JP2883903B2 - Novel bioactive peptide and its use - Google Patents

Description

The present invention relates to a novel bioactive peptide and its use, and more particularly, to a human brain natriuretic peptide and hypertension, edema disease containing the same, The present invention relates to a therapeutic agent for cardiovascular diseases such as heart failure and renal failure.

[Conventional technology]

Recently, a peptide has been isolated from pig brain and its structure has been determined. Its structure and bioactivity are very similar to those of atrial natriuretic peptide (hereinafter abbreviated as ANP), so brain natriuretic peptide (Brain Natriuretic Peptide; [Sudo et al., Nature, 332, 78-8]
0 (1988)]. BNP is involved in the regulation of body fluid volume, electrolyte balance and blood pressure in living organisms, which
And that there is a dual regulatory mechanism by BNP.

Meanwhile, the cDNA of this porcine BNP was cloned and
The structure of NP precursors has been elucidated (Maekawa et al .; Bioch
em. Biophys. Res. Commun., 157 (1), 410-416 (198
8)]. Furthermore, using the porcine BNP cDNA as a probe, human BN
CDNA of P was cloned and its structural analysis
The amino acid sequence of NP has been deduced.

[Problems to be solved by the invention]

However, there is no report that human BNP has been isolated or synthesized, and it is unclear what active area has activity. Therefore, synthesizing this human BNP, comparing its biological activity with a known natriuretic peptide, and exploring its usefulness is important in drug development from the viewpoint of avoiding side effects such as antibody production.

[Means for solving the problem]

The present inventors have synthesized a human BNP derivative which is a novel substance based on the estimation of the amino acid sequence of the human BNP, and further studied its pharmacological action.These substances have a known natriuretic peptide. They have found that they have a smooth muscle relaxing action and a natriuretic action, and have completed the present invention.

That is, the present invention (Wherein X is H, H-Gly-Ser-Gly- or H-Ser-Pr
o-Lys-Met-Val-Gln-Gly-Ser-Gly-) or a salt thereof, and a therapeutic agent for circulatory diseases containing the same.

In the present invention, the peptide (I) in which X is H-Gly-Ser-Gly- is referred to as human BNP-26, and X is H-Ser-Pro
The peptide (I) that is -Lys-Met-Val-Gln-Gly-Ser-Gly- may be referred to as human BNP-32.

In addition, in this specification, the abbreviation in a peptide is what is generally used in the said field | area, and 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 Try: L-Tyrosine The peptide (I) of the present invention can be prepared 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 .; Ed. “Biochemistry Experiment Course (I) / Protein Chemistry”, vol. 4, pp. 208-495,
1977, Tokyo Chemical Doujinshi, etc.].

For example, when the peptide (I) of the present invention is synthesized by a solid phase method, the α-amino group of the amino acid used is a 9-fluorenylmethyloxycarbonyl group (Fmoc group), and the β-carboxyl group of aspartic acid is tert-butyl. Group (tBu
Group), 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), histidine imidazole group is trityl group (Trt group), lysine Is an tert-butyloxycarbonyl group (Boc
Group). The insoluble resin used is a p-alkoxybenzyl alcohol resin (Wang
Resin) is preferred. Condensation of the protected amino acid is preferably performed using 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. For example, the α-amino group of the amino acid used may be protected with a tert-butyloxycarbonyl group (Boc group).

The peptide (I) of the present invention is produced by the solid phase method, for example, as follows. First, a protected derivative Fmoc-His (Trt) -OH of His, which is a C-terminal amino acid, 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 (TFA)
Treatment with piperidine and trimethylsilyl bromide (TMSBr) [Chem. Pharm. Bull., 35 (9), 3
880 (1987)] and cleavage of peptide from resin and A
The protecting group other than the cm group is simultaneously removed to obtain 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, gel filtration, ion exchange chromatography, reverse phase high performance liquid chromatography (HPLC), or the like.

In addition, the peptide (I) of the present invention includes inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, formic acid, acetic acid, citric acid, tartaric acid, fumaric acid,
An acid addition salt can be prepared using an organic acid such as maleic acid according to a conventional method.

[Action and effect of the invention]

The peptide of the present invention thus obtained has a smooth muscle relaxing action and the like. The results of studying these effects are shown below.

