JP3025672B2 - Novel bioactive peptides and their uses - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel physiologically active peptide and its use, and more particularly, to a human brain natriuretic peptide and circulating hypertension, edema disease, heart failure, renal failure and the like containing the same. The present invention relates to a therapeutic agent for organ diseases.

[0002]

2. Description of the Related Art Recently, a peptide has been isolated from pig brain and its structure has been determined. Since its structure and bioactivity are very similar to atrial natriuretic peptide (hereinafter abbreviated as ANP), cerebral natriuretic peptide Brain Natriureti
c Peptide; hereinafter abbreviated as BNP) [Sudo et al., Na
Nature, 332, 78-80 (1988)]. BNP is involved in the regulation of body fluid volume, electrolyte balance and blood pressure in living organisms, indicating that there is a dual regulation mechanism by ANP and BNP in living organisms.

On the other hand, the cDNA of this porcine BNP has been cloned, and the structure of the precursor of porcine BNP has been elucidated [Maekawa et al .; Biochem. Biophys. Res. Commun., 157 (1),
410-416 (1988)]. Furthermore, cDNA of human BNP has been cloned using the cDNA of porcine BNP as a probe, and the amino acid sequence of human BNP has been estimated by structural analysis.

[0004]

[Problems to be solved by the invention] However, human BN
There is no report that P was isolated or synthesized, and it is unclear what action or activity it has. Therefore, to synthesize this human BNP, compare its biological activity with known natriuretic peptides, and explore its usefulness,
It is important in drug development to avoid side effects such as antibody production.

[0005]

Means for Solving the Problems The present inventors have proposed the above-mentioned human
Based on the deduced amino acid sequence of BNP, we synthesized a new substance, human BNP derivative, and further investigated its pharmacological action.These substances showed smooth muscle relaxing action and natriuretic action of known natriuretic peptides. And completed the present invention.

That is, the present invention relates to the following formula (1)

[0007]

Embedded image

[0008] It is intended to provide a physiologically active peptide represented by the following or a salt thereof, and a remedy for circulatory diseases containing the same.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, the peptide (1) of the present invention is sometimes referred to as human BNP-32.

[0010] In the present specification, abbreviations in peptides are those generally used in the art.
It has the following meaning:

Asp: L-aspartic acid Ser: L-serine Gly: glycine Cys: L-cysteine Phe: L-phenylalanine Arg: L-arginine Leu: L-leucine Ile: L-isoleucine Asn: L-asparagine Val: L -Valine Tyr: L-Tyrosine

The peptide (1) of the present invention can be produced by a solid phase method or a liquid phase method commonly used for peptide synthesis [for example, Nobuo Izumiya et al., “Peptide Synthesis”, 1984, published by Maruzen Co., Ltd .; Biochemistry Experiment Course (I) / Protein Chemistry ", Volume 4, 20
8 to 495, published by Tokyo Chemical Dojin, 1977].

For example, the peptide (1) of the present invention is prepared by a solid phase method.
When synthesizing, the α-amino group of the amino acid used is 9-
Fluorenylmethyloxycarbonyl group (Fmoc group), β-carboxyl group of aspartic acid is tert-butyl group (tB
guanidino group of arginine is 4-methoxy-2,3,6-
Trimethylbenzenesulfonyl group (Mtr group), serine hydroxyl group is tert-butyl group (tBu group), cysteine thiol group is acetamidomethyl group (Acm group), histidine imidazole group is trityl group (Trt group), and lysine ε The -amino group is preferably protected with a tert-butyloxycarbonyl group (Boc group). The insoluble resin used is
P-alkoxybenzyl alcohol resin (Wang Resin) is preferred. For the condensation of the protected amino acid, it is preferable to use a dicyclohexylcarbodiimide (DCC) method, an active ester method using 1,3-diisopropylcarbodiimide (DIC), an acid anhydride method using DCC, a diphenylphosphoric acid azide method (DPPA) method, or the like. However, the protecting group of the amino acid is not limited to those described above.
-Amino group is tert-butyloxycarbonyl group (Boc group)
May be protected.

The peptide (1) of the present invention is produced by the solid phase method, for example, as follows. First, a protected derivative of His which is a C-terminal amino acid, Fmoc-His (Trt) -OH, is introduced into a p-alkoxybenzyl alcohol resin, and then the corresponding protected amino acids are sequentially bonded to synthesize a protected peptide resin. Then piperidine and trifluoroacetic acid (TF
A), piperidine and trimethylsilyl bromide (TMSBr) [Chem. Pharm. Bull., 35
(9), 3880 (1987)], etc., simultaneously perform cleavage of the peptide from the resin and removal of the protecting group other than the Acm group, and a peptide having an Acm group at the thiol group of cysteine (CYS (Acm)-
Peptide]. Next, this is oxidized with iodine to remove the protective group of the thiol, and at the same time, to form a disulfide bond by the thiol group of two cysteines in the peptide molecule to obtain a crude synthetic peptide.

