JP7218906B2 - Method for detecting vascular function, method for determining progression stage of hypertension, kit for determining progression stage for hypertension, and method for predicting therapeutic effect of hypertension - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for determining the advanced stage of hypertension, a kit for determining the advanced stage of hypertension, and a method for predicting the therapeutic effect of hypertension.

Hypertension is called a silent killer because it causes diseases such as stroke and myocardial infarction that are directly linked to death, and prevention and treatment of this are desired.

Many physiologically active peptides exhibiting various physiological actions have been found from enzymatic digests of food proteins. It is known that the onset of arteriosclerotic diseases is triggered by functional deterioration of blood vessels, particularly the vascular endothelium, and attention has been focused on the function of physiologically active peptides on blood vessels.

Patent Literatures 1 and 2 and Non-Patent Literature 1 disclose peptides having effects such as appetite suppression, anti-obesity, arterial relaxation, blood pressure lowering, and metabolic syndrome prevention.

International Publication WO2012/070554 JP 2018-184367

Kagebayashi T et al. Mol Nutr Food Res. 2012 Sep; 56(9):1456-63.

An object of the present invention is to provide a method for determining the progression stage of hypertension using a novel peptide combination, a kit for determining the progression stage of hypertension, and a method for predicting the therapeutic effect of hypertension.

The present inventors have made intensive studies to solve the above problems, and found that peptides of 2 to 20 amino acid residues long containing SY are highly effective in the early stage of hypertension, and 2 to 20 amino acid residues long containing NA. peptide was found to be highly effective in the late stage of hypertension. The present invention has been completed through further studies based on such findings.

That is, the present invention includes the following aspects.

Item 1, a method for detecting vascular function in hypertensive patients, comprising:
(A) A peptide with a length of 2 to 20 amino acid residues containing SY, and (B) A peptide with a length of 2 to 20 amino acid residues containing NA were administered to hypertensive patients, respectively. A method that uses the degree of change in the index as an index.

Item 2. The method according to Item 1, wherein peptide (A) is dipeptide SY and peptide (B) is dipeptide NA.

Item 3, a method for determining the progression stage of hypertension, comprising:
(A) a 2- to 20-amino acid residue-long peptide containing SY, and (B) a 2- to 20-amino acid residue-long peptide containing NA were administered to hypertensive patients, respectively, to measure changes in blood pressure,
If the drop in blood pressure when the peptide (A) is administered is large, it is determined to be in the early stage of hypertension,
A method for judging that the onset of hypertension is in the late stage when the decrease in blood pressure is large when the peptide (B) is administered.

Item 4. The method according to Item 3, wherein peptide (A) is dipeptide SY and peptide (B) is dipeptide NA.

Item 5. A kit for determining the progression stage of hypertension, comprising (A) a peptide of 2 to 20 amino acid residues long containing SY, and (B) a peptide of 2 to 20 amino acid residues long containing NA.

Item 6. The kit according to Item 5, wherein peptide (A) is dipeptide SY and peptide (B) is dipeptide NA.

Item 7, a method for predicting the therapeutic effect of hypertension,
Predicting that administration of a peptide having a length of 2 to 20 amino acid residues containing (A) SY has a high therapeutic effect when a hypertensive patient is in the early stage of hypertension,
(B) A method for predicting that administration of a peptide containing NA with a length of 2 to 20 amino acid residues will have a high therapeutic effect when a hypertensive patient is in the late stage of onset of hypertension.

Item 8. The method of Item 7, wherein peptide (A) is dipeptide SY and peptide (B) is dipeptide NA.

The present invention provides a novel method for detecting vascular function, a method for determining the progression stage of hypertension, a kit for determining the progression stage of hypertension, and a method for predicting the therapeutic effect of hypertension.

The results of arterial relaxation experiments in the early stage of hypertension (Early stage) and the late stage of hypertension (Advanced stage) are shown. (A) Administration of dipeptide SY. (B) Administration of dipeptide NA. The evaluation results of the antihypertensive action are shown. The evaluation results of the antihypertensive action are shown.