<Smooth muscle relaxing action> Test method The rectum of a chick aged 4 to 7 days was excised and used as a 1.5 cm-long muscle specimen. This is mixed with 95% O ₂ -5 in a 3 ml organ bath.
2.5 ml of a Krebs-Hensleit nutrient solution (containing carbachol (2 × 10 ⁸ M)) kept at 32 ° C. through% CO ₂ gas
Dipped in. The muscle specimen was subjected to a static pressure of 0.5 g, and allowed to stand for about 30 minutes to stabilize the automatic movement of the muscle. 100 ng of human BNP-26 was administered as a test substance, and the muscle was relaxed for 6 to 8 minutes after administration. Was measured. Immediately after measurement, wash the organ bath.
After 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.

Results The results are shown in FIGS. 1-A to 1-B. As a result, the peptide of the present invention showed a strong smooth muscle relaxing activity at a dose of 100 to 200 ng.

As described above, the peptide of the present invention or a salt thereof has an excellent smooth muscle relaxing action, and further has a diuretic and natriuretic action,
It has a hypotensive effect and is highly safe because it is derived from humans, for example, therapeutic agents for cardiac edema, renal edema, hepatic edema, pulmonary edema, hypertension, congestive heart failure, acute and chronic renal failure, etc. Useful as

The administration method can be the method used for administration of peptide-based drugs, that is, intravenous injection, intramuscular injection, subcutaneous injection, or sublingual administration, intranasal administration, or rectal administration.

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.

[Examples] Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Synthesis of Peptides Human BNP-26 and Human BNP-32: Synthesis of Protected Peptide Resin In the synthesis of the protected peptide resin, all α-amino groups of the constituent amino acids were 9-fluorenylmethyloxycarbonyl. Group (Fmoc group)
The β-carboxyl group of aspartic acid is a tert-butyl group (tBu group), and the guanidino group of arginine is a 4-methoxy-2,3,6-trimethylbenzenesulfonyl group (Mtr group)
The hydroxyl group of serine is a tert-butyl group (tBu group), and the thiol group of cysteine is an acetamidomethyl group (Acm group)
And the imidazole group of histidine is a trityl group (Trt
), The ε-amino group of lysine was protected with a tert-butyloxycarbonyl group (Boc group). As the resin, 0.1 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 amino group at the terminal of the protected peptide bound to the resin, with piperidine for 6 minutes at room temperature was repeated twice to remove almost completely. Subsequently, the amino group released by the de-Fmoc-decomposition 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, Fmoc
-1 mmol of an amino acid in the presence of 1-hydroxybenztriazole in 1,3-diisopropylcarbodiimide (DIC)
And condensed. 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.

In this manner, Fmoc-Gly-Ser (tBu) -Gly-Cys (A
cm) -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 (Mtr) -Arg (Mtr) -Hi
s (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 amino group at the terminal of the protected peptide, was removed according to the method described above, and H-Gly-Ser
(TBu) -Gly-Cys (Acm) -Phe-Gly-Arg (Mtr) -Ly
s (Boc) -Met-Asp (tBu) -Arg (Mtr) -Ile-Ser (t
Bu) -Ser (tBu) -Ser (tBu) -Ser (tBu) -Gly-Leu
-Gly-Cys (Acm) -Lys (Boc) -Val-Leu-Arg (Mt
r) -Arg (Mtr) -His (Trt) -resin (hereinafter referred to as protected human B)
670 mg of NP-26 resin).

The remainder was further subjected to an N-terminal extension reaction, and Fmoc-Ser (tB
u) -Pro-Lys (Boc) -Met-Val-Gln-Gly-Ser (tB
u) -Gly-Cys (Acm) -Phe-Gly-Arg (Mtr) -Lys (B
oc) -Met-Asp (tBu) -Arg (Mtr) -Ile-Ser (tBu)
-Ser (tBu) -Ser (tBu) -Ser (tBu) -Gly-Leu-Gl
y-Cys (Acm) -Lys (Boc) -Val-Leu-Arg (Mtr) -A
rg (Mtr) -His (Trt) -resin was obtained. Then, 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) was removed.
-Met-Val-Gln-Gly-Ser (tBu) -Gly-Cys (Acm)
-Phe-Gly-Arg (Mtr) -Lys (Boc) -Met-Asp (tB
u) -Arg (Mtr) -Ile-Ser (tBu) -Ser (tBu) -Ser
(TBu) -Ser (tBu) -Gly-Leu-Gly-Cys (Acm) -Ly
s (Boc) -Val-Leu-Arg (Mtr) -Arg (Mtr) -His (T
rt) -resin (hereinafter referred to as protected human BNP-32 resin) 1.5 g
Obtained.

