ES2320057B1 - USE OF EXAPEPTIDES TO PREPARE INHIBITING FORMULATIONS OF THE CONVERSING ENGINE OF ANGIOTENSIN I. - Google Patents
USE OF EXAPEPTIDES TO PREPARE INHIBITING FORMULATIONS OF THE CONVERSING ENGINE OF ANGIOTENSIN I. Download PDFInfo
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- ES2320057B1 ES2320057B1 ES200600880A ES200600880A ES2320057B1 ES 2320057 B1 ES2320057 B1 ES 2320057B1 ES 200600880 A ES200600880 A ES 200600880A ES 200600880 A ES200600880 A ES 200600880A ES 2320057 B1 ES2320057 B1 ES 2320057B1
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- Organic Chemistry (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
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Abstract
Uso de hexapéptidos para preparar formulaciones inhibidoras de la enzima conversora de angiotensina I.Use of hexapeptides to prepare formulations angiotensin I converting enzyme inhibitors I.
La invención consiste en la utilización de hexapétidos sintéticos como productos bioactivos. Se trata de un péptido de seis residuos de aminoácidos como inhibidores efectivos de la enzima conversora de la angiotensina (ECA). Los péptidos objeto de la patente se pueden obtener químicamente, biotecnológicamente. Son de especial interés los sintetizados exclusivamente a partir de los estereoisómeros D- de los aminoácidos naturales ya que así se consigue más estabilidad además de su actividad inhibidora de la enzima convertidora de la angiotensina in vitro y/o ex vivo, medida como la disminución de la contracción de arterias carótidas de conejo inducida por exposición a angiotensina I. Estos productos nutracéuticos, ya sea como péptido biactivos, son tanto útiles para la industria alimentaría como para la farmacéutica.The invention consists in the use of synthetic hexapétides as bioactive products. It is a peptide of six amino acid residues as effective inhibitors of the angiotensin converting enzyme (RCT). The peptides object of the patent can be obtained chemically, biotechnologically. Of particular interest are those synthesized exclusively from the D- stereoisomers of natural amino acids since this provides more stability in addition to its inhibitory activity of the angiotensin converting enzyme in vitro and / or ex vivo , measured as the decrease in The contraction of rabbit carotid arteries induced by exposure to angiotensin I. These nutraceutical products, either as biactive peptides, are both useful for the food industry and for the pharmaceutical industry.
Description
Uso de hexapéptidos para preparar formulaciones inhibidoras de la enzima conversora de angiotensina I.Use of hexapeptides to prepare formulations angiotensin I converting enzyme inhibitors I.
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Control de la hipertensión; Industria agroalimentaria; Ingredientes bioactivos; Farmacología; Alimentos funcionales.Control of hypertension; Industry agrifood; Bioactive ingredients; Pharmacology; Foods functional.
La hipertensión, que consiste en un aumento de la presión sanguínea superior a la deseable para la 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 ictus, constituye, después de las enfermedades isquémicas cardíacas y del cáncer, la tercera causa de mortalidad y la 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 la oclusión aguda por un trombo ("trombosis") o un émbolo ("embolia") de una de las principales arterias cerebrales, lo que origina un descenso en la perfusión sanguínea ("isquemia") y consiguiente necrosis ("infarto") de la región cerebral irrigada por dicha arteria. El resto de accidentes cerebrovasculares (15%) son de tipo "hemorrágico", originados por la rotura de un vaso sanguíneo en el propio parénquima cerebral ("hemorragia intracerebral") o en la superficie cerebral ("hemorragia subaracnoidea").Hypertension, which consists of an increase in blood pressure higher than 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 stroke , constitutes, after ischemic heart disease and cancer, the third leading cause of death 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. which causes a decrease in blood perfusion ("ischemia") and consequent necrosis ("infarction") of the brain region irrigated by said artery. The remaining strokes (15%) are of the "hemorrhagic" type, caused by the rupture of a blood vessel in the brain parenchyma itself ("intracerebral hemorrhage") or on the cerebral surface ("subarachnoid hemorrhage").
