.000 description 2
- 208000005189 Embolism Diseases 0.000 description 2
- 108010011756 Milk Proteins Proteins 0.000 description 2
- 102000014171 Milk Proteins Human genes 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108090001109 Thermolysin Proteins 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 241001261506 Undaria pinnatifida Species 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000003276 anti-hypertensive effect Effects 0.000 description 2
- 239000002220 antihypertensive agent Substances 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 235000004611 garlic Nutrition 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 235000021239 milk protein Nutrition 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000036454 renin-angiotensin system Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 208000019553 vascular disease Diseases 0.000 description 2
- 239000003071 vasodilator agent Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- USSUMSBPLJWFSZ-TUFLPTIASA-N (2s)-2-[[2-[[(2s)-1-[(2s)-1-[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]pyrrolidine-2-carbonyl]amino]acetyl]amino]-3-phenylpropanoic acid Chemical group NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(O)=O)CCC1 USSUMSBPLJWFSZ-TUFLPTIASA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 1
- 208000028185 Angioedema Diseases 0.000 description 1
- 101710129690 Angiotensin-converting enzyme inhibitor Proteins 0.000 description 1
- 102000004881 Angiotensinogen Human genes 0.000 description 1
- 108090001067 Angiotensinogen Proteins 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 101710086378 Bradykinin-potentiating and C-type natriuretic peptides Proteins 0.000 description 1
- 101710170564 Bradykinin-potentiating peptide Proteins 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 241000238366 Cephalopoda Species 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108010082495 Dietary Plant Proteins Proteins 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108010061435 Enalapril Proteins 0.000 description 1
- 244000025361 Ficus carica Species 0.000 description 1
- 235000008730 Ficus carica Nutrition 0.000 description 1
- 108010028690 Fish Proteins Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000001953 Hypotension Diseases 0.000 description 1
- 206010061216 Infarction Diseases 0.000 description 1
- 150000008575 L-amino acids Chemical class 0.000 description 1
- 240000002605 Lactobacillus helveticus Species 0.000 description 1
- 235000013967 Lactobacillus helveticus Nutrition 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- UWWDHYUMIORJTA-HSQYWUDLSA-N Moexipril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CC2=CC(OC)=C(OC)C=C2C1)C(O)=O)CC1=CC=CC=C1 UWWDHYUMIORJTA-HSQYWUDLSA-N 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 108010093625 Opioid Peptides Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 238000004617 QSAR study Methods 0.000 description 1
- 241000269851 Sarda sarda Species 0.000 description 1
- 208000032851 Subarachnoid Hemorrhage Diseases 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108010046377 Whey Proteins Proteins 0.000 description 1
- 229920002494 Zein Polymers 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 206010000891 acute myocardial infarction Diseases 0.000 description 1
- 229960002478 aldosterone Drugs 0.000 description 1
- 239000002333 angiotensin II receptor antagonist Substances 0.000 description 1
- 239000002416 angiotensin derivative Substances 0.000 description 1
- 229940125364 angiotensin receptor blocker Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940030600 antihypertensive agent Drugs 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000004872 arterial blood pressure Effects 0.000 description 1
- 208000037849 arterial hypertension Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000013060 biological fluid Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000010352 biotechnological method Methods 0.000 description 1
- 230000008081 blood perfusion Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229960000830 captopril Drugs 0.000 description 1
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000001627 cerebral artery Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000002999 depolarising effect Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 208000017574 dry cough Diseases 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229960000873 enalapril Drugs 0.000 description 1
- GBXSMTUPTTWBMN-XIRDDKMYSA-N enalapril Chemical compound C([C@@H](C(=O)OCC)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(O)=O)CC1=CC=CC=C1 GBXSMTUPTTWBMN-XIRDDKMYSA-N 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 238000010228 ex vivo assay Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000008246 gaseous mixture Substances 0.000 description 1
- 235000003869 genetically modified organism Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000005802 health problem Effects 0.000 description 1
- 201000010235 heart cancer Diseases 0.000 description 1
- 208000024348 heart neoplasm Diseases 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- VPUGDVKSAQVFFS-UHFFFAOYSA-N hexabenzobenzene Natural products C1=C(C2=C34)C=CC3=CC=C(C=C3)C4=C4C3=CC=C(C=C3)C4=C2C3=C1 VPUGDVKSAQVFFS-UHFFFAOYSA-N 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000001631 hypertensive effect Effects 0.000 description 1
- 230000036543 hypotension Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000007574 infarction Effects 0.000 description 1
- 208000020658 intracerebral hemorrhage Diseases 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 229940054346 lactobacillus helveticus Drugs 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 229960005170 moexipril Drugs 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- LVRLSYPNFFBYCZ-VGWMRTNUSA-N omapatrilat Chemical compound C([C@H](S)C(=O)N[C@H]1CCS[C@H]2CCC[C@H](N2C1=O)C(=O)O)C1=CC=CC=C1 LVRLSYPNFFBYCZ-VGWMRTNUSA-N 0.000 description 1
- 229950000973 omapatrilat Drugs 0.000 description 1
- 238000011369 optimal treatment Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001175 peptic effect Effects 0.000 description 1
- 229960002582 perindopril Drugs 0.000 description 1
- IPVQLZZIHOAWMC-QXKUPLGCSA-N perindopril Chemical compound C1CCC[C@H]2C[C@@H](C(O)=O)N(C(=O)[C@H](C)N[C@@H](CCC)C(=O)OCC)[C@H]21 IPVQLZZIHOAWMC-QXKUPLGCSA-N 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 229940080237 sodium caseinate Drugs 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000011699 spontaneously hypertensive rat Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 238000009369 viticulture Methods 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
- 239000005019 zein Substances 0.000 description 1
- 229940093612 zein Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/55—Protease inhibitors
- A61K38/556—Angiotensin converting enzyme inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
Definitions
- Acute stroke also called idus, constitutes, after ischemic heart disease and cancer, the third cause of mortality and the first permanent disability in advanced western societies.
- the majority of acute strokes (85%) are of the "ischemic” type, and have their origin in acute occlusion due to a thrombus ("thrombosis") or an embolus ("embolism”) of one of the main cerebral arteries, Io which causes a decrease in blood perfusion (“ischemia”) and consequent necrosis ("infarction”) of the brain region irrigated by said artery.
- renin-angiotensin system a complex regulatory system
- renin the enzyme convertor of angiotensin (ACE), aldosterone, and angiotensins I and II.
- SRA is a circulating hormonal system
- renin and the RCT are two peptidases that are part of it and that act sequentially on a series of small peptides, ultimately regulators of blood pressure.
- renin is released in the kidney and ACE is mainly present in vascular endothelial cells, in the lungs, in the kidneys and in the brain.
- This renin-angiotensin system is activated in certain situations by means of the action of the renin on a precursor peptide called angiotensinogen (of hepatic origin), which becomes the angiotensin I decapeptide.
- angiotensinogen of hepatic origin
- This angiotensin I inactive from the biological point of view, It is in turn transformed by the action of ACE into angiotensin Il when the dipeptide is separated from its C-terminal end.
- the angiotensin Il generated is a potent vasoconstrictor that exerts its action after binding to its specific receptors called “ATY receptors.” This action translates into the contraction of blood vessels that consequently produces an increase in blood pressure.
- the ECA also acts on another circulating peptide, the nonapeptide called bradykinin, a potent vasodilator agent that loses this characteristic when hydrolyzed.
- SRA could have beneficial effects in the treatment of vascular disorders associated with hypertension.
- the inhibition of the ECA activity would allow to reduce the formation of angiotensin II in addition to reducing the loss of functionality of the bradykinin, thus avoiding the vasoconstrictor action of the first and enhancing the vasodilator action of the second.
- the efficacy of ACE inhibitors in reducing morbidity and mortality in patients with heart failure, cardio-metabolic syndrome and diabetes has been shown in numerous studies.
- ACE inhibitor drugs Despite its proven efficacy in the treatment of cardiovascular diseases associated with hypertension, the currently available ACE inhibitor drugs cannot be considered as definitive option Due to their lack of specificity, these drugs are not well tolerated by some patients in whom undesirable side effects such as dry cough and angioedema occur; In addition, they do not completely block the synthesis of angiotensin Il since it follows other synthetic routes that do not depend on the RCT. Therefore, it is necessary to find new ACE inhibitors with greater specificity and that can be co-administered with other drugs, such as "angiotensin receptor blockers", for the optimal treatment of vascular disorders of hypertensive origin linked to ARS. while minimizing the aforementioned side effects. Even and depending on the characteristics of the selected inhibitors, a more natural approach to treatment could be achieved by adding said inhibitors to food, which would produce a positive effect both on said treatment and on the prevention of symptoms inherent in hypertension.
- LKPNM a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44, 123-127; Pihlanto-Leppálá, A. (2001).
- the present invention describes the amino acid sequence of other peptides, different from those mentioned above, characterized by having ACE inhibitory activity. Said inhibition of the ECA activity is manifested in in vitro tests, measuring the inhibition of the conversion of artificial (Hipuril-Histidyl-Leucine) and natural (Angiotensin I) mediated ACA mediated substrates, and in ex vivo tests, measuring the decrease of the contraction of basilar or carotid arteries of rabbit induced by exposure to Angiotensin I.
- the present invention is related to the pharmacological and agri-food industry sectors and consists in the identification and characterization of certain peptides (called PIECA, Angiotensin Conversion Enzyme Inhibitor Peptides) as effective inhibitors of the Conzyme Enzyme of the Angiotensin (RCT) that is involved in the formation of the compound Angiotensin Il which is a vasoconstrictor responsible, among other causes / mechanisms, for hypertension.
- PIECA Angiotensin Conversion Enzyme Inhibitor Peptides
- the residue sequences of amino acids of the peptides identified are the following, from the amino terminal to the carboxy terminal end: (peptide PIECA32; SEQ ID NO 1) and (peptide PIECA34, SEQ ID NO 2) that have been described in patent application ES200001973 as PAF32 and PAF34 respectively.
- the inhibitory activity of the peptides is manifested by a reduction of the ACE activity determined in in vitro assays, as well as by a reduction of the ECA-dependent contraction ex vivo using artery segments. It is demonstrated that the inhibitory activity of the peptides corresponds to a characteristic amino acid sequence, since other peptides related to the above and with a similar, but not identical amino acid residue sequence, do not exhibit said activity.
- the ACE inhibitor peptides as bioactive compounds for the control of hypertension is described.
- the ACE inhibitory activity of said peptides is described when Hipuril-Histidyl-Leucine (HHL) and Angiotensin I are used as substrates.
- HHL Hipuril-Histidyl-Leucine
- Angiotensin I Angiotensin I
- the identification and characterization of new peptides with inhibition activity of the Angiotensin Conversion Enzyme (RCT), involved in the blood pressure control mechanisms is described.
