JP3349528B2 - peptide - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a peptide having opioid activity and a salt thereof, and an agent having the opioid activity containing the peptide and its salt as an active ingredient.

[0002]

2. Description of the Related Art It is widely known that there are a large number of bioregulators involved in higher life activities in foods, and these bioregulators are used to promote health, prevent diseases, and so on. In recent years, various attempts have been made to positively utilize such treatments and prevent aging. As such bioregulatory factors, various factors acting on the nervous system, digestive absorption system, circulatory system, biological defense / immune system, endocrine system, cell differentiation / proliferation system, etc. are known. Among them, some food proteins act on the nervous system to cause morphine-like anesthesia,
It has been found that there is a peptide unit having an analgesic activity or the like (so-called opioid activity), and such a peptide is generally called an opioid peptide.

[0003] Opioid peptides are known to have an effect on the digestive tract and on the secretion of gastrointestinal hormones. Prolonged retention of food in the intestine when insulin degraded from wheat gluten is administered orally, insulin in blood And have been reported to exhibit opioid activity such as an increase in the amount of somatostatin. Based on such findings, the present inventors can hydrolyze wheat gluten and fractionate a novel peptide having an opioid activity from the product, and have filed a prior application (Japanese Patent Application No. 3-171879). issue).

[0004]

Under the circumstances described above, the present inventors have further studied on peptides having opioid activity. And SEQ ID NO: 1 having an amino acid sequence different from that of the peptide in Japanese Patent Application No. 3-171879.
And chemically synthesizing a novel peptide in which the terminal carboxyl group is amidated and measuring its opioid activity, it was found that the peptide has extremely high opioid activity.

Accordingly, the present invention provides a peptide represented by SEQ ID NO: 1 (where Xaa is Gly,
From and the N-terminus represents Pro or Val fourth carboxyl group of the amino acid Xaa at is amidated) The
Is its salt.

[0006] The above-mentioned invention of SEQ ID NO: 1
Peptides or their salts have high opioid activity
You .

The peptide represented by SEQ ID NO: 1 has its N
Three amino acids of tyrosine (Tyr), proline (Pro) and isoleucine (Ile) are bonded in order from the terminal, a predetermined amino acid (Xaa) is bonded to the fourth, and the fourth amino acid (Xaa) is bonded. Is a tetrapeptide represented by the formula: Try-Pro-Ile-Xaa-NH _{2 in} which the carboxyl group is amidated.

In SEQ ID NO: 1, the fourth amino acid
The acid (Xaa) is glycine (Gly), proline (Pro) or valine (Val) as described above .

In the peptide of the present invention represented by SEQ ID NO: 1 in which the carboxyl group of amino acid (Xaa) is amidated, the four amino acids constituting the peptide may be either L-amino acids or D-amino acids. For example, 4
Even if all of the amino acids consist of L-amino acids,
Alternatively, it may consist of only D-amino acids, or 4
Any of the amino acids may be L-amino acids and the remainder may be D-amino acids. Both peptides can be easily produced by chemical synthesis. In particular, in the peptide of the present invention in which Xaa is proline (Pro) or valine (Val), the peptide to which D-proline or D-valine is bound as Xaa has high opioid activity.

[0010] The term "agent having opioid activity" as used herein refers to analgesia, anesthesia, sedation, emotion, respiration,
An agent having an activity recognized as so-called opioid activity involved in pulsation, body temperature, gastrointestinal function, feeding, immunity, regulation of secretion of hormones such as insulin and somatostatin, promotion of electrolyte absorption, regulation of contraction of myocardium, etc. .

The peptide of the present invention and a salt thereof can be produced by any method as long as it has the amino acid sequence represented by SEQ ID NO: 1 and the carboxyl group of the fourth amino acid (Xaa) is amidated. May be. An example of the method for producing the peptide of the present invention is a peptide represented by SEQ ID NO: 1, wherein the fourth amino acid (Xaa) is represented by SEQ ID NO: 2 which is glycine (Gly), and the carboxyl group of the glycine is amidated. This will be described below by taking the case of a peptide as an example.