Peptide The peptide according to the present invention includes (A) a 2 to 20 amino acid residue long peptide containing SY (also referred to as "peptide (A)"), and (B) a 2 to 20 amino acid residue long peptide containing NA. It is a peptide (also referred to as "peptide (B)").

The peptide (A) and peptide (B) are 2 to 20 amino acid residues, preferably 2 to 10 amino acid residues, more preferably 2 to 5 amino acid residues, particularly preferably 2, 3, 4 or 5 It has an amino acid residue.

When the peptide has an amino acid residue length of 3 or more, the peptide (A) is on the N-terminal side and/or C-terminal side of the amino acid sequence SY, and the peptide (B) is on the N-terminal side and/or the C-terminal side of the amino acid sequence NA. /or has any amino acid residue on the C-terminal side. Amino acid residues may be natural or non-natural amino acids. The natural amino acids that make up proteins are alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. and other amino acid residues such as selenocysteine, N-formylmethionine, pyrrolysine, pyroglutamine, and the like.

In one of the preferred embodiments of the present invention, said peptide (A) is dipeptide SY. In one preferred embodiment of the present invention, the peptide (B) is dipeptide NA.

The amino acids constituting the peptide (A) and/or peptide (B) are L-form amino acids, D-form amino acids, or DL-form amino acids (if the amino acids are a mixture of D-form and L-form, they are either racemic or any of the amino acids in excess of either enantiomer) can be used. Peptides consisting of only L-amino acids or only D-amino acids, particularly peptides consisting of only L-amino acids, are preferred.

Moreover, when the peptide (A) and/or peptide (B) contains two or more asymmetric carbon atoms, it may be in the form of each enantiomer or diastereomer, or a mixture thereof at any ratio. Separation of enantiomers or diastereomers can be carried out by a conventional column method, by using an optically active column, by introducing an optically active group and optically resolving it in the form of a derivative, and then by removing the optically active group. Any known method such as a method of optical resolution by forming a salt with an active acid or base can be used.

The peptide (A) and peptide (B) can have modifications. The amino terminus (N-terminus) of the peptide may be a free amino group (NH ₂ —) or modified such as an acetyl group (CH ₃ CO—). The carboxy terminus (C terminus) of the peptide may be a free carboxyl group (--COOH) or modified such as an amide group. The amino acid residues of the peptide may be unmodified or modified with phosphate groups, sugar chains and the like.

The above peptide (A) and peptide (B) may be salts (acid addition salts or base salts). Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, and perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, and p-toluenesulfonic acid. , benzenesulfonic acid, methanesulfonic acid, and trifluoroacetic acid. Base salts include alkali metal salts such as sodium, potassium and lithium, alkaline earth metal salts such as calcium and magnesium, and the like.

The peptide (A) and peptide (B) may be solvates. Solvates include water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethylsulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. things are mentioned.

The above peptide (A) and peptide (B) can be obtained by hydrolysis of natural proteins or polypeptides, or by chemical synthesis.

Chemical synthesis includes peptide synthesis methods, ie, liquid phase methods or solid phase methods, which are commonly used methods for peptide synthesis. Specific examples include a method using an active ester such as HBTU, a method using a coupling agent such as carbodiimide, and the like, which combine a raw material having a reactive carboxyl group and a raw material having a reactive amino group. If the resulting peptide has a protecting group, it can also be produced by removing the protecting group.

Functional groups that should not participate in the reaction in this reaction step are protected by protective groups. Examples of amino-protecting groups include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of carboxyl group-protecting agents include groups capable of forming alkyl esters, benzyl esters, and the like. It is bound to a carrier such as an 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 protective group is removed, and in the case of the solid-phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptides of the invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reversed phase liquid chromatography, affinity chromatography and the like. Synthesis of the synthesized peptide is analyzed by a protein sequencer, GC-MS, etc., which reads the amino acid sequence from the C-terminus by the Edman degradation method.

