JP2513439B2 - New peptide - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel peptide having a binding property to an opioid receptor and therefore expected as an analgesic anesthetic, a mild hypnotic agent, a mild stimulant, an anti-anesthetic agent or an anti-shock agent. .

[0002]

2. Description of the Related Art Analgesic anesthetics (opiates) such as morphine
From the study of the mechanism of action, it was found that there are opioid receptors to which these substances specifically bind, in various organs including the brain. Furthermore, it has been found that there are peptides that bind to this receptor and have an analgesic effect in the animal body, and are collectively referred to as endogenous opioid peptides.
It has been shown that these peptides are involved not only in the analgesic action but also in the regulation of secretion of various hormones and the regulation of feeding.

On the other hand, a substance which has an affinity for an opiate receptor but which does not exhibit an analgesic action by itself and interferes with the action of an analgesic anesthetic such as morphine is called an opiate antagonist, and as such a substance, synthesis of naloxone and the like is possible. There is a compound. Naloxone and naltrexone are known to have an effect of improving shock symptoms due to various causes and an effect of promoting brain development. Recently, it was found that an opiate antagonist is present in coffee for the first time in the natural world, and it has been pointed out that this substance may be involved in the afferent action of coffee together with caffeine. That is, the substance having a binding property to the opiate receptor is an opioid or an opiate antagonist,
They are useful substances having the above-mentioned physiological effects.

In 1979, Teschmache
r) et al., a dairy product was searched by a guinea pig ileal longitudinal plexus contraction inhibition test, and β-casomorphin (heptapeptide) having opioid activity was isolated from milk casein peptone (Hoppe-Seyler's., Z. Physiol). . Ch
See em., 360, 1211, and 1217 (1979)). Then, the structure of β-casomorphin 7 (heptapeptide) was studied, and β-casomorphin 6, β-casomorphin 5 and β-casomorphin 4 amide (having about 100 times the activity of β-casomorphin 7). Have been synthesized (Chang, KJ et al., Science 212, 75 (1981);
Life Science., 30 1547 (1982)). Also, Geodrow
(Zioudrou) et al. Confirmed the presence of opioid-active pepsin degradation products of milk α-casein. This is called α-casein exorphin. (J. Biol. Ch
em., 254, 2446 (1979)).

If a similar peptide were produced from human milk casein, it would be desirable for humans, especially infants, to ingest it. It has also been shown that there is a possibility that an exogenous peptide having opioid activity may be present in other food proteins such as wheat gluten and soybean protein.

The present inventors isolated or synthetically obtained a fragment peptide containing a tyrosine residue present in human milk protein, cow milk protein and soybean protein, and screened for opioid activity. Was discovered, and the present invention was completed.

[0007]

The development and production of novel peptides that can be used as medicines such as analgesics, hypnotics, stimulants, antianesthetics or antishock agents are expected.

[0008]

SUMMARY OF THE INVENTION We have the general formula T
We have succeeded in newly producing the peptide represented by yr-Gly-X, found that these novel peptides have opioid activity, and can be expected to be used in the above-mentioned pharmaceuticals, and completed the present invention based on this discovery. Came to do.

In the above general formula, X is Phe-Leu-P.
represents ro or Leu-Phe-NH ₂ .

The novel peptide of the present invention may be obtained by, for example, extracting and separating a peptide having a binding property to rat brain opioid receptor from an enzymatic degradation product of a protein by an activity test by a receptor assay method by a liquid chromatography operation as described later. . Alternatively, based on these, it is also possible to chemically bond the constituent amino acids by sequentially linking the constituent amino acids using a conventionally-used peptide synthesis method. The amino acid that constitutes the novel peptide of the present invention may be either L-form or D-form. The novel peptide of the present invention is produced based on the examples described below and by utilizing a conventional peptide synthesis method (see, for example, Izumiya et al., Synthetic Chemistry Series "Peptide Synthesis" published by Maruzen Co., Ltd., 1975). be able to. The same applies to the method of protecting with a protecting group or the method of removing it. Among the novel peptides of the present invention, the amide derivative is a conventional method, for example, a method in which the amide is used instead of the amino acid serving as the C-terminal to similarly perform peptide synthesis, or a corresponding peptide ester is treated with ammonia to amidate Can be manufactured by.

