WO2001068113A1 - Peptides antihypertenseurs - Google Patents

Peptides antihypertenseurs Download PDF

Info

Publication number
WO2001068113A1
WO2001068113A1 PCT/US2001/007479 US0107479W WO0168113A1 WO 2001068113 A1 WO2001068113 A1 WO 2001068113A1 US 0107479 W US0107479 W US 0107479W WO 0168113 A1 WO0168113 A1 WO 0168113A1
Authority
WO
WIPO (PCT)
Prior art keywords
asn
pro
lys
met
subject
Prior art date
Application number
PCT/US2001/007479
Other languages
English (en)
Inventor
Zhonghong Guan
Foe Siong Tjoeng
Wei Li
Kathy Mandrell
Min Liu
Xiangna Chen
Original Assignee
Monsanto Technology Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Monsanto Technology Llc filed Critical Monsanto Technology Llc
Priority to AU2001245537A priority Critical patent/AU2001245537A1/en
Publication of WO2001068113A1 publication Critical patent/WO2001068113A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • C07K5/06043—Leu-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
    • C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to short oligopeptides, two to four amino acid residues in length, derived from food protein digest products or chemically synthesized, which exhibit antihypertensive activity. These novel antihypertensive oligopeptides may be used as active ingredients in pharmaceutical compounds, dietary supplements and food ingredients.
  • the present invention also relates to methods of treatment and prophylaxis of hypertension and disease states such as myocardial infarction, left ventricular systolic dysfunction, diabetes mellitus, progressive renal failure and congestive heart failure caused by or associated with hypertension using such novel antihypertensive oligopeptides.
  • Hypertension generally defined as abnormally increased blood pressure, is a disease which is estimated to affect approximately 50 million people in the United States and 170 million people worldwide. It is clinically recognized as an elevation of systolic arterial blood pressure of 140 mm Hg or greater and an elevation of diastolic arterial blood pressure of 90 mm Hg or higher. Hypertension may be caused by a number of different physiological mechanisms and no single or specific cause is known for the hypertension referred to as primary (essential) hypertension.
  • Primary hypertension is also associated with various disease states for example, obesity, sleep apnea, physical inactivity, alcohol intake, smoking, diabetes mellitus, polycythemia and gout. Secondary forms of hypertension may arise from oral contraceptive use and parenchymal renal disease, for example, renovascular hypertension may be caused by atherosclerotic disease, tumors (renin-secretory tumors), Cushing's Syndrome, heart surgery and pregnancy. Chronic hypertension and renal disease during pregnancy may progress into eclampsia which is a primary cause of fetal death. Furthermore, primary hypertension is a relatively common disease state in humans and has been associated with the early onset of coronary disease, kidney failure and stroke. Primary hypertension is generally asymptomatic and has been termed a silent killer.
  • angiotensinogen is secreted by the liver and is cleaved by renin to yield the biologically inactive angiotensin I (Nsp-Arg-Nal-Tyr-Ile-His-Pro-Phe-His-Leu) (SEQ ID NO: 1).
  • angiotensin II Asp-Arg-Nal-Tyr-Ile-His- Pro-Phe
  • ACE angiotensin converting enzyme
  • angiotensin II increases blood pressure by contracting the smooth muscles of the blood vessel walls and promoting secretion of aldosterone by action on the adrenal cortex.
  • angiotensin II further acts to increase blood pressure by decomposing and inactivating bradykinin, a peptide which lowers blood pressure.
  • ACE not only produces a peptide which increases blood pressure (angiotensin II) but also decomposes and inactivates a peptide which decreases blood pressure (bradykinin), inhibiting the activity of ACE is an effective way of depressing blood pressure.
  • antihypertensive compounds may reduce blood pressure through other mechanisms or through a combination of physiologic mechanisms.
  • hypertension is often treated with the use of anti-hypertensive pharmaceutical products including calcium channel blockers, beta blockers, diuretics, alpha blockers, central alpha agonists, angiotensin II antagonists and ACE inhibitors, such as Captopril and Enalapril.
  • While such pharmaceutical products are effective in decreasing elevated blood pressure, they often have negative side effects associated with their use. Some common side effects include increased cholesterol and glucose levels associated with diuretics, sedation and dry mouth associated with central alpha agonists, bronchospasm and bradycardia associated with beta blockers, and conduction defects associated with calcium agonists. Furthermore, sudden blood pressure elevation and/or other unsafe side effects may occur after one ceases taking such antihypertensive pharmaceuticals.
  • ACE inhibitory substances from natural sources such as milk protein, soybean protein or fish meat protein are proposed for practical use as antihypertensive agents having low toxicity and great safety.
  • Proteins from natural resources are important for supplying nutrients and energy and these functions have been defined as a primary function of protein.
  • proteins perform other functions relating to physiological regulation.
  • peptides from natural sources provide other beneficial effects such as promoting calcium absorption and regulating serum cholesterol.
  • Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) have been derived from lactic acid bacteria-fermented milk. See ⁇ akamura et al., J. Dairy Sci. 78: 777-783 (1995). Furthermore, these tripeptides have been reported to exhibit strong antihypertensive effect in spontaneously hypertensive rats (SHR). See ⁇ akamura et al, J. Dairy Sci., 78: 1253-1257 (1995). However, since the tripeptides are produced by proteinase which is produced by lactic acid bacteria as lactic acid fermentation proceeds in milk, the resulting amount of tripeptides tends to vary depending on the conditions of fermentation. It is thus difficult to obtain a consistent amount of the tripeptides.
  • LBP ⁇ M Leu-Lys-Pro-Asn-Met
  • SEQ ID NO: 3 Another antihypertensive peptide, Leu-Lys-Pro-Asn-Met (LKP ⁇ M) (SEQ ID NO: 3), has been isolated from dried bonito digested in thermolysin.
  • Dried bonito is a traditional Japanese seasoning made of skipjack tuna (bonito) muscle.
  • bonito meat is treated with fungi.
  • Thermolysin digests of non-dried bonito protein does not produce the LKPNM oligopeptide that is found in the thermolysin digest of dried bonito. See Yokoyama et al., Biosci. Biotech. Biochem. 56: 1541-1545 (1992).
  • LKPNM After LKPNM enters the body, it is believed to be further converted into the peptide LKP and NM in the digestive organs or blood.
  • the LKP peptide fragment exhibits the greatest anti-hypertensive properties and is therefore believed to be a principal active form of LKPNM.
  • LKP has been isolated from digestion offish muscle, corn protein, soybeans, and milk products. Other peptides obtained from natural sources may also demonstrate antihypertensive activities.
  • LKPN SEQ ID NO: 4
  • LKPN has been isolated from the enzymatic digestion offish muscle and soybeans.
  • an object of the present invention is to provide compositions comprising a peptide selected from the group consisting of Lys-Pro-Asn-Met (KPNM) (SEQ ED NO: 5), Lys-Pro-Asn (KPN), Pro-Asn-Met (PNM), Leu-Lys (LK), Pro-Asn (PN) and Asn-Met (NM) or their acceptable acid or base addition salt, together with a pharmaceutically suitable diluent.
  • KPNM Lys-Pro-Asn-Met
  • KPN Lys-Pro-Asn-Met
  • PPM Pro-Asn-Met
  • LK Leu-Lys
  • PN Pro-Asn
  • NM Asn-Met
  • Another object of the present invention is to provide edible compositions comprising a foodstuff comprising a peptide selected from the group consisting -of KPNM, KPN, PNM, LK, PN and NM and their acceptable acid or base addition salts. It is yet another object of the present invention to provide edible compositions comprising a nutritional substance and an oligopeptide selected from the group consisting of KPNM, KPN, PNM, LK, PN and NM or their acceptable acid or base addition salt.
  • Yet a further object of the present invention is to provide methods of reducing various diseases such as myocardial infarction, left ventricular systolic dysfunction, diabetes mellitus, progressive renal failure and congestive heart failure caused by or associated with hypertension which comprises administering an antihypertensively effective amount of an oligopeptide selected from the group consisting of KPNM, KPN, PNM, LK, PN and NM or their pharmaceutically acceptable acid or base addition salt.
  • an oligopeptide selected from the group consisting of KPNM, KPN, PNM, LK, PN and NM or their pharmaceutically acceptable acid or base addition salt.
  • Figure 1 is a graph representing the effect of a 10 ⁇ mol intravenous dose of LKPNM and its peptide fragments on systolic blood pressure in spontaneous hypertensive rats.
  • Figure 2 is a graph representing the effect of a 5 ⁇ mol intravenous dose of
  • Figure 3 is a graph representing the effect of a 10 ⁇ mol oral dose of LKPNM and its peptide fragments on systolic blood pressure in 27 week old spontaneous hypertensive rats.
  • Figure 4 is a graph representing the effect of a 10 ⁇ mol oral dose of LKPNM and its peptide fragments on systolic blood pressure in 13 week old spontaneous hypertensive rats.
  • a and Ala represent alanine
  • R and Arg represent arginine
  • N and Asn represent asparagine
  • D and Asp represent aspartic acid
  • C and Cys represent cysteine
  • Q and Gin represent glutamine
  • E and Glu represent glutamic acid
  • G and Gly represent glycine
  • H and His represent histidme
  • I and He represent isoleucine
  • L and Leu represent leucine
  • K and Lys represent lysine
  • M and Met represent methionine
  • F and Phe represent phenylalanine
  • P and Pro represent proline
  • S and Ser represent serine
  • T and Thr represent threonine
  • W and Trp represent tryptophan
  • Y and Tyr represent tyrosine
  • V and Val represent valine.
  • ACE angiotensin converting enzyme
  • edible composition is defined as compositions which may be ingested by a mammal such as foodstuffs, nutritional substances and pharmaceutical compositions.
  • foodstuffs refer to substances that can be used or prepared for use as food for a mammal and include substances that may be used in the preparation of food (such as frying oils) or food additives.
  • foodstuffs include animals used for human consumption or any product therefrom, such as, for example, eggs.
  • Typical foodstuffs include but are not limited to beverages, (e.g. , soft drinks, carbonated beverages, ready to mix beverages), infused foods (e.g.
  • sauces condiments, salad dressings, fruit juices, syrups, desserts (e.g., puddings, gelatin, icings and fillings, baked goods and frozen desserts such as ice creams and sherbets), soft frozen products (e.g., soft frozen creams, soft frozen ice creams and yogurts, soft frozen toppings such as dairy or non-dairy whipped toppings), oils and emulsified products (e.g. , shortening, margarine, mayonnaise, butter, cooking oil, and salad dressings) and intermediate moisture foods (e.g., rice and dog foods).