The above peptide (A) and peptide (B) can also be synthesized by an enzymatic method using L-amino acid ligase (see International Publication WO2003/010307).

When administering the above peptide (A) and peptide (B), the route of administration is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted. It can be administered parenterally. Oral administration is preferred.

The dosage for each of the above peptide (A) and peptide (B) is usually 0.01 mg/kg to 500 mg/kg, preferably 0.05 mg/kg to 100 mg/kg, more preferably 0 mg/kg per day for adults. .1-30 mg/kg.

The above peptide (A) and peptide (B) can be administered in the form of a pharmaceutical composition prepared by mixing each with pharmaceutical carriers. As a pharmaceutical carrier, a substance commonly used in the pharmaceutical field and which does not react with the peptide of the present invention is used.

Specifically, pharmaceutical carriers, e.g. carriers for oral intake, diluents or excipients such as substances such as lactose, glucose, mannite, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate , synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light silicic anhydride, stearic acid. Magnesium, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate, glycerin, fatty acid glycerin ester, refined lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, Examples include nonionic surfactants, propylene glycol, water, and the like.

Dosage forms include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like. These formulations are prepared according to a conventional method. Liquid formulations may be dissolved or suspended in water or other suitable solvents at the time of use. Moreover, tablets and granules may be coated by a known method. Injections are prepared by dissolving the peptide of the present invention in water, but if necessary, they may be dissolved in physiological saline or glucose solution, and buffers and preservatives may be added. good.

These formulations may contain the peptides of the invention in proportions of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.

In order to produce a solid preparation for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate, silicic anhydride, etc. to form a powder, or if necessary, sucrose, A binder such as hydroxypropyl cellulose or polyvinylpyrrolidone, a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium, and the like are added and wet or dry granulated to form granules. Tablets can be produced by compressing these powders and granules as they are or after adding a lubricant such as magnesium stearate and talc. These granules or tablets are coated with an enteric base such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc. to form an enteric preparation, or ethyl cellulose, carnauba wax, hydrogenated oil, etc. to form a sustained release preparation. can also In order to manufacture capsules, hard capsules are filled with powders or granules, or active ingredients are dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and coated with a gelatin film to form soft capsules. can be done.

Liquid preparations for oral administration can be prepared by dissolving the active ingredient and a sweetening agent such as sucrose, sorbitol or glycerin in water to prepare a clear syrup, or by adding essential oils, ethanol or the like to prepare an elixir. Gum arabic, tragacanth, polysorbate 80, sodium carboxymethylcellulose, etc. may be added to form emulsions or suspensions. Flavoring agents, coloring agents, preservatives and the like may be added to these liquid preparations as desired.

The above peptides (A) and (B) have hypotensive and arterial relaxant effects.

As shown in the examples below, the peptide (A) has a higher antihypertensive effect and an arterial relaxation effect in the early stage of hypertension. On the other hand, the above peptide (B) has higher antihypertensive and arterial relaxant effects in the late stage of hypertension.

Since the action of the peptide (A) is inhibited by the nitric oxide synthase inhibitor L-NAME, the action is thought to be mediated through the nitric oxide (NO) pathway (nitric oxide production).

Since the action of the above peptide (B) is inhibited by lorglumide, which is a _CCK1 receptor (cholecystokinin receptor) antagonist, the action is thought to be mediated by activation of the _CCK1 receptor.

A method for detecting vascular function in hypertensive patients One aspect of the present invention is
A method for detecting vascular function in a hypertensive patient, comprising:
(A) A peptide with a length of 2 to 20 amino acid residues containing SY, and (B) A peptide with a length of 2 to 20 amino acid residues containing NA were administered to hypertensive patients, respectively. is a method in which the degree of change in is used as an index.

In the method of the present invention, vascular function of a hypertensive subject is detected.

Hypertension in the present invention refers to a condition in which systolic blood pressure (highest blood pressure) is maintained at 140 mmHg or higher or diastolic blood pressure (lowest blood pressure) is maintained at 90 mmHg or higher, preferably essential hypertension.