Synthesis of Cys (Acm) -human BNP-26 600 mg of protected human BNP-26 resin was placed in a reactor with 2.4 ml of thioanisole and 20 ml of trifluoroacetic acid (TFA) was added.
2.6 ml of trimethylsilyl bromide (TMSBr) and 2.4 ml of ethanedithiol were added and reacted at 0 ° C. for 3 hours. After completion of the reaction, anisole was removed by washing with 200 ml of ether, and the product was extracted with 20 ml of 1N-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 adjusted again 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 (Chemco)]
After washing well with 0.1N-acetic acid, elution was performed with 200 ml of 60% acetonitrile containing 0.1% TFA. Acetonitrile was distilled off under reduced pressure, lyophilized, and 300 mg of crude Cys (Acm) -human BNP-
I got 26.

This was dissolved in 1N-acetic acid (9 ml), and the mixture was divided into 9 portions and the reversed phase H was added.
PLC (column: nucleosyl 120-5C18, 20 × 250
mm, flow rate 5 ml / min, solvent system: (A) water: acetonitrile: 10
% TFA = 90: 10: 1, (B) water: acetonitrile: 10% TFA = 4
0: 60: 1, (A) :( B) = 90: 10 to (A) :( B) = 5
120 minute linear gradient up to 5:45]. 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) -human BNP-26 96.
0 mg was obtained.

Synthesis of human BNP-26 Dissolve 227 mg of iodine in 50 ml of 95% acetic acid, and add
(Hereinafter referred to as solution A) was prepared.

Also, 2.1 g of citric acid and 575 mg of L-ascorbic acid were added in 2N.
-Dissolved in 10 ml of sodium hydroxide and added water to make 50 ml (hereinafter referred to as solution B).

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.
Stirred for another minute. 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 × 9.5 cm) packed with 30 ml of ODS resin [LC-Sorb (Chemco)]. 0.1N−
After washing well with acetic acid, elution was carried out 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 one
The residue was dissolved in 4 ml of N-acetic acid and subjected to reverse phase HPLC in four portions [column: nucleosyl 120-5C18, φ20 × 250 mm, flow rate 5 m
l / min, solvent system; (A) water: acetonitrile: 10% TFA = 9
0: 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 four times, and a main peak at 62 to 66 minutes was collected. After acetonitrile was distilled off under reduced pressure, this fraction was lyophilized to give 25
mg of human BNP-26 was obtained.

Synthesis of Cys (Acm) -human BNP-32 700 mg of protected human BNP-32 was desorbed and deprotected from the resin with TFA, TMSBr and ethanediol together with thioanisole in the same manner as described above, followed by reverse phase HPLC. Purification yielded 60.0 mg of Cys (Acm) -human BNP-32.

Synthesis of human BNP-32 Cys (Acm) -human BNP-32 (60.0 mg) was de-Acmized with iodine and cyclized in the same manner as described above to obtain crude human BNP-32.
20.0 mg were obtained. Next, this was dissolved in 1N-acetic acid (4 ml) and subjected to reversed phase HPLC in four portions [column: nucleosyl 120-5C18, φ20 × 250 mm, flow rate 5 ml / 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 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.

(2) Physicochemical properties The physicochemical properties of human BNP-26 and 32 obtained in (1) above 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) Acidity, neutrality, basicity and basicity (d) Amino acid composition As shown in Table 1.

[Brief description of the drawings]

FIG. 1-A shows that human BNP-26 of the present invention was added to a chick rectal specimen.
FIG. 1-B is a graph showing the time-dependent change in the relaxation length when 200 ng of the human BNP-32 of the present invention was administered to a chick rectal specimen.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Atsushi Izumi 5-5-12, Nippondaira, Sumida-ku, Tokyo Daiichi Chemical Co., Ltd. Tokyo Technical Center (72) Inventor Mika Takashima 5-5-Norihira, Sumida-ku, Tokyo No. 12 Daiichi Kagaku Co., Ltd. Tokyo Technical Center (72) Inventor Toshiyuki Matsuo 6553 Kihara, Kiyotake-cho, Miyazaki-gun, Miyazaki Prefecture (56) References Table 3-505280 (JP, A)

Claims (1)

(57) [Claims] 1. A protected histidine derivative is introduced into an insoluble resin, and a protected amino acid is sequentially bonded thereto to synthesize a protected peptide resin. Then, the protecting group of amino acids other than cysteine is removed and the peptide is cleaved from the resin. And then oxidizing it with iodine to remove the protecting group of cysteine and simultaneously form a disulfide bond by the thiol group of two cysteines in the peptide molecule. (Wherein X is H, H-Gly-Ser-Gly- or H-Ser-Pr
o-Lys-Met-Val-Gln-Gly-Ser-Gly-).