JPH0613553B2 - New peptide - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel peptide which can be expected to be used as a medicine such as an analgesic agent and a hypnotic agent.

2. Description of the Related Art β-casomorphin, a peptide having opioid activity (morphine-like activity), has been isolated from milk casein peptone (Hoppe-Seyler's Z.Physiol.Chem., 360 , 1211).
And 1217, 1979), however, there is no report from human milk β-casein regarding peptides having opioid activity as in the present invention.

Problems to be Solved by the Invention Development and production of peptides that can be used as medicines such as analgesics and hypnotics are expected.

Means for Solving the Problems The present inventors have found that a peptide represented by the general formula Tyr-Pro-Phe-Val-Glu- (R) n,
We succeeded in synthesizing the amide and ester derivatives, and found that these novel peptides have opioid activity and can be expected to be used in the above-mentioned pharmaceuticals, and based on this discovery, the present invention was completed. It was

The amino acids constituting the novel peptide of the present invention are L-form, D
-Any body.

The novel peptide of the present invention is based on the examples described below, and further utilizing a conventional peptide synthesis method (for example, Izumiya et al., Synthetic Chemistry Series "Peptide Synthesis" published by Maruzen Co., Ltd., Showa
See 50 years. ) Can be manufactured. 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 prepared by a conventional method, for example, a method in which the amide is used instead of the amino acid serving as the C-terminal, or a corresponding peptide ester is treated with ammonia to amidate. It can be manufactured.

When the novel peptide of the present invention is used as a sedative or hypnotic as an active ingredient, it can be in a free form or a pharmaceutically acceptable non-toxic salt and acid addition salt.

In the present invention, the pharmaceutically acceptable non-toxic salt may be a commonly used organic or inorganic acid addition salt such as hydrochloric acid, sulfuric acid, sulfonic acid, citric acid, phosphoric acid or benzoic acid. Good. On the other hand, alkali metal salts such as Na and K and ammonium salts are included.

The novel peptide of the present invention is effective as a sedative or hypnotic agent for mammals including humans, for example, gallstone colic, renal stone colic, pain such as cancer, not only removal of various pains such as pain in the postoperative period, Due to its hypnotic effect, it is also effective as a hypnotic drug.

Upon administration, it may be formulated as a tablet, capsule solution or elixir for oral administration, or as a sterile solvent solution or suspension for parenteral injection or suppository.

In addition, physiologically acceptable vehicles, carriers, excipients,
It is needless to say that they can be mixed and administered together with a binder, a preservative, a stabilizer, a flavoring agent and the like in a unit dosage form generally required for the implementation of the preparation. 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.

Abbreviations in the present specification are as follows. Tyr: Tyrosine, Pro: Proline, Phe: Phenylalanine, Val: Valine, Glu: Glutamic acid, Ile: Isoleucine, Boc:
t- butyloxycarbonyl, OBzl: benzyloxy, _Cl 2 -Bzl: 2,6- dichlorobenzene benzine, TFA: trifluoroacetic acid, ODS: octadecylsilane, HEPES: N-2- hydroxyethylpiperazine--N'-2- Ethanesulfonic Acid Examples The present invention will be described in detail below with reference to Examples.

Example 1 Tyr-Pro-Phe-Val-Glu-Pro and Tyr-Pro-Phe-V
Synthesis of al-Glu-Pro-Ile-Pro 1. Introduction of Boc-L-Pro into chloromethyl resin After dissolving 2 g of Boc-L-Pro by adding 12 ml of ethanol and 4 ml of water, the mixture was neutralized with an aqueous solution of cesium bicarbonate and dried to obtain a cesium salt. . This was dissolved in 65 ml of dimethylformamide, 7.2 g of chloromethyl polystyrene resin (1.28 meq. Gl / g) was added, and the mixture was stirred at 50 ° C. overnight. After the reaction was completed, add 300 ml of dimethylformamide to the resin,
Wash sequentially with 00 ml 90% dimethylformamide, 300 ml dimethylformamide and 600 ml ethanol, B
An oc-L-Pro-resin (0.86 mmol Pro / g) was obtained.