- RCT Angiotensin Conversion Enzyme
- the use of said peptides as ACE inhibitors is described through the use of artificial substrates such as Hipuril-Histidyl-Leucine (HHL) or natural substrates such as Angiotensin I, as well as their inhibitory effect on ECA-dependent contraction of rabbit artery segments.
- HHL Hipuril-Histidyl-Leucine
- Angiotensin I angiotensin I
- the inhibitors described in the present invention are peptides with a sequence of six amino acids characteristic SEQ ID NO 1 (PIECA32 peptide) and SEQ ID NO 2 (PIECA34 peptide), Figure 1, and different from the previously known ACE inhibitor peptides [Guan- Hong Li, Guo-Wei Le,
- said hexapeptides were chemically synthesized with the L-natural stereoisomers of the amino acids (PIECA32L and PIECA34L) and then purified, following usual procedures for all those skilled in the area of knowledge of the present invention [Fields, GB and Noble, RL (1990). SoNd phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34, 161-214].
- PIECA peptides were synthesized and purified using the D-stereoisomers of the constituent amino acids (PIECA32D and PIECA34D), which are not natural but have the property of conferring the resulting peptides greater stability and resistance to Ia degradation by proteases present in biological fluids.
- the inhibitory activity of the PIECA peptides described in the present invention is manifested with a reduction in the activity of the RCT when performing the in vitro tests under the established conditions, as demonstrated by the experimental tests described in the present invention. These tests also demonstrate that the PIECA have a characteristic and specific amino acid sequence, since related peptides of similar sequence - but not identical - to PIECA32L, PIECA34L, PIECA32D and PIECA34D do not exhibit the said inhibitory activity.
- the inhibitory activity of the PIECA peptides described in the present invention is also manifested with a reduction of the ECA-dependent contraction of rabbit, carotid and basilar arteries, induced by the addition of Angiotensin I in ex vivo assays.
- P26D SEQ ID NO 3 and P36D SEQ ID NO 4 (used as negative controls in the described tests).
- the sequences are written from the amino terminal end (on the left) to the carboxy terminal end (on the right).
- the peptides are acetylated at their amino terminal (Ac-) end and amidated at their carboxy terminal (Am-) end.
- PIECA34D, PIECA34L and P36D SEQ ID NO 4) were chemically synthesized on solid phase following usual procedures using the group
- Peptides P26D and P36D were designed as a negative control in the assays described below.
- the N-terminal end of the peptides is acetylated (Ac) and the C-terminal amidated end (NH2), as a consequence of the synthesis procedure.
- the Peptides were purified by RP-HPLC (reversed phase high performance liquid chromatography) and their identity was confirmed by MALDI-TOF mass spectrometry (English, matrix-assisted laser desorption / ionization time-of-flight). All these procedures are common for all those experts in the area of knowledge of the present invention.
- L peptides composed of natural amino acids (L- stereoisomers) described in the present invention by means of strategies derived from biotechnology. It is obvious, for all those experts in the area of knowledge, that the production of the peptides through biotechnological procedures, which include the methodologies of recombinant DNA and the genetic transformation of organisms, would mean an improvement in production costs, and that Therefore, said production is an important aspect in the context of the industrial applicability of the present invention.
- the sequence of the peptide produced by a genetically modified organism would be encoded by a DNA fragment according to the laws of the genetic code [Sambrook, J., Fritsch, EF, and Maniatis, T. ( 1989). Molecular cloning: A laboratory manual, 2nd edition. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY.],
- the reaction mixture has a volume of 225 ⁇ l and is made up of 50 ⁇ l of 25 mM HHL in 200 mM Tris HCI buffer pH 8.3 with 600 mM NaCI and 10 ⁇ M ZnCI 2 , 75 ⁇ l of an ACE solution in the same buffer corresponding to 1.5 thousand activity, and 100 ⁇ l of peptide (dissolved in 10 mM MOPS buffer pH 7) at different concentrations according to the final concentration desired in the assay.
- the enzyme and the inhibitor are pre-incubated for 15 minutes at 37 ° C and then the substrate is added by incubating the whole 30 minutes at said temperature.
- the reaction is stopped by adding 25 ⁇ l of 6M HCI.
- PIECA 32L and SEQ ID NO 2 PIECA 34L being these of 10.7 and 8.1 ⁇ M respectively.
- the experimental preparation consisted of obtaining cylindrical segments (3 mm) of isolated arteries (basilar and carotid arteries of New Zealand white rabbit), which were arranged in an organ bath designed to record the changes of isometric tension in the vascular wall.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention consists in using synthetic hexapeptides as bioactive products. These are peptides with six amino acid residues as effective inhibitors of angiotensin converting enzyme (ACE). The peptides that are the subject of the patent may be obtained chemically or biotechnologically. Of special interest are those synthesized exclusively from the D-stereoisomers of natural amino acids since this method affords greater stability in addition to their in vitro and/or ex vivo angiotensin converting enzyme inhibitory activity measured as the reduction in the contraction of rabbit carotid arteries induced by exposure to angiotensin 1. These nutraceutical products, possibly in the form of bioactive peptides, are useful both in the food industry and in the pharmaceutical industry.
Description
TítuloTitle
Uso de hexapéptidos para preparar medicamentos de la enzima conversora de angiotensina IUse of hexapeptides to prepare angiotensin I converting enzyme medications
Sector de Ia TécnicaTechnical Sector
Control de Ia hipertensión; Industria agroalimentaria; Ingredientes bioactivos; Farmacología; Alimentos funcionalesControl of hypertension; Agri-food industry; Bioactive ingredients; Pharmacology; Functional Foods
Estado de Ia TécnicaState of the Technique
La hipertensión, que consiste en un aumento de Ia presión sanguínea superior a Ia deseable para Ia salud, es un problema sanitario bastante serio ya que está relacionada con un alto riesgo de complicaciones cardio- y cerebro- vasculares. El accidente cerebrovascular agudo, también denominado idus, constituye, después de las enfermedades isquémicas cardíacas y del cáncer, Ia tercera causa de mortalidad y Ia primera de discapacidad permanente en las sociedades occidentales avanzadas. La mayoría de accidentes cerebrovasculares agudos (85%) son de tipo "isquémico", y tienen su origen en Ia oclusión aguda por un trombo ("trombosis") o un émbolo ("embolia") de una de las principales arterias cerebrales, Io que origina un descenso en Ia perfusión sanguínea ("isquemia") y consiguiente necrosis ("infarto") de Ia región cerebral irrigada por dicha arteria. El resto de accidentes cerebrovasculares (15%) son de tipo "hemorrágico", originados por Ia rotura de un vaso sanguíneo en el propio parénquima cerebral ("hemorragia intracerebral") o en Ia superficie cerebral ("hemorragia subaracnoidea").Hypertension, which consists of an increase in blood pressure higher than that desirable for health, is a very serious health problem since it is related to a high risk of cardio- and cerebrovascular complications. Acute stroke, also called idus, constitutes, after ischemic heart disease and cancer, the third cause of mortality and the first permanent disability in advanced western societies. The majority of acute strokes (85%) are of the "ischemic" type, and have their origin in acute occlusion due to a thrombus ("thrombosis") or an embolus ("embolism") of one of the main cerebral arteries, Io which causes a decrease in blood perfusion ("ischemia") and consequent necrosis ("infarction") of the brain region irrigated by said artery. The rest of the cerebrovascular accidents (15%) are of the "hemorrhagic" type, caused by the rupture of a blood vessel in the cerebral parenchyma itself ("intracerebral hemorrhage") or in the cerebral surface ("subarachnoid hemorrhage").
De Io dicho anteriormente se desprende que en el desarrollo de estas enfermedades falla Ia correcta regulación de Ia presión arterial sistémica, en Ia que interviene un complejo sistema regulador llamado sistema renina- angiotensina (SRA), y del que forman parte Ia renina, Ia enzima conversora de Ia angiotensina (ECA), Ia aldosterona, y las angiotensinas I y II.
Este SRA es un sistema hormonal circulante, y concretamente Ia renina y Ia ECA son dos peptidasas que forman parte del mismo y que actúan secuencialmente sobre una serie de pequeños péptidos, reguladores en última instancia de Ia presión sanguínea. En los seres humanos, Ia renina se libera en el riñon y Ia ECA se encuentra presente principalmente en las células endoteliales vasculares, en los pulmones, en los ríñones y en el cerebro.From the aforementioned it follows that in the development of these diseases the correct regulation of the systemic arterial pressure fails, in which a complex regulatory system called the renin-angiotensin system (ARS) intervenes, and of which the renin, the enzyme convertor of angiotensin (ACE), aldosterone, and angiotensins I and II. This SRA is a circulating hormonal system, and specifically the renin and the RCT are two peptidases that are part of it and that act sequentially on a series of small peptides, ultimately regulators of blood pressure. In humans, renin is released in the kidney and ACE is mainly present in vascular endothelial cells, in the lungs, in the kidneys and in the brain.
Este sistema renina-angiotensina se activa en determinadas situaciones mediante Ia actuación de Ia renina sobre un péptido precursor denominado angiotensinógeno (de procedencia hepática), el cual se convierte en el decapéptido angiotensina I. Esta angiotensina I, inactiva desde el punto de vista biológico, se transforma a su vez por acción de Ia ECA en angiotensina Il al separarse el dipéptido a partir de su extremo C-terminal. La angiotensina Il generada es un potente vasoconstrictor que ejerce su acción tras Ia unión a sus receptores específicos denominados "receptores ATY'. Dicha acción se traduce en Ia contracción de los vasos sanguíneos que como consecuencia produce un aumento de Ia presión sanguínea. Además de contribuir a Ia formación de Ia angiotensina II, Ia ECA también actúa sobre otro péptido circulante, el nonapéptido llamado bradiquinina, potente agente vasodilatador que pierde esta característica al ser hidrolizado.This renin-angiotensin system is activated in certain situations by means of the action of the renin on a precursor peptide called angiotensinogen (of hepatic origin), which becomes the angiotensin I decapeptide. This angiotensin I, inactive from the biological point of view, It is in turn transformed by the action of ACE into angiotensin Il when the dipeptide is separated from its C-terminal end. The angiotensin Il generated is a potent vasoconstrictor that exerts its action after binding to its specific receptors called “ATY receptors.” This action translates into the contraction of blood vessels that consequently produces an increase in blood pressure. At the formation of angiotensin II, the ECA also acts on another circulating peptide, the nonapeptide called bradykinin, a potent vasodilator agent that loses this characteristic when hydrolyzed.