De lo dicho anteriormente se desprende que en el desarrollo de estas enfermedades falla la correcta regulación de la presión arterial sistémica, en la que interviene un complejo sistema regulador llamado sistema renina-angiotensina (SRA), y del que forman parte la renina, la enzima conversora de la angiotensina (ECA), la aldosterona, y las angiotensinas I y II.From the aforementioned it follows that in the development of these diseases fails the correct regulation of the systemic blood pressure, in which a complex is involved regulatory system called system renin-angiotensin (SRA), and of which the renin, the angiotensin converting enzyme (RCT), the aldosterone, and angiotensins I and II.
Este SRA es un sistema hormonal circulante, y concretamente la renina y la ECA son dos peptidasas que forman parte del mismo y que actúan secuencialmente sobre una serie de pequeños péptidos, reguladores en última instancia de la presión sanguínea. En los seres humanos, la renina se libera en el riñón y la ECA se encuentra presente principalmente en las células endoteliales vasculares, en los pulmones, en los riñones y en el cerebro.This SRA is a circulating hormonal system, and specifically renin and RCT are two peptidases that form part of it and acting sequentially on a series of small peptides, ultimately pressure regulators blood In humans, renin is released in the kidney and ECA is present mainly in cells vascular endothelials, in the lungs, in the kidneys and in the brain.
Este sistema renina-angiotensina se activa en determinadas situaciones mediante la actuación de la 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 la ECA en angiotensina II al separarse el dipéptido a partir de su extremo C-terminal. La angiotensina II generada es un potente vasoconstrictor que ejerce su acción tras la unión a sus receptores específicos denominados "receptores AT_{1}". Dicha acción se traduce en la contracción de los vasos sanguíneos que como consecuencia produce un aumento de la presión sanguínea. Además de contribuir a la formación de la angiotensina II, la 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 it is activated in certain situations by acting on the renin on a precursor peptide called angiotensinogen (from liver origin), which becomes the decapeptide angiotensin I. This angiotensin I, inactive from the point of biological view, in turn is transformed by action of the ECA in angiotensin II when the dipeptide is separated from its end C-terminal The angiotensin II generated is a powerful vasoconstrictor that exerts its action after binding to its specific receptors called "AT1 receptors". Bliss action results in the contraction of blood vessels that as a result it causes an increase in blood pressure. In addition to contributing to the formation of angiotensin II, the RCT It also acts on another circulating peptide, the nonapeptide called bradykinin, a potent vasodilator agent that loses this characteristic when hydrolyzed.
Por todo lo expuesto anteriormente, la interferencia farmacológica con el SRA podría tener efectos beneficiosos en el tratamiento de los desórdenes vasculares asociados con la hipertensión. La inhibición de la actividad ECA permitiría disminuir la formación de angiotensina II además de reducir la pérdida de funcionalidad de la bradiquinina, evitando de esta manera la acción vasoconstrictora de la primera y potenciando la acción vasodilatadora de la segunda. A este respecto se ha puesto de manifiesto en numerosos estudios la eficacia de los inhibidores de ECA reduciendo la morbilidad y la mortalidad en pacientes con fallo cardíaco, síndrome cardio-metabólico y diabetes.For all the above, the Pharmacological interference with the ARS could have effects beneficial in the treatment of vascular disorders associated with hypertension. ACE activity inhibition would allow to reduce the formation of angiotensin II in addition to reduce the loss of bradykinin functionality, avoiding this way the vasoconstrictor action of the first and enhancing the vasodilator action of the second. In this regard it has revealed in numerous studies the effectiveness of ACE inhibitors reducing morbidity and mortality in patients with heart failure syndrome cardio-metabolic and diabetes.
A pesar de su demostrada eficacia en el tratamiento de las enfermedades cardiovasculares asociadas a la hipertensión, los fármacos inhibidores del ECA disponibles en la actualidad no pueden considerarse la 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 la síntesis de angiotensina II ya que ésta sigue otras vías de síntesis que no dependen del ECA. Es necesario, por lo tanto, encontrar nuevos inhibidores del ECA con mayor especificidad y que puedan ser co-administrados 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 la 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, lo cual produciría un efecto positivo tanto sobre dicho tratamiento como sobre la prevención de los síntomas inherentes a la hipertensión.Despite its proven effectiveness in the treatment of cardiovascular diseases associated with hypertension, the ACE inhibitor drugs available in the Currently they cannot be considered the definitive option. For his lack of specificity these drugs are not well tolerated by some patients with side effects undesirable such as dry cough and angioedema; besides, they don't block completely the synthesis of angiotensin II as it continues other routes of synthesis that do not depend on the RCT. It is necessary, for therefore, find new ACE inhibitors with higher specificity and that can be co-administered with other drugs, such as "receptor blockers of angiotensin ", for the optimal treatment of disorders vascular of hypertensive origin linked to the ARS at the same time as Minimize the aforementioned side effects. Even and according to characteristics of the selected inhibitors, could get a more natural approach to the treatment by adding said food inhibitors, which would produce an effect positive about both treatment and prevention of the symptoms inherent in hypertension.