2. Extension of peptide chain on Boc-L-Pro resin 1.6 g of the above Boc-L-Pro resin was added to 40 ml of the de-Boc agent (55% T).
FA, 5% anisole, 40% methylene chloride mixture, each v /
(v%) at room temperature for 30 minutes, remove the Boc group, wash with 60 ml of 33% dioxane, then 67% methylene chloride mixture, and neutralize with 20 ml of 10% triethylamine 90% methylene chloride mixture. , Washed with 100 ml of methylene chloride. 13.8 mmol of dicyclohexylcarbodiimide corresponding to 10 times equivalent of Pro resin was added to 13.8 mmol of Boc-L-Ile.
It was dissolved in methylene chloride (ml), added to Pro resin and stirred overnight at room temperature.

The reaction-completed resin was washed with methylene chloride, and it was confirmed that there were no unreacted amino groups in the nihydrin reaction. Thereafter, similarly, Boc-L-Pro, Boc-L-Glu (OBzl), Boc-L-Va
l, Boc-L-Phe, Boc-L-Pro, Boc-L-Tyr (Cl 2 -
Bzl) was attached in this order to give the octapeptide-resin.

In addition, Boc-L-Glu (OBz
l), Boc-L-Val, Boc-L-Phe, Boc-L-Pro Boc
-L-Tyr and _(Cl 2 -Bzl) were combined in this order, to obtain a hexapeptide resin.

3. Cleavage of peptide from resin and removal of protecting groups To the above octapeptide resin, 1.1 ml of m-cresol, 10 ml of 1M trifluoromethanesulfonic acid, and 1M thioanisole in TFA were added to 1.25 g, and the mixture was added at 0 ° C. for 30 minutes, then. After reacting at room temperature for 2 hours, 190 ml of ether was added and the mixture was centrifuged. The precipitate obtained was subjected to the same operations as above and then suction-filtered to obtain a filtrate. 190 ml to this
Ether was added to precipitate and collect the peptide, which was dissolved in water, neutralized with Animonia, and then lyophilized. The same treatment was performed for hexapeptide.

4. Purification of Peptide The peptide obtained as described above was subjected to gel filtration with a Biogel P-2 column equilibrated with 0.1 M ammonia acetate to obtain a fraction which was eluted the earliest. This fraction was subjected to reverse phase chromatography on an ODS column (Cosmosil 5 C ₁₈ ) and eluted with an acetonitrile gradient containing 0.1% TFA. In both octapeptide and hexapeptide, one main peak having an absorption at 280 nm was obtained and eluted with 29% and 26.5% acetonitrile, respectively. When these peaks were dried by concentrating centrifugation and amino acid analysis was performed, the predicted amino acid compositions were shown. Yield: Octapeptide 300mg, Hexapeptide 2
50 mg.

5. Preparation of Pentapeptide by Treatment of Hexapeptide with Carboxypeptidase 50 mg of hexapeptide obtained as described above was added to 10 ml of 100 mM HE
Dissolve in PES buffer (pH 7.0) and add 0.5 mg of carboxypeptidase Y (130 unit / mg, manufactured by Oriental Yeast Co., Ltd.), stop the reaction at 100 ° C after 5 minutes, and OD the reaction solution.
It was applied to an S column and developed with an acetonitrile gradient containing 5 mM phosphate buffer (pH 7.0). The peak eluted with 24% acetonitrile is Tyr: Pro: Phe: Val:
The target pentapeptide had an amino acid composition of Clu = 1: 1: 1: 1: 1.