In the method of the present invention, changes in blood pressure are measured when peptide (A) and peptide (B) are administered to hypertensive patients. The order of administration of peptide (A) and peptide (B) is not limited. The administration interval between the peptide administered first and the peptide administered later is desirably one day or more, ideally about 3 to 10 days.

The doses, administration methods, formulations, etc. of peptide (A) and peptide (B) can be as described in the above "Peptides" section.

Measurement of blood pressure change can be performed by a conventional method. From the viewpoint of convenience, a method using a sphygmomanometer with a cuff wrapped around the extremities (for example, the upper arm) can be used. The blood pressure value measured may be either systolic (highest) or diastolic (lowest).

Blood pressure changes are measured by measuring the blood pressure immediately before administration and 2 to 6 hours after administration of the peptide, and calculating the difference between them. Since both peptide (A) and peptide (B) have antihypertensive and arterial relaxant effects, administration of any of them is expected to lower the blood pressure level.

In the method of the present invention, the degree of change in blood pressure, that is, the measurement results of changes in blood pressure when peptide (A) and peptide (B) are administered, is used as an index to detect vascular function in hypertensive patients. Specifically, the change in blood pressure when peptide (A) is administered is compared with the decrease in blood pressure when peptide (B) is administered, and the decrease in blood pressure when peptide (A) is administered is If the decrease in blood pressure is greater than that caused by administration of peptide (B), the function of blood vessels, preferably the function of vascular endothelial cells, more preferably the function of producing nitric oxide, which is a vascular endothelial-derived relaxing factor, is maintained. (Hypertension is in the early stage of onset of hypertension) is detected. When the decrease in blood pressure upon administration of peptide (B) is greater than the decrease in blood pressure upon administration of peptide (A), vascular function, preferably vascular endothelial cell function, more preferably vascular It is detected that the function of producing nitric oxide, which is an endothelium-derived relaxing factor, is reduced (that hypertension is in the late stage of hypertension).

When peptide (A) and peptide (B) decrease blood pressure to the same extent, the function of vascular endothelial cells, especially the function of producing nitric oxide, which is a vascular endothelium-derived relaxing factor, is maintained. It can detect that there is a high risk of decline even though the For the sake of convenience, it can also be assumed that the function of vascular endothelial cells, particularly the function of producing nitric oxide, which is a vascular endothelium-derived relaxing factor, is reduced.

Since the patient who is the test subject has hypertension, medical intervention such as antihypertensive drugs is desirable regardless of the detection result. When it is detected that vascular function, particularly vascular endothelial cell function, is maintained, for example, antihypertensive agents based on nitric oxide-dependent mechanisms can be administered. For example, cholecystokinin-dependent antihypertensive agents can be administered when vascular function, particularly vascular endothelial cell function, is detected to be impaired.

In one embodiment, when it is detected that vascular function is maintained, the peptide (A) (for example, a pharmaceutical composition containing the peptide (A) as an active ingredient) can be administered continuously. can. When a decrease in vascular function is detected, the peptide (B) (for example, a pharmaceutical composition containing the peptide (B) as an active ingredient) can be administered continuously.

- A method for determining the progression stage of hypertension Another aspect of the present invention comprises:
A method for determining the progression stage of hypertension, comprising:
(A) a 2- to 20-amino acid residue-long peptide containing SY, and (B) a 2- to 20-amino acid residue-long peptide containing NA were administered to hypertensive patients, respectively, to measure changes in blood pressure,
If the drop in blood pressure when the peptide (A) is administered is large, it is determined to be in the early stage of hypertension,
A method for judging that the patient is in the late stage of onset of hypertension when there is a large drop in blood pressure when the peptide (B) is administered.

In the method of the present invention, it is determined whether the stage of progression of hypertension in a subject patient is early hypertension or late hypertension.

