JP3430177B2 - New bioactive peptide - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a novel physiologically active peptide having the following structure, which is useful for pharmaceuticals, biochemical reagents, etc. having arterial relaxation activity. Available. H-Tyr-Pro-Phe-Pro-Pro-Leu
BACKGROUND OF THE INVENTION In recent years, it has been known that peptides derived from foods have various physiological activities, and many physiologically active peptides have been published. Conventionally, it is widely known that peptides have arterial relaxation activity, for example, bradykinin, substance P, neurotensin, and catoxin D are typical ones. In general, these substances are considered to exhibit a relaxing activity by releasing prostaglandins such as prostacyclin as a second messenger or nitric oxide from vascular endothelial cells.
Nitric oxide is produced using L-arginine as a substrate and is produced from nerve cells such as the brain [Bredt, D. et al. S. , Et al. N
atature 347 768-770 (1990)] or macrophage [Stuhr, D, J. et al. , Et
al. J. Exp. Med. 169 1543-15
45 (1989)]. It is known that it is produced as a relaxing factor derived from endothelial cells in blood vessels, exhibits extremely potent arterial relaxing activity, and is deeply involved in blood pressure regulation. [Moncada, S .; , Et al. Pro
c. Natl. Acad. Sci. USA 86 33
75-3378 (1989)]. Therefore, peptides having the activity of releasing nitric oxide may be potent antihypertensive agents. [0003] Among the known peptides having arterial relaxation activity, Casoxin D and its analogs, and peptides obtained from the pepsin digest of ovalbumin all have some kind of prostaglandin. It has been shown to show its activity via On the other hand, bradykinin and substance P exhibit arterial relaxation activity via nitric oxide, but are not generally used because they have side effects such as painful action or inflammatory action. Therefore, in the present invention, arterial relaxation activity by releasing nitric oxide,
It is another object of the present invention to provide a peptide which exhibits a hypotensive action but does not exhibit any side effects. [0004] In the course of studying the amino acid sequence of human milk casein, the present inventors have found a novel peptide having strong arterial relaxation activity and hypotensive action without side effects, and casomokinin L. It was named.
This peptide is H-Tyr-Pro-Phe-Pro-
It is a hexapeptide having a Pro-Leu-OH sequence. [0005] The above-mentioned Tyr means tyrosine, Pro means proline, Phe means phenylalanine, and Leu means leucine, and all such amino acids are in L-form. The peptide of the present invention is a method commonly used for peptide synthesis,
That is, a raw material having a reactive carboxyl group corresponding to one of two types of fragments bisected at an arbitrary position of a peptide bond by a liquid phase method or a solid phase method, and a raw material having a reactive amino group corresponding to the other fragment Is condensed using a carbodiimide method, an active ester method, or the like, and when the resulting condensate has a protective group, it can be produced by removing the protective group. [0006] In this reaction step, functional groups that should not participate in the reaction are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl, t-
Butyloxycarbonyl, p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl and the like. Examples of the carboxyl-protecting group include groups capable of forming an alkyl ester, a benzyl ester, and the like.In the case of the solid phase method, the C-terminal carboxyl group is a chloromethyl resin, an oxymethyl resin,
It is bound to a carrier such as P-alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester. After completion of the condensation reaction, the protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography,
Examples include reversed-phase liquid chromatography and affinity chromatography. The amino acid sequence of the peptide thus obtained can be identified by sequence analysis using a protein sequencer and acid species analysis using an amino acid analyzer. The administration route of the peptide used in the present invention may be any of oral administration, parenteral administration and rectal administration, but oral administration is preferred. The dose of the peptide of the present invention varies depending on the type of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.001 to 1000 mg, preferably 0.01 to 10 mg once to 3 times per day. It is. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. [0008] As the carrier for pharmaceutical preparations, substances commonly used in the field of pharmaceutical preparations and which do not react with the peptide of the present invention are used. Specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium metasilicate aluminate, synthetic aluminum silicate, sodium carboxymethylcellulose,
Hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose,
Gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid,
Magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, Fluorocarbons, nonionic surfactants, propylene glycol, water and the like. [0009] Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like.