Por todo Io expuesto anteriormente, Ia interferencia farmacológica con elFor all the above, the pharmacological interference with the
SRA podría tener efectos beneficiosos en el tratamiento de los desórdenes vasculares asociados con Ia hipertensión. La inhibición de Ia actividad ECA permitiría disminuir Ia formación de angiotensina Il además de reducir Ia pérdida de funcionalidad de Ia bradiquinina, evitando de esta manera Ia acción vasoconstrictora de Ia primera y potenciando Ia acción vasodilatadora de Ia segunda. A este respecto se ha puesto de manifiesto en numerosos estudios Ia eficacia de los inhibidores de ECA reduciendo Ia morbilidad y Ia mortalidad en pacientes con fallo cardíaco, síndrome cardio-metabólico y diabetes.SRA could have beneficial effects in the treatment of vascular disorders associated with hypertension. The inhibition of the ECA activity would allow to reduce the formation of angiotensin II in addition to reducing the loss of functionality of the bradykinin, thus avoiding the vasoconstrictor action of the first and enhancing the vasodilator action of the second. In this regard, the efficacy of ACE inhibitors in reducing morbidity and mortality in patients with heart failure, cardio-metabolic syndrome and diabetes has been shown in numerous studies.
A pesar de su demostrada eficacia en el tratamiento de las enfermedades cardiovasculares asociadas a Ia hipertensión, los fármacos inhibidores del ECA disponibles en Ia actualidad no pueden considerarse Ia
opción definitiva. Por su falta de especificidad estos fármacos no son bien tolerados por algunos pacientes en los que se presentan efectos secundarios indeseables como tos seca y angioedema; además, no bloquean completamente Ia síntesis de angiotensina Il ya que ésta sigue otras vías de síntesis que no dependen del ECA. Es necesario, por Io tanto, encontrar nuevos inhibidores del ECA con mayor especificidad y que puedan ser coadministrados con otros fármacos, como por ejemplo los "bloqueadores del receptor de angiotensina", para el tratamiento óptimo de los desórdenes vasculares de origen hipertensivo ligados al SRA a Ia vez que se minimizan los efectos secundarios antes citados. Incluso y según las características de los inhibidores seleccionados, podría conseguirse una aproximación mas natural del tratamiento al añadir dichos inhibidores a los alimentos, Io cual produciría un efecto positivo tanto sobre dicho tratamiento como sobre Ia prevención de los síntomas inherentes a Ia hipertensión.Despite its proven efficacy in the treatment of cardiovascular diseases associated with hypertension, the currently available ACE inhibitor drugs cannot be considered as definitive option Due to their lack of specificity, these drugs are not well tolerated by some patients in whom undesirable side effects such as dry cough and angioedema occur; In addition, they do not completely block the synthesis of angiotensin Il since it follows other synthetic routes that do not depend on the RCT. Therefore, it is necessary to find new ACE inhibitors with greater specificity and that can be co-administered with other drugs, such as "angiotensin receptor blockers", for the optimal treatment of vascular disorders of hypertensive origin linked to ARS. while minimizing the aforementioned side effects. Even and depending on the characteristics of the selected inhibitors, a more natural approach to treatment could be achieved by adding said inhibitors to food, which would produce a positive effect both on said treatment and on the prevention of symptoms inherent in hypertension.
Hasta Ia fecha se han publicado numerosos trabajos bibliográficos relacionados con Ia inhibición de ECA mediante el empleo de pequeños péptidos sintéticos como principales responsables de dicha inhibición. Estos péptidos presentan una gran variabilidad pues tienen diferentes longitudes y estructuralmente difieren en las secuencias de los aminoácidos que los constituyen [Patchett, A.A., Harris, E., Tristram, E.W., Wyvratt, MJ. , Wu, M.T., Taub, D., Peterson, E. R., Ikeler, TJ. , Broeke, J.Ten., Payne, L. G., Ondeyka, D. L., Thorsett, E. D., Greenlee, WJ., Lohr, N.S., Hoffsommer, R.D., Joshua, H., Ruyle, W.V., Rothrock, J.W., Áster, S. D., Maycock, A.L., Robinson, F. M., Hirschmann, R., Sweet, CS. , UIm, E. H., Gross, D. M., Vassil, T.C. y Stone, CA. (1980). A new class of angiotensin-converting enzyme inhibitors. Nature 288, 280-283; Ondetti, M.A., Rubin, B. y Cushman, D.W. (1977). Design of specific Inhibitors of angiotensin-converting enzyme: New class of orally active antihypertensive agents. Science 196, 441-444; Cushman, D. W., Cheung, H. S., Sabo, E. F. y Ondetti, M.A. (1977). Design of potent competitive inhibitors of angiotensin-converting enzyme. carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry 16 (25), 5484-5491 ; Cushman, D.W., Cheung, H. S., Sabo, E. F. y Ondetti, M.A. (1981). Angiotensin converting enzyme inhibitors. evolution
of a new class of antihypertensive drugs. En: Angiotensin converting enzyme inhibitors. Mechanisms of action and clinical implications. Section I, pp3-25. Ed. Horovitz, ZP. , Urban & Schwarzenberg (Baltimor-Munich); Edling, O., Bao, G., Feelisch, M., Unger, T. y Gohlke, P. (1995). Moexipril, a new angiotensin- converting enzyme (ACE) inhibitor: Pharmacological characterization and comparison with enalapril. Journal of Pharmacology and Experimental Therapeutics 275 (2), 854-863; Gómez-Ruiz, J.A., Recio, I. y Belloque, J. (2004). ACE-Inhibitory activity and structural properties of peptide Asp-Lys-lle- His-Pro [β-CN f(47-51)]. Study of the peptide forms synthesized by different methods. Journal of Agricultura! and Food Chemistry 52 (20), 6315-6319; Cotton, J., Hayashi, M.A.F., Cuniasse, P., Vazeux, G., Lanzer, D., De Camargo, A. C. M. y Dive, V. (2002). Selective inhibition of the c-domain of angiotensin i converting enzyme by bradykinin potentiating peptides. Biochemistry 41 (19), 6065-6071 ; Lau, C-P., Tse, H-F., Ng, W., Chan, K-K., Li, S-K., Keung, K-K., Lau, Y-K., Chen, W-H., Tang, Y-W. y Leung, S-K. (2002). Comparison of Perindopril Versus Captopril for Treatment of Acute Myocardial Infarction. American Journal of Cardiology 89 (15), 150-154; Smith, A.I., Lew, R.A., Shrimpton, C. N., Evans, R.G. y Abbenante, G. (2000). A Novel Stable Inhibitor of Endopeptidases EC 3.4.24.15 and 3.4.24.16 Potentiates Bradykinin-lnduced Hypotension. Hypertension 35, 626-630; Azizi, M., Massien, C, Michaud, A. y Corvol, P. (2000). In vitro and in vivo inhibition of the 2 active sites of ace by omapatrilat, a vasopeptidase inhibitor. Hypertension 35, 1226-1231 ; Hou, W-C, Chen, H-J. y Lin, Y-H. (2004). Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. Journal of Agricultura! and Food Chemistry 51 (6), 1706-1709].To date, numerous bibliographic works related to the inhibition of RCTs have been published through the use of small synthetic peptides as the main responsible for such inhibition. These peptides have great variability because they have different lengths and structurally differ in the sequences of the amino acids that constitute them [Patchett, AA, Harris, E., Tristram, EW, Wyvratt, MJ. , Wu, MT, Taub, D., Peterson, ER, Ikeler, TJ. , Broeke, J.Ten., Payne, LG, Ondeyka, DL, Thorsett, ED, Greenlee, WJ., Lohr, NS, Hoffsommer, RD, Joshua, H., Ruyle, WV, Rothrock, JW, Aster, SD, Maycock, AL, Robinson, FM, Hirschmann, R., Sweet, CS. , UIm, EH, Gross, DM, Vassil, TC and Stone, CA. (1980). A new class of angiotensin-converting enzyme inhibitors. Nature 288, 280-283; Ondetti, MA, Rubin, B. and Cushman, DW (1977). Design of specific Inhibitors of angiotensin-converting enzyme: New class of orally active antihypertensive agents. Science 196, 441-444; Cushman, DW, Cheung, HS, Sabo, EF and Ondetti, MA (1977). Design of potent competitive inhibitors of angiotensin-converting enzyme. carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry 16 (25), 5484-5491; Cushman, DW, Cheung, HS, Sabo, EF and Ondetti, MA (1981). Angiotensin converting enzyme inhibitors. evolution of a new class of antihypertensive drugs. In: Angiotensin converting enzyme inhibitors. Mechanisms of action and clinical implications. Section I, pp3-25. Ed. Horovitz, ZP. , Urban & Schwarzenberg (Baltimor-Munich); Edling, O., Bao, G., Feelisch, M., Unger, T. and Gohlke, P. (1995). Moexipril, a new angiotensin-converting enzyme (ACE) inhibitor: Pharmacological characterization and comparison with enalapril. Journal of Pharmacology and Experimental Therapeutics 275 (2), 854-863; Gómez-Ruiz, JA, Recio, I. and Belloque, J. (2004). ACE-Inhibitory activity and structural properties of peptide Asp-Lys-lle-His-Pro [β-CN f (47-51)]. Study of the peptide forms synthesized by different methods. Journal of Agriculture! and Food Chemistry 52 (20), 6315-6319; Cotton, J., Hayashi, MAF, Cuniasse, P., Vazeux, G., Lanzer, D., De Camargo, ACM and Dive, V. (2002). Selective inhibition of the c-domain of angiotensin and converting enzyme by bradykinin potentiating peptides. Biochemistry 41 (19), 6065-6071; Lau, CP., Tse, HF., Ng, W., Chan, KK., Li, SK., Keung, KK., Lau, YK., Chen, WH., Tang, YW. and Leung, SK. (2002). Comparison of Perindopril Versus Captopril for Treatment of Acute Myocardial Infarction. American Journal of Cardiology 89 (15), 150-154; Smith, AI, Lew, RA, Shrimpton, CN, Evans, RG and Abbenante, G. (2000). A Novel Stable Inhibitor of Endopeptidases EC 3.4.24.15 and 3.4.24.16 Potentiates Bradykinin-lnduced Hypotension. Hypertension 35, 626-630; Azizi, M., Massien, C, Michaud, A. and Corvol, P. (2000). In vitro and in vivo inhibition of the 2 active sites of ace by omapatrilat, a vasopeptidase inhibitor. Hypertension 35, 1226-1231; Hou, WC, Chen, HJ. and Lin, YH. (2004). Antioxidant peptides with angiotensin converting enzyme inhibitory activities and applications for angiotensin converting enzyme purification. Journal of Agriculture! and Food Chemistry 51 (6), 1706-1709].