The obtained crude synthetic peptide can be purified by a conventional method.
For example, it can be performed by gel filtration, ion exchange chromatography, reversed phase high performance liquid chromatography (HPLC), or the like.

The peptide (1) of the present invention can be prepared from inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, formic acid, acetic acid, citric acid, tartaric acid,
Using an organic acid such as fumaric acid and maleic acid, an acid addition salt can be formed according to a conventional method.

When the peptide of the present invention or a salt thereof is administered, the method used for the administration of peptide-based drugs, that is, intravenous injection, intramuscular injection, subcutaneous injection, or sublingual administration, intranasal administration, or rectal administration. Can be adopted. The amount that can be administered without causing any dangerous or harmful side effects is preferably in the range of 0.5 μg / kg to 100 mg / kg.

[0018]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto.

Example 1 [A] Synthesis of peptides human BNP-26 and human BNP-32:

(1) Synthesis of Protected Peptide Resin In the synthesis of the protected peptide resin, all α-amino groups of each constituent amino acid are protected by a 9-fluorenylmethyloxycarbonyl group (Fmoc group) to form an active side chain. Of these, the β-carboxyl group of aspartic acid is a tert-butyl group (tBu
The guanidino group of arginine is 4-methoxy-2,3,6
-Trimethylbenzenesulfonyl group (Mtr group), serine hydroxyl group is tert-butyl group (tBu group), cysteine thiol group is acetamidomethyl group (Acm group), and histidine imidazole group is trityl group (Trt group) Where the ε-amino group of lysine is a tert-butyloxycarbonyl group (BO
C group). As the resin, 1.0 g of a p-alkoxybenzyl alcohol resin into which protected His was introduced was used.

In the condensation of the protected amino acid, the treatment of the Fmoc group, which is the protecting group of the terminal amino group of the protected peptide bound to the resin, with piperidine for 6 minutes at room temperature was repeated twice to remove almost completely. . Then this Fmoc removal
The amino group released by the hydrolysis was condensed with the carboxyl group of the Fmoc-protected derivative of the next amino acid in the amino acid sequence of the target peptide. In the condensation of this protected amino acid, 1 mmol of Fmoc-amino acid was condensed by treating it with 1,3-diisopropylcarbodiimide (DIC) in the presence of 1-hydroxybenztriazole. When the reaction was not completed by this operation, the same operation was repeated. The progress and completion of the reaction were monitored by a Kaiser test using ninhydrin.

Thus, Fmoc-Gly-Ser (tBu) -Gly-
Cys (Acm) -Phe-Gly-Arg (Mtr) -Lys (Boc) -Met-Asp (tBu) -Ar
g (Mtr) -Ile-Ser (tBu) -Ser (tBu) -Ser (tBu) -Ser (tBu) -Gly
-Leu-Gly-Cys (Acm) -Lys (Boc) -Val-Leu-Arg (Mtr) -Arg (Mt
r) -His (Trt) -resin was synthesized. At this stage, a part of this product was removed, and the Fmoc group, which is a protecting group for the terminal amino group of the protected peptide, was removed according to the method described above, and H-Gly-Ser (tB
u) -Gly-Cys (Acm) -Phe-Gly-Arg (Mtr) -Lys (Boc) -Met-Asp
(tBu) -Arg (Mtr) -Ile-Ser (tBu) -Ser (tBu) -Ser (tBu) -Ser
(tBu) -Gly-Leu-Gly-Cys (Acm) -Lys (Boc) -Val-Leu-Arg (Mt
670 mg of r) -Arg (Mtr) -His (Trt) -resin (hereinafter referred to as protected human BNP-26 resin) was obtained.

