JPH05140185A - Peptide and its salt - Google Patents

Abstract

PURPOSE:To provide a new peptide having specific reactivity and high binding performance to diuretic hormone and useful as a treating agent or clinical examination agent for hypertension and circulatory diseases and an inhibitor for the physiological and biochemical investigation of diuretic hormone. CONSTITUTION:A peptide and its salt (e.g. the peptide of formula III) bindable to diuretic hormone and containing a continuous sequence of at least 4 amino acids existing in the amino acid sequence of formula I or formula II. The peptide can be produced e.g. by chemical synthesis using a solid-phase process or liquid-phase process or by biological synthesis comprising the preparation of a DNA vector containing a DNA chain capable of coding the peptide, the transformation of a cultured procaryotic cell or eucaryotic cell with the vector and the production of the peptide by the transformant.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a medicinally useful peptide having a binding specificity for diuretic hormone and a salt thereof which is acceptable for pharmaceuticals, diagnostics or preparation of organism-derived substances. More specifically, a DNA encoding human type A diuretic hormone (hereinafter abbreviated as human ANP)
Pairing of strands (or RNA strands transcribed from the DNA strand) D
NA chain (or RNA chain transcribed from the paired DNA chain)
The present invention relates to a peptide consisting of a partial sequence of a peptide sequence defined by a sequence and having a binding affinity to diuretic hormone, and a salt thereof which is acceptable for pharmaceuticals, diagnostics or preparation of organism-derived substances. Further, the peptides and salts thereof of the present invention are clinical test agents or physiological, diuretic hormone detection reagents useful for biochemical research,
Further, it is used as a drug used for the purpose of modifying the physiological activity of diuretic hormone or an inhibitor used in the study of physiological biochemistry.

[0002]

2. Description of the Related Art ANP, or Atrial Natriuretic Peptide, is a peptide hormone that is secreted mainly from the atrium and exhibits a strong natriuretic action, vasodilatory action and hypotensive action. , Β-type, γ-type. The α-type is a single-chain polypeptide chain composed of 28 amino acids and has one disulfide bond [Cys (7) -Cys (23)] in the molecule (Biochemistry and Biotechnology). Physics Research Communication (Biochem. Biophys. Res. Commun., 118 , 131-1
39, 1984)). β-type is an antiparallel dimer of α-type, and γ
The type contains 126 type α sequence in the C-terminal region [99-126].
It is a high-molecular-weight protein composed of 4 amino acids and has been suggested to be a precursor for biosynthesis (Nature (Nat
ure), 313 , 397-400, 1985).

In patients with essential hypertension and secondary hypertension, and in animal models of inherited and secondary hypertension, the blood ANP concentration generally increases (hypertension (Hypertension)).
tension) 11, I212-I216, 1988: Circulation Research (Circ. Res.), 62 , 926-930, 1988). It is considered that this is because ANP secretion is compensatoryly enhanced as a result of increased load on the atrium due to hypertension. Also, A
The blood concentration of NP is well correlated with the atrial pressure and increases, so that it is also an index of congestive heart failure, for example (Journal
Of Clinical Investigation (J. Clin.
Invest.), 81 , 1962-1970, 1988: J. Clin. Inves
t., 83 , 298-305, 1989: J. Clin. Invest., 83 , 46-5
1, 1989). That is, the clinically essential significance of measuring the hormone lies in the predictive diagnosis of heart diseases including this disease, the diagnosis of hypertension, and the follow-up observation.

The current assay method for ANP is to use an antibody as a detection reagent (Science, 228 , 323-32).
5, 1985: Nature, 314 , 264-266, 1985
: Biochem. Biophys. Res. Commun., 124, 815-821, 1
984: Biochemistry and Biophysics
Research Communication (Biochem. Biophys. Re
s. Commun.), 124 , 663-668, 1984: Biochem. Biophy
s. Res. Commun., 125 , 315-323, 1984), and recent technological advances in cell biology have facilitated the production of monoclonal antibodies (Life Science
i.), 38 , 1991-1997: Molecular Immunology (Mol.Immunol.), 24 , 127-132, 1987: Endocrinology, 121 , 843-852, 1987: Hybridoma, 6 , 433 -440, 1987: Kohei
2-16997), an enzyme immunoassay method using the antibody as a detection reagent has been reported (JP-A 64-61500: JP-A-1-61500: JP-A 1-221399). Furthermore, in the field of basic medicine, monoclonal antibodies are used as research reagents used to elucidate the physiological significance of ANP in hypertension models (Biochemistry and Biochemistry).
Biophysics Research Communication (Bi
ochem. Biophys. Res. Commun.), 151 , 1277-1284, 19
88: Journal of Clinical Investigation (J. Clin. Invest.), 84, 145-154, 1989).