Así mismo, también se han llevado a cabo estudios con el fin de aislar e identificar inhibidores de carácter natural presentes en los alimentos. En numerosos casos se ha llegado a identificar como responsables ciertos péptidos naturales llegando incluso a Ia determinación de su secuencia. De esta manera se han podido sintetizar Ia mayoría de ellos con el fin de confirmar su actividad. Como materia prima, se emplean proteínas tanto de origen animal como vegetal [WO2005012355 Bioactive peptides derived from the proteins of
egg white by means of enzymatic hydrolysis; Li, G-H., Le, G-W., Shi, Y-H. y Shrestha S. (2004). Angiotensin l-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrítion Research 24, 469-486; Pripp, A.H., Isaksson, T., Stepaniak, L. y Sorhaug, T. (2004). Quantitative structure-activity relationship modelling of ACE-inhibitory peptides derived from milk proteins. European Food Research and Technology. 219, 579-583; Robert, M-C, Razaname, A., Mutter, M. y Juillerat, M.A. (2004). Identification of angiotensin l-converting enzyme inhibitory peptides derived from sodium caseinate hydrolysates produced by Lactobacillus helveticus NCC 2765. Journal of Agricultural and Food Chemistry 52 (23), 6923-6931 ; Chen, T- L., Lo, Y-C, Hu, W-T., Wu, M-C, Chen, S-T. y Chang, H-M. (2003). Microencapsulation and Modification of synthetic peptides of food proteins reduces the blood pressure of spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry 51 (6), 1671 -1675; Fujita, H. y Yoshikawa, M. (1999). LKPNM: a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44, 123-127; Pihlanto-Leppálá, A. (2001 ). Bioactive peptides derived from bovine whey proteins: opioid and Ace-inhibitory peptides. Trends in Food Science & Technology 11 , 347-356; Suetsuna, K. y Nakano, T. (2000). Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). Journal of Nutritional Biochemistry 11 , 450-454; Yokoyama, K., Chiba, H. y Yoshikawa, M. (1992). Peptide inhibitors for angiotensin i-converting enzyme from thermolysin digest of dried bonito. Bioscience Biotechechnology and Biochemistry 56 (10), 1541 -1545; Yano, S., Suzuki, K. y Funatsu, G. (1996). Isolation from α-zein of thermolysin peptides with angiotensin i-converting enzyme inhibitory activity. Bioscience Biotechnology and Biochemistry 60 (4), 661 -663; Wako, Y., Ishikawa, S. y Muramoto, K. (1996). Angiotensin l-converting enzyme inhibitors in autolysates of squid liver and mantle muscle. Bioscience Biotechnology and Biochemistry 60 (8), 1353-1355; Suetsuna, K. (1998). Isolation and characterization of angiotensin l-converting enzyme inhibitor dipeptides derived from Allium sativum L (garlic). Journal of Nutritional Biochemistry 9, 415-419; Pihlanto- Leppálá, A., Rokka, T. y Coronen, H. (1998). Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. International Dairy Journal
8, 325-331 ; Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M. y Mimura, T. (1988). Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochemical and Biophysical Research Communications 155 (1 ), 332-337. Maruyama, S., Miyoshi, S. y Tanaka, H. (1989). Angiotensin I- converting enzyme inhibitors derived from Ficus carica. Agricultural and . Biológica! Chemistry 53 (10), 2763-2767; Ariyoshi, Y. (1993). Angiotensin- converting enzyme inhibitors derived from food proteins. Trends in Food Science & Technology 4, 139-144; Takayanagi, T. y Yokotsuka, K. (1999). Angiotensin I converting enzyme-inhibitory peptides from wine. American Journal of Enology and Viticulture 50 (1 ), 65-68; Fuglsang, A., Nilsson, D. y Nyborg, N. C. B. (2003). Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo. Journal of Enzyme Inhibition and Medical Chemistry 18 (5), 407-412 ].Likewise, studies have also been carried out in order to isolate and identify natural inhibitors present in food. In many cases, certain natural peptides have been identified as responsible, even determining their sequence. In this way, most of them have been synthesized in order to confirm their activity. As a raw material, both animal and vegetable proteins are used [WO2005012355 Bioactive peptides derived from the proteins of egg white by means of enzymatic hydrolysis; Li, GH., Le, GW., Shi, YH. and Shrestha S. (2004). Angiotensin l-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrítion Research 24, 469-486; Pripp, AH, Isaksson, T., Stepaniak, L. and Sorhaug, T. (2004). Quantitative structure-activity relationship modeling of ACE-inhibitory peptides derived from milk proteins. European Food Research and Technology. 219, 579-583; Robert, MC, Razaname, A., Mutter, M. and Juillerat, MA (2004). Identification of angiotensin l-converting enzyme inhibitory peptides derived from sodium caseinate hydrolysates produced by Lactobacillus helveticus NCC 2765. Journal of Agricultural and Food Chemistry 52 (23), 6923-6931; Chen, T-L., Lo, YC, Hu, WT., Wu, MC, Chen, ST. and Chang, HM. (2003). Microencapsulation and Modification of synthetic peptides of food proteins reduce the blood pressure of spontaneously hypertensive rats. Journal of Agricultural and Food Chemistry 51 (6), 1671-1675; Fujita, H. and Yoshikawa, M. (1999). LKPNM: a prodrug-type ACE-inhibitory peptide derived from fish protein. Immunopharmacology 44, 123-127; Pihlanto-Leppálá, A. (2001). Bioactive peptides derived from bovine whey proteins: opioid and Ace-inhibitory peptides. Trends in Food Science & Technology 11, 347-356; Suetsuna, K. and Nakano, T. (2000). Identification of an antihypertensive peptide from peptic digest of wakame (Undaria pinnatifida). Journal of Nutritional Biochemistry 11, 450-454; Yokoyama, K., Chiba, H. and Yoshikawa, M. (1992). Peptide inhibitors for angiotensin i-converting enzyme from thermolysin digest of dried bonito. Bioscience Biotechechnology and Biochemistry 56 (10), 1541-1545; Yano, S., Suzuki, K. and Funatsu, G. (1996). Isolation from α-zein of thermolysin peptides with angiotensin i-converting enzyme inhibitory activity. Bioscience Biotechnology and Biochemistry 60 (4), 661-663; Wako, Y., Ishikawa, S. and Muramoto, K. (1996). Angiotensin l-converting enzyme inhibitors in autolysates of squid liver and mantle muscle. Bioscience Biotechnology and Biochemistry 60 (8), 1353-1355; Suetsuna, K. (1998). Isolation and characterization of angiotensin l-converting enzyme inhibitor dipeptides derived from Allium sativum L (garlic). Journal of Nutritional Biochemistry 9, 415-419; Pihlanto- Leppálá, A., Rokka, T. and Coronen, H. (1998). Angiotensin I converting enzyme inhibitory peptides derived from bovine milk proteins. International Dairy Journal 8, 325-331; Kohama, Y., Matsumoto, S., Oka, H., Teramoto, T., Okabe, M. and Mimura, T. (1988). Isolation of angiotensin-converting enzyme inhibitor from tuna muscle. Biochemical and Biophysical Research Communications 155 (1), 332-337. Maruyama, S., Miyoshi, S. and Tanaka, H. (1989). Angiotensin I- converting enzyme inhibitors derived from Ficus carica. Agricultural and. Biological! Chemistry 53 (10), 2763-2767; Ariyoshi, Y. (1993). Angiotensin-converting enzyme inhibitors derived from food proteins. Trends in Food Science & Technology 4, 139-144; Takayanagi, T. and Yokotsuka, K. (1999). Angiotensin I converting enzyme-inhibitory peptides from wine. American Journal of Enology and Viticulture 50 (1), 65-68; Fuglsang, A., Nilsson, D. and Nyborg, NCB (2003). Characterization of new milk-derived inhibitors of angiotensin converting enzyme in vitro and in vivo. Journal of Enzyme Inhibition and Medical Chemistry 18 (5), 407-412].
En Ia presente invención se describe Ia secuencia de aminoácidos de otros péptidos, distinta a Ia de los mencionados anteriormente, caracterizados por tener actividad inhibidora de Ia ECA. Dicha inhibición de Ia actividad ECA se manifiesta en ensayos realizados in vitro, midiendo Ia inhibición de Ia conversión de sustratos artificiales (Hipuril-Histidil-Leucina) y naturales (Angiotensina I) mediada por ECA, y en ensayos realizados ex vivo, midiendo Ia disminución de Ia contracción de arterias basilares ó carótidas de conejo inducida por exposición a Angiotensina I.The present invention describes the amino acid sequence of other peptides, different from those mentioned above, characterized by having ACE inhibitory activity. Said inhibition of the ECA activity is manifested in in vitro tests, measuring the inhibition of the conversion of artificial (Hipuril-Histidyl-Leucine) and natural (Angiotensin I) mediated ACA mediated substrates, and in ex vivo tests, measuring the decrease of the contraction of basilar or carotid arteries of rabbit induced by exposure to Angiotensin I.
Breve descripción de Ia invención La presente invención está relacionada con los sectores farmacológico y de Ia industria agroalimentaria y consiste en Ia identificación y caracterización de unos determinados péptidos (llamados PIECA, Péptidos Inhibidores de Ia Enzima Conversora de Angiotensina) como inhibidores efectivos de Ia Enzima Conversora de Ia Angiotensina (ECA) que se halla implicada en Ia formación del compuesto Angiotensina Il que es un vasoconstrictor responsable, entre otras causas/mecanismos, de Ia hipertensión. Las secuencias de residuos de
aminoácidos de los péptidos identificados son las siguientes, desde el extremo amino terminal al extremo carboxi terminal: (péptido PIECA32; SEQ ID NO 1 ) y (péptido PIECA34, SEQ ID NO 2) que se hayan descritos en Ia solicitud de patente ES200001973 como PAF32 y PAF34 respectivamente. La actividad inhibidora de los péptidos se manifiesta por una reducción de Ia actividad ECA determinada en ensayos in vitro, así como por una reducción de Ia contracción ECA dependiente ex vivo empleando segmentos de arterias. Se demuestra que Ia actividad inhibidora de los péptidos se corresponde con una secuencia de aminoácidos característica, ya que otros péptidos relacionados con los anteriores y con una secuencia de residuos de aminoácidos parecida, pero no idéntica, no presentan dicha actividad. Se describe Ia potencial utilización de los péptidos inhibidores de ECA como compuestos bioactivos para el control de Ia hipertensión. En un ejemplo particular, se describe Ia actividad inhibidora de ECA de dichos péptidos cuando se emplean como sustratos Hipuril-Histidil- Leucina (HHL) y Angiotensina I. La actividad inhibidora se ha demostrado en condiciones in vitro empleando ECA purificada a partir de riñon de cerdo y ex vivo empleando segmentos de arterias carótida y basilar de conejo.BRIEF DESCRIPTION OF THE INVENTION The present invention is related to the pharmacological and agri-food industry sectors and consists in the identification and characterization of certain peptides (called PIECA, Angiotensin Conversion Enzyme Inhibitor Peptides) as effective inhibitors of the Conzyme Enzyme of the Angiotensin (RCT) that is involved in the formation of the compound Angiotensin Il which is a vasoconstrictor responsible, among other causes / mechanisms, for hypertension. The residue sequences of amino acids of the peptides identified are the following, from the amino terminal to the carboxy terminal end: (peptide PIECA32; SEQ ID NO 1) and (peptide PIECA34, SEQ ID NO 2) that have been described in patent application ES200001973 as PAF32 and PAF34 respectively. The inhibitory activity of the peptides is manifested by a reduction of the ACE activity determined in in vitro assays, as well as by a reduction of the ECA-dependent contraction ex vivo using artery segments. It is demonstrated that the inhibitory activity of the peptides corresponds to a characteristic amino acid sequence, since other peptides related to the above and with a similar, but not identical amino acid residue sequence, do not exhibit said activity. The potential use of the ACE inhibitor peptides as bioactive compounds for the control of hypertension is described. In a particular example, the ACE inhibitory activity of said peptides is described when Hipuril-Histidyl-Leucine (HHL) and Angiotensin I are used as substrates. The inhibitory activity has been demonstrated in in vitro conditions using purified RCT from kidney of pig and ex vivo using carotid and basilar rabbit artery segments.