When the novel peptide of the present invention is used as an active ingredient as an analgesic, hypnotic or stimulant, anti-anesthetic, anti-shock agent, a non-toxic salt or acid addition salt in free form or pharmaceutically acceptable. Can be In the present invention, pharmaceutically acceptable non-toxic salts include commonly used organic and inorganic acid addition salts such as hydrochloric acid,
An addition salt of sulfuric acid, sulfonic acid, citric acid, phosphoric acid or benzoic acid may be used. On the other hand, it also includes alkali metal salts such as Na and K and ammonium salts.

The novel peptide of the present invention is effective as an analgesic or hypnotic agent for mammals including humans, and only eliminates various pains such as gallstone colic, renal stone colic, cancer pain, and postoperative pain. It is also effective as a hypnotic drug due to its hypnotic action. On the other hand, an opiate antagonist is effective as a drug used for releasing anesthesia and improving various shock symptoms.

For administration, it may be formulated as a tablet, capsule or elixir for oral administration or as a sterile solvent solution or suspension for parenteral administration. In addition, it is of course possible to mix and administer it in a unit dosage form generally required for the implementation of the formulation together with physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavoring agents and the like. . The amount of active substance used in these compositions or preparations is such that a suitable dosage in the indicated range is obtained. The dose of the active ingredient is determined by considering the severity of disease, weight and age of the patient or other factors.

The abbreviations used in this specification are as follows.
Tyr: Tyrosine, Pro: Proline, Phe: Phenylalanine, Ser: Serine, Gly: Glycine, Le
u: leucine, Boc: t-butyloxycarbonyl,
Bzl: benzyl, Cl ₂ -Bzl: 2,6-dichlorobenzyl, TFA: trifluoroacetic acid, ODS: octadecylsilane, HOBt: 1-hydroxybenzotriazole, Tos: tosyl, DCCD: dicyclohexylcarbodiimide.

[0015]

The present invention will be described in detail below with reference to examples.

Reference Example Tyr-Pro-Ser-Phe
Synthesis of -NH ₂ which is an amide compound of the peptides corresponding to 44 of SEQ from 41 th human β- casein.

Production Example In 5 g of benzhydrylamine resin (0.89 mmole / g) washed with dimethylformamide, Boc-Phe, HOBt and DCCD corresponding to 3 equivalents of the amino groups of the resin were dissolved in a small amount of dimethylamide. After that, they were added in this order and reacted at room temperature overnight. After the reaction, the resin was washed with dimethylformamide, methylene chloride, ethanol and methanol three times each. Unreacted amino groups contained in the resin were acetylated by shaking the resin twice in 50 ml of pyridine containing 10% acetic anhydride for 5 minutes each, and after reaction, the resin was treated with 50 ml of ethanol and 50 ml of methanol, respectively. It was washed twice to obtain Boc-Phe benzhydrylamine resin. 2.3 g of Boc-Phe
-After washing benzhydrylamine resin (corresponding to 2 g of benzhydrylamine resin) four times with methylene chloride, 55
De-B by shaking in methylene chloride containing 10% TFA, 10% anisole for 2 minutes and 30 minutes.
oc was performed. Resin is methylene chloride, methylene chloride containing 33% dioxane, and methylene chloride at 3 each
After washing twice, the mixture was shaken in methylene chloride containing 10% triethylamine for 5 minutes for neutralization, and washed three times with methylene chloride to obtain a Phe-benzhydrylamine resin. After the resin was washed with dimethylformamide three times, 3 equivalents of Boc-Ser (Bzl), HOBt, and DCCD dissolved in a small amount of dimethylformamide were added in this order, and the mixture was shaken at room temperature for 5 hours and cupped. A ring reaction was performed. After the reaction, the resin is dimethylformamide,
Each was washed with methylene chloride, ethanol, and methanol three times to obtain Boc-Ser (Bzl) -Phe-benzhydrylamine resin. After confirming that there is no unreacted amino group by ninhydrin reaction, the same procedure as in Boc-Pr was performed.
o and Boc-Tyr (Cl ₂ -Bzl) in this order to couple Boc-Tyr (Cl ₂ -Bzl) -P
A ro-Ser (Bzl) -Phe-benzhydrylamine resin was obtained.

