JPH03271300A - New peptide - Google Patents

Abstract

NEW MATERIAL:A peptide expressed by the formula, ester thereof, amide thereof, acylated product thereof or salt thereof. USE:An antiulcer agent, antiinfectious agent and antiphlogistics. PREPARATION:For example,alpha-tert. butyloxycarbonyl-bromobenzyloxy carbonyl-L-tyrosin of C end protected amino acid is bonded to polystyrene solid phase carrier by a solid phase synthetic method and treated with 50% trifluoroacetic acid to remove the tert. butyloxycarbonyl group of alpha-amino protecting group and then operation for binding a protecting amino acid thereto according to amino acid sequence of peptide and then removing alpha-amino protect ing group is repeated to synthesize decapeptide chain on a solid phase carrier and the resultant peptide chain is treated with trifluoromethanesulfonic acid, etc., to carry out elimination of peptide from the carrier and removal of whole protecting group and then purified by subjecting it to reverse phase high speed liquid chromatography to provide the peptide expressed by the formula.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel peptide having human interleukin-1 activity, its ester, its amide, its acylated product, and salts thereof.

(Background of the Invention) When a foreign substance 2, such as a bacteria or virus, invades a living body, an inflammatory reaction is triggered at the site of the invasion, and furthermore, a biological defense reaction by an immune response is induced. Factors produced by macrophages have attracted attention as substances involved in a series of these biological reactions, and their biological effects differ, such as lymphocyte activating factors, B cell activating factors, and endogenous pyrogens. They were called because they have similar physicochemical properties.

It was unified and named as interleukin-1 (IL-1).

IL-1 was initially thought to be produced by monocytes, macrophages, or monocytic leukemia strains.

Later, as it became clear that it is produced by cells derived from many tissues, such as skin keratinocytes, brain astrocytes, glial cells, renal glomerular mesangial cells, and eye corneal cells, its diverse roles have attracted attention. Ta.

Two genes encoding IL-1 and its precursors have been cloned, and their entire amino acid sequences have been determined (IL-1α and IL-1β) [Nature 312,
458 (1984), Proc, Natl, Ac.
ad, Sci, USA.

81, 7907 (1984), Nature, 31
5.641 (1985) rNucleic Ac1d
Rea, 13.5869 (1985).

Currently, through genetic recombination, 7' can be produced uniformly in large quantities.
fIL-1 has become available, and various biological actions of IL-1 have been recognized. Its main effects include suppression of tumor growth and complete regression [J, Im
Munol, 136.3098 (1986).

Jpn, J, C11n, Immunol, 9
．． 330 (1986), Prevention of radiation damage [J,
Immunol, 136.2483 (1986)].

Protective effect against opportunistic infections [Infect-Immu
In addition, IL-1 is expected to be an endogenous pyrogen, and prostaglandin E2 (P G It has been reported that it has the effect of enhancing the production of E2). Recently,

IL-1 has been reported to be involved in exacerbation of arteriosclerosis because it promotes the proliferation of fibroblasts, and it may also have a detrimental effect on the treatment of β-cell diseases.

This complicates the medicinal use of IL-1.

Recently, some IL-1α derivatives (JP-A-62-185
097) and IL-1β derivatives (JP-A-63-152)
398), partially synthetic peptide of IL-1 (Japanese Unexamined Patent Publication No. 6
2-185095) has been reported, and attempts have been made to extract and improve the effects of IL-1. However, the present inventors found that the IL-1β derivatives that had already been prepared had improved IL-1 activity compared to natural human IL-1β, and could be usefully used as pharmaceuticals.

A patent application (Japanese Patent Application No. 63-272019) has been filed.

(Solution Means) As a result of various researches based on this technical level, the present inventors found that
The inventors have found that a decapeptide represented by the following formula (I), which has not been described in any literature, its ester, its amide, its acylated product, and its salt have IL-1 activity, and have completed the present invention.

Ala-Leu-Gly-Leu-Lys-Glu-L
ys-Asn-Leu-Tyr (I), that is, 2 The present invention comprises a peptide represented by the above formula (1), an ester thereof, an amide thereof, an acylated product thereof, or a salt thereof.

The object of the present invention is to provide the above-mentioned novel compounds.