Hasta la fecha se han publicado numerosos trabajos bibliográficos relacionados con la 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, M.J., Wu, M.T., Taub, D., Peterson, E.R., Ikeler, T.J., Broeke, J.Ten., Payne, L.G., Ondeyka, D.L., Thorsett, E.D., Greenlee, W.J., Lohr, N.S., Hoffsommer, R.D., Joshua, H., Ruyle, W.V., Rothrock, J.W., Aster, S.D., Maycock, A.L., Robinson, F.M., Hirschmann, R., Sweet, C.S., Ulm, E.H., Gross, D.M., Vassil, T.C. y Stone, C.A. (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, Z.P., 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-Ile-His-Pro [\beta-CN f(47-51)]. Study of the peptide forms synthesized by different methods. Journal of Agricultural 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-Induced 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 Agricultural and Food Chemistry 51 (6), 1706-1709].To date, numerous bibliographical works related to ACE inhibition 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, Ulm, 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-Ile-His-Pro [β-CN f (47-51)]. Study of the peptide forms synthesized by different methods. Journal of Agricultural 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-Induced 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 Agricultural 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 la determinación de su secuencia. De esta manera se han podido sintetizar la 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 I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition 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 I-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-Leppälä, 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 \alpha-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 I-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 I-converting enzyme inhibitor dipeptides derived from Allium sativum L (garlic). Journal of Nutritional Biochemistry 9, 415-419; Pihlanto-Leppälä, 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 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. 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 they have been able to synthesize most of them 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 I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition 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 I-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, TL., 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-Leppälä, 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 I-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 I-converting enzyme inhibitor dipeptides derived from Allium sativum L (garlic). Journal of Nutritional Biochemistry 9, 415-419; Pihlanto-Leppälä, 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 la presente invención se describe la secuencia de aminoácidos de otros péptidos, distinta a la de los mencionados anteriormente, caracterizados por tener actividad inhibidora de la ECA. Dicha inhibición de la actividad ECA se manifiesta en ensayos realizados in vitro, midiendo la inhibición de la conversión de sustratos artificiales (Hipuril-Histidil-Leucina) y naturales (Angiotensina I) mediada por ECA, y en ensayos realizados ex vivo, midiendo la disminución de la contracción de arterias basilares ó carótidas de conejo inducida por exposición a Angiotensina I.In the present invention the amino acid sequence of other peptides, other than those mentioned above, characterized by having ACE inhibitory activity is described. Said inhibition of 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 rabbit contraction of basilar or carotid arteries induced by exposure to Angiotensin I.
La presente invención está relacionada con los sectores farmacológico y de la industria agroalimentaria y consiste en la identificación y caracterización de unos determinados péptidos (llamados PIECA, Péptidos Inhibidores de la Enzima Conversora de Angiotensina) como inhibidores efectivos de la Enzima Conversora de la Angiotensina (ECA) que se halla implicada en la formación del compuesto Angiotensina II que es un vasoconstrictor responsable, entre otras causas/mecanismos, de la 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 la solicitud de patente ES200001973 como PAF32 y PAF34 respectivamente. La actividad inhibidora de los péptidos se manifiesta por una reducción de la actividad ECA determinada en ensayos in vitro, así como por una reducción de la contracción ECA dependiente ex vivo empleando segmentos de arterias. Se demuestra que la 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 la potencial utilización de los péptidos inhibidores de ECA como compuestos bioactivos para el control de la hipertensión. En un ejemplo particular, se describe la 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 riñón de cerdo y ex vivo empleando segmentos de arterias carótida y basilar de conejo.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 Angiotensin Conversion Enzyme (RCT) ) that is involved in the formation of the compound Angiotensin II which is a vasoconstrictor responsible, among other causes / mechanisms, for hypertension. The amino acid residue sequences of the identified peptides are the following, from the amino terminal end to the carboxy terminal end: (PIECA32 peptide; SEQ ID NO 1) and (PIECA34 peptide, SEQ ID NO 2) described in the application of patent ES200001973 as PAF32 and PAF34 respectively. The inhibitory activity of the peptides is manifested by a reduction of the ECA activity determined in in vitro assays , as well as by a reduction of the ECA-dependent contraction ex vivo using arterial segments. It is shown that the inhibitory activity of the peptides corresponds to a characteristic amino acid sequence, since other peptides related to the above and a similar, but not identical, amino acid residue sequence do not exhibit such activity. The potential use of 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 vitro conditions using purified ECA from kidney of pig and ex vivo using carotid and basilar rabbit artery segments.