Descripción detallada de Ia invención En Ia presente invención se describe Ia identificación y caracterización de nuevos péptidos con actividad de inhibición de Ia Enzima Conversora de Angiotensina (ECA), implicada en los mecanismos de control de Ia presión arterial. En un ejemplo particular, se describe Ia utilización de dichos péptidos como inhibidores de ECA mediante el empleo de sustratos artificiales como el Hipuril-Histidil-Leucina (HHL) ó naturales como Ia Angiotensina I, así como su efecto inhibidor sobre Ia contracción ECA-dependiente de segmentos de arterias de conejo.DETAILED DESCRIPTION OF THE INVENTION In the present invention, the identification and characterization of new peptides with inhibition activity of the Angiotensin Conversion Enzyme (RCT), involved in the blood pressure control mechanisms, is described. In a particular example, the use of said peptides as ACE inhibitors is described through the use of artificial substrates such as Hipuril-Histidyl-Leucine (HHL) or natural substrates such as Angiotensin I, as well as their inhibitory effect on ECA-dependent contraction of rabbit artery segments.
Los inhibidores descritos en Ia presente invención son péptidos con una secuencia de seis aminoácidos característica SEQ ID NO 1 (péptido PIECA32) y SEQ ID NO 2 (péptido PIECA34), Figura 1 , y distinta de los péptidos inhibidores de ECA conocidos anteriormente [Guan-Hong Li, Guo-Wei Le,The inhibitors described in the present invention are peptides with a sequence of six amino acids characteristic SEQ ID NO 1 (PIECA32 peptide) and SEQ ID NO 2 (PIECA34 peptide), Figure 1, and different from the previously known ACE inhibitor peptides [Guan- Hong Li, Guo-Wei Le,
Yong-Hui Shi y Sundar Shrestha (2004). Angiotensin l-converting enzyme
inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrítion Research 24, 469-486; Dziuba, J., Minkiewicz, P., Nalecz, D. y Iwaniak, A. (1999). Datábase of biologically active peptide sequences. Nahrung 43, 190-195; Fujita, H., Yokoyama, K. y Yoshikawa, M. Classification and Antihypertensive Activity of Angiotensin I- Converting Enzyme Inhibitory Peptides Derived from Food Proteins. Journal of Food Science 65, 564-569; Reed, J. D., Edwards, D. L., y González, C. F. (1997)]. En un ejemplo particular de realización de Ia invención, dichos hexapéptidos se sintetizaron químicamente con los estereoisómeros L- naturales de los aminoácidos (PIECA32L y PIECA34L) y seguidamente se purificaron, siguiendo procedimientos habituales para toda aquella persona experta en el área de conocimiento de Ia presente invención [Fields, G. B. y Noble, R. L. (1990). SoNd phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34, 161 -214]. Para todo aquel experto en el tema, es conocido que las actividades biológicas de pequeños péptidos sintetizados con L-aminoácidos son también compartidas por los péptidos de Ia misma secuencia de aminoácidos construidos con los estereoisómeros D- de los aminoácidos constituyentes [Blondelle, S. E., Houghten, R. A., y Pérez-Payá, E. (1998b). Peptide inhibitors of calmodulin. United States Patent Number 5840697; Edwards, D. L. (1997). Synthetic Antibiotics. United States Patent Number 5602097]. Con los mismos procedimientos conocidos citados anteriormente, dichos péptidos PIECA se sintetizaron y purificaron utilizando los estereoisómeros D- de los aminoácidos constituyentes (PIECA32D y PIECA34D), que no son naturales pero tienen Ia propiedad de conferir a los péptidos resultantes mayor estabilidad y resistencia a Ia degradación por proteasas presentes en los fluidos biológicos.Yong-Hui Shi and Sundar Shrestha (2004). Angiotensin l-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrítion Research 24, 469-486; Dziuba, J., Minkiewicz, P., Nalecz, D. and Iwaniak, A. (1999). Date of biologically active peptide sequences. Nahrung 43, 190-195; Fujita, H., Yokoyama, K. and Yoshikawa, M. Classification and Antihypertensive Activity of Angiotensin I- Converting Enzyme Inhibitory Peptides Derived from Food Proteins. Journal of Food Science 65, 564-569; Reed, JD, Edwards, DL, and González, CF (1997)]. In a particular example of embodiment of the invention, said hexapeptides were chemically synthesized with the L-natural stereoisomers of the amino acids (PIECA32L and PIECA34L) and then purified, following usual procedures for all those skilled in the area of knowledge of the present invention [Fields, GB and Noble, RL (1990). SoNd phase peptide synthesis utilizing 9- fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34, 161-214]. For all those skilled in the art, it is known that the biological activities of small peptides synthesized with L-amino acids are also shared by the peptides of the same amino acid sequence constructed with the D-stereoisomers of the constituent amino acids [Blondelle, SE, Houghten , RA, and Pérez-Payá, E. (1998b). Peptide inhibitors of calmodulin. United States Patent Number 5840697; Edwards, DL (1997). Synthetic Antibiotics. United States Patent Number 5602097]. With the same known procedures mentioned above, said PIECA peptides were synthesized and purified using the D-stereoisomers of the constituent amino acids (PIECA32D and PIECA34D), which are not natural but have the property of conferring the resulting peptides greater stability and resistance to Ia degradation by proteases present in biological fluids.
En Ia presente invención se describen ensayos experimentales que ilustran Ia actividad de los dos estereoisómeros de SEQ ID NO 1 PIECA32L y PIECA32D y de SEQ ID NO 2 PIECA34L, y PIECA34D, en condiciones experimentales in vitro, utilizando ECA purificada de riñon de cerdo y tres sustratos distintos, uno artificial denominado HHL y dos naturales como son Ia Angiotensina I y Ia Bradiquinina. El primero de los sustratos permite llevar a
cabo una serie de ensayos encaminados a conocer Ia capacidad inhibidora de los péptidos, mientras que el uso de los dos últimos permite contrastar los resultados obtenidos con el anterior y además comprobar dicha capacidad inhibidora de ECA en el caso de utilizar sustratos naturales. La actividad inhibidora de los péptidos PIECA descritos en Ia presente invención se manifiesta con una reducción de Ia actividad de Ia ECA al llevar a cabo los ensayos in vitro en las condiciones establecidas, como queda demostrado mediante los ensayos experimentales descritos en Ia presente invención. Estos ensayos también demuestran que los PIECA tienen una secuencia de aminoácidos característica y específica, ya que péptidos relacionados de secuencia parecida -pero no idéntica- a PIECA32L, PIECA34L, PIECA32D y PIECA34D no presentan Ia mencionada actividad inhibidora.In the present invention experimental tests are described that illustrate the activity of the two stereoisomers of SEQ ID NO 1 PIECA32L and PIECA32D and SEQ ID NO 2 PIECA34L, and PIECA34D, in experimental conditions in vitro, using purified RCT of pig kidney and three different substrates, one artificial called HHL and two natural ones such as Angiotensin I and Bradiquinine. The first of the substrates allows to carry It carries out a series of tests aimed at knowing the inhibitory capacity of the peptides, while the use of the last two allows to contrast the results obtained with the previous one and also to verify said ACE inhibitory capacity in the case of using natural substrates. The inhibitory activity of the PIECA peptides described in the present invention is manifested with a reduction in the activity of the RCT when performing the in vitro tests under the established conditions, as demonstrated by the experimental tests described in the present invention. These tests also demonstrate that the PIECA have a characteristic and specific amino acid sequence, since related peptides of similar sequence - but not identical - to PIECA32L, PIECA34L, PIECA32D and PIECA34D do not exhibit the said inhibitory activity.
La actividad inhibidora de los péptidos PIECA descritos en Ia presente invención también se manifiesta con una reducción de Ia contracción ECA- dependiente de arterias de conejo, carótida y basilar, inducida mediante Ia adición de Angiotensina I en ensayos ex vivo.The inhibitory activity of the PIECA peptides described in the present invention is also manifested with a reduction of the ECA-dependent contraction of rabbit, carotid and basilar arteries, induced by the addition of Angiotensin I in ex vivo assays.
Considerando las propiedades de los PIECA descritos en Ia presente invención, es obvio para todo aquel experto en el tema su potencial utilización como aditivos alimentarios, compuestos ó fármacos de utilidad en Ia prevención ó tratamiento de enfermedades que tengan como causa ó sintomatología Ia hipertensión arterial.Considering the properties of the PIECAs described in the present invention, it is obvious to all those skilled in the art their potential use as food additives, compounds or drugs useful in the prevention or treatment of diseases that have arterial hypertension as a cause or symptomatology.
Es obvio para todo aquel experto en el área de Ia presente invención el interés, diseño y desarrollo de estrategias derivadas de Ia biotecnología, que incluyen Ia metodología del ADN recombinante y de Ia transformación genética de organismos, que pueden ser utilizadas para Ia producción y utilización de los PIECA32L y PIECA34L descritos en Ia presente invención y sus derivados para los fines descritos. En un ejemplo de realización de Ia invención se explica Ia producción a gran escala de los péptidos mediada por organismos transformados genéticamente.
