JP2001270897A - Biologically active peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new biologically active peptide exhibiting smooth muscle contractile activity and vascular permeability potentiating action. SOLUTION: This new biologically active peptide has a specific amino acid sequence synthesized on the basis of the amino acid sequence of Blomhotin prepared by purifying the venom of Agkistrodon blomhoffi.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel bioactive peptide having both smooth muscle contraction activity and vascular permeability enhancing action.

[0002]

2. Description of the Related Art A novel peptide has been found by purifying an active peptide from Japanese viper venom using the smooth muscle contraction activity as an index and is named Blomhotin (hereinafter referred to as BH) (Toxicon 37, 1761-1770 (1999)). )). Its amino acid sequence is as set forth in SEQ ID NO: 8 and is very similar to the amino acid sequence of known peptide BPP10d (or also referred to as potentiator A), and has a structure in which Arg is added to the C-terminal of BPP10d. ing. BPP10d is a peptide that exhibits inhibitory activity against angiotensin converting enzyme, which plays an important role in regulating blood pressure and body fluid volume, and promotes smooth muscle contraction by bradykinin, and is contained in Japanese viper venom like BH . However, BPP10d alone has no smooth muscle contractile activity. On the other hand, by synthesizing an analog in which amino acids have been removed from the N-terminus of BH, it has been revealed that a peptide consisting of four amino acids at the C-terminal side (PIPR) is the minimum unit showing contractile activity. This motif has no homology to conventionally known bioactive peptides, and is considered to be a novel family of bioactive peptides.

[0003]

An object of the present invention is to provide a peptide having a higher physiological activity than BH.

[0004]

Means for Solving the Problems The present invention provides SEQ ID NOs.
A physiologically active peptide having any one of the amino acid sequences described in Item 7, and a medicament containing the same as an active ingredient.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the peptide of the present invention having the amino acid sequence of SEQ ID NOS: 1 to 7, Gly is glycine, Arg is arginine, Pro is proline, and Ile.
Indicates isoleucine, Ala indicates alanine, and Tyr indicates tyrosine. These amino acids are all in L-form. C
The four amino acid sequences on the terminal side are different from the amino acid sequence of PIPR, which was considered as the minimum unit showing contractile activity as described above, and in this respect, the peptide of the present invention was completely unexpected. Was.

[0006] The peptide of the present invention can be synthesized by applying conventional means of peptide synthesis. That is, liquid phase method or solid phase method (for example, Nobuo Izumiya et al., “Peptide Synthesis” 1984)
Published by Maruzen Co., Ltd .; The Chemical Society of Japan
(I) / Protein Chemistry, Vol. 4, pp. 208-495, 1977,
Published by Tokyo Chemical Doujin; Development of continuum medicines 14 “Peptide synthesis”, published by Hirokawa Shoten, etc.), a raw material having a reactive carboxyl group corresponding to one of two fragments bisected at an arbitrary position of a peptide bond; The starting material having a reactive amino group corresponding to the other fragment was treated with 2- (1H-Be
nzotriazole-1-yl) -1,1,3,3-tetramethyluronium hexaf
When condensation is performed using a method using an active ester such as luorophosphate (HBTU) or a method using carbodiimide, and the resulting condensate has a protective group, it can be produced by removing the protective group.

[0007] Functional groups that should not participate in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), 9-
Fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting group include groups capable of forming an alkyl ester, a benzyl ester and the like. In the case of the solid phase method, the carboxyl group at the C-terminus is a chlorotrityl resin, a chloromethyl resin, an oxymethyl resin, 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 protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. Examples of the purification method include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. The synthesized peptide is a protein sequencer that reads the amino acid sequence from the C-terminal by Edman degradation method, FAB-M
It is analyzed by S, TOF-MS and the like. The peptide of the present invention can be prepared from an acid addition salt using an inorganic acid such as hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid such as formic acid, citric acid, tartaric acid, fumaric acid, maleic acid, acetic acid or trifluoroacetic acid according to a conventional method. It can be.

The peptide of the present invention has a high smooth muscle contracting activity and also has a vascular permeability enhancing action.

The peptide of the present invention can be administered orally or parenterally as it is or as a pharmaceutical composition mixed with known pharmaceutically acceptable carriers and excipients, and used as a hypotensive agent and the like. can do. The dose is
Although it depends on the age of the patient and the degree of the disease, it is generally 0.001 to 1000 mg / day for oral administration.
Even when administered intravenously, intramuscularly or subcutaneously, the dose is about 0.001 to 1000 mg / kg body weight per day. The frequency of administration is 1 to 3 times a day for oral administration and 1 to 2 times a day for injection.