Other objects of the invention will become apparent from the following description and examples.

Prior to peptide synthesis, the present inventors identified the active site of IL-1 protein. Human IL-1β protein has been crystallized and its three-dimensional structure has been revealed by X-ray analysis [EMBO.

J, 7.339 (1988). According to this, human IL-1β consists of amino acids constituting 12 β-sheets and amino acids constituting an α-loop connecting them, and a set of two antiparallel β-sheets forms one It has a tetrahedral structure with edges. Furthermore, the amino acid sequence postulated as the active part of IL-1β [J, Imm
unol, 137.3201 (1986)], but 4
It constitutes the first half of the fifth β-sheet from the second half of the second β-sheet [Proteins/Nucleic Acids/Enzymes, 33.1
793 (1988).

Furthermore, the amino acid sequence of IL-1 is human and bovine.

It is well conserved in mice and rabbits, and is known to have low bispecies specificity. Furthermore, it is known that many active sites of proteins are found in relatively highly hydrophilic regions.

The present inventors focused on these points, and found that this region is particularly well conserved among species and has relatively high hydrophilicity. A peptide (Pro
e, Natl, Acad, Sci.

USA, 81.7907 (1984) in the amino acid sequence of IL-1β, from position 175 to position 18.
4)).

(Compound) Nine compounds of the present invention will be described in detail below.

The ester of the peptide of formula (I) includes an ester formed by combining an alcohol component with one or both of the carboxyl group at the C-terminus and the γ-carboxyl group of Glu (glutamic acid residue), and such Examples of ester-forming alcohol components include Fi, straight-chain or branched lower alcohols having 1 to 6 carbon atoms such as methanol, ethanol, propanolwinpropertool, butanol, imbutanol, and tert-butanol, and aralkyl groups such as benzyl groups. can be mentioned.

The amide of the peptide of formula 9 (I) includes the C-terminal carboxyl group and the γ of Glu (glutamic acid residue).
Examples include compounds in which one or both of the -carboxyl groups are carbamoyl groups.

As an acylated form of the peptide of formula (I),

This includes acylated products formed by bonding some, all, or all of the amino group at the N-terminus and the ε-amino group of Lys (lysine residue) with an organic acid component; Examples include formic acid, acetic acid, myristic acid, succinic acid,
Examples include maleic acid and glucuronic acid.

The peptide of the present invention, its ester, its amide, and its acylated product may form acid addition salts. Such salts include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, carbonic acid, trifluoroacetic acid, propionic acid, oxalic acid, malonic acid, succinic acid.

fumaric acid, maleic acid, lactic acid, IJ malic acid,
Tartaric acid, methanesulfonic acid, ethanesulfonic acid.

Examples include acid addition salts with organic acids such as picric acid.

In addition, 9 peptides of the present invention, esters thereof, amides thereof,
The acylated product may form a salt with a base.

Such salts include salts with alkali metals such as potassium and sodium, alkaline earth metals such as calcium and magnesium, salts with valent metals such as aluminum, and ammonium salts.

There are many examples.

Since the compound of the present invention is decabegutide or a derivative thereof, it contains at least 10 asymmetric carbon atoms and has isomers. The present invention includes these isomers. Therefore, the amino acid residues constituting the compound of the present invention are not limited to L-configuration amino acid residues, but may be D-configuration or DL-configuration. Among these, preferable IL-1 activators of the two compounds of the present invention include natural types and those in which some of the 10 amino acid residues are D-type.

Note that the symbols for amino acid residues representing the peptide (I) of the present invention are expressed based on the three-letter abbreviation for amino acids commonly used in the art.

Ala: alanine, Leu: oisine, cty nigericin.

Lys: lysine, Glu: glutamic acid, Asn: asparagine.

Tyr: Tyrosine The respective residues are shown.

<Production method> The peptide of the present invention, its ester, its amide, its acylated product, or a salt thereof can be produced by a coupling method using various protecting groups, coupling reagents, etc., which is one of the peptide synthesis methods, and a C-terminus method. It is of course possible to synthesize using liquid phase methods such as activation method and N-terminal activation method, but solid phase method [Merrifield [
J.Am.