The amidated pep of the invention of SEQ ID NO: 2
Production of Pide Synthesis is performed using a SAM TWO-type automatic peptide synthesizer manufactured by Biosearch Inc., USA, according to the standard protocol of the same. That is, a commercially available benzhydrylamine resin was added to and reacted with a dimethylformamide solution of Boc (butoxycarbonyl) -Gly and a methylene chloride solution of diisopropylcarbodiimide to obtain a Boc-Gly-benzhydrylamine resin. Next, Gly-NH with the Boc group removed
₂ -coupling Boc-Ile to the resin in the presence of diisopropylcarbodiimide,
c-Pro-OH and Boc-Tyr and (Cl ₂ -Bzl) by sequentially _{coupling, Boc-Tyr (Cl 2 -Bzl} ) -Pro-Ile-Gly-NH 2
-Obtain the resin. This peptide resin is placed in hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 2 hours. After distilling off hydrogen fluoride, the residue was washed with ether and 30%
Extract the peptide with acetic acid. The peptide represented by SEQ ID NO: 2 is obtained by purifying the crude peptide obtained by extraction using high performance liquid chromatography on an ODS column. Further, other peptides encompassed in SEQ ID NO: 1 and in which the fourth carboxyl group is amidated can be obtained by synthesizing and purifying by the same operation.

The peptide of the present invention, which has the amino acid sequence represented by SEQ ID NO: 1 and in which the carboxyl group of the fourth amino acid (Xaa) is amidated, and a salt thereof have opioid activity. So, agents containing those peptides and salts thereof as an active ingredient,
For example, analgesics, hypnotics, gastrointestinal hormone enhancers,
As an electrolyte absorption enhancer, an agent for improving diarrhea by suppressing intestinal peristalsis, it can be administered to humans and various animals,
It is particularly effective as an analgesic or sedative anesthetic. At that time,
The peptide of the present invention and a salt thereof may be used alone or in the form of a mixture containing two or more peptides. The suitable dose of the agent having opioid activity of the present invention may vary depending on various conditions such as age, weight, sex, symptoms, animal type, etc. of the human or animal to be administered.

The agent having an opioid activity of the present invention can be administered orally or parenterally, and can be administered alone or in combination with a solid or liquid carrier commonly used in the pharmaceutical industry. They may be administered together or used in admixture or combination with other agents. The dosage form can be in any form such as tablets, pills, granules, capsules, powders, aqueous solutions, injections and the like. Hereinafter, the present invention will be described specifically by way of examples, but the present invention is not limited thereto. In the following examples, each amino acid is L unless otherwise specified.
-Means amino acid.

[0015]

【Example】

<< Example 1 >> A peptide having an amino acid sequence represented by SEQ ID NO: 2 and having a carboxyl group of the fourth amino acid (Gly) amidated was synthesized by the following method. Commercially available benzhydrylamine resin (substitution rate 0.3 m
eq / g) 0.4 g, 0.4 ml of a solution of Boc-Gly in dimethylformamide (0.4 ml) and 5 ml of a 0.4 M solution of diisopropylcarbodiimide in methylene chloride were mixed and reacted in a reaction vessel with stirring for 2 hours. A Boc-Gly-benzhydrylamine resin was obtained. To this resin, 20 ml of methylene chloride containing 45% trifluoroacetic acid and 2.5% anisole was used.
The reaction was carried out at room temperature for 25 minutes in a reaction vessel of AM TWO and B
The oc group was removed. Removed Gly-NH of the Boc group ₂ - resin was washed with methylene chloride, the filtrate was neutralized with methylene chloride containing 10% diisopropylethylamine was washed with additional methylene chloride. 0.4M Boc-I
5 ml of a solution of le in dimethylformamide and 5 ml of a 0.4 M solution of diisopropylcarbodiimide in methylene chloride were mixed, placed in a reaction vessel, and reacted at room temperature for 2 hours with stirring.

The resin obtained above is washed successively with dimethylformamide, methylene chloride, methylene chloride containing 10% of diisopropylethylamine, a methylene chloride and a mixed solution of methylene chloride / dimethylformamide.
Boc-Ile-Gly-NH ₂ -resin was obtained. Subsequently, the removal of the Boc group and the coupling of the Boc amino acid were repeated in the same manner as described above to obtain Boc-Tyr (Cl ₂ -Bzl) -Pro
-Ile-Gly-NH ₂ - to obtain a product made of resin.

The resin was placed in 20 ml of hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 2 hours to release the amidated peptide from the resin. Thereafter, hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and lyophilized to obtain a crude peptide. The obtained amidated peptide was converted to ODS-Cosmosil 5C ₁₈ -A, which is an octadecylsilane (ODS) column manufactured by Nacalai Tesque, Inc.
Purification by reverse phase chromatography using R provided 20 mg of the final product. Amino acid composition (molar ratio)
Was Tyr: Pro: Ile: Gly = 1: 1: 1: 1: 1. In addition, Applied System's protein sequencer 4
Analysis by type 77-A revealed that the peptide had the amino acid sequence of SEQ ID NO: 2 and the carboxyl group of glycine (Gly), the fourth amino acid, was amidated. When the Rf value was determined by thin-layer chromatography using a silica gel plate,
Butanol: acetic acid: pyridine: water = 15: 3: 10: 1
The Rf value of the product in thin layer chromatography when the developing solvent of No. 2 was used was 0.744.

