JPH05247089A - Peptide - Google Patents

Abstract

PURPOSE:To provide a peptide leaving a specific amino acid sequence, exhibiting an opioid activity, and permitting to expect its utilization as an analgestic anaesthetic agent, hypnotic agent, digestive tract hormone secretion-accelerating agent, electrolyte absorption-accelerating agent, diarrhea-improving agent due to gastrointestinal transport contraction, etc. CONSTITUTION:The objective peptide (salt) of the formula (Xaa is linear hydrophobic amino acid, the residue of an aromatic amino acid, etc.,) is obtained by treating a Boc (butoxycarbonyl)-Len-resin as a solid carrier with a solution containing trifluoroacetic acid to remove the Boc group, reacting the remained product with a compound of Boc-Ser (Bzl) (Bzl is benzyl), removing the Boc group from the product, reacting the remained product with a linear hydrophobic amino acid whose a-amino group is protected with a Boc group, with an aromatic amino acid, etc., subsequently with a compound of formula Boc-Pro-OH, further with a compound of formula Boc-Tyr (Cl2-Bzl), and subsequently treating the obtained protected peptide resin with a solution containing hydrogen fluoride.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide having an opioid activity and a salt thereof, and an agent having an opioid activity containing the peptide and a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art It has become widely known that many bioregulatory factors involved in higher-order life activities are present in foods, and these bioregulatory factors are used to promote health and prevent diseases. In recent years, various attempts have been made to positively utilize it for treatment and prevention of aging. As such biological regulators, various factors are known which act on the nervous system, digestive absorption system, circulation system, biological defense / immune system, endocrine system, cell differentiation / proliferation system and the like. Among them, some types of food proteins act on the nervous system to cause morphine-like anesthesia,
It has been revealed that there is a peptide unit having an analgesic effect or the like (so-called opioid activity), and such a peptide is generally called an opioid peptide.

It is known that opioid peptides have an effect on digestive tract and gastrointestinal hormone secretion, and when orally administered a decomposed product of wheat gluten, intestinal retention time of food and insulin in blood are increased. It has been reported to show opioid activity such as an increase in the amount of somatostatin and somatostatin. Based on such findings, the present inventors were able to hydrolyze wheat gluten and to fractionate a novel peptide represented by SEQ ID NO: 2 having opioid activity from the product, and filed a prior application. (Japanese Patent Application No. 3-1718
No. 79).

[0004]

Under the circumstances as described above, the present inventors further researched peptides having opioid activity. Then, in the peptide represented by SEQ ID NO: 2 above, isoleucine (Ile), which is the third amino acid from the N-terminus, was replaced with one amino acid of a linear hydrophobic amino acid and an aromatic amino acid, The resulting novel peptide is particularly effective in enhancing the activity of the mu (mu) receptor, which is known to have higher opioid activity and to be involved in analgesia, anesthesia, and sedation. I found something.

Therefore, the present invention is a peptide represented by SEQ ID NO: 1 and salts thereof (provided that SEQ ID NO: 1
In Xaa represents one amino acid selected from linear hydrophobic amino acids and aromatic amino acids). Furthermore, the present invention also includes an agent having opioid activity, which comprises one or more of the peptide represented by SEQ ID NO: 1 and salts thereof as an active ingredient.

Xa in the peptide represented by SEQ ID NO: 1
As mentioned above, a is one amino acid selected from linear hydrophobic amino acids and aromatic amino acids. Xaa may be any one of a straight-chain hydrophobic amino acid and an aromatic amino acid, and particularly, as the straight-chain hydrophobic amino acid, norvaline (Nva) or norleucine (Nle) is preferable, and as the aromatic amino acid, Tryptophan (Tr
p), phenylalanine (Phe) or tyrosine (Tyr) are preferred.

In the peptide represented by SEQ ID NO: 1,
The peptide of the present invention in which Xaa is norvaline is SEQ ID NO: 3.
And a peptide of the present invention in which Xaa is norleucine is represented by SEQ ID NO: 4. Norvaline, which is one of the amino acids constituting the peptide represented by SEQ ID NO: 3, is
As shown in the following chemical formula, valine has a side chain methyl group linearly bonded and can be produced by chemical synthesis.

[0008]

[Chemical 1]

Norleucine, which is one of the constituent amino acids of the peptide represented by SEQ ID NO: 4, is a leucine or isoleucine side chain methyl group that is linearly linked as shown in the following chemical formula. , Can be manufactured by chemical synthesis.