6. Physicochemical properties Tyr-Pro-Phe-Val-Glu Specific irradiance [α] _D = -49.3 ° (C = 0.3, methanol)
UVλmax = 277.5nm, Amino acid composition (6N HCl, hydrolyzed at 110 ° C. for 24 hours): Glu (1) 0.98, Pro (2) 2.04, Val (1) 1.00, Tyr (1) 0.97, Phe (1) 1.03 ODS column (Cosmosil 5 C ₁₈ , Elution from 4.6 × 150 mm, manufactured by Hanai Chemical Co., Ltd .: 0-50% acetonitrile containing 0.1% TFA Linear gradient / eluted with 29% acetonitrile in 50 ml Tyr-Pro-Phe-Val-Glu-Pro Specific irradiance [α ] _D = −64.0 ° (C = 0.3, methanol)
UVλmax = 277.5nm, Amino acid composition (hydrolysis under the same conditions as above) Clu (1) 0.99, Pro (2) 2.02, Val (1) 1.00, Tyr (1) 0.99, Phe (1) 1.02 Elution from ODS column: Under the same conditions as above Elution with 31% acetonitrile in Tyr-Pro-Phe-Val-Glu-Pro-Ile-Pro Specific irradiance [α] _D = -72.1 ° (C = 0.3, methanol)
UVλmax = 277.5nm, Amino acid composition (hydrolyzed under the same conditions above) Glu (1) 0.98, Pro (3) 2.95, Val (1) 1.00, Ile (1) 1.02, Tyr (1) 0.98, Phe (1) 1.02 from ODS column Elution: Elution with 35% acetonitrile under the same conditions as described above Example 2 Measurement of rat 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.,
70 , 2243 (1973). ).

Cerebral brain (1.1-1.3) of male Wistar rats (100-200g)
g) was extracted and 10 ml of 50 nM Tris-hydrochloric acid buffer (pH 7.4) was homogenized at 0 ° C. using a Potter homogenizer. This was diluted with the same buffer solution to 100 times its weight, and then centrifuged (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 and incubated at 35 ° C. for 5 minutes. Then, [ ³ H] -Naloxon (NEN, 37.7
Ci / mmol) to a final concentration of 1 nM (34.000 cpm), and incubated again at 35 ° C for 15 minutes.

Vacuum filtration was performed using a glass filter (Whatman GF / B, 2.4 cm) to retain the membrane component containing the receptor on the filter, and the filter was quickly washed 4 times with 4 ml of buffer solution (owning time 30 seconds ). This filter was put into a measuring vial, 1 ml of 10% sodium dodecyl sulfate (SDS) was added, and the mixture was left for 30 minutes or more. Then 10 ml
PSC (manufactured by Amsrsham) 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 sample activity is [ ³
The temperature of the sample required to inhibit the specific binding of [H] -naloxone by 50% (IC ₅₀ ) is indicated.

(2) Result Example 3 Guinea pig distension longitudinal muscle plexus contraction inhibition test (1) Experimental method Kosterlitz et al. (Kosterlitz et al Br. J. Phar)
macol., 33 , 266 (1968)).

Longitudinal muscle plexus preparations were prepared from ileum excised from 300-350 g guinea pigs. 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 (118nM NaCl, 4.7
5mM KCl, 1.19mM KH ₂ PO ₄ , 2.54mM CaC
l ₂ , 1.2 mM MgSO ₄ , 25 mM NaHCO ₃ , 11 mM
Glucose) into a Magnus tube and keep it at 37 ° C.
A gas mixture of% O ₂ -5% CO ₂ was aerated. Electrical stimulation (50 V 0.1 msec) once every 10 seconds to the electrodes in the Magnus tube
And the resulting contraction strength was recorded electronically. The concentration of the peptide required to inhibit contraction by 50% (IC ₅₀ ) was determined and the opiate agonistic action was evaluated.

(2) Result EFFECTS OF THE INVENTION As is clear from the above, the novel peptide of the present invention has mild morphine-like analgesic activity and can be expected as a pharmaceutical product.

Claims (1)

[Claims] 1. A peptide represented by the general formula Tyr-Pro-Phe-Val-Glu- (R) n. However, in the formula, n is 0 or 1 and R is Pro or Pro-
Represents Ile-Pro, respectively.