  • sauces condiments, salad dressings, fruit juices, syrups, desserts
  • desserts e.g., puddings, gelatin, icings and fillings, baked goods and frozen desserts such as ice creams and sherbets
  • soft frozen products e.g., soft frozen creams, soft frozen ice creams and yogurts, soft frozen toppings such as dairy or non-dairy
  • substantially pure when referring to oligopeptides, denotes those oligopeptides that are largely or wholly separated from the proteins or other contaminants with which they are naturally associated.
  • An oligopeptide is considered substantially pure when that oligopeptide makes up greater than about 50% of the total content of the composition containing the oligopeptide and typically, greater than about 60% of the total content of the composition containing the oligopeptide. More typically, a substantially pure oligopeptide will make up from about 75% to about 90% of the total composition containing the oligopeptide. Preferably, the oligopeptide will make up greater than about 90%, and more preferably, greater than about 95% of the total composition containing the oligopeptide.
  • anti-hypertensively effective amount shall mean an amount sufficient to reduce, inhibit, or prevent hypertension in a subject.
  • treatment relate to any treatment of hypertensive disease and include: (1) preventing hypertension from occurring in a subject who may be predisposed to the disease but who has not yet been diagnosed as having it; (2) inhibiting the disease, i.e., arresting its development; or (3) ameliorating or relieving the symptoms of the disease, i.e., causing regression of the hypertensive state.
  • the present invention is directed to a set of short oligopeptides Lys- Pro-Asn-Met (KPNM), Lys-Pro-Asn (KPN), Pro-Asn-Met (PNM), Leu-Lys (LK), Pro- Asn (PN) and Asn-Met (NM) which exhibit antihypertensive activity and effectively reduce blood pressure.
  • KPNM Lys- Pro-Asn-Met
  • KPN Lys-Pro-Asn
  • PPM Pro-Asn-Met
  • LK Leu-Lys
  • PN Pro- Asn
  • NM Asn-Met
  • the present invention also relates to compositions comprising an antihypertensively effective amount of any of the oligopeptides KPNM, KPN, PNM, LK, PN and NM or their pharmaceutically acceptable acid or base addition salt.
  • pharmaceutically acceptable acid addition salts include but are not limited to salts formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and salts formed with an organic acid such as acetic acid, propionic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, oxalic acid, fumaric acid, succinic acid, malonic acid, methanesulfonic acid, benzensulfonic acid, toluene-sulfonic acid.
  • oligopeptides have demonstrated significant antihypertensive activity and therefore, are useful in the treatment and prophylaxis of various disease states such as left ventricular systolic dysfunction, myocardial infarction, diabetes mellitus and progressive renal impairment/failure caused by or associated with hypertension.
  • the oligopeptides of the invention may be prepared by a process designed to hydrolyze or digest the selected protein with a cleaving agent such as a protease.
  • a source protein such as milk, soybeans, corn, wheat, rice, peanuts, beef muscle, lamb muscle, pork muscle, turkey muscle, chicken muscle, fish muscle other than dried bonito fish, yeast, bacteria, or products thereof is mixed with a suitable liquid such as sterilized water to form a homogeneous mixture.
  • a suitable liquid such as sterilized water
  • the pH of the homogeneous mixture is adjusted to a pH that is favorable for a selected cleaving agent.
  • the pH can, if necessary, be adjusted during the course of the reaction with an aqueous sodium hydroxide solution, hydrochloric acid or the like.
  • the homogeneous mixture is then boiled and the cleavage agent is added to digest the protein present in the mixture.
  • the temperature of the reaction mixture during the reaction is cooled and maintained within approximately 20°C to 50°C, preferably 30°C to 40°C, and most preferably about 37°C.
  • An endo type protease such as the ⁇ nolysin, pepsin, trypsin, chyrnotrypsin,
  • Proteinase E, Proteinase K and Actinase E are preferred for use to digest the protein, with thermolysin being particularly preferred.
  • the optimal concentration of the cleaving agent within the digestion mixture is dependent upon the cleaving agent selected. Although the addition amount of the cleavage agent depends on its titer, the amount is usually 0.01% by weight or more, preferably 0.1 to 10% by weight based on the protein, preferably a final concentration of approximately 880 ⁇ g/ml based on the amount of protein. It is also possible to add part of the cleavage agent during the course of the reaction.
  • Treatment of the protein with the cleavable agent is usually carried out in water or in a suitable buffer (for example, a Tris-HCl buffer or a phosphate buffer). While the concentration of the protein substrate is not particularly limiting so long as it is possible to carry out stirring and mixing, it is preferable that the concentration of the protein substrate is in the range of 2 to 20% (w/v).
  • the reaction time also varies depending on the cleavage agent used, addition amount of the enzyme, reaction temperature, reaction pH and the desired oligopeptide to be isolated but is preferably in the range of 1 to 5 hours.
  • Discontinuance of the hydrolysis reaction can be made according to a known method.
