HK1147210B - Anti-hypertensive agent - Google Patents

Description

Hypotensive agent

Technical Field

The present invention relates to an antihypertensive agent containing a peptide as an active ingredient.

Background

Hypertension and diseases caused by hypertension have become the 1 st cause of death all over the world, and it can be said that 1 person out of about 10 million adults worldwide suffers from these diseases in every 4 persons. In particular, diabetic patients are at a 2 to 4 times higher risk of cardiovascular complications from hypertension than non-diabetic patients, thus advocating the necessity of tight blood pressure control in the international diabetes consortium.

However, it is the actual situation that, despite the various treatments, about 70% of hypertensive patients, and also nearly 90% of patients with concurrent diabetes and hypertension, are unable to reduce blood pressure to a target value. Although it is recognized that hypertension is a major risk factor for cardiovascular diseases such as cerebral stroke and myocardial infarction, it is not widely known that a situation in which blood pressure cannot be sufficiently controlled (especially occult hypertension) is more dangerous. In fact, it has been reported that about 9 patients with hypertension who have developed cerebral infarction are undergoing blood pressure treatment before onset of disease, which indicates that cerebral apoplexy is caused by insufficient blood pressure reduction treatment. Further, from the results of clinical tests, it was also found that the occurrence of cerebral apoplexy can be reduced by about 40% by merely lowering the systolic blood pressure by 10mmHg, and it is also found that the blood pressure "10 mmHg higher" is very dangerous.

In the current hypertension medication, the following 6 drugs are generally used according to their action mechanisms:

(A) diuretic,

(B) A calcium antagonist,

(C) An angiotensin receptor antagonist,

(D) Angiotensin converting enzyme inhibitors,

(E) Beta-retarder,

(F) An alpha blocker.

In addition, in recent years, antihypertensive peptides having an action of lowering blood pressure have been attracting attention. Specifically, as the antihypertensive peptides, in addition to natriuretic peptides (ANP, BNP, CNP) and adrenomedullin (see non-patent documents 1 to 3) which are peptides produced in the body, an albumin enzymatic hydrolysate Arg-Pro-Leu-X-Pro-Trp (X is His, Lys or Arg) (seq id No. 7) (see patent document 1) and a soybean protein enzymatic hydrolysate (see non-patent document 5) can be cited.

At present, when dietary life is reevaluated, particularly, the peptides derived from albumin and soybean protein described in patent document 1 and non-patent document 5 are safe and, after ingestion of albumin or soybean, the peptides are digested in vivo, and thus, the use value thereof is considered to be high.

Non-patent document 1: yoshihara, et al, Plasma atomic peptidic conjugation in very enzymes with left enzymatic collagen in proteins with enzymatic hybridization. Plasma ANP as a reactive biochemical marker to predict the outer membrane of the macromolecular product.J Am colloidal catalyst, 39, 288-

Non-patent document 2: t.horio, et al.Gene expression, section, and analysis of C-type natural peptide in cut additive rates, Endocrinology, 144, 2279 + 2284, 2003

Non-patent document 3: takami, et al, diagnostic and diagnostic value of plasma biological peptide in non-physiological-dependent cyclic reaction, am J Kidney Dis, 44, 419-429, 2004-429

Non-patent document 4: yoshihara, et al, polymeric entrapment in hypoxia-induced acquired culture in cultured cardiac cyto-cells, Eur J Pharmacol, 436, 1-6, 2002

Non-patent document 5: songjing reserping, highly formed とそ anti-radiation and anti-radiation can be used for とそ physiological anti-radiation value of "7S グロブリンから lower blood pressure ペプチド SVY", soybean た one-serving Chinese lobelia study Vol.6(2003), 73-77

Patent document 1: japanese laid-open patent publication No. 2002-80496

Disclosure of Invention

The purpose of the present invention is to provide an antihypertensive agent containing a low-molecular-weight peptide, particularly a peptide derived from a globin decomposition product, as an active ingredient.

In view of the fact that diabetic patients are 2 to 4 times more dangerous for cardiovascular complications due to hypertension than patients who do not suffer from diabetes, it is also an object of the present invention to provide a hypotensive agent which has an antidiabetic effect in addition to an antihypertensive effect and can be suitably used particularly for ameliorating hypertension in diabetic patients or patients in the early stage of diabetes.

In order to achieve the above object, the present inventors have intensively studied and found that it is known that each peptide comprising an amino acid sequence of Val-Tyr-Pro (seq id No. 1), Trp-Gly-Lys-Val-Asn (seq id No. 2), Trp-Gly-Lys-Val (seq id No. 3), Trp-Gly-Lys (seq id No. 4), Ala-Trp-Gly-Lys (seq id No. 5) and Phe-Glu-Ser (seq id No. 6) having an action of suppressing blood glucose increase or insulin hypersecretion and being effective for the treatment of diabetes is a physiologically active peptide having an action of suppressing blood glucose increase or insulin hypersecretion.

The present inventors have also confirmed that, among the peptides, a peptide consisting of an amino acid sequence of Val-Tyr-Pro (seq id No. 1) and a globin degradation product comprising the peptide have an effect of inhibiting blood glucose increase or an insulin hypersecretion effect as described in WO2006/052031a1, and thus are physiologically active peptides that can be suitably used for improving hypertension in a diabetic patient or a patient in the pre-diabetic stage.

