JPH1171397A - New bioactive peptide - Google Patents

Abstract

(57) [Summary] (Modifications) [Problem] To provide a novel active peptide useful as an analgesic or a biochemical reagent which exhibits analgesic activity and does not decrease its effect even if used continuously every day. SOLUTION: General formula X ₁ -Ala-Ser-Lys-
Arg-Ala-Gly-Thr-Phe-Gly-G
ly-X ₂ becomes the new peptide. (X ₁ = Gln-Asn-
Ala-Leu-Lys-Arg, Asn-Ala-L
eu-Lys-Arg, Ala-Leu-Lys-Ar
g, Leu-Lys-Arg, Lys-Arg, Arg
Wherein X ₂ is -COOH of Phe
_{CH 2 OH, -CONH 2, -COOCH} 3, -COO
_{C 2 H 5, -COO (n} -C 3 H 7), - COO (is
_o-C 3 shows the amino acid was replaced with either _{H 7).} )

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a useful novel peptide having an analgesic action, and can be used for biochemical reagents, pharmaceuticals, and the like.

[0002]

2. Description of the Related Art Analgesic anesthetics are used to remove pain at the time of surgery or terminal cancer. Narcotic analgesics, such as morphine, are most often used, but there is a problem in the formation of tolerance that the effect is reduced when administered in the same amount every day. In addition, opioid peptides that have an analgesic effect through the same receptor as narcotic analgesics have similar weaknesses.

[0003]

However, no analgesic having the above-mentioned weakness has been found as an alternative to a narcotic analgesic, and analgesics having a new mechanism are being studied. In recent years, a receptor gene having similarity to an opioid receptor has been cloned, a peptide consisting of 17 residues has been isolated as its endogenous ligand, and named nociceptin. It has been reported that the peptide has a nociception enhancing effect.

[0004]

Means for Solving the Problems The present inventor has proposed nociceptin
As a result of diligent search for antagonists of the peptide, based on the assumption that the antagonist has analgesic properties,
Conversely sequence retro Nociceptin with of Nociceptin (the proviso N- terminus Phe needs a derivative X ₂ of Phe) Gln-Asn-Ala- Leu-Lys-Ar
g-Ala-Ser-Lys-Arg-Ala-Gly
-Thr-Phe-Gly-Gly- X 2, further X ₁ -A
la-Ser-Lys-Arg-Ala-Gly-Th
found r-Phe-Gly-Gly- X 2 comprising peptides to exhibit analgesic activity, yet the present invention has been completed found that an analgesic is not observed decrease in effect be administered daily.

The peptide of the present invention is X ₁ -Ala-Ser
-Lys-Arg-Ala-Gly-Thr-Phe-
It has a sequence of Gly-Gly-X ₂ , wherein X ₁ is Gln-Asn-Ala-Leu-Lys-Arg,
Asn-Ala-Leu-Lys-Arg, Ala-L
eu-Lys-Arg, Leu-Lys-Arg, Ly
It is any one of s-Arg and Arg, and is preferably Gln-Asn-Ala-Leu-Lys-Arg.

[0006] The peptide can also be produced by applying conventional means for peptide synthesis.
Gln mentioned above glutamine, Asn is asparagine, Leu is leucine, Lys is lysine, Arg is arginine, Ala is alanine, Ser serine, Gly is glycine, Thr is threonine, Phe is phenylalanine, X ₂ is the Phe - -CH ₂ OH and COOH, -C
_{ONH 2, -COOCH 3, -COOC 2} H 5, -COO
_{(N-C 3 H 7)} , - COO (iso-C 3 H 7) or a substituted amino acid in either. All such amino acids are in the L-form.

Specifically, X ₂ is phenylalaninol, phe
nylalanineamide, phenylalanine metylester, phenyla
Examples thereof include lanine ethylester and phenylalanine n-propylester. Phenylalaninol is preferable.

[0008] The peptide of the present invention can be obtained by a peptide synthesis method, and can be obtained by one of two types of fragments which are bisected at an arbitrary position of a peptide bond by a method usually used for peptide synthesis, that is, a liquid phase method or a solid phase method. A raw material having a corresponding reactive carboxyl group and a raw material having a reactive amino group corresponding to the other fragment were subjected to 2- (1H-Benzotriazole-
1-yl) -1,1,3,3-tetramethyluronium hexafluorophospha
When condensation is performed using a method using an active ester such as te (HBTU), a method using carbodiimide, or the like, and the resulting condensate has a protective group, it can also be produced by removing the protective group.

The functional groups which should not take part in the reaction in this reaction step are protected by protecting groups. Examples of the amino-protecting group include benzyloxycarbonyl (B
z), t-butyloxycarbonyl (Boc), p-biphenylisopropyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl-protecting group include groups capable of forming, for example, an alkyl ester and a benzyl ester.In the case of the solid phase method, the carboxyl group at the C-terminal is a chlorotrityl resin, a chloromethyl resin, an oxymethyl resin,
It is bound to a carrier such as P-alkoxybenzyl alcohol resin. The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.