Half amount of the tetrapeptide amide resin thus obtained was taken, 1 ml of anisole was added, and then 25
The mixture was stirred in 0 ml of hydrogen fluoride at 0 ° C. for 1 hour to remove the peptide from the resin and remove the protecting group. After removing the hydrogen fluoride, the peptide and the resin were washed with ether, and the peptide was extracted with 5 ml of TFA. The peptide was precipitated by adding 50 ml of ether to this extract, collected by centrifugation, dissolved in 5 ml of water, adjusted to pH 8.0 with aqueous ammonia, and stirred at room temperature for 1 hour. , N that may have occurred at the serine residue
→ The O-acyl rearrangement was restored to the peptide bond. The crude tetrapeptide amide thus obtained was purified by reverse phase liquid chromatography using an ODS column (“Cosmosil ₅ C ₁₈ ”, 1 × 25 cm, manufactured by Hanai Chemical Co., Ltd.).
Tyr-Pro-Scr-Phe-N with absorption at 0 nm
H ₂ (yield 110 mg) was obtained.

Analytical value Amino acid composition (6N-HCl, 110 ° C., 24 hours hydrolysis): Tyr0.95, Pro1.0, Ser0.94, Phe0.98, N
H ₃ 0.89. ODS column (“Cosmosil ₅ C ₁₈ ”, 0.46 × 15 cm
Elution conditions from Hanai Chemical Co., Ltd .: Elution with 27% acetonitrile was performed using an acetonitrile linear gradient (0 to 40% / 40 ml / 40 min) containing 0.1% TFA.

Example 1 Tyr-Gly-Phe-Le
Synthesis of u-Pro This is a peptide corresponding to the 59th to 63rd sequences of human β-casein.

[Production Example] 12 ml of 2 g of Boc-Pro
Ethanol (4 ml) and water (4 ml) were added and dissolved, and the mixture was neutralized with an aqueous cesium bicarbonate solution and dried to give a cesium salt. To this was added 8.9 g of chloromethyl resin (1.2
(8 mmole Cl / g) and 60 ml of dimethylformamide are added, and the mixture is stirred overnight at 50 ° C., and the resin is dimethylformamide, 90% dimethylformamide, 300 ml.
Was sequentially washed with dimethylformamide and ethanol to obtain Boc-Pro resin (0.60 mmol / g).
This resin was sequentially processed with Boc-L according to the same method as in Reference Example.
eu, Boc-Phe, Boc-Gly, Boc-Ty
The corresponding pentapeptide resin was obtained by coupling r (Cl ₂ -Bzl). This peptide resin was deprotected and purified in the same manner as in Reference Example to obtain Tyr-Gly-Phe-.
Leu-Pro (Sample 1, yield 120 mg / g resin) was obtained.

Analytical values (hydrolysis and elution conditions are the same as in the reference example) Amino acid composition: Tyr0.95, Gly0.99, Phe0.98, Leu0.96, P
Elution position from ro1.0 ODS column: 35% acetonitrile

Example 2 Tyr-Gly-Leu-Ph
Synthesis of e-NH ₂ This is 50% of human or bovine α-lactalbumin.
It is an amidation product of the peptide corresponding to the 53rd position.