En la presente invención se describe la identificación y caracterización de nuevos péptidos con actividad de inhibición de la Enzima Conversora de Angiotensina (ECA), implicada en los mecanismos de control de la presión arterial. En un ejemplo particular, se describe la utilización de dichos péptidos como inhibidores de ECA mediante el empleo de sustratos artificiales como el Hipuril-Histidil-Leucina (HHL) ó naturales como la Angiotensina I, así como su efecto inhibidor sobre la contracción ECA-dependiente de segmentos de arterias de conejo.In the present invention the identification and characterization of new peptides with activity of inhibition of Angiotensin Conversion Enzyme (RCT), involved in the mechanisms of blood pressure control. In a particular example describes the use of said peptides as ACE inhibitors through the use of substrates artificial like the Hipuril-Histidil-Leucine (HHL) or natural as Angiotensin I, as well as its inhibitory effect on ECA-dependent contraction of segments of Rabbit arteries
Los inhibidores descritos en la 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, Yong-Hui Shi y Sundar Shrestha (2004). Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition Research 24, 469-486; Dziuba, J., Minkiewicz, P., Nalecz, D. y Iwaniak, A. (1999). Database 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 Gonzalez, C. F. (1997)]. En un ejemplo particular de realización de la 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 la presente invención [Fields, G. B. y Noble, R. L. (1990). Solid 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 la 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 la propiedad de conferir a los péptidos resultantes mayor estabilidad y resistencia a la degradación por proteasas presentes en los fluidos biológicos.The inhibitors described in the present invention are peptides with a six amino acid sequence characteristic SEQ ID NO 1 (PIECA32 peptide) and SEQ ID NO 2 (PIECA34 peptide), Figure 1, and other than the previously known ACE inhibitor peptides [Guan- Hong Li, Guo-Wei Le, Yong-Hui Shi and Sundar Shrestha (2004). Angiotensin I-converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects. Nutrition Research 24 , 469-486; Dziuba, J., Minkiewicz, P., Nalecz, D. and Iwaniak, A. (1999). Database 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 Gonzalez, 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). Solid 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 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 degradation by proteases present in biological fluids.
En la presente invención se describen ensayos experimentales que ilustran la 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 riñón de cerdo y tres sustratos distintos, uno artificial denominado HHL y dos naturales como son la Angiotensina I y la Bradiquinina. El primero de los sustratos permite llevar a cabo una serie de ensayos encaminados a conocer la 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.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, under experimental conditions in vitro , using purified pig kidney ACE and three different substrates, one artificial called HHL and two natural ones such as Angiotensin I and Bradykinin. The first of the substrates allows to carry 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.