[0011]

Next, the present invention will be described more specifically with reference to examples. Example 1 [Synthesis of peptide] Commercially available Fmoc-A manufactured by Watanabe Chemical Industry
0.25 g of rg (Pbf) -Alko resin (Wang) (polystyrene resin terminated with p-alkoxybenzyl alcohol, substitution rate 0.5 meq / g) was added to ABI-
The fraction was collected in a reaction vessel of a 433-type peptide synthesizer (manufactured by Perkin Elmer) and synthesized as follows. Note that Pb
f represents a 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl group. First, the above resin was placed in a reaction vessel, and 0.25 mmol of Fmoc
-Ala and 0.25 mmol of HB as activator
TU was dissolved in 0.5 M dimethylformamide, added to the reaction vessel, 0.5 mmol of diisopropylethylamine was added, and the mixture was stirred and reacted at room temperature for 20 minutes. The resulting resin was gently stirred in N-methylpyrrolidone containing 20% piperidine to remove the Fmoc group.
Fmoc-amino acid was sequentially coupled from the C-terminal in the same manner as in the method of coupling moc-Ala,
Gly-Arg (Pbf) -Pro-Pro-Gly-
Pro-Pro-Ile-Ala-Arg (Pbf) resin was obtained. 100 mg of the resin was deprotected (1 ml
Trifluoroacetic acid, 0.1 ml thioanisole, 0.1
The mixture was stirred for 1 hour at room temperature in 0.1 ml of trimethylsilane, and the peptide was released from the resin.

[0012] To this was added 15 ml of cold ether to precipitate the peptide, which was further washed three times with cold ether to obtain a crude peptide. Use this with an ODS column (Inertsi
The peptide was developed and purified by reverse phase chromatography using 1Prep ODS (20 × 250 mm) with a linear concentration gradient of acetonitrile containing 0.1% trifluoroacetic acid to obtain a peptide having the amino acid sequence of SEQ ID NO: 1. The product was analyzed by MALDI-TOF-MS (manufactured by Kratos) to confirm that it was the desired product.

[Assay] Measurement of smooth muscle contractile activity A longitudinal muscle section was prepared from the fundus of 250-350 g of male Wistar rats and the specimen from which mucous membrane was removed was kept at 37 ° C.
It was hung on an organo bath containing ml of nutrient solution. After the tension of the specimen was stabilized, the test substance was added to an organo bath as an aqueous solution of 0.1 ml of a nutrient solution. The resulting contraction was recorded via a transducer. The shrinkage of the test substance was evaluated with the shrinkage caused by adding 10-6 M of serotonin as 100%. Table 1 shows the results. In addition, EC50 (μ
M) is the dose that contracts 50% of the maximal contraction of rat fundus smooth muscle by 10-6M serotonin.

Examples 2 to 7 In the same manner as in Example 1, a peptide having the amino acid sequence of SEQ ID NOs: 2 to 7 was synthesized, and MALDI-TOF was synthesized.
-Confirmed by MS. The peptide thus obtained was evaluated in the same manner as in Example 1. Table 1 shows the results.

Comparative Example 1 A peptide having the amino acid sequence of SEQ ID NO: 8 was synthesized in the same manner as in Example 1, and confirmed by FAB-MS. The peptide thus obtained was evaluated in the same manner as in Example 1. Table 1 shows the results.

[0016]

[Table 1]

[0017]

Industrial Applicability The novel physiologically active peptide of the present invention exhibits a smooth muscle contraction activity and a blood vessel permeation enhancing effect and is useful as a pharmaceutical.

[0018]

[Sequence List] SEQUENCE LISTING <110> Toray Research Center, Inc. <120> PHYSIOLOGICALLY ACTIVE PEPTIDE <130> 95A00010-A <160> 8 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220 > <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 1 Gly Arg Pro Pro Gly Pro Pro Ile Ala Arg 1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 2 Gly Arg Pro Tyr Gly Pro Pro Ile Pro Arg 1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 3 Gly Arg Pro Pro Gly Pro Ala Ile Pro Arg 1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth mu scle contraction activity <400> 4 Gly Arg Pro Pro Gly Pro Pro Ile Pro Arg 1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 5 Gly Arg Pro Pro Gly Pro Pro Ile Gly Arg 1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223 > Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 6 Gly Arg Pro Pro Gly Pro Pro Ile Gln Arg 1 5 10 <210> 7 <211> 10 <212> PRT <213 > Artificial Sequence <220> <223> Designed peptide based on the amino acid sequence of Blomhotin having an improved smooth muscle contraction activity <400> 7 Gly Arg Pro Tyr Gly Pro Ala Leu Ala Arg 1 5 10 <210> 8 <211 > 11 <212> PRT <213> Agkistrodon halys <400> 8 pGlu Gly Arg Pro Pro Gly Pro Pro Ile Pro Arg 1 5 10

[Sequence Listing Free Text] SEQ ID NO: 1: A peptide with improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 2: An improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 3: Peptide with improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 4: Peptide with improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 5: Peptide with improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 6: Peptide with improved smooth muscle contraction activity synthesized based on the amino acid sequence of Blomhotin SEQ ID NO: 7: Peptides with improved smooth muscle contraction activity synthesized based on amino acid sequence

 ────────────────────────────────────────────────── ─── Continued on the front page F term (reference) 4C084 AA01 AA07 BA01 BA17 BA23 CA59 DA35 DC40 MA52 MA66 ZA362 ZA422 ZA942 ZC162 4H045 AA10 AA30 BA15 CA53 EA20 EA23 FA33

Claims (2)

[Claims] A physiologically active peptide having the amino acid sequence of any one of SEQ ID NOs: 1 to 7. 2. A medicament comprising the physiologically active peptide according to claim 1 as an active ingredient.