Chem, Soc, 85.2185 (1963
It is advantageous to synthesize by applying the method first introduced by ) and improved since then. When synthesizing peptides by the solid phase method, excellent automatic peptide synthesizers9 such as the Applied Biosystems automatic peptide synthesizer 430A are commercially available, and the standard operating program for such devices is followed. Just do it. It goes without saying that the method for producing the peptide of the present invention should not be limited to the application of currently commercially available equipment.

Esters can be obtained by a conventional method in which the above-mentioned alcohol is applied to the peptide of the present invention or its C-terminal activated product in the presence or absence of a condensing agent, an acid, or a base catalyst; Each is synthesized by a conventional method in which ammonia or its salt is applied to the body. Also.

If a solid phase support to which benzhydrylamine is bound is used as an initial support during automatic peptide synthesis, the amide can be obtained as a C-terminal amide when released from the solid phase support. Furthermore, in the final stage of automatic peptide synthesis, the N-terminal amino group and Lys
It is possible to synthesize derivatives in which some or all of the ε-amino groups are acylated.

These synthetic peptides were subjected to 9-preparative reverse-phase high-performance liquid chromatography (HPLC) to further increase the degree of purification.
).

By applying this purification method, peptides can be isolated using an automated system. + Note that purity was established by analytical reverse phase HPLC.

Furthermore, in order to maintain the purity and stability of the 9 synthetic peptide, it is preferable to freeze-dry it. Note that the peptide of the present invention can also be produced by genetic engineering using DNA encoding this peptide (I).

(Effect of the invention) The novel peptide (I) of the present invention and its ester.

Its amides, its acylated forms, and their salts (hereinafter referred to as peptides) have pharmacological actions similar to those observed in IL-1β, but some actions are selective; The effect is improved. The novel peptides of the present invention have effects on immune system cells such as lymphocyte activation, interleukin-2 (IL-2) production enhancement, antibody production enhancement, and colony stimulating factor (C8F) enhancement effects. Improved IL-1 activity can be demonstrated by increasing the effect on IL-1, reducing the pyrogenic effect, and reducing the PGE and advanced effect on production.

Furthermore, the two novel peptides of the present invention, like IL-1, promote C8F production from macrophages, vascular endothelial cells, fibroblasts, etc. [J, C11n.

Invest, 81, 92 (1988); Blo
od, 68, 1316 (1986) and has an action to prevent radiation damage.

It is useful for preventing granulocytopenia and radiation damage after chemotherapeutic treatment and radiotherapy, and is also useful as an anti-infective and anti-inflammatory agent for opportunistic infections.

Furthermore, the novel peptides of the present invention may contain other physiologically active substances,
The therapeutic effect can be enhanced by using it in combination with chemotherapeutic agents and immune enhancers. For example, IL-2,
When used in combination with tumor necrosis factors (TNFα, TNFβ), interferon-gamma (IFNγ), etc., additive and synergistic effects are observed in immune-enhancing effects and anti-tumor effects. By using it in combination with chemotherapeutic agents, immune enhancers, etc., its antitumor effect can be enhanced and side effects can be reduced. In addition, it is expected to have a synergistic recovery effect on hematopoietic disorders caused by 5-FU when used in combination with G-C8F, and an enhancement effect on colonization ability in the lungs [J, ImmunoL, 140.
3830 (1988)].

(Example) The present invention will be described in further detail by referring to Examples below. Note that the examples do not limit the present invention.

Example 1 Peptide synthesis and purification Peptide synthesis was performed using an automatic peptide synthesizer model 430A manufactured by Applied Biosystems.

The test was carried out using reagents and solvents commercially available from the company, and according to the standard operating program for the machine. That is, α-t-phthyloxycarbonyl-promopenzyloxycarbonyl-L-
A polystyrene solid support bound to tyrosine (0.5 mmol) was treated with 50% trifluoroacetic acid-methylene chloride to remove the t-butyloxycarbonyl group and
The α-amino group of tyrosine was liberated. To this, 4 equivalents of α-t-butyloxycarbonyl-L-leucine was activated with 2 equivalents of dicyclohexylcarbodiimide, and the resulting active ester of L-leucine was condensed. Tyrosine was synthesized according to the program in these steps. Hereinafter, the elimination of t-butyloxycarbonyl with trifluoroacetic acid and the condensation of amino acids using dicyclohexylcarbodiimide as an activator will be described as L-asuno-ragin.