It is understood that the progression stage of hypertension progresses from the onset of hypertension to the late stage of hypertension over time from the early stage of hypertension. It is understood that the early stage of hypertension preferably refers to a stage in which the function of vascular endothelial cells, particularly the function of producing nitric oxide, which is a vascular endothelium-derived relaxing factor, is maintained. The late stage of hypertension is understood to refer to a stage in which hypertension progresses more than the early stage of hypertension, and preferably the above-mentioned vascular endothelial cell functions are diminished. Thus, vascular function can be assessed by the methods of the present invention.

In the method of the present invention, changes in blood pressure are measured when peptide (A) and peptide (B) are administered to hypertensive patients. The order of administration of peptide (A) and peptide (B) is not limited. The administration interval between the peptide administered first and the peptide administered later is desirably one day or more, ideally about 3 to 10 days.

The doses, administration methods, formulations, etc. of peptide (A) and peptide (B) can be as described in the above "Peptides" section.

Measurement of blood pressure change can be performed by a conventional method. From the viewpoint of convenience, a method using a sphygmomanometer with a cuff wrapped around the extremities (for example, the upper arm) can be used. The blood pressure value measured may be either systolic (highest) or diastolic (lowest).

Blood pressure changes are measured by measuring the blood pressure immediately before administration and 2 to 6 hours after administration of the peptide, and calculating the difference between them. Since both peptide (A) and peptide (B) have antihypertensive and arterial relaxant effects, administration of any of them is expected to lower the blood pressure level.

Changes in blood pressure when peptide (A) is administered and changes in blood pressure when peptide (B) is administered, based on the measurement results of blood pressure changes when peptide (A) and peptide (B) are administered respectively. to determine the progression stage of hypertension. If the decrease in blood pressure when peptide (A) is administered is greater than the change in blood pressure when peptide (B) is administered, it is determined to be in the early stage of hypertension, and peptide (B) is administered. When the decrease in blood pressure after administration of peptide (A) is large compared to the change in blood pressure after administration of peptide (A), it is judged to be in the late stage of onset of hypertension.

When the blood pressure is reduced to the same degree when peptide (A) is administered and when peptide (B) is administered, it can be determined that the risk of reaching the late stage of hypertension is high, although the latter stage of hypertension has not yet been reached. . For convenience, it can also be determined to be in the late stage of hypertension.

Since the subject patient has hypertension, medical intervention such as an antihypertensive drug is desirable regardless of the determination result. If the subject patient is determined to be in the early stage of hypertension, for example, a nitric oxide-dependent antihypertensive agent can be administered. If the subject patient is determined to be in the late stage of hypertension, for example, a cholecystokinin-dependent antihypertensive agent can be administered.

In one embodiment, when the subject patient is determined to be in the early stage of hypertension, the peptide (A) (for example, a pharmaceutical composition containing the peptide (A) as an active ingredient) is continuously administered. be able to. When the subject patient is determined to be in the late stage of hypertension, the peptide (B) (for example, a pharmaceutical composition containing the peptide (B) as an active ingredient) can be administered continuously.

-Kit Another aspect of the present invention is
A kit for determining the progression stage of hypertension, comprising (A) a 2- to 20-amino acid residue long peptide containing SY, and (B) a 2- to 20-amino acid residue long peptide containing NA.

The kit of the present invention is a kit for determining the advanced stage of hypertension. It can be suitably used for carrying out the method of determining the progression stage of hypertension of the present invention. However, the usage pattern is not limited to this.

The kit of the present invention contains peptide (A) and peptide (B). Formulations and the like of peptide (A) and peptide (B) can be as described in the "Peptide" section above.

In addition, the kit of the present invention can contain other components as needed. Examples include, but are not limited to, peptide-free formulations (which can be used as negative controls), means for measuring blood pressure (eg, sphygmomanometers), and the like. A document (procedure manual) describing the procedure for performing the method for determining the progress stage of hypertension can also be included.