Descripción de los dibujosIt is obvious to all those skilled in the area of the present invention the interest, design and development of strategies derived from biotechnology, which include the methodology of recombinant DNA and the genetic transformation of organisms, which can be used for the production and use of the PIECA32L and PIECA34L described in the present invention and their derivatives for the purposes described. In an embodiment of the invention, the large-scale production of the peptides mediated by genetically transformed organisms is explained. Description of the drawings
Figura 1. Secuencia de aminoácidos de los hexapéptidos SEQ ID NO 1 con sus correspondientes estereoisómeros PIECA32D, PIECA32L, yFigure 1. Amino acid sequence of the hexapeptides SEQ ID NO 1 with their corresponding stereoisomers PIECA32D, PIECA32L, and
SEQ ID NO 2 con sus estereoisómeros PIECA34D, PIECA34L (descritos como inhibidores de ECA en Ia presente invención) y deSEQ ID NO 2 with its stereoisomers PIECA34D, PIECA34L (described as ACE inhibitors in the present invention) and of
P26D SEQ ID NO 3 y P36D SEQ ID NO 4 (utilizados como controles negativos en los ensayos descritos). Las secuencias están escritas desde el extremo amino terminal (a Ia izquierda) al extremo carboxi terminal (a Ia derecha). Los péptidos se encuentran acetilados en su extremo amino terminal (Ac-) y amidados en su extremo carboxi terminal (Am-).P26D SEQ ID NO 3 and P36D SEQ ID NO 4 (used as negative controls in the described tests). The sequences are written from the amino terminal end (on the left) to the carboxy terminal end (on the right). The peptides are acetylated at their amino terminal (Ac-) end and amidated at their carboxy terminal (Am-) end.
Figura 2. Efecto de Ia concentración de PIECA32L sobre Ia actividad de Ia Enzima Conversora de Angiotensina I.Figure 2. Effect of the concentration of PIECA32L on the activity of the Angiotensin I Conversion Enzyme.
Figura 3. Efecto de Ia concentración de PIECA34L sobre Ia actividad de Ia Enzima Conversora de Angiotensina I.Figure 3. Effect of the concentration of PIECA34L on the activity of the Angiotensin I Conversion Enzyme.
Un ejemplo de realización de Ia invención.An example of embodiment of the invention.
1. Síntesis de péptidos. Los péptidos caracterizados y analizados en Ia presente invención (Figura 1 : P26D SEQ ID NO 3, SEQ ID NO 1 con sus estereoisómeros PIECA32D , PIECA32L, SEQ ID NO 2 con sus estereoisómeros1. Synthesis of peptides. The peptides characterized and analyzed in the present invention (Figure 1: P26D SEQ ID NO 3, SEQ ID NO 1 with its stereoisomers PIECA32D, PIECA32L, SEQ ID NO 2 with its stereoisomers
PIECA34D, PIECA34L y P36D SEQ ID NO 4) se sintetizaron químicamente sobre fase sólida siguiendo procedimientos habituales que utilizan el grupoPIECA34D, PIECA34L and P36D SEQ ID NO 4) were chemically synthesized on solid phase following usual procedures using the group
N-(9-fluorenyl) methoxycarbonyl (Fmoc) para Ia protección del grupo α- amino de los aminoácidos constituyentes [Fields, G. B. y Noble, R. L.N- (9-fluorenyl) methoxycarbonyl (Fmoc) for the protection of the α-amino group of the constituent amino acids [Fields, G. B. and Noble, R. L.
(1990). SoNd phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34, 161 -(1990). SoNd phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34, 161 -
214]. Los péptidos P26D y P36D se diseñaron como control negativo en los ensayos descritos a continuación. El extremo N-terminal de los péptidos se encuentra acetilado (Ac) y el extremo C-terminal amidado (NH2), como consecuencia del procedimiento de síntesis. Después de Ia síntesis, los
péptidos se purificaron mediante RP-HPLC (del inglés, reversed phase- high performance liquid chromatography, cromatografía líquida de alta resolución de fase inversa) y su identidad se confirmó mediante espectrometría de masas MALDI-TOF (del inglés, matrix-assisted láser desorption/ionization time-of-flight). Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de Ia presente invención.214]. Peptides P26D and P36D were designed as a negative control in the assays described below. The N-terminal end of the peptides is acetylated (Ac) and the C-terminal amidated end (NH2), as a consequence of the synthesis procedure. After the synthesis, the Peptides were purified by RP-HPLC (reversed phase high performance liquid chromatography) and their identity was confirmed by MALDI-TOF mass spectrometry (English, matrix-assisted laser desorption / ionization time-of-flight). All these procedures are common for all those experts in the area of knowledge of the present invention.
También es posible Ia producción de los péptidos L compuestos por aminoácidos naturales (estereoisómeros L-) descritos en Ia presente invención mediante estrategias derivadas de Ia biotecnología. Es obvio, para toda aquella persona experta en el área de conocimiento, que Ia producción de los péptidos mediante procedimientos biotecnológicos, que incluyen las metodologías del ADN recombinante y de Ia transformación genética de organismos, supondría una mejora en los costes de producción, y que por tanto dicha producción es un aspecto importante en el contexto de Ia aplicabilidad industrial de Ia presente invención. En el supuesto de Ia producción mediante biotecnología, Ia secuencia del péptido producido por un organismo modificado genéticamente sería codificada por un fragmento de ADN de acuerdo a las leyes del código genético [Sambrook, J., Fritsch, E. F., y Maniatis, T. (1989). Molecular cloning: A laboratory manual, 2nd edition. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY.],It is also possible to produce L peptides composed of natural amino acids (L- stereoisomers) described in the present invention by means of strategies derived from biotechnology. It is obvious, for all those experts in the area of knowledge, that the production of the peptides through biotechnological procedures, which include the methodologies of recombinant DNA and the genetic transformation of organisms, would mean an improvement in production costs, and that Therefore, said production is an important aspect in the context of the industrial applicability of the present invention. In the case of biotechnology production, the sequence of the peptide produced by a genetically modified organism would be encoded by a DNA fragment according to the laws of the genetic code [Sambrook, J., Fritsch, EF, and Maniatis, T. ( 1989). Molecular cloning: A laboratory manual, 2nd edition. CoId Spring Harbor Laboratory Press, CoId Spring Harbor, NY.],
Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de Ia presente invención.All these procedures are common for all those experts in the area of knowledge of the present invention.
2. Ensayos in vitro de inhibición de Ia actividad ECA sobre el sustrato artificial HHL. En estos ensayos, Ia capacidad inhibidora de los péptidos se determinó midiendo por HPLC (del inglés high performance liquid chromatography) el ácido hipúrico resultante de Ia hidrólisis del sustrato artificial HHL (Hipuril-Histidil-Leucina) basándose en el método propuesto en Ia literatura [Wu, J., Aluko, R.E. y Muir, A.D. (2002). Improved method for direct high performance liquid-chromatography assay of ACE catalyzed reactions. Journal of Chromatography A 950, 125-130]. La mezcla de reacción tiene un volumen de 225 μl y está constituida por 50 μl de HHL 25 mM en tampón Tris HCI 200 mM pH 8.3 con NaCI 600 mM y ZnCI2 10 μM,
75 μl de una solución de ACE en el mismo tampón que corresponden a 1.5 mil de actividad, y 100 μl de péptido (disuelto en tampón MOPS 10 mM pH 7) a diferentes concentraciones según Ia concentración final deseada en el ensayo. El enzima y el inhibidor se preincuban durante 15 minutos a 37°C y a continuación se añade el sustrato incubándose el conjunto 30 minutos a dicha temperatura. La reacción se detiene añadiendo 25 μl de HCI 6M. Para Ia determinación cromatográfica del ácido hipúrico liberado se utiliza una columna de fase inversa C18, Ia elución se lleva a cabo empleando un gradiente de acetonitrilo en agua con TFA 0.05% y se determina el ácido hipúrico midiendo Ia absorbancia a 228 nm. Los resultados aparecen en Ia2. In vitro assays for the inhibition of ECA activity on the artificial HHL substrate. In these tests, the inhibitory capacity of the peptides was determined by measuring by HPLC (high performance liquid chromatography) the hippuric acid resulting from the hydrolysis of the artificial substrate HHL (Hipuril-Histidil-Leucine) based on the method proposed in the literature [ Wu, J., Aluko, RE and Muir, AD (2002). Improved method for direct high performance liquid-chromatography assay of ACE catalyzed reactions. Journal of Chromatography A 950, 125-130]. The reaction mixture has a volume of 225 μl and is made up of 50 μl of 25 mM HHL in 200 mM Tris HCI buffer pH 8.3 with 600 mM NaCI and 10 μM ZnCI 2 , 75 μl of an ACE solution in the same buffer corresponding to 1.5 thousand activity, and 100 μl of peptide (dissolved in 10 mM MOPS buffer pH 7) at different concentrations according to the final concentration desired in the assay. The enzyme and the inhibitor are pre-incubated for 15 minutes at 37 ° C and then the substrate is added by incubating the whole 30 minutes at said temperature. The reaction is stopped by adding 25 μl of 6M HCI. For the chromatographic determination of the released hipuric acid a C18 reverse phase column is used, the elution is carried out using a gradient of acetonitrile in water with TFA 0.05% and the hippuric acid is determined by measuring the absorbance at 228 nm. The results appear in Ia
Tabla 1 pudiéndose observar una inhibición significativa para los péptidos PIECA 32 SEQ ID NO 1 y PIECA 34 SEQ ID NO 2, siendo mayor Ia inhibición para los PIECA 32 tanto L como D. Los P 26D SEQ ID NO 3 y P 36D SEQ ID NO 4 no mostraron una inhibición significativa. 3. Ensayos in vitro de inhibición de Ia actividad ECA sobre el sustrato natural Angiotensina I. El protocolo experimental descrito en el apartado 2 se repitió empleando como sustrato Ia Angiotensina I (cantidad final en el ensayo 20μg). La determinación del producto de reacción resultante, Angiotensina II, se llevó a cabo cromatográficamente utilizando una columna de fase inversa C18, un gradiente de acetonitrilo en agua con TFATable 1, a significant inhibition can be observed for the PIECA 32 SEQ ID NO 1 and PIECA 34 SEQ ID NO 2 peptides, the inhibition for the PIECA 32 being both L and D. being greater. 4 did not show a significant inhibition. 3. In vitro tests of inhibition of the ECA activity on the natural substrate Angiotensin I. The experimental protocol described in section 2 was repeated using as substrate Ia Angiotensin I (final amount in the 20μg test). The resulting reaction product, Angiotensin II, was determined chromatographically using a C18 reverse phase column, a gradient of acetonitrile in water with TFA
0.1% y midiendo Ia absorbancia a 214 nm. Los resultados obtenidos se muestran en Ia Tabla 2 y ponen de manifiesto una mayor inhibición en el caso de los péptidos con configuración L, tanto SEQ ID NO 1 , PIECA32 como SEQ ID NO 2 PIECA34. Con este sustrato natural los péptidos con configuración D tienen una capacidad inhibitoria inferior a Ia conseguida con HHL.0.1% and measuring the absorbance at 214 nm. The results obtained are shown in Table 2 and show a greater inhibition in the case of peptides with L configuration, both SEQ ID NO 1, PIECA32 and SEQ ID NO 2 PIECA34. With this natural substrate, peptides with D configuration have an inhibitory capacity lower than that achieved with HHL.