The present invention has been completed based on such findings, and has the following aspects.

(I) Hypotensive agent

(I-1) an antihypertensive agent containing, as an active ingredient, at least 1 peptide selected from the group consisting of peptides derived from a globin decomposition product and having amino acid sequences represented by the following (1) to (6), or a globin decomposition product containing at least 1 peptide selected from these peptides:

(1) Val-Val-Tyr-Pro (SEQ ID NO: 1)

(2) Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2)

(3) Trp-Gly-Lys-Val (SEQ ID NO: 3)

(4) Trp-Gly-Lys (SEQ ID NO: 4)

(5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5)

(6) Phe-Glu-Ser (SEQ ID NO: 6).

(I-2) the hypotensive agent according to (I-1) comprising, as an active ingredient, a peptide selected from the group consisting of (1) an amino acid sequence of Val-Val-Tyr-Pro (SEQ ID NO: 1) and a globin reduction product comprising the peptide.

(I-3) the hypotensive agent according to (I-1) or (I-2), wherein the hypotensive agent is orally administered to a patient with mild hypertension.

(I-4) the hypotensive agent according to (I-2), which is administered to a patient suffering from a disease caused by hyperglycemia or in a state preceding the disease.

(I-5) the hypotensive agent according to any one of (I-1) to (I-4), wherein the peptide consisting of the amino acid sequence shown in (1) to (6) is administered daily to an adult in a total amount of 1mg to 500mg per day, or the globin decomposition product is administered daily in a ratio of 0.1g to 5g per day, by an oral administration route.

(II) method for improving hypertensive state or preventing or treating diseases caused by hypertensive state Method of

(II-1) A method for improving a hypertensive state or preventing or treating a disease resulting from a hypertensive state, comprising the step of administering to a hypertensive patient a hypotensive agent containing, as an active ingredient, at least 1 peptide selected from the group consisting of peptides derived from a globin decomposition product and having amino acid sequences represented by the following (1) to (6), or a globin decomposition product containing at least 1 peptide selected from the group consisting of the peptides:

(1) Val-Val-Tyr-Pro (SEQ ID NO: 1)

(2) Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2)

(3) Trp-Gly-Lys-Val (SEQ ID NO: 3)

(4) Trp-Gly-Lys (SEQ ID NO: 4)

(5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5)

(6) Phe-Glu-Ser (SEQ ID NO: 6).

(II-2) A method for improving a hypertensive state or preventing or treating a disease caused by a hypertensive state, comprising the step of administering to a hypertensive patient suffering from a disease caused by a hyperglycemic state a hypotensive agent containing, as an active ingredient, (1) a peptide consisting of an amino acid sequence (SEQ ID NO: 1) of Val-Val-Tyr-Pro or a globin degradation product containing the peptide.

(II-3) the method according to (II-1) or (II-2), which is characterized by orally administering the compound to a patient suffering from mild hypertension.

(II-4) the method according to (II-2), which is characterized by administering the composition to a patient suffering from a disease caused by hyperglycemia or a hypertensive disorder in a pre-stage state thereof.

(II-5) the method according to any one of (II-1) to (II-4), wherein the peptide consisting of the amino acid sequence shown in (1) to (6) is administered daily to an adult in a total amount of 1mg to 500mg per day, or the globin decomposition product is administered daily in a ratio of 0.1g to 5g per day, by an oral administration route.

(III) use of peptides and globin decomposition products

(III-1) use of at least 1 peptide selected from the group consisting of peptides derived from a globin decomposition product and having amino acid sequences represented by the following (1) to (6), or a globin decomposition product containing at least 1 peptide selected from these peptides, for the preparation of an antihypertensive agent:

(1) Val-Val-Tyr-Pro (SEQ ID NO: 1)

(2) Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2)

(3) Trp-Gly-Lys-Val (SEQ ID NO: 3)

(4) Trp-Gly-Lys (SEQ ID NO: 4)

(5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5)

(6) Phe-Glu-Ser (SEQ ID NO: 6).

(III-2) use of a peptide comprising the amino acid sequence (SEQ ID NO: 1) of (1) Val-Val-Tyr-Pro or a globin reduction product comprising the peptide for the preparation of an antihypertensive agent for a patient suffering from a disease caused by a hyperglycemic state.

(III-3) at least 1 peptide selected from the group consisting of peptides derived from a globin decomposition product and having amino acid sequences represented by the following (1) to (6), or a globin decomposition product containing at least 1 peptide selected from the group consisting of the peptides:

(1) Val-Val-Tyr-Pro (SEQ ID NO: 1)

(2) Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2)

(3) Trp-Gly-Lys-Val (SEQ ID NO: 3)

(4) Trp-Gly-Lys (SEQ ID NO: 4)

(5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5)

(6) Phe-Glu-Ser (SEQ ID NO: 6).

(III-4) A peptide consisting of (1) a Val-Val-Tyr-Pro amino acid sequence (SEQ ID NO: 1), or a globin reduction product containing the peptide, which is used in a method for improving the hypertensive status of a hypertensive patient suffering from a disease resulting from a hyperglycemic status or a method for preventing or treating a disease resulting from a hypertensive status in the patient.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, it is possible to provide an antihypertensive agent capable of lowering blood pressure and improving the hypertensive state. In particular, the antihypertensive agent of the present invention containing, as an active ingredient, a peptide consisting of (1) the amino acid sequence of Val-Val-Tyr-Pro (SEQ ID NO: 1) or a globin reduction product containing the peptide has not only an antihypertensive effect but also an effect of inhibiting blood glucose elevation or an insulin hypersecretion effect, and therefore can be used more effectively for patients with diabetes or hypertension in its pre-stage state.