[0010]

[Chemical 2]

Further, in the peptide represented by SEQ ID NO: 1, the peptide of the present invention in which Xaa is tryptophan is represented by SEQ ID NO: 5, and the peptide of the present invention in which Xaa is phenylalanine is represented by SEQ ID NO: 6, and The peptide of the present invention in which Xaa is tyrosine is represented by SEQ ID NO: 7.

In the peptide of the present invention included in SEQ ID NO: 1, the five amino acids constituting the peptide are L-
Either an amino acid or a D-amino acid may be used. For example, all five amino acids may consist of L-amino acids, or D-amino acids only,
Alternatively, any of the five amino acids may be an L-amino acid and the rest may be D-amino acids. Both peptides can be easily produced by chemical synthesis.

Further, the "agent having opioid activity" in the present invention means analgesia, anesthesia, sedation, emotion, respiration, pulsation, body temperature, digestive tract function, feeding, immunity, secretion of hormones such as insulin and somatostatin. , Refers to agents having activity recognized as so-called opioid activity involved in the promotion of electrolyte absorption, regulation of myocardial contraction, etc. Particularly, the agent having opioid activity of the present invention is effective for analgesia, anesthesia, and sedation as described above. Μ commonly known to be involved
It has the activity of enhancing the activity of the receptor.

The peptide of the present invention and its salt may be produced by any method as long as it has the amino acid sequence of SEQ ID NO: 1. An example of the method for producing the peptide of the present invention will be described below by taking the peptide of SEQ ID NO: 3 in which Xaa is norvaline as an example.

Of the peptide of the present invention represented by SEQ ID NO: 3
Production method Synthesis is performed using a SAM TWO type automatic peptide synthesizer manufactured by US Biosearch Co., Ltd. according to the standard protocol of the same. That is, Boc (butoxycarbonyl)-
After treating the Leu-resin with a deblocking solution containing 45% trifluoroacetic acid, Boc-Ser (Bzl) is coupled in the presence of diisopropylcarbodiimide. Hereinafter, after deblocking as described above, Boc-Nva-OH, Bo
c-Pro-OH and Boc-Tyr (Cl ₂ -Bzl) and was successively _{coupling, Boc-Tyr (Cl 2 -Bzl} ) -Pro-Nva-Ser (Bzl)
-Leu-resin is obtained. The 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 is washed with ether and the peptide is extracted with 30% acetic acid. Then, the pH of the extract was adjusted to 7 to 8 with NaOH, stirred at room temperature for 1 hour, and the crude peptide obtained by extraction was purified using high performance liquid chromatography with an ODS column to give SEQ ID NO: 3. A peptide represented by The peptides represented by SEQ ID NO: 4 to SEQ ID NO: 7 can also be obtained by synthesizing and purifying by the same procedure.

The peptides of the present invention included in SEQ ID NO: 1, particularly the peptides of the present invention represented by SEQ ID NO: 3 to SEQ ID NO: 7 and salts thereof have opioid activity. The agent containing the peptide and its salt as an active ingredient is, for example, an analgesic anesthetic,
It can be administered to humans and various animals as a hypnotic agent, a gastrointestinal hormone promoter, an electrolyte absorption promoter, a diarrhea-improving agent by suppressing intestinal peristalsis, etc., and is particularly effective as a painkiller or a sedative anesthetic. In that case, the peptide of the present invention encompassed by SEQ ID NO: 1 and a salt thereof may be used alone or in the form of a mixture containing two or more kinds of peptides. Suitable dose of the agent having opioid activity of the present invention, the age of the human or animal to be administered, body weight,
It depends on various conditions such as sex, symptoms, and animal type.

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 carrier or liquid carrier usually used in the pharmaceutical industry. They may be administered together or they may be mixed or combined with other agents. Further, the administration form can be any form such as tablets, pills, granules, capsules, powders, aqueous solutions, injections and the like. Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto.

[0018]

【Example】

<< Example 1 >> The peptide represented by SEQ ID NO: 3 was synthesized by the following method. Commercially available Boc (Butoxycarbonyl)
-Leu-resin (substitution rate 0.64 meq / g) 0.16 g and 4
20 ml of methylene chloride containing 5% trifluoroacetic acid and 2.5% anisole was reacted at room temperature for 25 minutes in a reaction tank of a peptide synthesizer SAM TWO manufactured by Biosearch to remove the Boc group. Next, Leu- with the Boc group removed
The resin was washed with methylene chloride, then neutralized with methylene chloride containing 10% diisopropylethylamine, and then further washed with methylene chloride. 0.4M on this resin
5 ml of Boc-Ser (Bzl) in dimethylformamide,
5 ml of a 0.4M solution of diisopropylcarbodiimide in methylene chloride was mixed and put in a reaction tank, and the reaction was carried out at room temperature for 2 hours with stirring.