4. Ensayos in vitro de inhibición de Ia actividad ECA sobre el sustrato natural Angiotensina I para el cálculo del IC5O- El protocolo experimental descrito en el apartado anterior se repitió empleando diferentes concentraciones de péptido: 0, 0.5,2.5, 4, 5, 6, 10, 20, 40, 50 y 80 μM para los ensayos con SEQ ID NO 2 PIECA34L y 0, 2.5, 5, 10, 20, 40, y 80 μM para SEQ ID NO 1 PIECA32L. En cada caso se efectuaron siete series de
experimentos completos con todas las concentraciones, calculando para cada una de ellas Ia media y su desviación. La representación gráfica del valor medio correspondiente a Ia actividad residual para cada una de las concentraciones de péptidos ensayada se muestran en las figuras 2 y 3. A partir de estos resultados se calcularon los IC5O para SEQ ID NO 14. In vitro tests of inhibition of the ECA activity on the natural substrate Angiotensin I for the calculation of IC 5 O- The experimental protocol described in the previous section was repeated using different concentrations of peptide: 0, 0.5,2.5, 4, 5 , 6, 10, 20, 40, 50 and 80 μM for tests with SEQ ID NO 2 PIECA34L and 0, 2.5, 5, 10, 20, 40, and 80 μM for SEQ ID NO 1 PIECA32L. In each case, seven series of Complete experiments with all concentrations, calculating for each of them the mean and its deviation. The graphic representation of the mean value corresponding to the residual activity for each of the concentrations of peptides tested are shown in Figures 2 and 3. From these results, the IC 5 O for SEQ ID NO 1 was calculated.
PIECA 32L y SEQ ID NO 2 PIECA 34L siendo estos de 10.7 y 8.1 μM respectivamente.PIECA 32L and SEQ ID NO 2 PIECA 34L being these of 10.7 and 8.1 μM respectively.
5. Ensayos in vitro de inhibición de Ia actividad ECA sobre el sustrato natural Bradiquidina. El protocolo experimental descrito en el apartado 3 se repitió utilizando como sustrato Ia Bradiquinina. Se empleó Ia misma cantidad de sustrato, 20 μg en el ensayo y se detectó como producto final el fragmento de Bradiquinina 1-5. Los resultados obtenidos se muestran en Ia Tabla 3 y ponen de manifiesto que no existe inhibición por parte de ninguno de los péptidos SEQ ID NO 1 PIECA32 y SEQ ID NO 2 PIECA34 ensayados.5. In vitro assays of inhibition of ECA activity on the natural substrate Bradiquidine. The experimental protocol described in section 3 was repeated using as a substrate Ia Bradykinin. The same amount of substrate, 20 μg was used in the assay and the Bradykinin 1-5 fragment was detected as the final product. The results obtained are shown in Table 3 and show that there is no inhibition by any of the peptides SEQ ID NO 1 PIECA32 and SEQ ID NO 2 PIECA34 tested.
6. Ensayos ex vivo de inhibición de Ia contracción de arterias aisladas.6. Ex vivo tests of inhibition of the contraction of isolated arteries.
La preparación experimental consistió en obtener segmentos cilindricos (3 mm) de arterias aisladas (arterias basilar y carótida de conejo blanco New Zealand), los cuales se dispusieron en un baño de órganos diseñado para registrar los cambios de tensión isométrica en Ia pared vascular. El medioThe experimental preparation consisted of obtaining cylindrical segments (3 mm) of isolated arteries (basilar and carotid arteries of New Zealand white rabbit), which were arranged in an organ bath designed to record the changes of isometric tension in the vascular wall. The middle
(solución Ringer-Locke) en el que se hallan inmersos los segmentos arteriales se mantiene termostatizado a 370C y continuamente burbujeado con una mezcla gaseosa de 95% O2 y 5% CO2 que Ie confiere un pH de 7.3-7.4. Los experimentos comienzan tras un periodo de 30-60 min necesario para alcanzar Ia estabilización en el tono pasivo de 0.5 g para Ia arteria basilar y de 2 g para Ia carótida. Tras comprobar Ia viabilidad de los segmentos arteriales mediante contracción con una solución despolarizante (Ringer-Locke 50 mM KCI), cada segmento arterial se somete a una primera contracción ECA-dependiente con Angiotensina I (1 μM). Tras preincubar los segmentos durante 20 minutos con alguno de los péptidos objeto de estudio (SEQ ID NO 1 , PIECA32D y PIECA32L, SEQ ID NO 2, PIECA34D, y PIECA34L a 20 μM de concentración), se indujo una segunda contracción
con Angiotensina I. Como control, se dejaron algunos segmentos sin preincubar con PIECA. Los resultados obtenidos se muestran en Ia Tabla 4 y ponen de manifiesto efectos inhibitorios significativos en los casos de SEQ ID NO 1 PIECA32D y PIECA32L sobre arteria carótida.
(Ringer-Locke solution) in which the arterial segments are immersed is kept thermostated at 37 0 C and continuously bubbled with a gaseous mixture of 95% O2 and 5% CO2 that confers a pH of 7.3-7.4. The experiments begin after a period of 30-60 min necessary to achieve stabilization in the passive tone of 0.5 g for the basilar artery and 2 g for the carotid. After checking the viability of the arterial segments by contraction with a depolarizing solution (Ringer-Locke 50 mM KCI), each arterial segment undergoes a first ECA-dependent contraction with Angiotensin I (1 μM). After preincubating the segments for 20 minutes with any of the peptides under study (SEQ ID NO 1, PIECA32D and PIECA32L, SEQ ID NO 2, PIECA34D, and PIECA34L at 20 μM concentration), a second contraction was induced with Angiotensin I. As a control, some segments were left unincubated with PIECA. The results obtained are shown in Table 4 and show significant inhibitory effects in the cases of SEQ ID NO 1 PIECA32D and PIECA32L on the carotid artery.
Tabla 1.Table 1.
Efecto de los péptidos descritos en Ia presente invención como inhibidores de Ia Enzima Conversora de Ia Angiotensina I (SEQ ID NO 1 con sus estereoisómeros PIECA32D, PIECA32L y SEQ ID NO 2 con sus estereoisómeros PIECA34D y PIECA34L) y de los usados como control negativo (SEQ ID NO 4 P36D y P26D SEQ ID NO 3) sobre Ia actividad ECA, cuando esta enzima actúa sobre el sustrato artificial Hipuril-Histidin-Leucina. Se indican los porcentajes de actividad de Ia ECA para una concentración de péptido en el ensayo de 20 μM, expresándose estos valores como Ia media ± desviación estándar de un número de repeticiones (n).Effect of the peptides described in the present invention as inhibitors of the Angiotensin I Conversion Enzyme I (SEQ ID NO 1 with its stereoisomers PIECA32D, PIECA32L and SEQ ID NO 2 with its stereoisomers PIECA34D and PIECA34L) and those used as negative control ( SEQ ID NO 4 P36D and P26D SEQ ID NO 3) on the ECA activity, when this enzyme acts on the artificial substrate Hipuril-Histidin-Leucine. The percentages of activity of the RCT for a peptide concentration in the test of 20 μM are indicated, these values being expressed as the mean ± standard deviation of a number of repetitions (n).
Tabla 2.Table 2.
Efecto inhibidor de los péptidos descritos en Ia presente invención (SEQ ID NO 1 con sus estereoisómeros PIECA32D, PIECA32L y SEQ ID NO 2 con sus estereoisómeros PIECA34D y PIECA34L) y del P36D SEQ ID NO 4 usado como control negativo sobre Ia actividad ECA, cuando esta enzima actúa sobre Ia Angiotensina I. Se indican los porcentajes de actividad de Ia ECA para una concentración de péptido en el ensayo de 20 μM, expresándose estos valores como Ia media ± desviación estándar de un número de repeticiones (n).Inhibitory effect of the peptides described in the present invention (SEQ ID NO 1 with its stereoisomers PIECA32D, PIECA32L and SEQ ID NO 2 with its stereoisomers PIECA34D and PIECA34L) and of the P36D SEQ ID NO 4 used as a negative control on ECA activity, when this enzyme acts on the Angiotensin I. The percentages of activity of the RCT for a peptide concentration in the 20 μM assay are indicated, these values being expressed as the mean ± standard deviation of a number of repetitions (n).
Tabla 3.Table 3.
Efecto inhibidor de los péptidos descritos en Ia presente invención (SEQ ID NO 1 con sus estereoisómeros PIECA32D, PIECA32L, SEQ ID NO 2 con sus estereoisómeros PIECA34D y PIECA34L) sobre Ia actividad ECA, cuando esta enzima actúa sobre el sustrato Bradiquinina. Se indican los porcentajes de actividad de Ia ECA para una concentración de péptido en el ensayo de 20 μM, expresándose estos valores como Ia media ± desviación estándar de un número de repeticiones (n).Inhibitory effect of the peptides described in the present invention (SEQ ID NO 1 with its stereoisomers PIECA32D, PIECA32L, SEQ ID NO 2 with its stereoisomers PIECA34D and PIECA34L) on the ECA activity, when this enzyme acts on the Bradykinin substrate. The percentages of activity of the RCT for a peptide concentration in the test of 20 μM are indicated, these values being expressed as the mean ± standard deviation of a number of repetitions (n).
Tabla 4.Table 4
Efecto inhibidor de los péptidos descritos en Ia presente invención (SEQ ID NO 1 con sus estereoi someros PIECA32D, PIECA32L, SEQ ID NO 2 con sus estereoisómeros PIECA34D y PIECA34L) sobre Ia contracción ECA- dependiente con Angiotensina I en arterias aisladas de conejo. Los resultados indican Ia contracción en respuesta a Angiotensina I (1 μM), en % respecto a una respuesta previa en el mismo segmento arterial, y se expresan como Ia media ± desviación estándar de los experimentos realizados en (n) segmentos arteriales.Inhibitory effect of the peptides described in the present invention (SEQ ID NO 1 with its shallow stereois PIECA32D, PIECA32L, SEQ ID NO 2 with its stereoisomers PIECA34D and PIECA34L) on the ECA-dependent contraction with Angiotensin I in isolated rabbit arteries. The results indicate the contraction in response to Angiotensin I (1 μM), in% with respect to a previous response in the same arterial segment, and are expressed as the mean ± standard deviation of the experiments performed in (n) arterial segments.
Significativamente menor respecto a su control, P<0.01.
Significantly lower than its control, P <0.01.