Detailed Description

The hypotensive agent of the present invention is characterized by comprising at least 1 peptide selected from the group consisting of (1) a peptide consisting of an amino acid sequence of Val-Val-Tyr-Pro (SEQ ID NO: 1) (hereinafter, simply referred to as "peptide (VVYP)"), a peptide consisting of an amino acid sequence of Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2) (hereinafter, simply referred to as "peptide (WGKVN)"), a peptide consisting of (3) Trp-Gly-Lys-Val (SEQ ID NO: 3) (hereinafter, simply referred to as "peptide (WGKV)"), a peptide consisting of (4) Trp-Gly-Lys (SEQ ID NO: 4) (hereinafter, simply referred to as "peptide (WGK)"), a peptide consisting of (5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5) (hereinafter, simply referred to as "peptide (WGAAK)") and a peptide consisting of (6) Phe-Glu-Ser (SEQ ID NO: 6) (hereinafter, simply referred to as "peptide (FES)") Or a globin decomposition product containing at least 1 of these peptides as an active ingredient.

These peptides can be prepared by chemical synthesis using a known peptide synthesis method. Examples of The Peptide synthesis method include an azide method, an acid chloride method, an acid anhydride method, a mixed acid anhydride method, a DCC method, an active ester method, a carboimidazole (carboimidazole) method, an oxidation-reduction method, and a DCC-addition (HOMB, HOBt, HOSu) method (The Peptide 1 (1966), Schreder & Luhke, Academic Press, New York, USA, and The Peptide synthesis method of "ペプチド Synthesis", spring valley, Kabushiki Kaisha (1975)). These peptide synthesis methods can be carried out by either solid phase synthesis methods or liquid phase synthesis methods.

In the above peptide synthesis method, amino acids having side chain functional groups, such as tyrosine and threonine, are preferably protected. As the protecting group, a known protecting group can be used, and for example, benzyloxycarbonyl (Cbz-), tert-butoxycarbonyl (Boc-), benzyl (Bz-) and the like can be used. In the peptide synthesis step of the present invention, the protecting group can be deprotected by a known method.

In addition, since these peptides are contained in a hydrolysate of globin such as hemoglobin and myoglobin (hereinafter referred to as "globin degradation product"), they can be obtained by separation and purification from the globin degradation product. In addition, hydrolysates other than globin, such as fish protein, fish meal, corn protein (zein), etc., may be used as raw materials for separation and purification. When the globin is an animal protein, the source of supply is not particularly limited, and blood of cattle, pigs, sheep, humans, horses, and the like can be widely used. In addition, meat containing a large amount of myoglobin such as livestock meat or fish meat may be used as a supply source.

The operation of the globin hydrolysis can be carried out according to the method described in International publication No. WO 89/06970. The hydrolysis is usually carried out using 1 or 2 or more kinds of hydrolytic enzymes selected from acid protease, neutral protease and alkaline protease.

Specifically, when hydrolyzing globin, the following method can be exemplified: first, a globin-containing substance is added to water, dispersed to 5 to 30 wt% (as a solid content), adjusted to the optimum pH for protease with an acid or an alkali, and the protease is added at once or successively, and reacted at 20 to 70 ℃ for 3 to 48 hours.

The method for separating and producing the peptide of the present invention from the globin decomposition product thus obtained can employ a known purification method for proteins or peptides. For example, a salting-out method, a dialysis method, an ion exchange resin method, an ultrafiltration method, a reverse phase chromatography method, or the like is used, and if necessary, a component containing each peptide [ peptide (VVYP), peptide (WGKVN), peptide (WGKV), peptide (WGK), peptide (AAWGK), or peptide (FES) ] can be obtained by appropriately combining these methods, and each peptide can be separated from the component. Among the above purification methods, the reverse phase chromatography is preferably a reverse phase chromatography under an acidic condition, or a combination of the reverse phase chromatography under an acidic condition and a reverse phase chromatography under a neutral condition.

The amount of protein in the component (a) can be measured by a known protein quantification method such as the indetrione method. The amino acid sequence of the peptide contained in the screened fraction can be identified by a known method (amino acid analysis method), and the presence of the target peptide (VVYP), peptide (WGKVN), peptide (WGKV), peptide (WGK), peptide (AAWGK), or peptide (FES) can be confirmed. In addition, in the hypotensive agent of the present invention, the peptides may be mixed in the form separated and purified by the above-mentioned method, but may be mixed in the form of a crude purified product containing the peptides as long as it has an antihypertensive effect. Examples of such a crude purified product include a globin degradation product containing each of the above-mentioned peptides, or a component thereof.

The antihypertensive agent of the present invention may be one composed of each of the above peptides or the crude/purified product (globin decomposition product or component thereof) containing the peptide, but it is usually prepared together with a pharmaceutically acceptable carrier or additive in addition to the above active ingredient. When the antihypertensive agent is prepared in combination with such a pharmaceutically acceptable carrier or additive, the amount of each peptide to be incorporated therein is not particularly limited as long as it is an effective amount having an antihypertensive effect.