[0005] On the other hand, a report of a drug that modifies or suppresses various physiological activities of ANP for multiple organs by competing with ANP for a receptor binding reaction is reported in Rat ANP.
Of the synthetic ligand C-ANF (Science, 238 , 675-678,
1987) and human ANP [7-28] antiparallel dimer, in which a pair of disulfide bonds was replaced with L-α-aminosuberic acid (Analog) III (Fabs Letter (FEBS)
Lett.), 248 , 28-34, 1989). However, A
There is no report of an activator that enhances the physiological activity of ANP by binding to NP.

In addition, a paired DNA chain or complementary RN for the DNA chain or mRNA chain encoding the peptide (i)
The fact that the peptide (ii) obtained by arranging the amino acids defined by the genetic codons constituting A according to the order of the genetic codons has affinity for the original peptide (i) means that angiotensin (Proc. Natl. Acad. Sci. USA,
85, 2518-2522), luteinizing hormone-releasing hormone (Pro
c. Natl. Acad. Sci. USA, 83, 9714-9718), adrenocorticotropic hormone (Proc. Natl. Acad. Sci. USA, 82, 1372-1)
375) etc. have been reported as an example. Furthermore, regarding the method for designing the peptide (ii) defined by the non-coding strand sequence of DNA or the complementary RNA strand sequence, Bralock (Tokusho Sho 62-50)
No. 2513).

[0007]

As described above, it is general to use an anti-ANP antibody in the measurement of ANP by the existing method. Although various techniques related to cell biology or biochemistry have been advanced in recent years, the antibody molecule and its performance to be selected are basically governed by the chance of the organism or cell producing the antibody, which limits the use performance. receive. That is,
Although it is desired to produce an antibody having an extremely small binding constant, it is necessary to perform an operation of extracting and concentrating the test substance in the sample, that is, ANP, because an antibody that achieves the expected value cannot be obtained. ..

Although the chemical properties of antibody molecules have been well studied, it is after all a biosynthetic protein derived from an organism, and this property is extremely affected by temperature and time. This property often causes a deterioration in storage stability of the detection reagent. Furthermore, with the replacement of the individual organism or passage cell that produces the antibody molecule,
The antibody molecular species also changes, which is a major obstacle to homogenization of the detection reagent. Further, these are not limited to the problems relating to the detection reagents constituting the clinical diagnostic agents, but may also be problems in the research of physiology / biochemistry.

On the other hand, by connecting to ANP, ANP
A peptide-type drug is considered as one of the activators that enhances the binding reaction of saccharin to its receptor and stimulation of cGMP production by guanylyl cyclase. Regarding the method for designing the peptide (ii) having a high affinity for ANP, the method described above is used.
(Japanese Patent Publication No. 62-502513) is a general method, and does not specify an actual optimum peptide sequence. This is because the optimal design method of such a peptide (ii) is different for each original peptide (i) to be designed. That is, the design method is only one concept for designing the peptide (ii) having a higher affinity for the peptide (i), and further multistep research is required to achieve the object. In fact, there is also a report that the peptide designed by the above method does not have sufficient binding affinity to the original peptide (de Gasparo, M., Whitebread, S., Einsle, K. & Heusser,
C. (1989) Biochemical Journal (Biochem.J.) 26
1 , 310-311).

[0010]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have searched for a specific detection reagent or drug that substitutes for an antibody having a binding property to ANP by using a hormone receptor or a bioactive peptide receptor. Based on the hypothesis that the ligand-binding site of E. coli is evolutionarily defined by the genetic information encoded by the paired chain of the hormone gene or the physiologically active peptide gene, as a result of intensive research, a peptide sequence having a binding property to ANP was found. Then, they came to complete the invention of the usage.