A method for predicting the therapeutic effect of hypertension One of the aspects of the present invention is
A method for predicting the therapeutic effect of hypertension, comprising:
Predicting that administration of a peptide having a length of 2 to 20 amino acid residues containing (A) SY has a high therapeutic effect when a hypertensive patient is in the early stage of hypertension,
(B) A method for predicting that administration of a peptide containing NA with a length of 2 to 20 amino acid residues will have a high therapeutic effect when a hypertensive patient is in the late stage of hypertension.

As described above, peptides (A) and (B) have hypotensive and arterial relaxant effects. As shown in the examples below, the peptide (A) has a higher antihypertensive effect and an arterial relaxation effect in the early stage of hypertension. On the other hand, the above peptide (B) has higher antihypertensive and arterial relaxant effects in the late stage of hypertension.

The therapeutic effect can be evaluated by the degree of blood pressure reduction, the degree of sustained blood pressure reduction, and the like.

The doses, administration methods, formulations, and the like of peptide (A) and peptide (B) in therapeutic methods for which therapeutic effects are to be predicted can be as described in the "Peptides" section above.

Whether a hypertensive patient is in the early stage of hypertension and whether the hypertensive patient is in the late stage of hypertension can be determined, for example, by the above-described method for determining the stage of progression of hypertension of the present invention.

EXAMPLES Next, the present invention will be described in more detail with reference to examples. However, the following examples are not intended to limit the scope of the invention.

<Method>
The arterial relaxation experiment followed the methods described in Patent Documents 1 and 2 and Non-Patent Document 1.

(Arterial relaxation experiment)
(i) Animals used Male spontaneously hypertensive rats (SHRs/Izm) (manufactured by Japan SLC) were used. SHRs were housed in a controlled room at 23°C with a 12h/12h light/dark cycle. Solid SP feed (manufactured by Funabashi Farm Co., Ltd.) and CE-2 (manufactured by CLEA Japan, Inc.) were given as food, and the mice were allowed to ingest water ad libitum.

In SHR, rats aged 25 weeks or younger were used as early stage hypertensive rats. Rats older than 25 weeks of age were used as advanced stage hypertensive rats. For SHR, some antihypertensive agents are less effective after 25 weeks of age.
(ii) Experiment The mesenteric artery of SHR (15 to 40 weeks old) was excised and spirally dissected to prepare a specimen. Krebs-Henseleit nutrient solution ((120 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM _KH2PO4 , _2.5 mM CaCl2, 25 mM _NaHCO3 , 10 mM glucose), 37°C, This specimen was suspended in a Magnus tube filled with a 5% CO ₂ , 95% O ₂ mixed gas saturation), and changes in tension were recorded on a polygraph via a strain transducer (manufactured by Sanei Co., Ltd.).

The artery was preliminarily constricted with phenylephrine, and when the artery was stabilized, a sample was added and the degree of relaxation was measured. Vasorelaxation was calculated as the degree of contraction when the artery was completely relaxed with papaverine as a ratio of contraction to 100%.

(Evaluation of antihypertensive effect: noninvasive blood pressure measurement experiment (Tail-cuff method))
Systolic blood pressure was measured by the Tail-cuff method for SHR under non-anaesthesia. Blood pressure was measured using MK-2000 (manufactured by Muromachi Kikai Co., Ltd.). Animals trained with the Tail-cuff apparatus for about 3 weeks were used in this experiment. Each sample was dissolved in physiological saline and orally administered by gavage using a metal sonde. Blood pressure was measured immediately before administration and after the elapsed time (Time after administration) shown in each table (2 hours, 4 hours, and 6 hours).

Evaluation was performed by calculating the change in systolic blood pressure (ΔSystolic blood pressure) from immediately before administration.

<Manufacturing example>
(test substance (peptide))
Dipeptides SY and NA synthesized by a conventional method were used as test substances.
<Statistical analysis>
The data obtained by the test was expressed as the sum of the mean (Mean) of the number of trials n and the standard error of the mean (SEM). A t-test was used for comparison between two groups. For comparisons between three or more groups, data were analyzed by one-way ANOVA followed by Tukey-Kramer test for multiple comparisons. A significant difference was determined when p<0.05 compared to the control ("*" and "#" in the figure).