Further, the peptide (VVYP) or a globin proteolysate containing the peptide has an action of suppressing the increase in blood glucose level or an action of enhancing insulin secretion in addition to the hypotensive action. Therefore, when the antihypertensive agent of the present invention is prepared using the peptide (VVYP) or the globin reduction product containing the peptide as an active ingredient, the amount of the peptide to be incorporated is preferably an amount effective for reducing blood pressure and inhibiting the increase in blood glucose level or enhancing insulin secretion.

The carrier to be blended with the antihypertensive agent of the present invention may be appropriately selected from commonly used excipients, diluents, binders, wetting agents, disintegrants, disintegration inhibitors, absorption promoters, lubricants, solubilizers, buffers, emulsifiers, suspending agents and the like, depending on the formulation of the preparation. The additives may be appropriately selected from generally used stabilizers, preservatives, buffers, isotonic agents, chelating agents, pH adjusting agents, surfactants, coloring agents, flavors, flavoring agents, sweeteners, and the like, depending on the dosage form of the preparation.

The unit dosage form of such a hypotensive agent can be selected as appropriate depending on the route of administration, and these dosage forms are roughly classified into oral preparations, pulmonary preparations, nasal preparations, sublingual preparations, non-oral preparations (injections, drops), and the like. The blood pressure lowering agent can be made into solid dosage forms such as tablet, pill, powder, granule and capsule by conventional method; or liquid dosage forms such as solutions, suspensions, emulsions, syrups, and elixirs. In addition, the composition may be prepared as a dried product which can be made into a liquid state by adding an appropriate carrier at the time of use. These can be modulated according to a usual method.

The ratio of each peptide to be incorporated in the antihypertensive agent of the present invention is usually about 0.001 to 80% by weight, preferably about 0.1 to 50% by weight, and more preferably about 0.1 to 10% by weight, based on the total amount.

The dose of the thus obtained antihypertensive agent is appropriately selected depending on the purpose (the antihypertensive effect, the effect of inhibiting the increase in blood glucose level or the effect of enhancing insulin secretion and the antihypertensive effect), the method of administration of the antihypertensive agent, the dosage form, the age and body weight of the patient to be administered, the symptom (severity of the disease), and the like. Specifically, the dosage of the peptide of the present invention (total amount when 2 or more peptides are administered in combination) may be about 1 to 500mg, preferably about 2 to 100mg per day for an adult. The dose of the globin decomposition product of the present invention is about 0.1 to 5g, preferably about 0.5 to 3g per day for an adult.

The administration is not necessarily 1 time per day, and may be divided into 2 to 4 times per day. The preparations in various forms can be administered by an appropriate administration route according to the form thereof, for example, an injection form can be administered intravenously, intramuscularly, subcutaneously, intradermally, intraperitoneally, etc., and a pharmaceutical preparation in a solid preparation form can be administered orally, etc. Oral administration is preferred.

As shown in the experimental examples described below, the hypotensive agent of the present invention exerts an antihypertensive effect on patients in a hypertensive state, thereby improving the hypertensive state. Therefore, the hypotensive agent of the present invention is effective as a prophylactic or therapeutic agent for a disease caused by a hypertensive state.

In addition, the classification of hypertension specified by the Japan hypertension society and WHO/ISH is shown in Table 1.

[ Table 1]

Classification	Systolic blood pressure (mmHg) diastolic blood pressure (mmHg)
		Optimum blood pressure	<120 and < 80
Normal blood pressure	< 130 and < 85
		Normal high blood pressure	130 to 139 or 85 to 89
Mild hypertension	140 to 159 or 90 to 99
		Moderate hypertension	160 to 179 or 100 to 109
Severe hypertension	Not less than 180 or not less than 110
		Systolic hypertension	Not less than 140 and less than 90

(the Japanese society for hypertension treatment guidelines compiled by the Committee for hypertension treatment guidelines 2004)

The hypertensive patients to be treated by the present invention include patients belonging to mild hypertension, moderate hypertension, severe hypertension and systolic hypertension in the above-mentioned hypertension classification. Preferably, the patient is a patient suffering from mild hypertension, moderate hypertension or severe hypertension, and more preferably a patient suffering from mild hypertension. In particular, the peptide (VVYP) used in the present invention and a globin reduction product containing the peptide have a stronger effect on lowering diastolic blood pressure than systolic blood pressure. Therefore, the antihypertensive agent of the present invention containing such a peptide and a globin decomposition product containing the peptide as active ingredients is suitable for hypertension patients with relatively high diastolic blood pressure.