The inventors of the present invention have found that the Bralock method (Table 62-
502513), a peptide sequence complementary to ANP is used as a DN encoding the constituent amino acids of ANP.
It was designed by decoding the paired chain sequence of the A chain from the 5 ′ or 3 ′ direction, and further arranging the decoding amino acid from the N terminus or C terminus of the peptide sequence. Then, after that, the ANP-binding activity of the peptide of the partial sequence of the complementary peptide sequence thus obtained was measured, the high-affinity site for ANP was identified, and the peptide group containing the site was obtained.

That is, the present invention has a diuretic hormone-binding property and is represented by the following formula: Glu-Ala-Val-Thr-Ala-Gln or Gln-Ala-Thr-Val-Ala-Glu It relates to a peptide characterized in that it is selected from the group of peptides containing a contiguous sequence of at least 4 amino acids and a salt thereof, which is acceptable for pharmaceutical use, diagnostic use or preparation of organism-derived substances.

Specifically, Glu-Ala-Val-Thr and Ala-Val-T
hr-Ala, Val-Thr-Ala-Gln, Gln-Ala-Thr-Val, Ala-Thr-
Val-Ala, a tetrapeptide represented by Thr-Val-Ala-Glu,
Glu-Ala-Val-Thr-Ala, Ala-Val-Thr-Ala-Gln, Gln-Ala-
Thr-Val-Ala, Ala-Thr-Val-Ala-Glu pentapeptide, Glu-Ala-Val-Thr-Ala-Gln, Gln-Ala-Thr-Val-A
The hexapeptide represented by la-Glu is mentioned. In addition,
In the present specification, the abbreviations and sequences of the above amino acids are those conventionally used in the art, and all L-
It represents an amino acid.

The peptide of the present invention is a peptide containing at least 4 amino acids of the obtained sequence as described above. This is because the mathematical probability that the four amino acid sequences are specific to diuretic hormone is 1 in 160,000, which is a sufficient value to discuss biological specificity. Also, their analogs and derivatives are within the scope of the invention. That is, an amino acid having almost the same hydrophobicity at any amino acid rank, a peptide substituted with an appropriate amino acid with reference to the amino acid sequence of the ANP isopeptide or its receptor, or the N-terminal or C-terminal of the peptide Peptide optionally blocked or protected in either one or both, for example, a peptide chemically modified by acetylation or amidation at an arbitrary site, a protein having a higher molecular weight than the peptide, or a functional polymer compound The peptides and the like bound to are also included in the present invention. The ANP isopeptides include BNP and CNP. In addition, as those receptors, A of B type receptors is used.
Type, B type, and C type receptors.

Furthermore, the present invention also includes a salt of the peptide of the present invention, which is composed of an ion acceptable for use as a drug, a diagnostic agent, or a substance derived from an organism. Examples of such salts include acid addition salts and basic salts. Specific examples include salts of inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid and pyrophosphoric acid, acetic acid, lactic acid, palmitic acid, stearic acid and propione. Acid addition salts such as organic acid salts such as acids, citric acid, tartaric acid, malic acid, ascorbic acid, oxalic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc., alkali metals such as sodium salt, potassium salt, etc. salt,
Examples thereof include alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts, salts such as triethylamine salts. The peptide of the present invention can be prepared by a known method, for example,
Chemical synthesis by a solid phase method and a liquid phase method, a DNA vector containing a DNA chain capable of encoding the peptide is prepared, and a cultured prokaryotic cell or a eukaryotic cell is transformed therewith, and then the peptide is produced by the transformant. It can be produced by a biosynthesis method or the like.

As a high molecular weight protein to which the peptide of the present invention is bound, when an antibody against the peptide is obtained,
There are shellfish hemocyanin, bovine serum albumin, bovine thyroglobulin, avian gamma globulin, etc. used as immunogens, and when used for immunochemical analysis, horseradish peroxidase, alkaline phosphatase, glucose oxidase, glucose hexaphosphate used as detection enzymes. Examples thereof include dehydrogenase and galactose degrading enzyme, which are different depending on the use of the peptide and are not particularly limited. Examples of the functional polymer compound to which the peptide of the present invention is bound include polyvinylpyrrolidone used as an immunogen when an antibody against the peptide is obtained, and ¹²⁵ I used as a substance for detection when used for immunochemical analysis. There is a radioisotope such as TFTC, a fluorescent substance such as FITC, magnetized dextran, latex particles and the like, but it is not particularly limited.