  • Such methods which may be used to inactivate the hydrolysis reaction include but are not limited to inactivation of the cleavage agent either by heating of the hydrolyzate; altering the pH with the addition of an organic acid such as citric acid or malic acid, an inorganic acid such as hydrochloric acid or phosphoric acid or an alkali such as sodium hydroxide or potassium hydroxide; or the separation of the cleaving agent from the hydrolyzate by filtration using an ultrafiltration membrane.
  • the digestion mixture is cooled and the resulting hydrolyzate solution is subjected to solid-liquid separation (for example, centrifugation or filtration) and the resulting liquid is fractionated by ultrafiltration, gel filtration or other commonly used methods in order to obtain a liquid portion containing a fraction having a molecular weight of 10,000 or less.
  • This liquid or its concentrate (for example, freeze-dried product) contains the oligopeptides of the invention and is further fractionated to obtain each desired oligopeptide.
  • the liquid containing the desired oligopeptide is further separated into solid and liquid phases by centrifugation.
  • the supernatant is removed and may be transferred to another centrifuge tube to be centrifuged a second time.
  • the solid phases obtained by centrifugation are dried and a mobile phase solvent is added to resuspend the dried contents in the centrifuge tubes in order to form a mobile phase mixture.
  • the mobile phase mixture is then centrifuged and the resulting supernatant containing the desired oligopeptides is fractionated by chromatographic methods such as liquid chromatography, HPLC, or the like.
  • the desired oligopeptides are then isolated according to their respective retention times.
  • the oligopeptides of the present invention can alternatively be prepared by other synthetic methods such as liquid-phase peptide synthesis or solid-phase peptide synthesis.
  • Approximate peptide synthesis methods are described in Nobuo Izumiya, Tetsuo Kato, Haruhiko Aoyagi and Michinori Waki, "The Foundations and Experiments of Peptide Synthesis” (Pepuchido Gosei no Kiso to Jikken) published by Maruzen Co., Ltd., Tokyo, 1985, incorporated herein by reference.
  • Methods for carrying out liquid phase synthesis of peptides coupled to a soluble oligomeric support are well known by those skilled in the art.
  • Solid-phase peptide synthesis entails the sequential assembly of the appropriate amino acids into a peptide of a desired sequence while the end of the growing peptide is linked to an insoluble support.
  • the carboxyl terminus of the peptide is linked to a polymer from which it can be liberated upon treatment with a cleavage reagent.
  • an amino acid is bound to a resin particle, and the peptide generated in a stepwise manner by successive additions of protected amino acids to produce a chain of amino acids.
  • Modifications of the technique described by Merrifield are commonly used. See, e.g., Merrifield, J. Am. Chem. Soc. 96: 2989-93 (1964), incorporated herein by reference.
  • peptides are synthesized by loading the carboxy-terminal amino acid onto an organic linker (e.g., PAM, 4-oxymethyl phenylacetamidomethyl), which is covalently attached to an insoluble polystyrene resin cross-linked with divinyl benzene.
  • organic linker e.g., PAM, 4-oxymethyl phenylacetamidomethyl
  • the terminal amine may be protected by blocking with t-butyloxycarbonyl. Hydroxyl- and carboxyl-groups are commonly protected by blocking with O-benzyl groups.
  • Synthesis is accomplished in a commercially available automated peptide synthesizer. See e.g., Model 430- A, Applied Biosystems, Foster City, Calif.
  • the product may be removed from the resin and the blocking groups are removed by using hydrofluoric acid or trifluoromethyl sulfonic acid according to established methods. See e.g., Bergot and McCurdy, Applied Biosystems Bulletin (1987) incorporated herein by reference.
  • a routine synthesis may produce 0.5 mniole of peptide-resin. Following cleavage and purification, a yield of approximately 60 to 70% is typically produced. Purification of the product peptides may be accomplished by crystallizing the peptide from an organic solvent such as methyl-butyl ether, dissolving in distilled water and using dialysis or reverse high-pressure liquid chromatography.
  • Purified peptide may be lyophilized and stored in a dry state until use. Analysis of the resulting peptides may be done using the common methods of analytical high pressure liquid chromatography (HPLC) and electrospray mass spectrometry (ES-MS).
  • HPLC high pressure liquid chromatography
  • ES-MS electrospray mass spectrometry
  • the present oligopeptides and their acid or base addition salts have antihypertensive activity and thus are effective for treatment and prophylaxis of hypertension of mammals including human beings.
  • Administration of the desired oligopeptide and/or its acid or base addition salts may be in the form of a pharmaceutical composition and at least one pharmaceutical auxiliary and shall be in an amount to effectively reduce hypertension in a subject.
  • the present oligopeptides and their acid or base addition salts can be administered parenterally, namely by intravenous injection, rectal administration or orally, and formulated into a form suitable for each administration method.
  • compositions comprising any of the antihypertensive oligopeptides KPNM, KPN, PNM, LK, PN and NM or their pharmaceutically acceptable acid or base addition salts may be administered to a subject in order to effectively reduce hypertension.