The antihypertensive agent of the present invention comprising as an active ingredient a peptide (VVYP) or a globin decomposition product containing the peptide has an insulin secretion promoting action in addition to the above hypotensive action, improves the hyperglycemic state caused by the decrease or lack of insulin action, and exerts a hypoglycemic action. Therefore, the antihypertensive agent is effective as an antihypertensive agent suitable for use in hypertensive patients suffering from various diseases resulting from a hyperglycemic state caused by a decrease or lack of insulin action. Here, diabetes and diabetic complications are listed as diseases caused by a hyperglycemic state. Here, the target diabetes includes both type II diabetes (non-insulin-dependent diabetes) and type I diabetes (insulin-dependent diabetes). Non-insulin dependent type II diabetes mellitus is suitable. According to the guidelines of the Japan diabetes society (1999), diabetes can be judged when the blood glucose level at random is 200mg/dL or more, the blood glucose level at fasting state is 126mg/dL or more, and the 2-hour value at 75g oral glucose load test is 200mg/dL or more. Further, when the above criteria were met 2 times in examination on different days, or when the characteristic symptoms of diabetes were observed even if 1 time was confirmed, HbA was observed_1c(hemoglobin A)_1c) When the content is more than 6.5%, or when diabetic retinopathy exists, the patient can be judged to be diabetic. In addition, the diabetic patients to be treated by the present invention also include those in the preceding stages thereofSegment, i.e. patients in the borderline region (borderline diabetic patients). In addition, when the blood glucose value in fasting state is between 110-125 mg/dL, or the 2 hour value in glucose load test is between 140-199 mg/dL, the diabetes can be judged as critical diabetes.

The diabetic complication is a systemic or local disease that is complicated by diabetes (preferably non-insulin-dependent type II diabetes) as a direct or indirect factor, and specifically includes diabetic acidosis, diabetic xanthoma, diabetic amyotrophic lateral sclerosis, diabetic ketosis, diabetic coma, diabetic gastropathy, diabetic gangrene, diabetic ulcer, diabetic complication, diabetic diarrhea, diabetic microangiopathy, diabetic uterine sclerosis, diabetic cardiomyopathy, diabetic neuropathy, diabetic nephropathy, diabetic bullous disease, diabetic cataract, diabetic skin disease, diabetic sclerosis, diabetic retinopathy, diabetic lipoprogressive ring death, and diabetic blood flow disorder.

Examples

The present invention will be described in more detail below with reference to preparation examples and experimental examples. However, these experimental examples are not intended to limit the present invention in any way. In the following experimental examples,% means% by weight unless otherwise specified.

Preparation example 1Production of globin decomposition product

The following shows details of a method for producing a degraded globin obtained using bovine red blood cells.

100kg of fresh bovine red blood cells were added to 250 liters of water to fully hemolyze the cells, phosphoric acid was added to adjust the pH to 2.8, and 2.6X 10 was added⁷The unit of Aspergillus niger acidic protease was reacted at 50 ℃ for 3 hours.

After the reaction, the reaction mixture was heated at 80 ℃ for 30 minutes to stop the reaction, an aqueous suspension of calcium hydroxide was added to adjust the pH to 6.5, 10kg of diatomaceous earth was added, the mixture was filtered through a filter press, and the obtained filtrate was spray-dried to obtain 23kg of a globin decomposition product powder. The molecular weight distribution of the resultant globin resolved product was analyzed using gel filtration chromatography under the following conditions.

< gel filtration chromatography >

The device comprises the following steps: high performance liquid chromatograph (LC-6A type manufactured by Shimadzu corporation)

Column: polyhydroxyhexyl A, 5 μm, 9.4X 200mm, manufactured by PolyC Inc

Eluting solvent: 50mM formic acid

Flow rate: 0.5 ml/min

And (3) detection: ultraviolet absorption 221nm

Fig. 1 shows a gel filtration chromatogram of the globin reduction product obtained by the gel filtration chromatography.

Preparation example 2Separation and purification of blood pressure lowering peptide

The peptide (VVYP) of the present invention can be obtained by treating the globin decomposition product prepared in preparation example 1 in the following order: (1) ion exchange, (2) ultrafiltration, (3) separation by reverse phase chromatography under acidic conditions, and (4) separation by reverse phase chromatography under neutral conditions.

(1) Ion exchange

To weakly acidic cation exchange resin (Amberlite IRC)₅₀，H⁺Type Organo corporation) 13.7g of a 10% aqueous solution of the globin decomposition product obtained in preparation example 1 was added, and after stirring for 1 hour to adsorb, an unadsorbed component was obtained.

(2) Ultrafiltration

The unadsorbed fraction obtained by the above ion exchange treatment was subjected to ultrafiltration using a stirred ultrafiltration apparatus (manufactured by Advantec corporation, UHP 90K) and an ultrafiltration membrane (manufactured by Advantec corporation, UIIH-1, molecular weight isolation 1000), and a liquid (residual liquid) remaining on the ultrafiltration membrane was collected. The resulting fractions were quantified by the indetrione method after acid hydrolysis. Acid hydrolysis was performed as follows: 1ml of hydrochloric acid having a final concentration of 6N was added to a test tube for a protein amount of 3 to 5mg, the tube was sealed under normal pressure, and the tube was heated at 110 ℃ for 22 hours. In addition, the indetrione method is performed as follows. That is, the hydrolyzed specimen was adjusted to pH5.0 with sodium hydroxide, reacted at 100 ℃ for 15 minutes using ninhydrin reagent containing 0.2M citric acid buffer (pH5.0), and the absorbance at 570nm was measured. Further, as a standard solution, an L-leucine aqueous solution (75, 150, 225, 300nmol/ml) was subjected to ninhydrin reaction, and a standard curve was obtained from the absorbance obtained to calculate the amount of amino groups corresponding to L-leucine in the specimen. The results of the quantification are shown in table 2.

(3) Reversed phase (acid) chromatography

The residual liquid obtained in the above ultrafiltration was subjected to reverse phase (acid) chromatography under the following conditions.