The resin obtained above was washed successively with dimethylformamide, methylene chloride and methylene chloride containing 10% of diisopropylethylamine, methylene chloride and a methylene chloride / dimethylformamide mixture.
Boc-Ser (Bzl) -Leu-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 above to repeat Boc-Tyr (Cl ₂ -Bzl) -Nva-Ser (Bzl) -Leu-
A product consisting of resin was obtained.

The resin was placed in 20 ml of hydrogen fluoride containing 10% anisole and stirred at 0 ° C. for 2 hours to release the peptide from the resin. After that, hydrogen fluoride was distilled off under reduced pressure, the residue was extracted with 30% acetic acid, and the extract was added with NaOH.
After adjusting H to 7 to 8, the mixture was stirred for 1 hour, and this extract was mixed with octadecylsilane (OD) manufactured by Nacalai Tesque, Inc.
S) Purification by reverse phase chromatography using a column ODS-Cosmosil 5C ₁₈ -AR gave 20 mg of the final product. Its amino acid composition (molar ratio) is Ty
The ratio was r: Pro: Nva: Ser: Leu = 1: 1: 1: 1: 1. Further analysis by a protein sequencer Model 477-A manufactured by Applied Systems Inc. revealed that it was a peptide having the amino acid sequence represented by SEQ ID NO: 3. Rf of the product obtained by thin layer chromatography
The value was 0.72.

Example 2 In the case of the peptide represented by SEQ ID NO: 4, the commercially available Boc-Leu-resin (substitution rate 0.64 meq) was carried out in the same manner as in the case of the peptide represented by SEQ ID NO: 3.
/ G) 0.16 g to give 15 mg of final product. Its amino acid composition (molar ratio) is Tyr: Pro: Nle: Ser: Leu.
= 1: 1: 1: 1: 1 and analyzed by the protein sequencer used above revealed a peptide having the amino acid sequence represented by SEQ ID NO: 4. The Rf value of this product by thin layer chromatography was 0.74.

Example 3 In the case of the peptide represented by SEQ ID NO: 5, the commercially available Boc-Leu-resin (substitution rate 0.64 meq) was used in the same manner as in the case of the peptide represented by SEQ ID NO: 3.
/ G) 0.16 g to give 25 mg of final product. Its amino acid composition (molar ratio) is Tyr: Pro: Trp: Ser: Leu.
= 1: 1: 1: 1: 1 and analyzed by the protein sequencer used above revealed a peptide having the amino acid sequence represented by SEQ ID NO: 5. The Rf value of this product by thin layer chromatography was 0.73.

Example 4 In the case of the peptide represented by SEQ ID NO: 6, the commercially available Boc-Leu-resin (substitution rate 0.64 meq) was carried out similarly to the case of the peptide represented by SEQ ID NO: 3.
/ G) 0.16 g to give 25 mg of final product. Its amino acid composition (molar ratio) is Tyr: Pro: Phe: Ser: Leu.
= 1: 1: 1: 1: 1 and analyzed by the protein sequencer used above revealed a peptide having the amino acid sequence represented by SEQ ID NO: 6. The Rf value of this product by thin layer chromatography was 0.77.

SEQ ID NO: 3 obtained in Examples 1 to 4 above
~ Peptide represented by SEQ ID NO: 6, and SEQ ID NO: 2
The opioid activity of each of the peptides was measured as follows.

Method for measuring opioid activity 0.2 g of ileal longitudinal muscle isolated from a male guinea pig weighing 250 to 300 g was connected to an isotonic transducer (TB612-T manufactured by Nihon Kohden Kogyo Co., Ltd.).
Apply tension. A Krebs isotonic solution (NaCl 118 mM, KCl) was placed in a 2 ml-volume Magnus tube kept at 36 ° C.
4.75 mM, CaCl ₂ 2.54 mM, MgSO ₄ 1.
2 mM, NaHCO ₃ 25 mM, KH ₂ PO ₄ 1.19 m
M and glucose 11 mM) were soaked in the ileal longitudinal muscle, and aerated (0 ₂ 95%: CO ₂ 5)
%). After stabilizing for about 2 hours in this way, electrical stimulation of 0.5 msec was applied at 30 V to electrically contract the longitudinal muscle of the ileum of the guinea pig. Inhibitor 5 including aminopeptidase inhibitor etc. after electric contraction is stabilized
0 μl was added, the peptides represented by SEQ ID NO: 2 to SEQ ID NO: 6 were added, and changes in electrical contraction of guinea pig ileal longitudinal muscle were recorded with a recorder. Opioid activity is suppressed by electrical contraction after addition of peptide and 20 μl of 10 ⁻⁴ M naloxone
It was judged by the release of suppression by addition. And the concentration that suppresses the electrical contraction of longitudinal muscle of guinea pig ileum by 50% (IC
₅₀ ) was measured.