  • Examples of the pharmaceutically acceptable acid addition salts include but are not limited to salts formed with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and salts formed with an organic acid such as acetic acid, propionic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, oxalic acid, fumaric acid, succinic acid, malonic acid, methanesulfonic acid, benzensulfonic acid, toluene-sulfonic acid.
  • an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid
  • salts formed with an organic acid such as acetic acid, propionic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, oxalic acid, fumaric acid, succinic acid
  • the acid addition salts of the present oligopeptides can be prepared according to a conventional method.
  • an acid addition salt can be obtained by reacting one of the present oligopeptides containing a basic amino acid residue with a suitable acid in one equivalent amount thereto in water and then freeze-drying the product.
  • alkali or alkaline earth metal salts, ammonium salts or organic base salts (hereinafter these are referred to as base salts) can also be prepared according to a conventional method.
  • a base salt can be obtained by reacting one of the present oligopeptides containing an acidic amino acid residue with a suitable base in one equivalent amount thereto in water and then freeze-drying the product.
  • the effective dosage of the desired oligopeptide will depend partly on the route of administration of the pharmaceutical composition. If the oral route is employed, the absorption of the composition will be an important factor.
  • the effective amount of the desired oligopeptide or its pharmaceutically acceptable or base addition salt in the antihypertensive composition is approximately 1 to 100% (w/w), preferably 10 to 100% (w/w).
  • an antihypertensively effective amount of the desired oligopeptide or its pharmaceutically acceptable addition salt is approximately 10 to 200 mg/kg/day when administered to a mammal including humans beings for the purpose of treatment or prophylaxis of hypertension.
  • Formulations may be prepared in a manner suitable for systemic administration or local administration.
  • Systemic formulations include those designed for injection (e.g., intramuscular, intravenous or subcutaneous inj ection) or may be prepared for transdermal, transmucosal, or oral administration.
  • the formulation will generally include a pharmaceutically acceptable diluent as well as, in some cases, adjuvants, buffers, preservatives and the like.
  • Pharmaceutical forms for injections usually include sterilized aqueous solutions.
  • Pharmaceutical formulations for injection can further contain pharmaceutical auxiliaries other than water such as a buffering and a pH adjusting agent (e.g., sodium hydrogenphosphate, citric acid), atonicity agent (e.g., sodium chloride, glucose), a preservative (methyl p-hydroxybenzoate, propyl-hydroxybenzoate or the like).
  • the pharmaceutical formulations can be sterilized by filtration through a bacteria-holding filter, incorporation of a sterilant into the composition, or irradiation or heating of the composition.
  • the pharmaceutical formulations can also be prepared by first preparing and sterilizing a solid pharmaceutical composition and dissolving the solid composition at the time of use in sterilized water for use as an injection.
  • Orally administered agents are prepared in a form suitable for absorption in gastrointestinal organs.
  • Tablets, capsules, granules, fine granules and powders can contain conventional pharmaceutical auxiliaries such as a binder (e.g., syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone or hydroxycellulose), an excipient (e.g., lactose, sucrose, corn starch, calcium stearate, sorbitol or glycine), a lubricant (e.g., magnesium stearate, talc, polyethylene glycol or silica), a disintegrant (potato starch or carboxymethylcellulose), or a wetting agent (e.g., sodium lauryl sulfate).
  • a binder e.g., syrup, gum arabic, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone or hydroxycellulose
  • Oral liquid agents may also contain additives such as a preservative (methyl p-hydroxybenzoate, propyl p-hydroxybenzoate or sorbic acid).
  • edible compositions containing the antihypertensive oligopeptides and their acid or base addition salts can also be ingested as an additive or supplement contained in foods and drinks.
  • These oligopeptides can be formulated together with a nutritional substance such as various vitamins and minerals and incorporated into substantially liquid compositions such as nutrient drinks, soymilks and soups; substantially solid compositions; and gelatins or used in the form of a powder to be incorporated into various foods.
  • the content of the antihypertensive effective ingredient in such a functional or health food can be similar to the dose contained in a typical pharmaceutical agent.
  • Example 1 Preparation of Antihypertensive OligoPeptides from Natural Bonito Fish Dried bonito, Calpis milk, and soybeans were digested and the oligopeptide sequences of LKPNM and its enzymatically cleaved subunits were isolated and the peptide quantities compared according to the procedure outlined below.
  • the enzymatic reaction was stopped at the end of each digestion period by boiling the mixture for 10 minutes to inactivate the thermolysin.
  • the homogenized mixture was transferred to centrifuge tubes and centrifuged at 3000 rpm at 4°C for a period of 15 minutes. 200 ⁇ l of the supernatant was collected and placed into a clean microfuge tube and centrifuged at 8000 rpm at 10-40°C for a period of 10 minutes.
  • the contents of the microfuge tube were dried down under nitrogen gas (N 2 ) at a temperature of 37 ° C.
  • the mixture was held at a temperature of 37 °C and enzymatically digested for a period of 0, 1, 3, and 5 hours.