When the peptide of the present invention or a salt thereof is used as a drug, its preferred pharmaceutical use is as a therapeutic drug for hypertension and cardiovascular diseases. Further, suitable diagnostic fields to be used as clinical test agents are hypertension and cardiovascular diseases, but their use in these fields is a non-limiting example. When the peptide of the present invention or a salt thereof is used as a medicine, an effective amount thereof is used, or when it is used as a diagnostic agent, a predetermined amount thereof is contained or bound to a carrier together with a therapeutically or diagnostically acceptable carrier. In diagnostic applications,
A predetermined amount of the peptide or its salt can be bound to the enzyme for detection, the substance for detection or the carrier for immobilization and used. As the carrier for immobilization, latex particles, agarose resin, acrylamide resin, polystyrene resin,
There are polyethylene resins and the like, but they are not particularly limited. Furthermore, when the peptide of the present invention is bound to the above-mentioned enzyme for detection, substance for detection and carrier for immobilization, a spacer structure may be interposed between these substance and the peptide by covalent bond or the like.

For pharmaceutical use, a powder containing a pharmaceutically acceptable carrier prepared from a predetermined amount equal to or less than the effective amount of the peptide of the present invention or a salt thereof,
It can take the form of elixirs, solutions, pills, capsules, pellets, tablets, sprays or olfactory agents.
Acceptable carriers for this purpose include starch, sugar,
There are proteins, talc, conventionally used synthetic or natural rubber, or water. Further, the administration method of these medicines is not particularly limited, but subcutaneously, intradermally, intramuscularly, intravenously, or in some cases injection or oral into the cerebrospinal cavity, transdermal, suppository, spray or eye drop, etc. There are transmucosal routes, etc.

[0019]

EXAMPLES The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention. The meanings of the abbreviations used in this example are shown below. tBu; t-butyl ester, OtBu; t-butyl ether, Boc; t
Butyloxycarbonyl, Mtr; 4-methoxy-2,3,6-trimethylbenzenesulfonyl, Trt; trityl, Oppf; pentafluorophenyl ester, ODhbt; dihydroxybenzotriazine ester, HMD; hexamethylenediamine, Fm
oc; 9-fluorenylmethyloxycarbonyl, HOBT; 1-hydroxy-benzotriazole.

[Synthesis Example by Solid Phase Synthesizer] Example 1: Synthesis of peptide represented by Glu-Ala-Val-Thr-Ala-Gln As a solid phase, Fmoc-Gln-Pep Syn KA resin column (Milligen
(Manufactured by MilliGen) was used to synthesize the above-mentioned peptide by the Fmoc-polyamide method using a 9050 type peptide synthesizer (manufactured by MilliGen). 2.2 g of the solid phase resin (0.09
meq / g) After equilibrating the column with dimethylformamide for 1 minute at a flow rate of 5 ml / min, the Fmoc group was added at a flow rate of 5 ml / min for 20 minutes.
% Piperidine / dimethylformamide (volume / volume) was removed for 10 minutes, and then the resin was further washed with dimethylformamide at a flow rate of 5 ml / min for 15 minutes. Then, 4 times the amount of the amino acid bound to the solid surface (mol
(Number) Fmoc-Ala Oppf was dissolved in 2.64 ml of 5% (weight / volume) HOBT / dimethylformamide and circulated in the resin column for 30 minutes to carry out an elongation reaction of amino acids. After the reaction, the resin column was washed with dimethylformamide and subjected to the next step. The one-step reaction described above required about 1 hour. Further, the above steps are described below. Opfp (Thr is ODhbt), which is the protected amino acid in a 4-fold amount (mol number) of the amino acid bound to the solid surface; Fmoc-Thr (tBu), Fmoc-Val, Fmoc -Ala, Fmoc-Glu (OtBu) was used in this order for repeated reaction, and then 20% piperidine / dimethylformamide was added for 10 minutes at a flow rate of 5 ml / min.
By sequentially washing with dimethylformamide at the same flow rate for 15 minutes and then with dichloromethane at the same flow rate for 15 minutes,
Peptide resin with Fmoc group removed; Glu (OtBu) -Ala-Val-T
hr (tBu) -Ala-Gln-Pep Syn KA-resin was obtained. The peptide resin obtained by the above reaction was removed from the column, 0.6 ml of m-cresol, 3.6 ml of thioanisole and 25.8 ml of trifluoroacetic acid were added, and the mixture was stirred at room temperature for 1 hour,
The peptide was eluted from the resin. The eluent was collected as a filtrate, and then the mixture was agitated with 10% ether.
0 ml was added to precipitate the peptide, which was collected by centrifugation at 3,000 xg for 5 minutes. Thereafter, the precipitate recovered by the above operation was washed with ether 4 times by centrifugation and then dried under reduced pressure to obtain the above-mentioned peptide.