< reversed phase (acid) chromatography >

The device comprises the following steps: high performance liquid chromatograph (LC-10A type manufactured by Shimadzu corporation)

Column: SuperPac Pep-S, 15 μm, 22.5X 250mm, produced by Pharmacia

Eluting solvent: aqueous acetonitrile solution containing 0.1% by volume of trifluoroacetic acid

Gradient: a linear concentration gradient from 2% by volume to 35% by volume of acetonitrile, the acetonitrile concentration varying to 1% by volume/min

Flow rate: 5 ml/min

Temperature: 40 deg.C

And (3) detection: ultraviolet absorption of 220nm

Time of dividing: 53.8 to 54.5 minutes (component A)

Fig. 2 shows a chromatogram obtained by the reversed-phase (acid) chromatography.

The resulting fractions were quantified by amino acid analysis after acid hydrolysis. Acid hydrolysis was performed as follows: 1ml of hydrochloric acid (final concentration: 6N) was added to a test tube for 3 to 5mg of protein, and the tube was sealed under reduced pressure and heated at 110 ℃ for 22 hours. In addition, the amino acid analysis was performed under the following conditions.

< analysis of amino acids >

The device comprises the following steps: high performance liquid chromatograph (LC-6A type manufactured by Shimadzu corporation)

Column: shim-pack ISC-07/S1504 Na, 7 μm, 4.0X 150mm, manufactured by Shimadzu corporation

Eluting solvent: amino acid mobile phase kit (Na type) produced by Kazuki Kaishangjin

Flow rate: 0.3 ml/min

Temperature: 55 deg.C

Reaction solution 1: OPA reagent of analytical kit produced by Shimadzu corporation

And (3) detection: fluorescence absorption (Ex 348nm, Em 450nm)

The solution obtained after the acid hydrolysis was concentrated and dried by a rotary evaporator, and further dried under reduced pressure for 12 hours or more to completely remove the hydrochloric acid. Next, each amino acid was dissolved in 0.2M citric acid buffer solution (pH2.2) so that the content of each amino acid was about 100nmol/ml, and the solution was filtered through a 0.45 μ M filter, and then 10. mu.l of the filtrate was injected. On the other hand, as a standard solution, the amino acid mixture standard solution 18 component H form (Wako pure chemical industries, Ltd.) was diluted 25-fold with 0.2M citric acid buffer solution (pH2.2), and then 10. mu.l (1 nmol/10. mu.l of each amino acid) was injected. The peak area of the amino acid was calculated from Chromatopac C-R4A (manufactured by Shimadzu corporation), and the amount of the amino acid was calculated from the ratio of the peak area to the peak area of the standard solution.

(4) Reversed phase (neutral) chromatography

The fraction eluted and separated by the reversed phase (acidic) chromatography was subjected to reversed phase (neutral) chromatography under the following conditions.

< reversed phase (neutral) chromatography >

The device comprises the following steps: high performance liquid chromatograph (LC-10A type manufactured by Shimadzu corporation)

Column: SuperPac Pep-S, 15 μm, 22.5X 250mm (produced by Pharmacia)

Eluting solvent: aqueous acetonitrile solution containing 20mM ammonium acetate buffer (pH6.5)

Gradient: a linear concentration gradient of acetonitrile concentration from 0 volume% to 25 volume%, the acetonitrile concentration changing to 0.5 volume%/min

Flow rate: 5 ml/min

Temperature: 40 deg.C

And (3) detection: ultraviolet absorption of 220nm

Time of dividing: 45.8 to 51.0 minutes (component B)

Fig. 3 shows a chromatogram obtained by the reverse phase (neutral) chromatography.

The obtained components were quantified in the same manner as in (3) above, and then further identified. The amino acid composition was calculated from the ratio of the amount of each amino acid obtained to the total amount of the amino acid content. As a result, it was found that component B was VVYP (Val-Val-Tyr-Pro). This was compared with the amino acid sequence of hemoglobin to confirm that any sequence was present. The results of the quantification are shown in table 2.

[ Table 2]

Peptides	Protein weight (g)
		Globin decomposition product	13.70
Ion exchange + ultrafiltration reverse phase chromatography [ component A ]][ component B]VVYP	4.240.390.006

Preparation example 3Separation and purification of antihypertensive peptides

The 10% aqueous solution containing 100g of the globin decomposition product prepared in preparation example 1 was adsorbed onto an open column (. PHI.100 mm. times.400 mm) packed with a reversed-phase resin (YMC ODS AQ 120S 50), and the concentration of ethanol was gradually increased by 5% by volume to 5-15% by volume using aqueous ethanol as an eluent, thereby eluting the column. Subsequently, ethanol 5 vol% and 15 vol% of the elution component were subjected to reverse phase (acid) chromatography under the following conditions, respectively.

< reversed phase (acid) chromatography >

The device comprises the following steps: high performance liquid chromatograph (Waters Alliance 2695-2996(Waters products))

Column: nucleosil 5C18Phi 4.0X 250mm (produced by Chemo corporation)

Eluting solvent: aqueous acetonitrile solution containing 0.1% by volume of trifluoroacetic acid

Gradient: 0 min-5 min: the concentration of the acetonitrile is 5 percent by volume,

5 minutes to 30 minutes: linear concentration gradient of acetonitrile concentration from 5% to 30% by volume (acetonitrile concentration change of 1% by volume/min)

Flow rate: 0.8 mL/min

Temperature: 40 deg.C

And (3) detection: ultraviolet absorption of 210nm

Injection amount: 20 μ L

Fig. 4 and 5 show reverse phase chromatograms of ethanol at 5 vol% and 15 vol% elution components, respectively.