The remainder was further subjected to an N-terminal extension reaction to obtain Fmoc
-Ser (tBu) -Pro-Lys (Boc) -Met-Val-Gln-Gly-Ser (tBu) -Gl
y-Cys (Acm) -Phe-Gly-Arg (Mtr) -Lys (Boc) -Met-Asp (tBu)-
Arg (Mtr) -Ile-Ser (tBu) -Ser (tBu) -Ser (tBu) -Ser (tBu) -G
ly-Leu-Gly-Cys (Acm) -Lys (Boc) -Val-Leu-Arg (Mtr) -Arg
(Mtr) -His (Trt) -resin was obtained. Next, the Fmoc group, which is a protecting group for the terminal amino group of the protected peptide, was removed according to the method described above, and H-Ser (tBu) -Pro-Lys (Boc) -Met-Val-Gln-Gly-
Ser (tBu) -Gly-Cys (Acm) -Phe-Gly-Arg (Mtr) -Lys (Boc) -Me
t-Asp (tBu) -Arg (Mtr) -Ile-Ser (tBu) -Ser (tBu) -Ser (tBu)
-Ser (tBu) -Gly-Leu-Gly-Cys (Acm) -Lys (Boc) -Val-Leu-Ar
g (Mtr) -Arg (Mtr) -His (Trt) -resin (hereinafter protected human BNP-32
1.5 g of resin).

(2) Synthesis of Cys (Acm) -human BNP-26 600 mg of protected human BNP-26 resin was placed in a reactor together with 2.4 ml of thioanisole, 20 ml of trifluoroacetic acid (TFA), and 2.6 parts of trimethylsilyl bromide (TMSBr). ml and ethanedithiol 2.4 ml were added and reacted at 0 ° C. for 3 hours. After completion of the reaction, the anisole was removed by washing with 200 ml of ether.
The product was extracted with 20 ml of N acetic acid. The resin and insolubles were removed by centrifugation, 1 ml of 1M sodium fluoride (NaF) was added while cooling with ice, the pH was adjusted to about 8 on a universal test paper with 5% aqueous ammonia, and the mixture was allowed to stand for 30 minutes. Thereafter, the pH was readjusted to 5 by adding 1N acetic acid again, and further diluted 10-fold with water. This was adsorbed on a column (φ3 cm × 8.5 cm) packed with 60 ml of ODS resin [LC-Sorb (manufactured by Chemco)], washed well with 0.1N acetic acid, and eluted with 200 ml of 60% acetonitrile containing 0.1% TFA. . After acetonitrile was distilled off under reduced pressure,
Lyophilization yielded 300 mg of crude Cys (Acm) -human BNP-26.

This was dissolved in 9 ml of 1N acetic acid and subjected to reverse-phase HPLC in 9 portions [column: Nucleosyl 120-5C]
18, φ20mm × 250mm, flow rate 5ml / min, solvent system: (A) water:
Acetonitrile: 10% TFA = 90: 10: 1, (B) Water: acetonitrile: 10% TFA = 40: 60: 1, (A) :( B) = 90: 10
To (A) :( B) = 55: 45 linear gradient for 120 minutes]. This operation was repeated 9 times, and the main peak eluted at 57 to 61 minutes was collected. After acetonitrile was distilled off under reduced pressure, the fraction was freeze-dried, and Cys (Acm)-
96.0 mg of human BNP-26 was obtained.

(3) Synthesis of human BNP-26 A solution prepared by dissolving 227 mg of iodine in 50 ml of 95% acetic acid and adding 80 μl of 1N hydrochloric acid (hereinafter referred to as solution A) was prepared.

Further, citric acid 2.1 g, L-ascorbic acid 5
Dissolve 75mg in 10ml 2N sodium hydroxide and add water
A 50 ml solution (hereinafter referred to as solution B) was prepared.

A solution prepared by dissolving 89.0 mg of Cys (Acm) -human BNP-26 in 5 ml of 90% acetic acid was added dropwise to 30 ml of solution A while stirring at room temperature, and further stirred for 20 minutes. Thereafter, solution B was added dropwise until the brown color of iodine disappeared. This was diluted with 500 ml of water and adsorbed on a column (φ2 cm × 9.5 cm) packed with 30 ml of ODS resin [LC-Sorb (manufactured by Chemco)]. This was thoroughly washed with 0.1N acetic acid and eluted with 60 ml of 60% acetonitrile containing 0.1% TFA. After acetonitrile was distilled off under reduced pressure, the residue was freeze-dried to obtain 60.0 mg of crude human BNP-26. This was dissolved in 4 ml of 1N acetic acid and subjected to reversed-phase HPLC in four portions [column: Nucleosyl 120-5C18, φ20 mm × 250 m
m, flow rate 5 ml / min, solvent system; (A) water: acetonitrile: 1
0% TFA = 90: 10: 1, (B) water: acetonitrile: 10% TF
A = 40: 60: 1, (A) :( B) = 90: 10 to (A) :( B) = 55: 4
120-minute linear gradient up to 5]. This operation was repeated four times, and a main peak at 62 to 66 minutes was collected. After acetonitrile was distilled off under reduced pressure, the fraction was freeze-dried,
25 mg of human BNP-26 was obtained.