  • the enzymatic reaction was stopped at the end of each digestion period by boiling the mixture for 10 minutes to inactivate the cleaving agent.
  • the mixture was transferred to centrifuge tubes and centrifuged at 1500 rpm at a temperature of about 30°C for a period of 10 minutes.
  • the supernatant was removed and analyzed using liquid chromatography/mass spectroscopy (LC/MS) to quantify the presence of oligopeptide fragments.
  • the supernatant was diluted 1 : 10 in deionized water with 0.1% trifluoroacetic acid
  • TFA LC/MS injections
  • 10 ⁇ g samples were injected into an LC/MS (Hewlett-Packard 1100 HPLC/MSD System, Palo Alto, CA) operated in the positive ion electrospray ionization (ESI) mode.
  • ESI positive ion electrospray ionization
  • Components of the samples were separated using a reverse phase HPLC method and a Waters C 1S Symmetry column operated at 70°C.
  • Positive ESI mass spectra of standard reference materials showed that the major ions formed were MH + . This mass was monitored to quantify oligopeptides present in the samples.
  • Water and acetonitrile contained 0.1% TFA.
  • the water/acetonitrile flow rate was 1.0 mL/min with a gradient that began at 0% acetonitrile which increased linearly to 100%) acetonitrile between 4 and 10 minutes. A 2 minute post time was used to allow the column to equilibrate between inj ections .
  • oligopeptide concentrations were calculated using the following formula:
  • Tables 1-6 summarize the quantities of the oligopeptides identified from the pepsin, chymotrypsin, trypsin, and thermolysin digestions of the protein.
  • Table 1 Oligopeptides Released from Enzyme Digested Pork
  • Example 3 Liquid Phase Synthesis of Leucyl-Lysyl-Proline fLKP Step 1: Preparation of N ⁇ -Fmoc-N ⁇ -Boc-Lysyl-Proline tert-Butyl Ester
  • N,N'-Disuccinimidyl carbonate 3 g, 12 mmol
  • Fmoc-Lys(Boc)-OH 4.5 g, 10 mmol
  • Step 3 Preparation of Leucyl-Lysyl-Proline Boc-Leucyl-N ⁇ -Boc-Lysyl-Proline tert-Butyl Ester prepared in step 2 was de- protected by treating it with 90% TFA in water (10 ml) for 1 hour. The final desired oligopeptide was purified by prep HPLC and lyophilized to give a white powder (1.46 g).
  • Example 4 Synthesis of Lysyl-Prolyl-Asparaginyl-Methionine CKPNM KPNM (494 mg, 69% yield) was prepared using substantially the same procedure as described in Example 3. The synthesis was started with a Fmoc-Met resin (1.0 mmol) and subsequent coupling of Fmoc-Asn(Mtt), Fmoc-Pro and Fmoc-Lys(Boc).
  • Example 5 Synthesis of Lysyl-Prolyl-Asparagine (KPN) KPN (434 mg, 92% yield) was prepared using substantially the same procedure as described in Example 3. The synthesis was started with a Fmoc-Asn(Trt) resin (1.0 mmol) and subsequent coupling of Fmoc-Pro and Fmoc-Lys(Boc).
  • KPN Lysyl-Prolyl-Asparagine
  • Example 6 Synthesis of Leucyl-Lysine (LK) LK was prepared using substantially the same procedure as described in Example 3. The synthesis was started with a Fmoc-Lys(Boc) resin (1.0 mmol) and coupled with Fmoc-Leu. After synthesis, the resulting peptide resin was treated with TFA (20 mL), for 1 h. The desired product (389 mg, 80% yield) was obtained as a white powder.
  • Example 8 In vitro ACE inhibition Assay KPNM, KPN, PNM, LK, PN and NM were synthesized as discussed above and analyzed using an in vitro ACE inhibition assay using a 96-well plate format. N-(3-[2- furyl]acryloyl)-Phe-Gly-Gly (FAPGG) was used as the substrate, which had an absorbance at 340 nm. The decrease in absorbance of FAPGG was followed during the reaction to determine the enzyme activity.
  • FAPGG N-(3-[2- furyl]acryloyl)-Phe-Gly-Gly
  • the concentration of oligopeptide at the inhibition rate of 50% of reaction control is referred to as the IC 50 value.
  • KP ⁇ M, KP ⁇ , P ⁇ M, LK, P ⁇ and ⁇ M were synthesized as described above, dissolved in physiological saline and aseptically filtered through a filter. Catheters were placed in the femoral veins of the animals for intravenous infusion. The resulting filtrate containing the desired oligopeptide was intravenously administered to the animals.
  • Example 10 Effect of Oral Administration of Oligopeptides on Systolic Blood Pressure
  • SHR spontaneous hypertensive rats
  • KPNM, KPN, PNM, LK, PN and NM were synthesized as described above in the Examples above, dissolved in distilled water and aseptically filtered through a filter to obtain a filtrate containing the desired oligopeptide to be administrated orally. 10 ⁇ moles/rat of each of the oligopeptides was orally admimstered to each animal of group.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne de nouveaux oligopeptides, des résidus de deux à quatre acides aminés de longueur, obtenus à partir de produits de digestion de protéines alimentaires ou synthétisés chimiquement, qui présentent une activité d'antihypertenseur. Ces oligopeptides antihypertenseurs peuvent être utilisés comme ingrédients actifs dans des composés pharmaceutiques, des compléments alimentaires et des ingrédients alimentaires. L'invention concerne également des méthodes de traitement thérapeutique et prophylactique, au moyen de ces nouveaux oligopeptides antihypertenseurs, de maladies telles que l'infarctus du myocarde, le dysfonctionnement systolique ventriculaire gauche, le diabète sucré, l'insuffisance rénale progressive et l'insuffisance cardiaque congestive provoqués par ou associés à l'hypertension.
PCT/US2001/007479 2000-03-10 2001-03-09 Peptides antihypertenseurs WO2001068113A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001245537A AU2001245537A1 (en) 2000-03-10 2001-03-09 Anti-hypertensive peptides