[Production Example] Boc is the same as in the reference example.
-Phe-Benzhydrylamine resin with Boc-Le
u, Boc-Gly and Boc-Tyr (Cl ₂ -B
zl) was coupled in this order to give the corresponding tetrapeptide amide resin. Further deprotection with similar hydrogen fluoride when the reference example, Tyr-Gly-Leu-Phe -NH 2 was purified by reverse phase liquid chromatography
(Sample 2, yield 125 mg) was obtained.

Analytical values (same conditions as in the reference example) Amino acid composition: Tyr0.96, Gly1.03, Leu0.98, Phe1.0, N
Elution position from H ₃ 0.94 ODS column: 33.5% acetonitrile

Example 3 Rat Brain Opioid Receptor
Assay measurement (1) Experimental method The opioid activity of the peptides prepared in the above examples was measured.
The method of Snyder et al. (Proc.Natl.Acad.Sci.USA.,7
0, 2243 (1973). ). Male wista
Cerebrum (1.1-1 ..
3g) and remove it using a Potter homogenizer
10 ml of 50 mM Tris-HCl buffer (pH 7.4) 0
Homogenized at ℃. This is the same buffer solution with 1 of brain weight
After diluting to 00 times, centrifuge (1,000 rpm, 5 minutes, 0
(° C) to remove the precipitate. A sample is added to 1.7 ml of the obtained solution.
Or add morphine hydrochloride (Takeda Pharmaceutical Co., Ltd.),
Incubated at 35 ° C for 5 minutes. continue,〔³H]
-Final concentration with naloxone (NEN, 37.7Cl / mmol)
Add 1nM (34,000c.p.m.) So that
Incubated at 35 ° C for 15 minutes. Glass filter
(Whatman GF / B, 2.4 cm)
Keep the membrane components with scepters on the filter and
Quickly wash the filter 4 times with 4 ml of buffer (at the time of possession
30 seconds). Put this filter in the measurement vial,
Add 1 ml of 10% sodium dodecyl sulfate (SDS)
Left for 30 minutes or more. After that, 10 ml of PSC (Amars
ham) and shake well to obtain liquid scintillation.
It was measured with a counter. However, a large excess of non-radioactive
Subtracting the amount of binding that can be seen even in the presence of Roxon
It was defined as the amount of specific binding. The activity of the sample is [³H] -Naloxo
Concentration of sample required to inhibit 50% specific binding
(IC₅₀).

[0027]

[Table 1]

EXAMPLE 4 Guinea pig ileum longitudinal muscle plexus yield
Shrinkage suppression test (1) Experimental method Basically, the method of Kosterlitz et al. (Br. J. Pharmacol.,3
3, 266 (1968)). 300-350g model
Prepare longitudinal muscle plexus specimens from the ileum excised with lumot
Made. One end of the specimen goes through the thread and isometric trans
Connected to a juicer, the other is the bottom of a Magnus tube with an internal volume of 2 ml.
Fixed to. Nutrient solution (118mM NaCl, 4.75mM KCl, 11
9 mM KH₂PO_Four, 2.54mM CaCl₂, 1.2
mM MgSO_Four, 25 mM NaHCO₃, 11mM Gluc
ose) in a Magnus tube, keep it at 37 ℃,₂
-5% CO₂The mixed gas was aerated. Electrodes in Magnus tube
Electrical stimulation once every 10 seconds (50 V, 0.1 mse
c) The resulting contraction strength was recorded electronically. Income
The concentration of the peptide (I
C₅₀) Was evaluated and the opiate agonistic effect was evaluated.

[0029]

[Table 2]

[0030]

As is clear from the above description, the novel peptide of the present invention has a mild morphine-like analgesic activity and can be expected as a drug.

Claims (1)

(57) [Claims] 1. A peptide represented by the general formula: Tyr-Gly-X. (In the formula, X is Phe-Leu-
Represents Pro or Leu-Phe-NH ₂ . )