As a result, the IC ₅₀ of the peptide of the present invention represented by SEQ ID NO: 3 to SEQ ID NO: 6 obtained in the above Examples was as shown in Table 1 below. For reference, the IC ₅₀ when the opioid activity of the peptide represented by SEQ ID NO: 2 is measured in the same manner as above is also shown in Table 1.

[0027]

Table 1 [Opioid activity of peptides (IC ₅₀ )] Peptide IC ₅₀ (μM) Peptide of SEQ ID NO: 3 (present invention) 32 Peptide of SEQ ID NO: 4 (present invention) 25 Peptide of SEQ ID NO: 5 (present Invention) 15 Peptide of SEQ ID NO: 6 (Invention) 32 Peptide of SEQ ID NO: 2 40

From the results of Table 1, SEQ ID NO: 3 to SEQ ID NO: 6
It can be seen that the peptide of the present invention can achieve an IC ₅₀ at a lower concentration than the peptide obtained from wheat gluten represented by SEQ ID NO: 2.

In the longitudinal muscle of the ileum of the guinea pig used as the measurement system this time, there are many μ receptors that are involved in analgesia, anesthesia, and sedation as opioid receptors. Therefore, from the results of Table 1 above, SEQ ID NO: 3 to SEQ ID NO: 6
It can be seen that the opioid activator of the present invention comprising the peptide represented by is particularly effective for enhancing the activity of the μ receptor involved in analgesia, anesthesia, and sedation.

On the other hand, the mouse vas deferens, which has been widely used as a conventional measuring system, has many δ (delta) receptors, and it is known that these δ receptors are particularly involved in emotions. Then, when IC ₅₀ was measured using a mouse vas deferens in the same manner as above, it was 14 μM for the peptide represented by SEQ ID NO: 2, whereas the IC ₅₀ was compared with SEQ ID NO: 3
And 15 to 2 for the peptide represented by SEQ ID NO: 4
0 μM, and the peptides represented by SEQ ID NO: 5 and SEQ ID NO: 6 are both 40 μM. From this point as well, the peptides of the present invention represented by SEQ ID NO: 3 to SEQ ID NO: 6 have the activity of enhancing the activity of the aforementioned μ receptor. It turns out that is large.
Incidentally, the conventionally known opioid peptide, α-casein exorphin, has an IC ₅₀ of 70 μM when using mouse vas deferens, and has a lower opioid activity than any of the peptides of SEQ ID NO: 2 to SEQ ID NO: 6.

[0031]

The peptide of the present invention encompassed by SEQ ID NO: 1, particularly the peptide of the present invention represented by SEQ ID NO: 3 to SEQ ID NO: 7 and salts thereof have opioid activity. Agents containing peptides and their salts as active ingredients can be administered in extremely small amounts to reduce pain, anesthesia, emotion, respiration, pulsation, body temperature, gastrointestinal function, food intake, immunity, secretion of hormones such as insulin and somatostatin, and electrolytes. Since it may have a function of promoting absorption and regulating contraction of myocardium, it is an analgesic anesthetic, hypnotic, gastrointestinal hormone secretagogue,
It can be expected to be used as an electrolyte absorption enhancer and a diarrhea improver by gastrointestinal transport muscle contraction, and is particularly effective as an analgesic anesthetic and a sedative. Since the peptide of the present invention and its salt are low molecular weight compounds having an extremely simple structure in which only 5 amino acids are arranged, they can be easily produced by chemical synthesis.

[0032]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 2 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 4 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 5 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 6 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Topology: Linear Sequence type: Peptide

Claims (2)

[Claims] 1. A peptide represented by SEQ ID NO: 1 and a salt thereof (provided that in the peptide represented by SEQ ID NO: 1, Xaa represents one amino acid selected from a linear hydrophobic amino acid and an aromatic amino acid). 2. A peptide represented by SEQ ID NO: 1 and a salt thereof (provided that in the peptide represented by SEQ ID NO: 1,
Xaa is an agent having opioid activity containing one or more kinds of linear amino acids selected from a hydrophobic amino acid and an aromatic amino acid) as an active ingredient.