Example 2 In the peptide represented by SEQ ID NO: 1, Xaa is D-proline (D-Pro). In the case of the peptide represented by SEQ ID NO: 3, the peptide represented by SEQ ID NO: 2 In the same manner as in the above, using 0.4 g of a commercially available benzhydrylamine resin (substitution rate: 0.3 meq / g), D-Pro, Ile, Pro, and Tyr were sequentially bonded thereto, and the fourth amino acid was obtained. 25 mg of the final product represented by SEQ ID NO: 3 in which the carboxyl group of D-Pro was amidated. Its amino acid composition (molar ratio) is Ty
r: Pro: Ile: D-Pro = 1: 1: 1: 1, and when analyzed by the protein sequencer used in Example 1, it is represented by SEQ ID NO: 3 and the fourth amino acid, D- Pro was found to be a peptide having an amino acid sequence in which the carboxyl group of amid was amidated. The Rf value of the product determined by the same method as in Example 1 was 0.734 by thin layer chromatography.

Example 3 In the case of the peptide represented by SEQ ID NO: 1 in which Xaa is D-valine (D-Val) in the peptide represented by SEQ ID NO: 1, the peptide represented by SEQ ID NO: 2 In the same manner as in the above, using 0.4 g of a commercially available benzhydrylamine resin (substitution ratio: 0.3 meq / g), D-Val, Ile, Pro and Tyr were successively bonded thereto, and the fourth amino acid was obtained. 20 mg of the final product represented by SEQ ID NO: 4 in which the carboxyl group of D-Val was amidated. Its amino acid composition (molar ratio) is Ty
r: Pro: Ile: D-Val = 1: 1: 1: 1, and when analyzed by the protein sequencer used in Example 1, it is represented by SEQ ID NO: 4 and the fourth amino acid, D- The carboxyl group of Val was found to be a peptide having an amidated amino acid sequence. Further, the Rf value of the product obtained in the same manner as in Example 1 in thin layer chromatography was 0.658.

Example 4 In the peptide represented by SEQ ID NO: 3 in which the fourth amino acid, D-Pro, is replaced with L-Pro and the carboxyl group of L-Pro is amidated Similarly, in the same manner as in the case of the peptide represented by SEQ ID NO: 2, 0.4 g of a commercially available benzhydrylamine resin (substitution ratio: 0.3 meq / g) was used, and L-Pro, Ile, Pro, and By coupling Tyr, 25 mg of the final product in which the carboxyl group of the fourth amino acid, L-Pro, was amidated was obtained. Its amino acid composition (molar ratio) is Tyr: Pro: Ile: Pro = 1: 1: 1: 1
And analysis by the protein sequencer used in Example 1 reveals that the peptide is represented by SEQ ID NO: 3 and has an amino acid sequence in which the carboxyl group of L-Pro, the fourth amino acid, is amidated. There was found. Further, the Rf value of the product obtained in the same manner as in Example 1 by thin layer chromatography was 0.772.

The opioid activity of each of the peptides obtained in Examples 1 to 4 was measured as follows.

Measurement method of opioid activity The longitudinal muscle of the ileum extracted from a male guinea pig weighing 250 to 300 g was connected to an isotonic transducer (manufactured by Nihon Kohden Kogyo Co., Ltd .: TB612-T) and 0.2 g.
Apply tension. Krebs isotonic solution (NaCl 118 mM, KCl 4.75) was placed in a 2 ml Magnus tube kept at 36 ° C.
mM, CaCl ₂ 2.54 mM, MgSO ₄ 1.2 mM, NaHCO ₃
(25 mM, KH ₂ PO ₄ 1.19 mM, glucose 11 mM)
Soak ileum longitudinal muscle among those satisfying the vent from the cylinder _{(0 2 95%: CO 2} 5%) was. After stabilizing for about 2 hours in this way, electrical stimulation was applied at 30 V for 0.5 msec to electrically contract guinea pig ileum longitudinal muscle. After the electric contraction was stabilized, 50 μl of an inhibitor containing an aminopeptidase inhibitor or the like was added, the peptide represented by SEQ ID NO: 2 to SEQ ID NO: 4 was added, and changes in electric contraction of the guinea pig ileum longitudinal muscle were recorded by a recorder. The opioid activity was determined to be 10
Judgment was made by releasing suppression by adding 20 μl of ^-4 M naloxone. Then, the concentration (IC ₅₀ ) at which the electrical contraction of the guinea pig ileum longitudinal muscle was inhibited by 50% was measured.