These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good. These preparations can contain the peptide of the present invention in an amount of 0.01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable components. The novel physiologically active peptide of the present invention has an arterial relaxing activity and can be used for pharmaceuticals, biochemical reagents and the like. Now, the present invention will be described more specifically below with reference to working examples. [Synthesis of Peptide] 0.55 g of commercially available Boc (butoxycarbonyl) -Leu-O-Resin [benzyl resin (substitution rate: 0.55 meq / g)] was placed in a reaction tank of a peptide synthesizer SAM2 manufactured by Biosearch. And synthesized as follows. After removing the Boc group by reaction in methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole for 25 minutes, washing with methylene chloride, neutralization with methylene chloride containing 10% diisopropylethylamine, and methylene chloride Was performed. This and 5ml of 0.4M Boc-Pro
Was mixed with 5 ml of a 0.4 M solution of 0.4M diisopropylcarbodiimide in methylene chloride, and the mixture was added to the reaction vessel and reacted with stirring at room temperature for 2 hours. The resulting resin is washed with dimethylformamide, methylene chloride, methylene chloride containing 10% diisopropylethylamine, a mixed solution of methylene chloride and a mixture of methylene chloride and dimethylformamide, and Boc-Pro
-Leu-resin was obtained. Subsequently, the same removal of the Boc group and the coupling of the Boc group and the amino acid are repeated to obtain Ty.
r (Cl ₂ -Bzl) -Pro-Phe-Pro-Pr
An o-Leu resin was obtained. The resin was stirred in 20 ml of hydrogen fluoride (containing 10% anisole) at 0 ° C. for 1 hour,
The peptide was released from the resin. Hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and lyophilized to obtain a crude peptide. This was purified by reverse phase chromatography using an ODS column (Cosmosil 5C ₁₈ ), and H-Tyr was purified.
-Pro-Phe-Pro-Pro-Leu-OH (yield 110 mg) was obtained. The product was analyzed using the same protein sequencer as described above, and as a result, the product was found to have the above composition. The physical properties of the peptide are as follows. mp. 150 ° C. Specific rotation [α] ²⁵ _D : −13.4 (C = 0.5, water) TLC [n-butanol: acetic acid: pyridine: water = 15:
3:10:12] Rf = 0.40 (Measurement of relaxing action of canine mesenteric artery) Magnus tube maintained at 37 ° C. by filling a spiral strip of canine mesenteric artery with a Krebs-hanzelite solution And one end was connected to an isometric transducer. Peptides of the present invention to mesenteric arteries were contracted by 0.5μM prostaglandin F ₂ alpha of showed relaxation effect of persistence.
The EC _{50 at} that time was 10 ⁻¹¹ M. Since this relaxation reaction is inhibited by 10 ^-5 M nitroarginine methyl ester, which is a nitric oxide synthesis inhibitor, it is mediated by the release of nitric oxide as an endothelium-derived relaxing factor. (Measurement of blood pressure lowering effect by intravenous administration)
Using a 250 g male spontaneously hypertensive rat, a cannula was pushed into the carotid artery under α-chloralose anesthesia, and this cannula was connected to a pressure transducer to record on a polygraph. The peptide of the present invention was administered from the femoral vein, and a decrease in blood pressure was observed. At a dose of 0.5 mg / kg, a blood pressure of 180 mmHg was observed to drop to 150 mmHg. (Measurement of Blood Pressure Lowering Effect by Oral Administration) A decrease in blood pressure was observed by a non-invasive method using hypertensive rats (SHR). That is, using a 300 g hypertensive rat, a cuff is installed on the tail under no anesthesia to measure the blood pressure. Fluctuations in blood pressure were measured every 2 to 3 hours after oral administration of the physiological saline solution of the sample using a UR-5000 small animal blood pressure drop measuring device manufactured by Ueda Seisakusho. When 30 mg / kg of the peptide of the present invention was administered, 30 mm / kg after 8 hours.
A decrease in Hg blood pressure was observed. According to the present invention, there is provided a novel peptide which exhibits an arterial relaxation activity by releasing nitric oxide and further exhibits an antihypertensive effect.

Claims (1)

(57) [Claims] [Claim 1] H-Tyr-Pro-Phe-Pro-P
A novel bioactive peptide consisting of ro-Leu-OH.