La actividad inhibidora de los péptidos PIECA descritos en la presente invención se manifiesta con una reducción de la actividad de la ECA al llevar a cabo los ensayos in vitro en las condiciones establecidas, como queda demostrado mediante los ensayos experimentales descritos en la 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 la mencionada actividad inhibidora.The inhibitory activity of the PIECA peptides described in the present invention is manifested by a reduction in the activity of the RCT in carrying out the in vitro tests under the established conditions, as demonstrated by the experimental assays described in the present invention. These tests also demonstrate that PIECAs 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 la presente invención también se manifiesta con una reducción de la contracción ECA-dependiente de arterias de conejo, carótida y basilar, inducida mediante la adición de Angiotensina I en ensayos ex vivo.The inhibitory activity of the PIECA peptides described in the present invention is also manifested by a reduction in 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 la presente invención, es obvio para todo aquel experto en el tema su potencial utilización como aditivos alimentarios, compuestos ó fármacos de utilidad en la prevención ó tratamiento de enfermedades que tengan como causa ó sintomatología la hipertensión arterial.Considering the properties of PIECA described in the present invention, it is obvious to all subject matter expert its potential use as additives food, compounds or drugs useful in prevention or treatment of diseases that have as cause or symptomatology high blood pressure
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Es obvio para todo aquel experto en el área de la presente invención el interés, diseño y desarrollo de estrategias derivadas de la biotecnología, que incluyen la metodología del ADN recombinante y de la transformación genética de organismos, que pueden ser utilizadas para la producción y utilización de los PIECA32L y PIECA34L descritos en la presente invención y sus derivados para los fines descritos. En un ejemplo de realización de la invención se explica la producción a gran escala de los péptidos mediada por organismos transformados genéticamente.It is obvious to all those experts in the area of the present invention the interest, design and development of strategies derived from biotechnology, which include the recombinant DNA methodology and genetic transformation of organisms, which can be used for production and use of the PIECA32L and PIECA34L described herein invention and its derivatives for the purposes described. In an example of realization of the invention the production is explained in large peptide scale mediated by transformed organisms genetically
Figura 1. Secuencia de aminoácidos de los hexapéptidos SEQ ID NO 1 con sus correspondientes estereoisómeros PIECA32D, PIECA32L, y SEQ ID NO 2 con sus estereoisómeros PIECA34D, PIECA34L (descritos como inhibidores de ECA en la presente invención) y de 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 la izquierda) al extremo carboxi terminal (a la derecha). Los péptidos se encuentran acetilados en su extremo amino terminal (Ac-) y amidados en su extremo carboxi terminal (Am-).Figure 1. Amino acid sequence of hexapeptides SEQ ID NO 1 with their corresponding stereoisomers PIECA32D, PIECA32L, and SEQ ID NO 2 with its PIECA34D stereoisomers, PIECA34L (described as ACE inhibitors herein invention) and of P26D SEQ ID NO 3 and P36D SEQ ID NO 4 (used as negative controls in the described trials). The sequences are written from the amino terminal end (on the left) to carboxy terminal end (on the right). The peptides are found acetylated at its amino terminal (Ac-) end and amidated at its carboxy terminal end (Am-).
Figura 2. Efecto de la concentración de PIECA32L sobre la actividad de la Enzima Conversora de Angiotensina I.Figure 2. Effect of the concentration of PIECA32L on the activity of Angiotensin I Conversion Enzyme I.
Figura 3. Efecto de la concentración de PIECA34L sobre la actividad de la Enzima Conversora de Angiotensina I.Figure 3. Effect of PIECA34L concentration on the activity of Angiotensin I Conversion Enzyme I.
Un ejemplo de realización de la invención.An example of embodiment of the invention.
1. Síntesis de péptidos. Los péptidos caracterizados y analizados en la presente invención (Figura 1: P26D SEQ ID NO 3, SEQ ID NO 1 con sus estereoisómeros PIECA32D, PIECA32L, SEQ ID NO 2 con sus estereoisómeros PIECA34D, PIECA34L y P36D SEQ ID NO 4) se sintetizaron químicamente sobre fase sólida siguiendo procedimientos habituales que utilizan el grupo N-(9-fluorenyl)methoxycarbonyl (Fmoc) para la protección del grupo \alpha-amino de los aminoácidos constituyentes [Fields, G. B. y Noble, R. L. (1990). Solid 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 (NH_{2}), como consecuencia del procedimiento de síntesis. Después de la 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 laser desorption/ionization time-of-flight). Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de la presente invención.1. 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 and P36D SEQ ID NO 4) were chemically synthesized on solid phase following usual procedures using the N- (9-fluorenyl) methoxycarbonyl (Fmoc) group for the protection of the α-amino group of the constituent amino acids [Fields, GB and Noble, RL (1990). Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. International Journal of Peptide and Protein Research 34 , 161-214]. 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 synthesis, the peptides were purified by RP-HPLC ( reversed phase high performance liquid chromatography ) and their identity was confirmed by MALDI-TOF mass spectrometry. 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 la producción de los péptidos L compuestos por aminoácidos naturales (estereoisómeros L-) descritos en la presente invención mediante estrategias derivadas de la biotecnología. Es obvio, para toda aquella persona experta en el área de conocimiento, que la producción de los péptidos mediante procedimientos biotecnológicos, que incluyen las metodologías del ADN recombinante y de la 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 la aplicabilidad industrial de la presente invención. En el supuesto de la producción mediante biotecnología, la 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. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY], Todos estos procedimientos son habituales para toda aquella persona experta en el área de conocimiento de la presente invención.It is also possible to produce L-peptides composed of natural amino acids (L- stereoisomers) described in the present invention by biotechnology derived strategies. It is obvious, for all those experts in the area of knowledge, that the production of the peptides by means of 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 such production is an important aspect in the context of the industrial applicability of the present invention. In the case of biotechnology production, the peptide sequence 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. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY], All these procedures are common for all those skilled in the area of knowledge of the present invention.