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US18849900P 2000-03-10 2000-03-10
US60/188,499 2000-03-10

Publications (1)

Publication Number Publication Date
WO2001068113A1 true WO2001068113A1 (fr) 2001-09-20

Family

ID=22693404

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2001/007479 WO2001068113A1 (fr) 2000-03-10 2001-03-09 Peptides antihypertenseurs

Country Status (2)

Country Link
AU (1) AU2001245537A1 (fr)
WO (1) WO2001068113A1 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005097163A1 (fr) * 2004-04-08 2005-10-20 Biotempt, B.V. Oligopeptides permettant de reduire une concentration elevee d'uree sanguine
US6994987B1 (en) 1999-11-11 2006-02-07 Calpis Co., Ltd. Process for producing tripeptides
US7029670B2 (en) 2000-12-28 2006-04-18 Calpis Co., Ltd. Medicines for relieving intestinal disorders
US7282354B2 (en) 1997-09-26 2007-10-16 Calpis Co., Ltd. Method for producing fermented milk product
US7358330B2 (en) 2001-03-29 2008-04-15 Biotempt B.V. Immunoregulatory compositions
US7402322B2 (en) 1998-05-20 2008-07-22 Biotempt B.V. Methods of treatment for septic shock with urine extract
US7501391B2 (en) 2001-12-21 2009-03-10 Biotempt B.V. Treatment of transplant survival
US7517529B2 (en) 2003-04-08 2009-04-14 Biotempt B.V. Treatment of type I diabetes
US7524820B1 (en) 2001-10-04 2009-04-28 Biotempt B.V. Compounds of therapeutic value in the treatment of multiple sclerosis and other diseases wherein foamy cells are involved in the disease etiology
US7576174B2 (en) 2000-03-29 2009-08-18 Biotempt B.V. Compositions capable of reducing elevated blood urea concentration
US7662776B2 (en) 2005-07-05 2010-02-16 Biotempt B.V. Treatment of tumors using short peptides from human chorionic gonadotropin (HCG)
US7759108B2 (en) 1999-01-11 2010-07-20 Calpis Co., Ltd. Method for producing fermented milk containing angiotensin converting enzyme inhibitory peptide and method for producing whey
US7786084B2 (en) 2001-12-21 2010-08-31 Biotempt B.V. Treatment of burns
US7795226B2 (en) 2006-03-07 2010-09-14 Biotempt B.V. Control of radiation injury
US7820617B2 (en) 1998-05-20 2010-10-26 Biotempt B.V. Methods of selecting immunoregulator peptides obtained from gonadotropins
WO2011074731A1 (fr) * 2009-12-18 2011-06-23 주식회사 연세 Peptide ayant une capacité d'abaissement de la pression artérielle
KR101106303B1 (ko) * 2008-10-20 2012-01-18 주식회사 연세 혈압저하능을 가진 펩타이드
USRE43140E1 (en) 2000-03-29 2012-01-24 Biotempt B.V. Immunoregulator
USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
US8216998B2 (en) 2001-12-21 2012-07-10 Biotempt B.V. Treatment of ischemic events
US8680059B2 (en) 1998-05-20 2014-03-25 Biotempt B.V. Oligopeptide acetate and formulations thereof
CN112135619A (zh) * 2018-03-27 2020-12-25 三福有限公司 生物活性青口贝提取物及其用途
CN117534727A (zh) * 2023-12-21 2024-02-09 中国药科大学 一种食源性降压肽的制备方法和应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0469397A (ja) * 1990-07-06 1992-03-04 Nippon Synthetic Chem Ind Co Ltd:The 新規ペプチド及び用途
JPH08225593A (ja) * 1995-02-21 1996-09-03 Suetsuna Yoko 新規なテトラペプチド、ペンタペプチドおよびアンジオテンシン変換酵素阻害剤
US5691310A (en) * 1987-09-29 1997-11-25 Vesely; David L. Methods of treatment using proANF peptides
US5854029A (en) * 1996-08-02 1998-12-29 Calpis Co., Ltd. Method for preparing antihypertensive agent
JP2000060489A (ja) * 1998-08-24 2000-02-29 Nippon Synthetic Chem Ind Co Ltd:The ペプチド含有食品

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5691310A (en) * 1987-09-29 1997-11-25 Vesely; David L. Methods of treatment using proANF peptides
JPH0469397A (ja) * 1990-07-06 1992-03-04 Nippon Synthetic Chem Ind Co Ltd:The 新規ペプチド及び用途
JPH08225593A (ja) * 1995-02-21 1996-09-03 Suetsuna Yoko 新規なテトラペプチド、ペンタペプチドおよびアンジオテンシン変換酵素阻害剤
US5854029A (en) * 1996-08-02 1998-12-29 Calpis Co., Ltd. Method for preparing antihypertensive agent
JP2000060489A (ja) * 1998-08-24 2000-02-29 Nippon Synthetic Chem Ind Co Ltd:The ペプチド含有食品

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CASARINI ET AL.: "Angiotensin converting enzymes from human urine of mild hypertensive untreated patients resemble the N-terminal fragment of human angiotensin I-converting enzyme", INT. J. BIOCHEM. CELL BIOLOGY, vol. 33, January 2001 (2001-01-01), pages 75 - 85, XP002944499 *
NAKAMURA ET AL.: "Antihypertensive effect of sour milk and peptides isolated from it that are inhibitors to angiotensin I-converting enzyme", J. DIARY SCI., vol. 78, no. 6, 1995, pages 1253 - 1257, XP002944500 *
SAITO T. ET AL.: "Isolation and structural analysis of antihypertensive peptides that exist naturally in Gouda cheese", J. DAIRY SCIENCE, vol. 83, no. 7, July 2000 (2000-07-01), pages 1436 - 1439, XP002942635 *