The peptide thus obtained is YM
0.1% trifluoroacetic acid aqueous solution-acetonitrile (5% -23% gradient, 15 minutes, 0.5 minutes) as an eluent on C-ODS-5 (diameter 4.6 mm × 150 mm, manufactured by Yamamura Chemical Co., Ltd.)
Retention time of 21 minutes was shown on HPLC (ml / min). A peak fraction with a retention time of 21 minutes obtained by injecting 10 μg / 5 μl of an aqueous peptide solution was placed in a tube and dried under reduced pressure, and then 1% phenol / 6 was added to a reaction tank of a PICO TAG workstation (Waters).
N hydrochloric acid (volume / volume) is vaporized under reduced pressure (500 mTorr), and 1
The peptide was acid-decomposed by treatment at 50 ° C. for 1 hour. Then, the wet peptide is dried under reduced pressure, ethanol,
The mixture was dissolved in 10 μl of a 2: 2: 1 (volume: volume: volume) mixture of water and triethylamine, and dried again under reduced pressure. After that, 20 μl of a 7: 1: 1: 1 (volume: volume: volume: volume) mixture of ethanol, triethylamine, water and phenylisothiocyanate was further added, reacted at room temperature for 25 minutes, and then eluted with 50 μl of PTC-amino acid. Liquid A (Wako Pure Chemical Industries, Ltd.) / PTC-amino acid eluent B (Wako Pure Chemical Industries, Ltd.) (PTC-amino acid eluent B volume ratio 0%-
Amino acids were separated by HPLC using 70% gradient, 15 minutes, 1 ml / min) as a developing solution, and the amino acid composition of the synthetic peptide was determined from the peak height of an amino acid standard solution treated in the same manner. As a result, Glx 1.90 (2 ), Ala 2.08 (2), Val
It was 1.02 (1) and Thr 0.95 (1).

Example 2 [Synthesis by Pin Technology Method for Evaluation of Binding Property of Peptide] In order to evaluate the binding property of the peptide of the present invention, the N-terminal of the peptide was protected with an acetyl group and the C-terminal was changed to another. The peptide was synthesized by the pin technology method, which was bound to the solid phase via the amino acid derivative of. According to the PT-02-3000 manual of Cambridge Research Biochemicals (CRB), the CRB software is started on the NEC APC H5020 computer, and the amount of amino acid and the amount of reagent required for peptide synthesis are set. And the pin placement was calculated. For the synthesis of peptides, refer to the above manual and literature [Geysen H M .: Use of peptide synthesis to pr
obe viral antigens for epitopes to a resolution of
asingle amino acid: Proc.Natl.Acad.Sci.USA., 81 , 3
998-4002, (1984)], the CRB Mimotope
Pin block and Fmoc-L- included in the design kit
Performed with amino acids. All amino acids except serine and threonine were protected with an Fmoc group at the amino terminus (-Opf
p) and serine and threonine are protected at the amino terminus with an Fmoc group (-ODhbt). The side chain protecting groups for serine, threonine, and tyrosine are (-tBu), aspartic acid,
The side chain protecting group for glutamic acid is (-OtBu), the side chain protecting group for lysine and histidine is (Boc-), and the side chain protecting group for arginine is
(Mtr-), the side chain protecting group of cystine is (Trt-). In addition, the pins used for synthesis have Fmoc-β alanine (bAla) bound to a polyethylene support having acrylic acid graft-polymerized on its surface, using hexamethylenediamine (HMD) as a spacer. A specific synthesis example is shown below.