L-lysine, L-glutamic acid, L-lysine.

L-leucine, L-glycine, L-leucine.

Automatically repeats with L-alanine, but the N-terminus is t
-I use one protected with Boc.

The lysine e-ano group was protected with a p-chlorobenzyloxycarbonyl group, and the γ-carboxyl group of glutamic acid was protected with a benzyl group), and a decapeptide was synthesized on a solid support. However, 4 equivalents of dicyclohexylcarbodiimide and 4 equivalents of N-hydroxybenzotriazole were used for the activity of L-asunochragin. Desorption of synthetic peptides from resin is performed using 430A
This was achieved by treatment with trifluoromethanesulfonic acid according to the procedure described in the user's manual, yielding 425 μ of crude peptide after lyophilization. The obtained crude peptide was purified by preparative HPLC using a reverse phase column. That is, the crude peptide was dissolved in a 50% acetonitrile aqueous solution, and a reverse phase column (Senshu Scientific Co., Ltd.) was used.

ODS-H-5251, t 20 x 250mm)
However, 5% of solution A (0.1% trifluoroacetic acid) and B
Elution was performed using a gradient from a mixture of 95% of solution (0.1% trifluoroacetic acid-70% acetonitrile) to a mixture of 40% of solution A and 60% of solution B. Fractions containing the peptide (identity was confirmed by analyzing absorption at 210 μm using a Shimadzu 5PD-6A instrument) were collected.

Dry under reduced pressure to obtain Alt-Leu-Gly-Lys-
A white powder was obtained as the trifluoroacetate of Glu-'Lys-Asn-Leu-Tyr.

The obtained peptide was dissolved in a 50% acetonitrile aqueous solution and applied to a 9 analytical reverse phase column (Senshu Kagaku, 0DS-
H-1251, r4.6X250mm) Shadow.

Flow rate 0.7 m 17 minutes, 100% liquid A to 100% liquid B
Uniformity was confirmed by HPLC using gradient elution for up to 35 minutes (a single peak was observed at a retention time of about 21 minutes). The obtained peptide was heated to 150°C.

After hydrolysis with 6N hydrochloric acid (containing 1% phenol) for 1 hour, it was identified by amino acid analysis. The residue values obtained by the analysis are shown in Table 1.

Table 1 Example 2 Measurement of lymphocyte activating factor (LAF) activity LAF activity
Mizel, S, B, et al, J, I
ImmunaL, 120.

1497 (1978)] is a biological activity that promotes the mitogenic action of mouse periclinal cells by mitogens, and the activity of the trifluoroacetate of peptide (I) obtained in Example 1 was measured using this activity.

Put 50 μl of the analysis solution into a 96-well flat-bottom multiplate,
Leg line cells (l x 107 cells/mt) collected from 4-6 week old male C3H/HeJ mice were used for each.
) and a cell suspension containing concanavalin A (0.6 μg/m7).

100μ was added and cultured for 48 hours in an incubator at 37°C containing 5% carbon dioxide. Then 3H-thymidine was added to 1.
After adding 85 kBq/20 μl/well and culturing for 18 hours, cells were collected on a glass fiber filter using Cerno-Pesta, and the amount of 3H-thymidine incorporated into the cells was measured using a liquid scintillation counter. The proliferation of leg line cells was measured by this method.

FIG. 1 is a graph showing the effect of the trifluoroacetate of peptide (I) obtained in Example 1 on the proliferation of leg line cells. The vertical axis shows the amount (cpm) of 3H-thymidine incorporated into thymus cells.

The concentration of this synthetic peptide is shown in μg/ml on the horizontal axis. It is shown as the net amount of 3H-thymidine uptake by the synthetic peptide. As is clear from FIG. 1, 9 synthetic peptides of the present invention (■) trifluoroacetate have mouse thymocyte division activity.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the influence of the trifluoroacetate of peptide (I) obtained in Example 1 of the present invention on proliferation of pericytes.

Claims (1)

[Claims] Formula Ala-Leu-Gly-Leu-Lys-Glu-L
A peptide represented by ys-Asn-Leu-Tyr,
Its ester, its amide, its acylated product, or a salt thereof.