After completion of the condensation reaction, the protecting group is removed,
In the case of the solid phase method, the bond between the C-terminal of the peptide and the resin is further cleaved. Further, the peptide of the present invention is purified according to a usual method. For example, ion exchange chromatography,
Examples include reversed-phase liquid chromatography and affinity chromatography. The synthesis of the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, or the like.

The peptide of the present invention is useful for biochemical reagents and pharmaceuticals. For example, when used as a biochemical reagent, research and development of opioids and orphan receptors, or
It is useful as a lead substance for the development of a drug having a novel mechanism of action using the receptor.

It has been found that the peptide of the present invention has an action mechanism via an opioid or orphan receptor. Hereinafter, a receptor assay showing that the receptor is mediated will be described. The receptor assay was radiolabeled with rat brain membrane fraction [ ¹²⁵ I-Tyr
¹⁴ ] Performed using nociceptin. First 50 mM Tris
0.3 nM [ ¹²⁵ I-Tyr in hydrochloric acid buffer (pH 7.4)
¹⁴ ] Nociceptin, the sample and the rat brain membrane fraction were incubated at 25 ° C for 30 minutes, and the radioactivity after washing with the same buffer at 0 ° C on a Whatman GF / C glass filter was changed to γ-
It was measured with a counter. The above peptide showed binding inhibition at an IC ₅₀ value of 40 μM, and thus had binding activity to the orphan receptor.

Next, the case of using as a medicine will be described. The administration route of the peptide used in the present invention includes:
Although any of oral administration, parenteral administration and rectal administration may be used, oral administration is preferred. The dose of the peptide of the present invention varies depending on the kind of the compound, the administration method, the symptoms and age of the patient, etc., but is usually 0.001 to 1000 mg, preferably 0.01 to 10 mg once to 3 times per day. is there. The peptide of the present invention is usually administered in the form of a preparation prepared by mixing with a preparation carrier. As the pharmaceutical carrier, a substance which is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.

More specifically, for example, lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium metasilicate aluminate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, calcium carboxymethylcellulose, ion exchange Resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light silicic anhydride, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate , Glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbe Door, macrogol, vegetable oils,
Examples include wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol, water and the like.

Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections and the like.
These preparations are prepared according to a conventional method. In the case of a liquid preparation, it may be in the form of being dissolved or suspended in water or another appropriate medium at the time of use. Tablets and granules may be coated by a known method. In the case of an injection, the peptide of the present invention is prepared by dissolving the peptide in water, but may be dissolved in a physiological saline solution or a glucose solution if necessary, or may be added with a buffer or a preservative. Good.

These preparations contain the peptide of the present invention in 0.1%.
It can be contained at a rate of 01% or more, preferably 0.5 to 70%. These formulations may also contain other therapeutically valuable components.

[0017]

Now, the present invention will be described more specifically below with reference to working examples. Example 1 [Synthesis of Peptide] Commercially available Phenylalaninol (Pheo
l) -2-chlorotrityl resin (replacement ratio 0.46 meq /
g)] 0.65 g of PS3 peptide synthesizer (Protein
Technologies Co., Ltd.) and synthesized as follows. First, put the above resin in a reaction vessel,
mmol of Fmoc-Gly and 1 m
A solution prepared by dissolving 10 mol of HBTU in 10 ml of dimethylformamide containing 0.4 M N-methylmorpholine was added to the reaction tank, and the mixture was stirred and reacted at room temperature for 20 minutes. The Fmoc group was removed from the obtained resin in 20 ml of dimethylformamide containing 20% by weight of piperidine, and then Fmoc-amino acids were sequentially coupled from the C-terminus in the same manner as in the above-described method of coupling Fmoc-Gly. , Gln-Asn-Ala-Leu-Lys-Arg
-Arg-Ala-Ser-Lys-Arg-Ala-
Gly-Thr-Phe-Gly-Gly-Pheol
A resin was obtained. The resin was added to 10 ml of a deprotection solution (82 vol%
Trifluoroacetic acid, 5% by volume thioanisole, 3% by volume
Ethanedithiol, 2% by volume ethyl methyl sulfide,
The peptide was released from the resin by stirring for 4 hours at room temperature in 3% by volume phenol, 5% by volume water).

To this was added 40 ml of cold ether to precipitate the peptide, which was further washed three times with cold ether to obtain a crude peptide. This is supplied to an ODS column (Cosmosi
15C ₁₈ -AR, 20 × 250 mm), developed and purified with a linear concentration gradient of acetonitrile containing 0.1% by weight of trifluoroacetic acid by reverse phase chromatography.
Gln-Asn-Ala-Leu-Lys-Arg-A
rg-Ala-Ser-Lys-Arg-Ala-Gl
y-Thr-Phe-Gly-Gly-Pheol was obtained. The product was analyzed using a protein sequencer (Model 477A, manufactured by Applied Biosystems), and as a result, the product was found to have the above composition.