Synthesis of Peptide Represented by Glu-Ala-Val-Thr-Ala-Gln Acrylic acid was graft-polymerized on a pin ball (diameter 4 mm) at the end of polyethylene rod, and α-amino group was protected by Fmoc group via HMD. Fmo of a resin with bound β-alanine
The c group was treated with 20% piperidine / dimethylformamide (volume / volume) for 30 minutes, washed with excess amounts of dimethylformamide and methanol, respectively, and then air-dried.
After equilibrating the resin further in dimethylformamide, 2
2 dissolved in 2.5 mM HOBT / dimethylformamide
Fmoc-Gln-bAla-HMD-resin was obtained by immersing in 100 μl of 0 mM Fmoc-Gln Opfp at 30 ° C. overnight, and then successively washing the resin with an excess amount of dimethylformamide and methanol. Subsequently, the protective group for the α-amino group of the peptide resin (Fmo
c) is removed by the above-mentioned treatment, and the above-mentioned step is further described below in Opfp (where Thr is ODh) of a protected amino acid of 20 mM.
bt); Fmoc-Ala, Fmoc-Thr (tBu), Fmoc-Val, Fmoc-Ala, Fmoc-
Protected peptide resin by repeatedly reacting with Glu (OtBu) in this order; Fmoc-Glu (OtBu) -Ala-Val-Thr (tBu) -Ala-Gln-bAla-H
MD-resin was obtained. Protecting group for α-amino group of the peptide resin
(Fmoc) was removed by the above treatment, and the resin was washed successively with excess amounts of dimethylformamide and methanol and then air-dried. Then, the peptide resin is immersed in a 5: 2: 1 (volume: volume: volume) mixed solution of dimethylformamide, acetic anhydride, and triethylamine to carry out an acetylation reaction at 30 ° C for 90 minutes, and similarly, an excess amount of dimethyl is added. The resin was washed successively with formamide and methanol and then air dried. Subsequently, trifluoroacetic acid, anisole, and ethanedithiol 95: 2.
The peptide resin was immersed in a mixed solution of 5: 2.5 (volume: volume: volume) and reacted at room temperature for 4 hours to remove the protective groups, and after air-drying, 0.1% (volume / volume) hydrochloric acid, 50% ( The peptide was thoroughly ultrasonically washed in a mixture of (volume / volume) methanol to obtain Ac-Glu-Ala-Val-Thr-Ala-Gln-bAla-HMD-resin.

Example 3 [Measurement of Diuretic Hormone Binding Activity of Peptide] Various hexapeptides were produced by the method shown in Example 2 above, and the binding activity to diuretic hormone was evaluated by the method shown below. The results are shown in Table 1. a: [Enzyme Immunocompetitive Inhibition Assay (EIA Inhibitory Activity)] The peptide resin obtained in Example 2 was coated with a 0.2% skim milk powder solution on the easily adsorbable site, and then 200 μl of human A-type diuretic hormone solution ( 1 μg / ml) [prepared by dissolving in Dulbecco's phosphate buffered saline containing 0.1% skim milk powder and 0.1% Tween], and reacting for 3 hours at room temperature with gentle stirring The solution of was used as the sample solution (i). Separately, the wells of the microplate for ELISA were filled with human type A diuretic hormone solution (0.5 μg / ml).
It was treated with 00 μl at 4 ° C. overnight, and the next day, a 0.2% non-fat dry milk solution was used to protect unreacted sites on the plate to prepare human-A type diuretic hormone adsorption plate (ii). Wash the human-A type diuretic hormone adsorption plate (ii) with a washing buffer [0.1
% Tween-containing Dulbecco's Phosphate Buffered Saline Solution], then add 50 μl of the sample solution (i), and immediately thereafter dilute the anti-human A-type diuretic hormone rabbit antibody solution 1 / 10,000 [for dilution] 0.1% as a buffer
200 μl of Dulbecco's phosphate buffered saline containing skim milk powder and 0.1% Tween was used] and a competitive reaction was carried out at room temperature for 2 hours. After the competitive reaction, the plate was thoroughly washed with a washing buffer, and diluted to 1/2000 with alkaline phosphatase-labeled anti-rabbit IgG, A,
200 μl of M goat antibody solution was added, and a binding reaction was performed with the anti-human type A diuretic hormone rabbit antibody captured on the plate at room temperature for 2 hours. After that, the plate was washed in the same manner, and the substrate solution (1 mg p-nitrophenyl phosphate / ml 1
200 μl of (M diethanolamine buffer) is added, and released by the labeling enzyme remaining on the plate
The amount of p-nitrophenol was determined by measuring the absorbance at 405 nm, and the amount of human-type A diuretic hormone in the sample solution (i) was calculated from a separately prepared calibration curve. Table 1 shows the binding inhibitory activity of the peptide (ratio of the antigenic activity of human type A diuretic hormone in the sample solution (i) compared to before the binding reaction to the peptide resin pin).