The respective peaks obtained in the respective chromatograms were separated, and according to the method described in preparation example 2, amino acid analysis was performed after acid hydrolysis, and it was judged that in the reversed phase chromatogram of 5 vol% eluted components (fig. 4), the eluted peak with a retention time of 12.55 minutes was a peak corresponding to the peptide (FES)) consisting of the amino acid sequence Phe-Glu-Ser (seq id No. 6), and in the reversed phase chromatogram of 15 vol% eluted components (fig. 5), the eluted peaks with retention times of 18.16 minutes and 19.53 minutes were peaks corresponding to the peptide (AAWGK)) consisting of the amino acid sequence Ala-Trp-Gly-Lys and the peptide (WGKVN)) consisting of Trp-Gly-Lys-Val-Asn, respectively.

Experimental example 1

The peptide (VYP) prepared in preparation example 2 was orally administered to naturally-occurring hypertensive rats (SHR/Izm, male, 9-week-old, 266g in body weight, Japan SLC corporation) (6 rats) 1 time at a dose of 10 mg/kg. From the 2 nd and 6 th hours after the start of administration, systolic blood pressure (-O-) was measured by the Tail-cuff method without anesthesia using a non-warmed non-invasive sphygmomanometer (Chamber tone machine, MK-2000). As a control group, SHR mice (6 mice) were orally administered with a solvent (distilled water) 1 time in the same manner, and the systolic blood pressure (- ● -) was measured in the same manner. The results are shown in FIG. 6. As is clear from FIG. 6, it was confirmed that the peptide (VYP) of the present invention has a blood pressure lowering effect.

Experimental example 2

The peptide (VVYP) -containing globin compound prepared in preparation example 1 was orally administered to naturally-occurring hypertensive rats (SHR/Izm, male, 14 weeks old, initial administration weight range of 309g to 359g, Japan SLC corporation) (8 rats) at an administration dose of 1g/kg for 10 weeks. The systolic blood pressure (-O-) was measured by the Tail-cuff method without anesthesia using a non-warming non-invasive sphygmomanometer (Chamber tone machine, MK-2000) every 2 weeks from the start of administration. As a control group, SHR mice (8 mice) were orally administered with a solvent (distilled water) for 10 weeks in the same manner, and the systolic blood pressure (- ● -) was measured in the same manner. The results are shown in FIG. 7.

As shown in FIG. 7, it was found that the globin reduction product containing the peptide (VVYP) of the present invention had an effect of suppressing the increase in blood pressure.

Experimental example 3

89 mild hypertensive patients (systolic blood pressure: 140 to 159mmHg or diastolic blood pressure: 99 to 99mmHg) were randomly divided into 2 groups (44 and 45), and 500mg of the peptide (VVYP) -containing globin degradation product prepared in preparation example 1 was orally administered to the experimental group (45) 1 day 3 times for 10 weeks. On the other hand, an equivalent amount of lactose was administered to the control group (44) patients. Blood pressure (systolic blood pressure, diastolic blood pressure) was measured at weeks 3, 6, and 10 from the start of administration, and changes in blood pressure were evaluated.

The results are shown in FIG. 8. As shown in FIG. 8, by oral administration of the globin decomposition product of the present invention, the systolic blood pressure showed a tendency to decrease, and the diastolic blood pressure significantly decreased (-. O-) after the 6 th week from the start of administration. In addition, the difference from the control group (- ● -) at the 10 th week from the start of administration was about 5mmHg in systolic blood pressure and about 8mmHg in diastolic blood pressure. From the above results, it is clear that the globin degradation product containing peptide (VVYP) has a blood pressure lowering effect on humans.

Experimental example 4

The following peptides were synthesized according to a usual method based on the amino acid sequence of the peptide isolated and identified from the globin decomposition product in preparation example 3. The peptide (WGKV) of (3) and the peptide (WGK) of (4) correspond to peptides obtained by deleting 1C-terminal amino acid from the amino acid sequence of the peptide (WGKVN) of (2) at a time.

(2) Trp-Gly-Lys-Val-Asn (SEQ ID NO: 2)

(3) Trp-Gly-Lys-Val (SEQ ID NO: 3)

(4) Trp-Gly-Lys (SEQ ID NO: 4)

(5) Ala-Ala-Trp-Gly-Lys (SEQ ID NO: 5)

(6) Phe-Glu-Ser (SEQ ID NO: 6)

Each of the synthetic peptides ((2) to (6)) prepared as described above was orally administered to naturally-occurring hypertensive rats (SHR/Izm, male, 12-week-old, body weight 285g, Japan SLC corporation) 1 time at a dose of 50 mg/kg. Systolic blood pressure was measured by the Tail-cuff method without anesthesia using a non-warming non-invasive sphygmomanometer (Km-2000) from the 2 nd, 4 th and 6 th hours from the start of administration. As a control group, the same solvent (distilled water) was orally administered to naturally-occurring hypertensive rats 1 time, and systolic blood pressure was measured in the same manner. The results are shown in Table 3. As is clear from Table 3, the peptides were all judged to have hypotensive effects.