A) Measurement of analgesic activity Approximately 20 g of ddy male mouse was injected with 100 nmol of the above peptide as a 10 μl physiological saline solution to 100 μg / mouse in the lateral ventricle of the mouse, and 5 minutes after administration and 20 minutes after administration, a pinch was placed on the tail. , The time required to feel pain and to bite into a pinch was measured and evaluated as follows. The results are shown in Table 2. ◎: 6 seconds or more ○: 4 seconds to less than 6 seconds △: 2 seconds to less than 4 seconds ×: less than 2 seconds

B) Evaluation of the effect when administered daily The relative analgesic activity on the first day was set to 100.0%,
The analgesic activity on days 4 and 5 is shown in Table 2 as relative values. The analgesic relative activity was calculated by plotting the vertical axis as the relative analgesic activity (as described above, the time until pain felt and looking back was plotted on the vertical axis, and the time after administration was plotted on the horizontal axis. (The area under the curve (AUC) was shown as a relative value.)

Examples 2 and 3 The steps of adding Fmoc amino acids in Example 1 were reduced to synthesize a peptide resin having 13 or 15 residues of peptide, and the peptides shown in Table 1 were synthesized in the same manner as in Example.
The sequence was confirmed with a protein sequencer. Example 1
The results are shown in Table 2.

Example 4 In Example 1, Fmoc-NH-polyethyle was used instead of Phenylalaniol-2-chlorotrityl resin (0.46 meq / g).
Using neglycole handle resin (0.16 meq / g)
After binding Fmoc-phenylalanine,
Fmoc-amino acids such as -Gly were sequentially linked from the N-terminus to synthesize the peptides shown in Table 1 in the same manner as in Example 1, and the sequence was confirmed using a protein sequencer. Evaluation was performed in the same manner as in Example 1, and the results are shown in Table 2.

Example 5 In Example 1, Fmoc-phenylalanine was used instead of Phenylalaniol-2-chlorotrityl resin (0.46 meq / g).
-tritylamidomethylester-polyethyleneglycolehandle
Retro nociceptin was synthesized in the same manner as in Example 1 using the resin (0.16 meq / g), and the sequence was confirmed using a protein sequencer. 100 mg of the present peptide was added to 0.
It was dissolved in anhydrous methanol containing 1 M hydrochloric acid, left overnight at room temperature, and dried to synthesize a retro nociceptin methyl ester shown in Table 1. Evaluation was performed in the same manner as in Example 1, and the results are shown in Table 2.

[0024]

Table 1 Peptide sequence Example 1 Gln-Asn-Ala-Leu-Lys-Arg-Arg-Ala-Ser-Lys-Arg-Ala-Gly-Thr-Phe-Gly-Gly-Pheol * Example 2 Arg- Arg-Ala-Ser-Lys-Arg-Ala-Gly-Thr-Phe-Gly-Gly-Pheol * Example 3 Leu-Lys-Arg-Arg-Ala-Ser-Lys-Arg-Ala-Gly-Thr-Phe -Gly-Gly-Pheol * Example 4 Gln-Asn-Ala-Leu-Lys-Arg-Arg-Ala-Ser-Lys-Arg -Ala-Gly-Thr-Phe-Gly-Gly-PheNH ₂ ** Example 5 Gln-Asn-Ala-Leu-Lys-Arg-Arg-Ala-Ser-Lys-Arg -Ala-Gly-Thr-Phe-Gly-Gly-PheOCH ₃ *** * Pheol: -COOH of Phe is -CH ₂ OH substituted amino acid ** PheNH ₂ : amino acid in which -COOH of Phe is substituted with -CONH ₂ *** PheOCH ₃ : amino acid in which -COOH of Phe is substituted with -COOCH ₃

[0025]

[Table 2] a) b) 1 Day 2 Day 3 Day 4 Day 5 Example 1 ◎ ◎ 100.0 125.3 84.8 106.3 100.0 Example 2 ○ ○ 62.0 60.1 56.1 60.2 59.5 Example 3 ◎ ○ 75.3 78.3 73.5 71.2 73.5 Example 4 ◎ ◎ 102.5 96.1 105.1 95.6 103.1 Example 5 ◎ ○ 70.5 65.1 71.5 75.9 72.1

[0026]

The novel physiologically active peptide of the present invention exhibits an analgesic activity and has an analgesic effect such that the effect does not decrease even if it is used daily, or is useful as a drug.

Claims (2)

[Claims] 1. A novel bioactive peptide represented by the following general formula (1). X ₁ -Ala-Ser-Lys-Arg-Ala-Gly-Thr-Phe-Gly-Gly-X ₂ (1) (provided that X ₁ = Gln-Asn-Ala-Leu-Lys)
-Arg, Asn-Ala-Leu-Lys-Arg,
Ala-Leu-Lys-Arg, Leu-Lys-A
rg, Lys-Arg, any one of Arg,
X ₂ represents -COOH of Phe by -CH ₂ OH, -CONH
_{2, -COOCH 3, -COOC 2 H} 5, -COO (n-C 3
H _7), - a substituted amino acid in either the _{COO (iso-C 3 H 7} ). ) 2. X ₁ is Gln-Asn-Ala-Leu.
The novel bioactive peptide according to claim 1, which is -Lys-Arg.