B: [Radioligand binding activity] The peptide resin obtained by the method of Example 3 was coated with 0.2% non-fat dry milk solution on the easily adsorbed site, and then 100 ml of ¹²⁵
I-Human A type diuretic hormone solution (specific activity ~ 74TBq /
mmol) solution (0.131 ng / ml) [dissolved in Dulbecco's phosphate buffered saline containing 0.1% nonfat dry milk and 0.1% Tween] and allowed to react at room temperature for 2 hours. After the binding reaction, the peptide resin was thoroughly washed with a washing buffer [Dulbecco's phosphate buffered saline containing 0.1% Tween], and the radioactivity bound to the peptide resin was measured with a γ counter. As a result, the amount of human A-type diuretic hormone bound to the peptide resin was calculated. The results are shown in Table 1.

[0026]

[Table 1]

Example 4; In addition, when the peptides shown below were synthesized by the method shown in the above Example 2 and evaluated by the method shown in Example 3, all peptides showed sufficient binding activity. Glu-Ala-Val-Thr, Glu-Ala-Val-Thr-Ala, Gln-Al
a-Thr-Val-Ala-Glu

[0028]

INDUSTRIAL APPLICABILITY The peptide of the present invention has specific reactivity and high binding ability for diuretic hormone. Therefore, it is very useful as a detection reagent used for measuring a diuretic hormone and a precursor containing a diuretic hormone structure or a part of diuretic hormone, and a drug such as a therapeutic drug that activates or modifies these physiological functions. Is.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Glu Ala Val Thr 1

SEQ ID NO: 2 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Ala Val Thr Ala 1

SEQ ID NO: 3 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Val Thr Ala Gln 1

SEQ ID NO: 4 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Ala Thr Val 1

SEQ ID NO: 5 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Thr Val Ala 1

SEQ ID NO: 6 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Thr Val Ala Glu 1

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Ala Val Thr Ala 15

SEQ ID NO: 8 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Val Thr Ala Gln 15

SEQ ID NO: 9 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Gln Ala Thr Val Ala 15

SEQ ID NO: 10 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Ala Thr Val Ala Glu 15

SEQ ID NO: 11 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Glu Ala Val Thr Ala Gln 1 5

SEQ ID NO: 12 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence Gln Ala Thr Val Ala Glu 15

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Internal reference number FI Technical display location G01N 33/53 B 8310-2J C07K 99:00 (72) Inventor Yasuhisa Odagawa Wadai, Tsukuba, Ibaraki Address 48 Hitachi Chemical Co., Ltd. Tsukuba Development Laboratory (72) Inventor Kenzo Baba 48 Wadai, Tsukuba City, Ibaraki Prefecture Hitachi Chemical Co., Ltd. Tsukuba Development Laboratory

Claims (2)

[Claims] 1. A diuretic hormone-binding agent having at least 4 amino acid sequences specified by the following formula: Glu-Ala-Val-Thr-Ala-Gln or Gln-Ala-Thr-Val-Ala-Glu. A peptide selected from the group of peptides containing a continuous sequence of amino acids and a salt thereof, which is acceptable for pharmaceuticals, diagnostics, or preparation of a substance derived from an organism. 2. The peptide according to claim 1, wherein either one or both of the N-terminal and C-terminal of the peptide is optionally blocked or protected, and the peptide is acceptable as a pharmaceutical, a diagnostic agent, or a preparation of an organism-derived substance. That salt that will be.