2. Ensayos in vitro de inhibición de la actividad ECA sobre el sustrato artificial HHL. En estos ensayos, la capacidad inhibidora de los péptidos se determinó midiendo por HPLC (del inglés high performance liquid chromatography) el ácido hipúrico resultante de la hidrólisis del sustrato artificial HHL (Hipuril-Histidil-Leucina) basándose en el método propuesto en la 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 \mul y está constituida por 50 \mul de HHL 25 mM en tampón Tris HCI 200 mM pH 8.3 con NaCl 600 mM y ZnCl_{2} 10 \muM, 75 \mul de una solución de ACE en el mismo tampón que corresponden a 1.5 mU de actividad, y 100 \mul de péptido (disuelto en tampón MOPS 10 mM pH 7) a diferentes concentraciones según la concentración final deseada en el ensayo. El enzima y el inhibidor se preincuban durante 15 minutos a 37ºC y a continuación se añade el sustrato incubándose el conjunto 30 minutos a dicha temperatura. La reacción se detiene añadiendo 25 \mul de HCl 6M. Para la determinación cromatográfica del ácido hipúrico liberado se utiliza una columna de fase inversa C18, la elución se lleva a cabo empleando un gradiente de acetonitrilo en agua con TFA 0.05% y se determina el ácido hipúrico midiendo la absorbancia a 228 nm. Los resultados aparecen en la 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 la 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.2. In vitro assays for inhibition of ECA activity on the artificial HHL substrate . In these assays, 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-Histidyl-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 constituted by 50 µL of 25 mM HHL in 200 mM Tris buffer pH 8.3 with 600 mM NaCl and 10 µM ZnCl 2, 75 µl of a solution of ACE in the same buffer corresponding to 1.5 mU of 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 HCl. For the chromatographic determination of the released hipuric acid a C18 reverse phase column is used, elution is carried out using a gradient of acetonitrile in water with 0.05% TFA and the hippuric acid is determined by measuring the absorbance at 228 nm. The results appear in Table 1, showing a significant inhibition for the PIECA 32 SEQ ID NO 1 and PIECA 34 SEQ ID NO 2 peptides, the inhibition for the PIECA 32 both L and D. being greater. The P 26D SEQ ID NO 3 and P 36D SEQ ID NO 4 did not show a significant inhibition.
3. Ensayos in vitro de inhibición de la actividad ECA sobre el sustrato natural Angiotensina I. El protocolo experimental descrito en el apartado 2 se repitió empleando como sustrato la Angiotensina I (cantidad final en el ensayo 20 \mug). 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 TFA 0.1% y midiendo la absorbancia a 214 nm. Los resultados obtenidos se muestran en la 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 PIECA34.3. In vitro assays for inhibition of ECA activity on the natural substrate Angiotensin I. The experimental protocol described in section 2 was repeated using Angiotensin I as a substrate (final amount in the 20 µg test). The resulting reaction product, Angiotensin II, was determined chromatographically using a C18 reverse phase column, a gradient of acetonitrile in water with 0.1% TFA 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.