As a result, I of the peptide of the present invention represented by SEQ ID NO: 2 to SEQ ID NO: 4 obtained in the above Examples 1 to 4
_C50 was as shown in Table 1 below. For reference,
Table 1 also shows the IC ₅₀ when the opioid activity of the conventionally known opioid peptide derived from bovine β-casein represented by SEQ ID NO: 5 was measured in the same manner as described above.

[0024]

[Table 1] [Opioid activity of peptide (IC ₅₀ )] Peptide IC ₅₀ (μM) Peptide of Example 1 (SEQ ID NO: 2) 2.1 Peptide of Example 2 (SEQ ID NO: 3; however, D-Pro at position 4) 1.2 Peptide of Example 3 (SEQ ID NO: 2) No. 4; however, position 4 is D-Val) 1.6 Peptide of Example 4 (SEQ ID NO: 3; however, position 4 is L-Pro) 30 Peptide of SEQ ID NO: 5 (derived from bovine β-casein) 240

From the results in Table 1, the peptides of the present invention of Examples 1 to 3 represented by SEQ ID NOs: 2 to 4 in which the carboxyl group of the fourth amino acid is amidated are represented by SEQ ID NO: 5. It can be seen that the IC ₅₀ can be achieved at an extremely low concentration, and the opioid activity is extremely high, as compared with the bovine β-casein-derived opioid peptide to be used.

The longitudinal muscle of the guinea pig ileum used as a measurement system this time contains many opioid receptors, particularly μ receptors involved in analgesia, anesthesia, and sedation. Therefore, from the results in Table 1 above, SEQ ID NO: 2 to SEQ ID NO: 4
It can be seen that the opioid activator composed of the peptide represented by is effective for analgesia, anesthesia, and sedation.

[0027]

EFFECT OF THE INVENTION It is a peptide represented by SEQ ID NO: 1 wherein the carboxyl group of the fourth amino acid Xaa from the N-terminal is amidated or a salt thereof .
In particular, the peptide of the present invention represented by SEQ ID NO: 2 to SEQ ID NO: 4 wherein the fourth amino acid is glycine (Gly), proline (Pro) or valine (Val), or a salt thereof,
Having high opioid activity, the peptide or
Is an agent containing its salt as an active ingredient in a very small dose, which can be used for analgesia, anesthesia, emotion, respiration, pulsation, body temperature, gastrointestinal function, feeding, immunity, regulation of hormones such as insulin and somatostatin, and absorption of electrolytes. It may be used as an analgesic anesthetic, a hypnotic, a gastrointestinal hormonal secretion enhancer, an electrolyte absorption enhancer, and a diarrhea ameliorating agent due to gastrointestinal transport muscle contraction, It is particularly effective as an analgesic and sedative. Since the peptide of the present invention or a salt thereof is a low molecular weight compound having a very simple structure in which four amino acids are arranged, it can be easily produced by chemical synthesis.

[0028]

[Sequence list]

SEQ ID NO: 1 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Characteristic symbol: modified-site Location: 4 Method for determining characteristics: E Other information: Carboxyl group of 4th position Xaa is amidated

SEQ ID NO: 2 Sequence length: 4 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Characteristic symbol: modified-site Location: 4 Method for determining characteristics: E Other information: Carboxyl group of 4th position Gly is amidated

SEQ ID NO: 3 Sequence length: 4 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Characteristic symbol: modified-site Location: 4 Method for determining characteristics: E Other information: Carboxyl group of 4th position Pro is amidated

SEQ ID NO: 4 Sequence length: 4 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence characteristics Characteristic symbol: modified-site Location: 4 Method for determining characteristics: E Other information: Carboxyl group of Val at position 4 is amidated

SEQ ID NO: 5 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

──────────────────────────────────────────────────の Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 5/10-5/20 BIOSIS (DIALOG) CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A peptide represented by SEQ ID NO: 1 (wherein Xaa represents Gly, Pro or Val , and the carboxyl group of the fourth amino acid Xaa from the N-terminus is amidated) or Its salt.