<Experiment and result>
(Example 1: Arterial relaxation experiment)
Using the dipeptides SY and NA as test substances, the arterial relaxation effect was evaluated by an arterial relaxation experiment (n=3-5).

The results are shown in FIG. In the t-test, the vasorelaxation rate in the early stage of hypertension was used as a control, and the vasorelaxation rate in the late stage of hypertension was analyzed, and it was determined that there was a significant difference when p<0.05 ("*" in the figure). rice field.

The dipeptide SY exhibited a strong vasorelaxant effect in the early stage of hypertension, and the effect was significantly attenuated in the late stage of hypertension. On the other hand, dipeptide NA showed strong vasorelaxation in the late stage of hypertension. Dipeptide NA significantly enhanced the vasorelaxant activity in the late stage of hypertension compared to the early stage of hypertension.

(Example 2: Antihypertensive effect)
Using dipeptide SY as a test substance, 5 mg/kg was orally administered, and the antihypertensive effect was evaluated using SHR in the early stage of hypertension (SY group). A control group was administered with only physiological saline as a vehicle. Furthermore, in addition to the dipeptide, the hypotensive effect was also evaluated when L-NAME (20 mg/kg), a nitric oxide synthase inhibitor (NOS Inhibitor), was administered (SY+L-NAME group ).

The results are shown in Figure 2 (n=14-16). Dipeptide SY is presumed to exhibit a vasorelaxant effect in a system mediated by the production of nitric oxide. Therefore, there is a possibility that the action may be weakened in the late stage of hypertension when the vascular function (NO production function) is reduced.

(Example 3: Antihypertensive effect)
Using dipeptide NA as a test substance, 1.5 mg/kg was orally administered, and the antihypertensive effect was evaluated using SHR in the late stage of hypertension (NA group). A control group was administered with only physiological saline as a vehicle. Furthermore, in addition to the dipeptide, the CCK ₁ receptor antagonist (CCK ₁ R antagonist) lorglumide (0.3 mg/kg) was added and administered to evaluate the antihypertensive effect (NA + lorglumide group). .

The results are shown in Figure 3 (n = 4-7). It is speculated that dipeptide NA exhibits vasorelaxant effects via activation of the _CCK1 receptor. Because it is independent of NO production, it may exert a vasorelaxant effect even in the late stage of hypertension when vascular function is impaired.

From the results of the above Examples, it has become clear that the dipeptides SY and NA exhibit potent arteriolytic activity at different onset stages of hypertension. It was also revealed that the administration results of dipeptides SY and NA can be used as indicators for detecting vascular function.

Claims (4)

A method for detecting vascular function in a hypertensive patient, comprising:
(A) Dipeptide SY , and (B) Dipeptide NA
is administered to hypertensive patients, and changes in blood pressure are measured, and the degree of change in blood pressure is used as an index. A method for identifying the progression stage of hypertension comprising:
(A) Dipeptide SY , and (B) Dipeptide NA
to measure changes in blood pressure when administered to hypertensive patients,
As a result of comparing the change in blood pressure when peptide (A) was administered with the change in blood pressure when peptide (B) was administered,
If the drop in blood pressure upon administration of peptide (A) is greater than the drop in blood pressure upon administration of peptide (B), it is identified as being in the early stage of hypertension,
A method for specifying late onset of hypertension when the decrease in blood pressure upon administration of peptide (B) is greater than the decrease in blood pressure upon administration of peptide (A) . (A) Dipeptide SY , and (B) Dipeptide NA
A kit for determining the progression stage of hypertension, comprising: A method for predicting the therapeutic effect of hypertension, comprising:
If the hypertensive patient is in the early stage of hypertension, administration of (A) dipeptide SY is expected to have a high therapeutic effect,
A method for predicting that administration of (B) dipeptide NA will have a high therapeutic effect when a hypertensive patient is in the late stage of onset of hypertension.

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