JPH07149793A - New peptide - Google Patents

Abstract

PURPOSE:To obtain a new peptide having a specific amino acid sequence, exhibiting bondability to opioid receptor and mild morphine-like analgesic action and useful as pharmaceuticals such as analgesic anesthetic agent, hypnotic agent, psychostimulant, narcotic antagonist and antishock agent. CONSTITUTION:A new peptide is expressed by formula Tyr-X-Phe-NH2 (X is Leu-Leu or Val-Ser), has bondability to opioid receptor, exhibits mild morphine-like analgesic action, and is useful as pharmaceuticals such as analgesic anesthetic agent, mild hypnotic agent, mild psychostimulant, narcotic antagonist and antishock agent. The peptide can be produced by successively coupling amino acids having protected alpha-amino group and side-chain functional group from the C-terminal side according to the amino acid sequence of the peptide by a solid-phase synthesis using a benzhydrylamine resin, etc., as a carrier and treating the obtained protected peptide resin with hydrogen fluoride, etc., to effect the separation from the resin and the removal of the protecting groups.

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 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. 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
Based on this finding, we succeeded in newly producing the peptide represented by yr-X-Phe-NH ₂ , and found that these novel peptides have opioid activity and can be expected to be used in the above-mentioned pharmaceuticals. The invention was completed.

In the above general formula, X represents Leu-Leu or Val-Ser.

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. It is also possible to mix and administer in a unit dosage form generally required for the implementation of a formulation together with physiologically acceptable vehicles, carriers, excipients, binders, preservatives, stabilizers, flavors, etc. . 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, Leu: leucine, Va.
1: valine, Boc: t-butyloxycarbonyl, B
zl: benzyl, Cl ₂ -Bzl: 2,6- di-chlorobenzyl, 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-Ser-Phe-N with absorption at 0 nm
H ₂ (yield 110 mg) was obtained.

Analytical value Amino acid composition (6N-HCl, 110 ° C., hydrolysis for 24 hours): 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-Leu-Leu-Ph
Synthesis of e-NH ₂ This is from the 102nd position of bovine β-lactoglobulin 1.
It is an amidation product of the peptide corresponding to the 05th sequence.

[Production Example] As in the case of Reference Example, Boc
-Phe-Benzhydrylamine resin with Boc-Le
u, Boc-Leu, Boc- Tyr (Cl 2 -Bz
l) were coupled in this order to give the corresponding tetrapeptide amide resin. Further deprotection with similar hydrogen fluoride in Example 1, and purified by reverse-phase liquid chromatography Tyr-Leu-Leu-Phe- NH 2
(Sample 1, yield 130 mg) was obtained.

Analytical value (under the same conditions as in the reference example) Amino acid composition: Tyr0.97, Leu2.0, Phe1.01,
Elution position of NH ₃ 0.92 ODS column: 35% acetonitrile

Example 2 Tyr-Val-Ser-Ph
Synthesis of e-NH ₂ This is an amidated peptide of the soybean glycinin B _1a subunit corresponding to the 121st to 124th sequences.

[Production Example] Boc is the same as in the reference example.
-Phe-Benzhydrylamine resin Boc-Ser
(Bzl), Boc-Val, Boc-Tyr (Cl ₂
-Bzl) was coupled in this order to give the corresponding tetrapeptide amide resin. Further, in the same manner as in Example 1, deprotection with hydrogen fluoride and purification by reverse phase liquid chromatography were performed to perform Tyr-Val-Ser-Phe-.
NH ₂ (Sample 2, yield 107 mg) was obtained.

Analytical values (same conditions as in the reference example) Amino acid composition: Tyr0.94, Val0.96, Ser0.95, Phe1.0, N
Elution position from H ₃ 0.92 ODS column: 34.5% acetonitrile

Example 3 Measurement of Rat Brain Opioid Receptor Assay (1) Experimental Method The opioid activity of the peptides prepared in the above Examples was determined by the method of Snyder et al. (Proc. Natl. Acad. Sci. USA., 7 ).
0 , 2243 (1973). ). Cerebral brains (1.1-1..g) of male Wistar rats (100-200g).
3 g) was extracted and 10 ml of 50 mM Tris-hydrochloric acid buffer solution (pH 7.4) 0 was extracted using a Potter homogenizer.
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 or morphine hydrochloride (manufactured by Takeda Pharmaceutical Co., Ltd.) was added to 1.7 ml of the obtained solution,
Incubated at 35 ° C for 5 minutes. Then, [ ³ H]
-Naloxone (NEN, 37.7 Cl / mmol) was added to a final concentration of 1 nM (34,000 c.pm) and again incubated at 35 ° C for 15 minutes. Vacuum filtration was carried out using a glass filter (Whatman GF / B, 2.4 cm), the membrane component with the receptor was retained on the filter, and the filter was quickly washed 4 times with 4 ml of buffer solution (owning time 30 seconds ). Put this filter in the measurement vial,
1 ml of 10% sodium dodecyl sulfate (SDS) was added and left for 30 minutes or longer. After that, 10 ml of PSC (Amars
ham) was added, and the mixture was shaken well and measured with a liquid scintillation counter. However, the specific binding amount was obtained by subtracting the binding amount found even in the presence of a large excess of non-radioactive naloxone. The activity of the sample was expressed as the concentration of the sample required to inhibit the specific binding of [ ³ H] -naloxone by 50% (IC ₅₀ ).

[0027]

[Table 1]

Example 4 Guinea pig ileum longitudinal muscle plexus contraction inhibition test (1) Experimental method Basically, the method of Kosterlitz et al. (Br. J. Pharmacol., 3
3 , 266 (1968)). A longitudinal plexus plexus preparation was prepared from the ileum excised with a guinea pig of 300 to 350 g. One end of the sample was connected to an isometric transducer via a thread, and the other was fixed to the bottom of a Magnus tube having an internal volume of 2 ml. Nutrient solution (118mM NaCl, 4.75mM KCl, 11
9 mM KH ₂ PO ₄ , 2.54 mM CaCl ₂ , 1.2
mM MgSO ₄ , 25 mM NaHCO ₃ , 11 mM Gluc
ose) in a Magnus tube and kept at 37 ° C, 95% O ₂
The -5% CO ₂ gas mixture was vented. Electrical stimulation (50 V, 0.1 mse) was applied to the electrodes in the Magnus tube once every 10 seconds.
c) The resulting contraction strength was recorded electronically. Concentration of peptide required to inhibit contraction by 50% (I
C ₅₀₎ were evaluated opiate agonist action determined.

[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.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display area A61K 38/00 ABN A61K 37/18 ABN

Claims (1)

[Claims] 1. A peptide represented by the general formula: Tyr-X-Phe-NH ₂ . (In the formula, X represents Leu-Leu or Val-Ser.)