[ Table 3]

^*：p＜0.05 ^**：p＜0.01

Experimental example 5Safety test

The peptide (VVYP) prepared in preparation example 2 was orally administered to male and female ICR mice at a dose of 10g/kg or more (maximum dose) but there were no cases of death. Thus confirming the safety of the peptide.

Drawings

FIG. 1 shows a gel filtration chromatogram of a globin decomposition product (preparation example 1).

FIG. 2 shows the results (chromatogram) of reversed-phase (acid) chromatography performed in preparation examples 2 and 3.

FIG. 3 shows the results (chromatogram) of reversed-phase (neutral) chromatography performed in preparation examples 2 and 4.

FIG. 4 shows the results (chromatogram) of reversed phase (acid) chromatography of a globin decomposition product ethanol 5 vol% elution component performed in preparation example 3.

FIG. 5 shows the results (chromatogram) of reversed phase (acid) chromatography of a 15 vol% eluate of a globin decomposition product ethanol performed in preparation example 3.

FIG. 6 is a graph showing the change of blood pressure with time when a peptide (VVYP) was orally administered to naturally-occurring hypertensive rats (Experimental example 1).

Fig. 7 shows a graph of the change over time in blood pressure when a globin proteolysate containing peptide (VVYP) was orally administered to naturally-occurring hypertensive rats (experimental example 2).

Fig. 8 shows a graph of the change with time of blood pressure (systolic blood pressure, diastolic blood pressure) when a globin proteolysate containing peptide (VVYP) is orally administered to a human with mild hypertension (experimental example 3).

Sequence Listing free text

The sequence numbers 1 to 6 represent amino acid sequences of peptides contained in a hydrolysate of globin (a globin degradation product).

Sequence No. 7 shows the amino acid sequence of a peptide contained in an enzymatic hydrolysate of albumin.

Sequence listing

<110> love Meujian pharmaceutical Co.

<120> hypotensive agent

<130>P08-98

<140>JP2007-204929

<141>2007-08-07

<160>1

<170>PatentIn version 3.1

<210>1

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>1

Val Val Tyr Pro

1 4

<210>2

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>2

Trp Gly Lys Val Asn

1 5

<210>3

<211>4

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>3

Trp Gly Lys Val

1 4

<210>4

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>4

Trp Gly Lys

1 3

<210>5

<211>5

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>5

Ala Ala Trp Gly Lys

1 5

<210>6

<211>3

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in globin hydrolysate

<400>6

Phe Glu Ser

1 3

<210>7

<211>6

<212>PRT

<213> Artificial sequence

<220>

<223> peptides contained in albumin. Xaa represents His, Lys or Arg.

<400>7

Arg-Pro-Leu-Xaa-Pro-Trp

1 5

Claims (7)

1. An antihypertensive agent comprising, as an active ingredient, at least 1 peptide selected from the group consisting of peptides consisting of amino acid sequences represented by the following (2) to (5), or a globin degradation product containing at least 1 peptide selected from these peptides:

(2) Trp-Gly-Lys-Val-Asn of SEQ ID NO. 2

(3) Trp-Gly-Lys-Val of SEQ ID NO. 3

(4) Trp-Gly-Lys of SEQ ID NO. 4

(5) Ala-Ala-Trp-Gly-Lys of SEQ ID NO. 5.

2. The hypotensive agent according to claim 1, wherein:

is orally administered to patients with mild hypertension.

3. The hypotensive agent according to claim 1 or 2, wherein:

administering at least 1 of the peptides consisting of the amino acid sequences shown in (2) to (5) to an adult person daily at a rate of 1mg to 500mg in total per day, or administering a globin decomposition product containing at least 1 of these peptides at a rate of 0.1g to 5g per day, by oral administration route.

4. Use of at least 1 peptide selected from the peptides consisting of the amino acid sequences shown in the following (2) to (5), or a globin degradation product containing at least 1 peptide of these peptides, in the production of an antihypertensive agent:

(2) Trp-Gly-Lys-Val-Asn of SEQ ID NO. 2

(3) Trp-Gly-Lys-Val of SEQ ID NO. 3

(4) Trp-Gly-Lys of SEQ ID NO. 4

(5) Ala-Ala-Trp-Gly-Lys of SEQ ID NO. 5.

5. At least 1 peptide selected from peptides consisting of amino acid sequences represented by the following (2) to (5), or a globin decomposition product containing at least 1 peptide among these peptides, which is used in a method for improving a hypertensive state or a method for preventing or treating a disease caused by a hypertensive state:

(2) Trp-Gly-Lys-Val-Asn of SEQ ID NO. 2

(3) Trp-Gly-Lys-Val of SEQ ID NO. 3

(4) Trp-Gly-Lys of SEQ ID NO. 4

(5) Ala-Ala-Trp-Gly-Lys of SEQ ID NO. 5.

6. Use according to claim 4, characterized in that:

the hypotensive agent is orally administered to a patient with mild hypertension.

7. Use according to claim 4 or 6, characterized in that:

the antihypertensive agent is administered to an adult by an oral administration route by administering at least 1 peptide of the peptides consisting of the amino acid sequences shown in (2) to (5) in a total amount of 1mg to 500mg per day, or by administering a globin decomposition product containing at least 1 peptide of these peptides in a ratio of 0.1g to 5g per day.