(4) Synthesis of Cys (Acm) -human BNP-32 700 mg of the protected human BNP-32 resin was released from the resin with TFA, TMSBr and ethanediol together with thioanisole in the same manner as in (2) above. After deprotection, 60.0 mg of Cys (Acm) -human BNP-32 was obtained by purification by reverse phase HPLC.

(5) Synthesis of human BNP-32 60.0 mg of Cys (Acm) -human BNP-32 was de-Acmized with iodine and cyclized in the same manner as in (3), and crude human BNP-32 was converted to 20.0 mg. mg
Obtained. Next, this was dissolved in 4 ml of 1N acetic acid and subjected to reversed-phase HPLC four times [column: nucleosyl 120-5C1]
8, φ20mm × 250mm, flow rate 5ml / min, solvent system; (A) water: acetonitrile: 10% TFA = 90: 10: 1, (B) water: acetonitrile: 10% TFA = 40: 60: 1, (A ): (B) = 90: 10 to (A) :( B) = 55: 45 linear gradient over 120 minutes]. This operation was repeated four times, and a main peak at 61 to 64 minutes was collected. After acetonitrile was distilled off under reduced pressure, the residue was freeze-dried to obtain 5 mg of human BNP-32.

[B] Physicochemical properties The physicochemical properties of human BNP-26 and 32 obtained in the above [A] were as follows.

(A) Properties: white powder

(B) Solubility in solvent: soluble in water, acidic aqueous solution and acetic acid. Insoluble in chloroform, benzene, ethyl ether and hexane.

(C) Acidic, neutral or basic: basic

(D) Amino acid composition: as shown in Table 1.

[0036]

[Table 1]

Test Example 1 <Smooth Muscle Relaxing Activity> The results of examining the smooth muscle relaxing activity of the peptide of the present invention are shown below.

(1) Test Method The rectum of a chick aged 4 to 7 days was excised and used as a 1.5 cm-long muscle specimen. 95% O ₂ -5% in 3 ml organ bath
It was immersed in 2.5 ml of Krebs-Hensleit nutrient solution (containing carbachol (2 × 10 ⁸ M)) kept at 32 ° C. through CO ₂ gas. A 0.5 g static pressure is applied to this muscle specimen, and the muscle is allowed to stand for about 30 minutes. When the automatic movement of the muscle is stabilized, 100 ng of human BNP-26 is administered as a test substance, and the muscle is relaxed for 6 to 8 minutes after the administration. It was measured. Wash the organ bath immediately after measurement,
After 20 to 30 minutes, the above operation was repeated using 200 ng of human BNP-32 as a test substance. The test substance was used by dissolving a predetermined amount in physiological saline.

(2) Results As a result, as shown in FIGS. 1 and 2, the peptide of the present invention
The administration of 100-200 ng showed strong smooth muscle relaxing activity.

[0040]

Industrial Applicability The peptide of the present invention or a salt thereof has an excellent smooth muscle relaxing action, further has a diuretic and natriuretic action, a blood pressure lowering action, and is highly safe because it is derived from humans. Edema, renal edema, hepatic edema,
It is useful as a therapeutic agent for pulmonary edema, hypertension, congestive heart failure, acute and chronic renal failure and the like.

[Brief description of the drawings]

FIG. 1: 100 ng of human BNP-26 of the present invention in chick rectal specimen
It is a figure which shows the time-dependent change of the relaxation length at the time of administration.

FIG. 2: 200 ng of human BNP-32 of the present invention in chick rectal specimen
It is a figure which shows the time-dependent change of the relaxation length at the time of administration.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Mika Takashima Inventor, Tokyo Tech Center, Daiichi Chemicals Co., Ltd. 5-5-1-12, Sumida-ku, Tokyo (72) Inventor Toshiyuki Matsuo Oaza, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture No. 6653 Kihara (56) References Table of International Publication No. 3-505280 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07K 14/575 ZNA A61K 38/00 A61P 9/00 A61P 9 / 12 A61P 13/02 BIOSIS (DIALOG) CA (STN) REGISTRY (STN) WPIDS (STN)

Claims (2)

(57) [Claims] (1) Formula (1) Or a salt thereof. 2. A therapeutic agent for a cardiovascular disease, comprising the physiologically active peptide according to claim 1 or a salt thereof.