Con este sustrato natural los péptidos con configuración D tienen una capacidad inhibitoria inferior a la conseguida con HHL.With this natural substrate the peptides with D configuration have an inhibitory capacity less than achieved with HHL.
4. Ensayos in vitro de inhibición de la actividad ECA sobre el sustrato natural Angiotensina I para el cálculo del IC_{50}. 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 \muM para los ensayos con SEQ ID NO 2 PIECA34L y 0, 2.5, 5, 10, 20, 40, y 80 \muM 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 la media y su desviación. La representación gráfica del valor medio correspondiente a la actividad residual para cada una de las concentraciones de péptidos ensayada se muestran en las figuras 2 y 3.4. In vitro assays of inhibition of ECA activity on the natural substrate Angiotensin I for the calculation of IC 50 . 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 were carried out with all concentrations, calculating for each of them the mean and its deviation. The graphical representation of the mean value corresponding to the residual activity for each of the peptide concentrations tested are shown in Figures 2 and 3.
A partir de estos resultados se calcularon los IC_{50} para SEQ ID NO 1 PIECA 32L y SEQ ID NO 2 PIECA 34L siendo estos de 10.7 y 8.1 \muM respectivamente.From these results, the IC_ {50} for SEQ ID NO 1 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 la actividad ECA sobre el sustrato natural Bradiquidina. El protocolo experimental descrito en el apartado 3 se repitió utilizando como sustrato la Bradiquinina. Se empleó la misma cantidad de sustrato, 20 \mug en el ensayo y se detectó como producto final el fragmento de Bradiquinina 1-5. Los resultados obtenidos se muestran en la 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 for inhibition of ECA activity on the natural substrate Bradiquidine . The experimental protocol described in section 3 was repeated using Bradiquinine as a substrate. 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 la contracción de arterias aisladas. La preparación experimental consistió en obtener segmentos cilíndricos (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 la pared vascular. El medio (solución Ringer-Locke) en el que se hallan inmersos los segmentos arteriales se mantiene termostatizado a 37ºC y continuamente burbujeado con una mezcla gaseosa de 95% O_{2} y 5% CO_{2} que le confiere un pH de 7.3-7.4. Los experimentos comienzan tras un periodo de 30-60 min necesario para alcanzar la estabilización en el tono pasivo de 0.5 g para la arteria basilar y de 2 g para la carótida. Tras comprobar la viabilidad de los segmentos arteriales mediante contracción con una solución despolarizante (Ringer-Locke 50 mM KCl), cada segmento arterial se somete a una primera contracción ECA-dependiente con Angiotensina I (1 \muM). 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 \muM 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 la Tabla 4 y ponen de manifiesto efectos inhibitorios significativos en los casos de SEQ ID NO 1 PIECA32D y PIECA32L sobre arteria carótida.6. Ex vivo assays for inhibition of contraction of isolated arteries . The experimental preparation consisted of obtaining cylindrical segments (3 mm) of isolated arteries (basilar and carotid arteries of New Zealand white rabbit), which were placed in an organ bath designed to record the changes of isometric tension in the vascular wall. The medium (Ringer-Locke solution) in which the arterial segments are immersed is kept thermostated at 37 ° C and continuously bubbled with a gaseous mixture of 95% O2 and 5% CO2 which gives it 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 KCl), 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 cases of SEQ ID NO 1 PIECA32D and PIECA32L on the carotid artery.
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<110> Consejo Superior de Investigaciones Científicas<110> Higher Research Council Scientists
\hskip1cmUniversidad de Valencia
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<120> HEXAPEPTIDOS INHIBIDORES DE LA ENZIMA CONVERSORA DE ANGIOTENSINA I<120> INHIBITING HEXAPEPTIDES OF THE CONVERSORA OF ANGIOTENSINA I CONVERSORA
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PCT/ES2007/070071 WO2007113368A1 (en) | 2006-04-05 | 2007-04-09 | Use of hexapeptides for preparing angiotensin 1 converting enzyme medicinal products |
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Title |
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MIGUEL, M. et al. "{}Angiotensin I-converting enzyme inhibitory activity of peptides derived from egg white proteins by enzymatic hydrolysis"{}. JOURNAL OF FOOD PROTECTION. 2004, Vol. 67, N$^{o}$ 9, páginas 1914-1920, todo el documento. * |
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