Claims
1. Uso de un hexapéptido bioactivo como inhibidor de Ia enzima conversora de angiotensina I (ECA) in vitro y/o como inhibidores de Ia vasoconstricción ECA- dependiente ex vivo, caracterizado por Ia secuencia de aminoácidos1. Use of a bioactive hexapeptide as an inhibitor of angiotensin I converting enzyme (RCT) in vitro and / or as inhibitors of ex vivo ECA-dependent vasoconstriction, characterized by the amino acid sequence
SEQ ID NO 1 o SEQ ID NO 2.SEQ ID NO 1 or SEQ ID NO 2.
2. Uso de un hexapéptido bioactivo como inhibidor de Ia enzima conversora de angiotensina I (ECA) in vitro y/o como inhibidores de Ia vasoconstricción ECA- dependiente ex vivo, caracterizado por Ia secuencia de aminoácidos2. Use of a bioactive hexapeptide as an inhibitor of angiotensin I converting enzyme (RCT) in vitro and / or as inhibitors of ex vivo ECA-dependent vasoconstriction, characterized by the amino acid sequence
SEQ ID NO 1 o SEQ ID NO 2. porque su secuencia de aminoácidos sólo difiere de SEQ ID NO 1 o SEQ ID NO 2 según Ia reivindicación 1 por cambios conservativosSEQ ID NO 1 or SEQ ID NO 2. because its amino acid sequence only differs from SEQ ID NO 1 or SEQ ID NO 2 according to claim 1 due to conservative changes
3. Uso de un hexapéptido bioactivo como inhibidor de Ia enzima conversora de angiotensina I (ECA) in vitro y/o como inhibidores de Ia vasoconstricción ECA- dependiente ex vivo, según las reivindicaciones 1 y 2 caracterizado por haber sido sintetizado exclusivamente a partir de los estereoisómeros D- de los aminoácidos naturales3. Use of a bioactive hexapeptide as an inhibitor of the angiotensin I converting enzyme (RCT) in vitro and / or as inhibitors of the ECA-dependent vasoconstriction ex vivo, according to claims 1 and 2 characterized by having been synthesized exclusively from D- stereoisomers of natural amino acids
4. Uso de un hexapéptido bioactivo como inhibidor de Ia enzima conversora de angiotensina I (ECA) in vitro y/o como inhibidores de Ia vasoconstricción4. Use of a bioactive hexapeptide as an inhibitor of angiotensin I converting enzyme (RCT) in vitro and / or as vasoconstriction inhibitors
ECA- dependiente ex vivo, según las reivindicaciones 1 y 2 caracterizado por haber sido sintetizado a partir de una mezcla de aminoácidos naturales (estereoisómeros- L) y de estereoisómeros D- de los aminoácidos naturalesECA-dependent ex vivo, according to claims 1 and 2 characterized in that it has been synthesized from a mixture of natural amino acids (L-stereoisomers) and D- stereoisomers of natural amino acids
5. Uso de un péptido bioactivo como inhibidor de Ia enzima conversora de angiotensina I (ECA) in vitro y/o como inhibidores de Ia vasoconstricción5. Use of a bioactive peptide as an inhibitor of angiotensin I converting enzyme (RCT) in vitro and / or as vasoconstriction inhibitors
ECA- dependiente ex vivo, según las reivindicaciones 1 a 4 caracterizado porque su secuencia de aminoácidos contiene las SEQ ID NO 1 o SEQ ID NO 2 según la reivindicación 1 o cambios conservativos en las mismas según las reivindicación 2ECA-dependent ex vivo, according to claims 1 to 4, characterized in that its amino acid sequence contains SEQ ID NO 1 or SEQ ID NO 2 according to claim 1 or conservative changes therein according to claim 2
6. Aditivo, ingrediente o suplemento alimentario funcional caracterizada por comprender al menos alguno de los productos bioactivos con actividad IECA in vitro y/o como inhibidores de Ia vasoconstricción ECA- dependiente ex vivo indicados en las reivindicaciones 1 a 56. Additive, ingredient or functional food supplement characterized by comprising at least some of the bioactive products with ACEI activity in vitro and / or as inhibitors of ex vivo ECA-dependent vasoconstriction indicated in claims 1 to 5
7. Composición farmacéutica caracterizada por comprender al menos alguno de los productos bioactivos con actividad IECA in vitro y/o ex vivo indicados en las reivindicaciones 1 a 57. Pharmaceutical composition characterized by comprising at least some of the bioactive products with ACEI activity in vitro and / or ex vivo indicated in claims 1 to 5
8. Producto alimentario funcional caracterizada por comprender al menos alguno de los productos bioactivos con actividad IECA in vitro y/o como inhibidores de Ia vasoconstricción ECA- dependiente ex vivo indicados en las reivindicaciones 1 a 58. Functional food product characterized by comprising at least some of the bioactive products with ACEI activity in vitro and / or as inhibitors of ex vivo ECA-dependent vasoconstriction indicated in claims 1 to 5
9. Uso de Ia composición farmacéutica según Ia reivindicación 7 en Ia elaboración de un medicamento indicado contra Ia hipertensión.9. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament indicated against hypertension.
10. Uso de aditivo, ingrediente, alimentario funcional según Ia reivindicación 6 en Ia elaboración de un producto alimentario funcional favorable para reducir Ia hipertensión. 10. Use of additive, ingredient, functional food according to claim 6 in the preparation of a favorable functional food product to reduce hypertension.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| ES200600880A ES2320057B1 (en) | 2006-04-05 | 2006-04-05 | USE OF EXAPEPTIDES TO PREPARE INHIBITING FORMULATIONS OF THE CONVERSING ENGINE OF ANGIOTENSIN I. |
| ESP200600880 | 2006-04-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2007113368A1 true WO2007113368A1 (en) | 2007-10-11 |
Family
ID=38563146
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/ES2007/070071 WO2007113368A1 (en) | 2006-04-05 | 2007-04-09 | Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products |
Country Status (2)
| Country | Link |
|---|---|
| ES (1) | ES2320057B1 (en) |
| WO (1) | WO2007113368A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2387435B1 (en) * | 2011-02-04 | 2013-07-31 | Consejo Superior De Investigaciones Cientificas (Csic) | USE OF HEPTAPEPTIDES FOR HYPERTENSION CONTROL |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2191516A1 (en) * | 2000-07-28 | 2003-09-01 | Consejo Superior Investigacion | Anti-fungal antibiotic of peptide kind is inhibitor of germination and growth of phytopathogenic fungi |
-
2006
- 2006-04-05 ES ES200600880A patent/ES2320057B1/en not_active Expired - Fee Related
-
2007
- 2007-04-09 WO PCT/ES2007/070071 patent/WO2007113368A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2191516A1 (en) * | 2000-07-28 | 2003-09-01 | Consejo Superior Investigacion | Anti-fungal antibiotic of peptide kind is inhibitor of germination and growth of phytopathogenic fungi |
Non-Patent Citations (2)
| Title |
|---|
| CENTENO J.M. ET AL.: "Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction", AGRICULTURAL AND FOOD CHEMISTRY, vol. 54, no. 15, 26 July 2006 (2006-07-26), pages 5323 - 5329 * |
| MIGUEL M. ET AL.: "Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis", JOURNAL OF FOOD PROTECTION, vol. 67, no. 9, 2004, pages 1914 - 1920 * |
Also Published As
| Publication number | Publication date |
|---|---|
| ES2320057A1 (en) | 2009-05-18 |
| ES2320057B1 (en) | 2010-03-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Guang et al. | Three key proteases–angiotensin-I-converting enzyme (ACE), ACE2 and renin–within and beyond the renin-angiotensin system | |
| Forghani et al. | Purification and characterization of angiotensin converting enzyme-inhibitory peptides derived from Stichopus horrens: Stability study against the ACE and inhibition kinetics | |
| Fernández-Musoles et al. | Antihypertensive effects of lactoferrin hydrolyzates: Inhibition of angiotensin-and endothelin-converting enzymes | |
| ES2331312T3 (en) | BIOLOGICALLY NOT DEGRADABLE PEPTIDE, INHIBITOR OF THE ENZYME THAT CONVERTS ANGIOTENSIN, PHARMACO AND FUNCTIONAL FOOD. | |
| Yamada et al. | Novel angiotensin I-converting enzyme inhibitory peptide derived from bovine casein | |
| Lee et al. | Purification of novel angiotensin converting enzyme inhibitory peptides from beef myofibrillar proteins and analysis of their effect in spontaneously hypertensive rat model | |
| AU2012257774C1 (en) | High-affinity, dimeric inhibitors of PSD-95 as efficient neuroprotectants against ischemic brain damage and for treatment of pain | |
| US8815806B2 (en) | Yellow pea seed protein-derived peptides | |
| JP2004000208A5 (en) | ||
| US20080242837A1 (en) | Peptide compositions | |
| WO2018006667A1 (en) | Antihypertensive peptide and antihypertensive protein and use thereof | |
| KR20140026295A (en) | Pharmaceutical composition containing peptide with angiotensin-i converting enzyme inhibitory activity for preventing or treating cardiovascular disease | |
| ES2286398T3 (en) | USE OF AT LEAST ONE PEPTIDE OF THE CASEINA "ALPHA S2" WITH INHIBITING ACTIVITY OF I'ACE FOR THE PREPARATION OF MEDICINES AND FOODS. | |
| ES2606954B1 (en) | PROCEDURE FOR OBTAINING A HYDROLYZED CHICKEN FOOT CLAWS WITH ANTI-HANDLING ACTIVITY, HYDROLYZED OBTAINED AND PEPTIDES CONTAINED | |
| Shukla et al. | Current trends and applications of food-derived antihypertensive peptides for the management of cardiovascular disease | |
| ES2320057B1 (en) | USE OF EXAPEPTIDES TO PREPARE INHIBITING FORMULATIONS OF THE CONVERSING ENGINE OF ANGIOTENSIN I. | |
| ES2321358B1 (en) | USE OF PEPTIDES DERIVED FROM LACTOFERRINE TO PREPARE INHIBITING FORMULATIONS OF THE CONVERSING ENZYME OF ANGIOTENSIN I. | |
| US20140094415A1 (en) | High-affinity, dimeric inhibitors of psd-95 as efficient neuroprotectants against ischemic brain damage and for treatment of pain | |
| JP4278028B2 (en) | Peptide having inflammatory cytokine production inhibitory activity | |
| ES2387435B1 (en) | USE OF HEPTAPEPTIDES FOR HYPERTENSION CONTROL | |
| CA2801038A1 (en) | Lactadherin-derived peptides as antiviral agents | |
| JP2014500245A (en) | peptide | |
| KR20210093958A (en) | Analogs targeting mitochondrial diseases | |
| JP2003024012A (en) | Angiotensin i converting enzyme inhibitor and antihypertensive functional food | |
| KR101648330B1 (en) | Composition containing collagen hydrolyzate having enhanced proliferation of osteoblast |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 07730509 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 07730509 Country of ref document: EP Kind code of ref document: A1 |