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7282354B2 (en) 1997-09-26 2007-10-16 Calpis Co., Ltd. Method for producing fermented milk product
US7402322B2 (en) 1998-05-20 2008-07-22 Biotempt B.V. Methods of treatment for septic shock with urine extract
US7820617B2 (en) 1998-05-20 2010-10-26 Biotempt B.V. Methods of selecting immunoregulator peptides obtained from gonadotropins
US8680059B2 (en) 1998-05-20 2014-03-25 Biotempt B.V. Oligopeptide acetate and formulations thereof
US7759108B2 (en) 1999-01-11 2010-07-20 Calpis Co., Ltd. Method for producing fermented milk containing angiotensin converting enzyme inhibitory peptide and method for producing whey
US6994987B1 (en) 1999-11-11 2006-02-07 Calpis Co., Ltd. Process for producing tripeptides
USRE43309E1 (en) 2000-03-29 2012-04-10 Biotempt B.V. Immunoregulatory compositions
USRE43140E1 (en) 2000-03-29 2012-01-24 Biotempt B.V. Immunoregulator
USRE43279E1 (en) 2000-03-29 2012-03-27 Biotemp B.V. Compositions capable of reducing elevated blood urea concentration
US7576174B2 (en) 2000-03-29 2009-08-18 Biotempt B.V. Compositions capable of reducing elevated blood urea concentration
US7029670B2 (en) 2000-12-28 2006-04-18 Calpis Co., Ltd. Medicines for relieving intestinal disorders
US7358330B2 (en) 2001-03-29 2008-04-15 Biotempt B.V. Immunoregulatory compositions
US7524820B1 (en) 2001-10-04 2009-04-28 Biotempt B.V. Compounds of therapeutic value in the treatment of multiple sclerosis and other diseases wherein foamy cells are involved in the disease etiology
US8216998B2 (en) 2001-12-21 2012-07-10 Biotempt B.V. Treatment of ischemic events
US7786084B2 (en) 2001-12-21 2010-08-31 Biotempt B.V. Treatment of burns
US7501391B2 (en) 2001-12-21 2009-03-10 Biotempt B.V. Treatment of transplant survival
US7517529B2 (en) 2003-04-08 2009-04-14 Biotempt B.V. Treatment of type I diabetes
AU2005230403B2 (en) * 2004-04-08 2009-05-21 Biotempt, B.V. Oligopeptides for reducing elevated blood urea concentration
KR101217025B1 (ko) 2004-04-08 2013-01-02 바이오템프트, 비.브이. 증가된 혈중 요소 농도를 감소시키기 위한 올리고펩티드
WO2005097163A1 (fr) * 2004-04-08 2005-10-20 Biotempt, B.V. Oligopeptides permettant de reduire une concentration elevee d'uree sanguine
US7662776B2 (en) 2005-07-05 2010-02-16 Biotempt B.V. Treatment of tumors using short peptides from human chorionic gonadotropin (HCG)
US7795226B2 (en) 2006-03-07 2010-09-14 Biotempt B.V. Control of radiation injury
US8288341B2 (en) 2006-03-07 2012-10-16 Biotempt B.V. Control of radiation injury
KR101106303B1 (ko) * 2008-10-20 2012-01-18 주식회사 연세 혈압저하능을 가진 펩타이드
WO2011074731A1 (fr) * 2009-12-18 2011-06-23 주식회사 연세 Peptide ayant une capacité d'abaissement de la pression artérielle
CN112135619A (zh) * 2018-03-27 2020-12-25 三福有限公司 生物活性青口贝提取物及其用途
CN117534727A (zh) * 2023-12-21 2024-02-09 中国药科大学 一种食源性降压肽的制备方法和应用
CN117534727B (zh) * 2023-12-21 2024-04-19 中国药科大学 一种食源性降压肽的制备方法和应用

Also Published As

Publication number Publication date
AU2001245537A1 (en) 2001-09-24

Similar Documents

Publication Publication Date Title
WO2001068113A1 (fr) Peptides antihypertenseurs
EP1938832B1 (fr) Peptides inhibiteurs d'une enzyme de conversion de l´angiotensine
US9061003B2 (en) Therapeutic or preventive agent for diabetes
US20180170961A1 (en) Pharmaceutical composition for the prevention and treatment of cardiovascular disease comprising the peptide having the ability to inhibit angiotensin-1 converting enzyme as an active ingredient
US20090131331A1 (en) Novel Nutraceutical Compositions
CN101305017A (zh) 奶酪蛋白的酶水解产物中鉴定的生物活性肽及其生产方法
US9617300B2 (en) Dipeptidyl peptidase-IV inhibitor
WO2001068114A1 (fr) Nouveaux peptides a activite hypotensive
JP5417405B2 (ja) アンジオテンシン変換酵素阻害性降圧ペプチド組成物の製造方法
JP5416964B2 (ja) 新規トリペプチドおよびそれらトリペプチドの製造法、ならびにアンジオテンシン変換酵素阻害物質の製造方法
AU2004303647B2 (en) Peptide inhibiting angiotensin converting enzyme
WO2014002571A1 (fr) Dipeptide inhibant l'enzyme de conversion de l'angiotensine
WO2013133032A1 (fr) Inhibiteur de dipeptidyl peptidase-iv
WO2001068115A1 (fr) Procede de production de sequences peptidiques a activite hypotensive
JP5877560B2 (ja) ジペプチジルペプチダーゼ−ｉｖ阻害剤
AU2006239562B2 (en) Compositions comprising tripeptides inhibiting ace
JPH0458947B2 (fr)
WO2014030977A1 (fr) Composition pharmaceutique comprenant, en tant que principes actifs, des peptides présentant une activité inhibitrice contre l'enzyme de conversion de l'angiotensine-i, utiles dans la prévention ou le traitement de maladies cardiovasculaires
JP2000505074A (ja) アンジオテンシン転換酵素阻害剤
JP3726106B2 (ja) アンジオテンシン変換酵素阻害剤及び血圧降下剤
JPH03284694A (ja) 新規トリペプチド及び血圧降下剤
Sagar et al. NATURAL SOURCES OF ANGIOTENSIN CONVERTING ENZYME INHIBITOR—A REVIEW
JP2001122802A (ja) アンジオテンシン変換酵素阻害剤及び血圧降下剤

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP