JPH03123795A - Novel tetrapeptide, its salt and anti-allergic agent - Google Patents

Abstract

NEW MATERIAL:The tetrapeptide of formula H-Ala-Ser-Gly-Lys-OH (Ala is L-alanine residue; Ser is L-serine residue; Gly is glycine residue; Lys is L-lysine residue) or its salt. USE:Anti-allergic agent active to inhibit the isolation of histamine and to suppress the formation of IgE antibody. PREPARATION:Boc-Gly-OH (Boc is t-butyloxycarbonyl) is bonded to Boc-Lys[Z(2 C1)] [Z(2C1) is 2-chlorobenzyloxycarbonyl] bonded to a carrier such as oxymethyl resin using a peptide-synthesizing apparatus in the presence of a condensation agent such as dicyclohexylcarbodiimide. Successively, amino acids are bonded to the N-terminal from Gly and, finally, the obtained peptide is separated from the resin and purified.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel tetrapeptide, its salt, and an antiallergic agent.

[Prior Art] Various drugs have been proposed and developed for the prevention and treatment of various allergic diseases, and some are already on the market.

Among allergy symptoms, immediate allergic reactions such as bronchial asthma, hives, and allergic rhinitis are classified as ■-type allergic reactions. This type 1 allergic reaction is generally considered to consist of the following three stages based on the onset mechanism and the action mechanism of the antiallergic agent. In other words, IgE antibodies are produced by the interaction of macrophages, T cells, and B cells against foreign antigens that first invade the body, and these IgE antibodies are activated by Fc receptors on mast cells in tissues and basophils in blood. It will stick and cause sensitization. This process is the first stage. Next, when the foreign antigen enters the body again, the foreign antigen binds to the IgE antibody that has adhered to the cell's Fc receptor, triggering an antigen-antibody reaction that activates cell membrane enzymes and releases calcium ions into the cell. Influx occurs, which causes biochemical changes such as enzymatic reactions, and histological changes such as degranulation. As a result, chemical mediators (chemical messengers) such as histamine and 5R8-A are released outside the cell. This process is the second stage. The chemical mediator liberated extracellularly in the second step promotes smooth muscle contraction, increased capillary permeability, and mucus secretion, causing various allergic symptoms. This process is the third
It is a stage.

Among conventionally known antiallergic agents, nonspecific desensitization therapy agents and antibody production inhibitors are drugs that act in the first step. However, there are no commercially available drugs that specifically act only on this first stage. Drugs that act in the second stage include sodium cromoglycate (hereinafter referred to as DSCG).
), chemical mediators such as tranilast
There are inhibitors. Antihistamines and bronchodilators are also drugs that act in the third stage. In addition, special public service 1986-23
No. 18 discloses anti-allergic peptides.

According to the above publication, this peptide is an IgE antibody-derived pentapeptide consisting of five amino acid residues in the Fc region of gE antibody, as shown by the following primary structural formula: %Formula %.

Although this peptide has not been confirmed to have the effect of suppressing IgE antibody production in the first stage, it can block the binding of IgE antibodies to mast cells that occurs at the beginning of the second stage, and also inhibit the binding of IgE antibodies that have already bound to mast cells in the second stage. It is thought that by substituting this peptide with this peptide, it has the property of blocking allergies.

[Problems to be Solved by the Invention] Conventional development of anti-allergic drugs has been directed to the development of drugs that act on one of the three stages of the onset of allergic symptoms described above, and it has been difficult to develop drugs that act on one of the three stages of the onset of allergic symptoms. Research has been carried out on therapies to prevent or treat allergic symptoms by blocking them at any stage. Through such research, the development of drugs that act on one of the three stages of the onset of allergic symptoms has led to the development of therapies that are expected to be somewhat effective.

However, these known chemotherapeutic agents do not completely block the above three-step chain. Therefore, by combining a drug that acts on one of the three stages with a drug that acts on the other, the idea is to completely break the chain, and multiple drugs are used in combination. Although some efforts have been made to do so, the effects are not always as expected.

Therefore, if a single drug that can act on multiple stages of the above three stages of allergic symptom onset is developed, its effectiveness as an anti-allergic drug could be dramatically increased. The development of such drugs is highly anticipated.

In addition, from the mechanism of the onset of allergic symptoms described above, Ig
It is thought that it is possible to obtain an excellent allergy agent by developing a peptide derived from the Fc region of antibody E or a peptide similar thereto, and the development of new peptides from this approach was also expected.

The purpose of the present invention is to synthesize various peptide portions of the Fc region of IgE antibodies or similar peptides thereof, and to provide anti-allergic peptides with excellent pharmacological activity.

[Means for Solving the Problems] In order to achieve the above object, the present inventors have developed an IgE antibody F.
Focusing on peptides found in the c region, we synthesized various oligopeptides and examined their antiallergic activity. As a result, we found that a tetrapeptide represented by H-Ala-Ser-Gly-Lys-OH inhibits histamine release. The present invention has been completed based on the discovery that this method also suppresses IgE antibody production.

That is, the present invention provides (1) the following formula [I] H-Ala-Ser-Gly-Lys-OH(I) (
(Ala is an L-alanine residue, Ser is an L-serine residue, Gly is a glycine residue, and Lys is an L-lysine residue) or a pharmaceutically acceptable salt thereof.

(2) An anti-allergic agent containing the peptide of (1) above or a pharmaceutically acceptable salt thereof as an active ingredient.

It is related to.

Pharmaceutically acceptable salts of the tetrapeptide represented by H-Ala-5er-Gly-Lys-OH of the present invention include inorganic acid salts such as hydrochloride, sulfate, and phosphate; acetate;
organic acid salts such as citrate, fumarate, tartrate, lactate; alkali metal salts such as sodium salt and potassium salt;
Examples include alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts, and the like.

The tetrapeptide of the present invention can be synthesized by a solid phase method or a liquid phase method commonly used for peptide synthesis. In other words, a raw material having a reactive carboxyl group corresponding to one of two fragments that is bisected at an arbitrary position of a peptide bond, and a raw material having a reactive amino group corresponding to the other fragment are combined into dicyclohexylcarbodiimide or the like. It can be produced by performing dehydration condensation using a condensing agent and, if the condensate has a protecting group, removing the protecting group.

In the above reaction, the functional groups that should not be involved in the reaction are:
Protect it with a protecting group in advance.

Protecting groups for amino groups include benzyloxycarbonyl,
There are t-butyloxycarbonyl, 9-fluorenylmethyloxycarbonyl, etc., and the protection of the carboxyl group is
In the solid phase method, it is carried out by bonding to a carrier such as chloromethyl resin, p-alkoxybenzyl alcohol resin, or oxymethyl resin, and in the liquid phase method, it is carried out by using benzyl ester, methyl ester, etc.

After the condensation reaction, the protecting group is removed, and in the solid phase method, the bond between the C-terminus of the peptide and the resin is cleaved.

Furthermore, the tetrapeptide of the present invention can be purified using conventional purification means such as ion exchange chromatography and reversed phase liquid chromatography.

A pharmaceutically acceptable salt of the tetrapeptide represented by formula (I) can be obtained by removing the protecting group in the final step of synthesis, and then adding an acid such as hydrochloric acid or acetic acid, or adding an acid such as sodium hydroxide or potassium hydroxide. It is also possible to make a corresponding salt by adding a base such as, or after isolating the tetrapeptide represented by formula (I),
It can also be made into a salt by adding an acid or a base in the same manner as above.

The structure and purity of the substance of the present invention are confirmed by high performance liquid chromatography, elemental analysis, amino acid analysis, etc.

The antiallergic agent of the present invention includes a formulation consisting of a pharmaceutically acceptable carrier or diluent and the present compound or a pharmaceutically acceptable salt thereof. Preferred examples of salts include inorganic acid salts such as hydrochloride, sulfate, and phosphate; organic acid salts such as acetate, citrate, fumarate, tartrate, and lactate; and alkali salts such as sodium salt and potassium salt. Examples include metal salts, alkaline earth metal salts such as calcium salts and magnesium salts, and ammonium salts. The formulation can be formulated to release the active ingredient rapidly, continuously or with a delay after administration to a patient.

The antiallergic agent of the present invention can be administered orally or parenterally as appropriate. Typical methods of administration include oral, rectal, percutaneous, subcutaneous, intravenous, intramuscular, inhalation, or intranasal routes.

In these administration methods, the antiallergic agent of the present invention can be administered in the form of various pharmaceutical preparations. The forms of these pharmaceutical preparations include tablets, hard capsules, soft capsules, granules, powders, troches, suppositories, syrups, creams, ointments, tablets, injections, suspensions, inhalants,
There are aerosols, etc. Also other anti-allergic agents,
It can also be made into double-layer tablets, multi-layer tablets, etc. together with other medicines. Furthermore, in the case of tablets, they can be coated with a conventional coating, for example, sugar-coated tablets or enteric-coated tablets.

When preparing solid preparations such as tablets, granules, and powders, known additives such as lactose, sucrose, glucose, crystalline cellulose, cornstarch, calcium phosphate, sorbitol, glycine, carboxymethyl cellulose, and hydroxypropyl cellulose are used in the preparation. , gum arabic,
Polyvinylpyrrolidone, polyethylene glycol, magnesium stearate, talc, etc. can be added.

For semi-solid formulations, materials such as vegetable waxes, microcrystalline waxes, fats such as tarolanoline can be added.

When preparing a liquid formulation, additives such as sodium chloride, sorbitol, glycerin, olive oil, almond oil, propylene glycol, ethylene glycol, and ethyl alcohol can be added.

The dosage of the peptide represented by formula [I] depends on the patient's age,
The dosage can be adjusted as appropriate depending on body weight, symptoms, etc., but the dosage for oral administration is 0.01 to Lomg per day.
/kg, intranasal dosage is 0.1-100mg
It is. For parenteral administration, the dose is 10 to 1 per day.
000μg/kg.

[Example] The present invention will be specifically explained with reference to the following example.

The abbreviations used here have the following meanings.

Ala: L-alanine residue Ser: L-serine residue Gly nigericin residue Lys: L-lysine residue Boc: t-butyloxycarbonyl group Z: penzyloxycarbonyl group Z (2[I]: 2-chlorobenzyl Oxycarbonyl group PAM: 4-(oxymethyl)phenylacetamidomethyl TFA:) Lifluoroacetic acid DCC dicyclohexylcarbodiimide H-Ala-
A peptide synthesizer Model 1990B manufactured by Beckman was used as a peptide synthesizer for Ser-Gl -L 5-OH. 1.50 g of Boc-Lys (Z(2C1)
]-PMA resin (containing 0.7 mmol/g of Boc-Lys (Z(2C1)), manufactured by Abride Biosystems) was taken and stirred in dichloromethane for 2 hours to swell.

Next, Boc-Gly-OH was reacted with the following procedure.

(1) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

(2) Wash 73 times for 2 minutes with methanol ZQml.

(3) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

(4) Wash once for 5 minutes with 45 wt% TFA and 20 ml of dichloromethane.

(5) 1 in 5 wt% TFA and dichloromethane ZOml
Wash 5 minutes/once.

(6) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

(7) Wash with 20 ml of methanol for 2 minutes/3 times.

(8) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

At this point, confirm that the amino group reaction with ninhydrin becomes positive.

(9) Wash once/2 minutes with 10 wt% TFA and 20 ml of dichloromethane.

(10) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

(11) A solution of 4.2 mmol of Boc-Gly-OH in dichloromethane (10 ml) and a solution of 2.1 mmol of DCC in dichloromethane (5 ml) were added together and reacted by shaking in ice water for 20 minutes. Dry the resulting precipitate and suction filtrate it with a glass filter.

(12) Wash with 20 ml of dichloromethane for 2 minutes/3 times, and confirm that the amino group reaction becomes negative with ninhydrin.

(13) Wash with 20 ml of methanol for 2 minutes/3 times.

(14) Wash with 20 ml of dichloromethane for 2 minutes/3 times.

The introduction of Boc-Gly-OH is thus completed.

Repeat the above steps (1) to (14) in sequence,
Add amino acids to the N-terminus from Gly. The protected amino acids added are in the following order in place of Boc-Gly-OH.

Boc-8et(OBzl)-01 (4.2 mmol B
When all operations using 2 mmol or more of oc-Ala-OH4 are completed, the next protected peptide is synthesized on the resin.

Boc-Ala-Ser(OBzl)-Gly-Lys
(Z(2[I])-Resin The protected peptide bound on this resin was filtered and dried overnight in a desiccator after carrying out the above steps (1) to (14). 610mg was obtained.Take this and add 2m of anisole.
l, dimethyl suberimidate dihydrochloride (dimeth
yl-suberimidate dihydroch
It was treated with 30 ml of hydrogen fluoride at 0° C. for 1 hour in the presence of 0.5 ml of hydrogen fluoride. Hydrogen fluoride was distilled off, washed with anhydrous ether/n-hexane (volume ratio 1:1) mixture, then with anhydrous ether, thoroughly dried, and then reduced to 10 wt%.
The peptide was dissolved in 50 ml of acetic acid, and the insoluble resin was removed.

The resulting solution was Dowex lX-2
The mixture was applied to a column (1, OX 15 cm, manufactured by Dow Chemicals), eluted with 2N acetic acid, filtered through a 0.22 μm Millipore filter, and lyophilized. 128 mg of crude peptide was obtained. This was further subjected to preparative high performance liquid chromatography under the conditions shown below.

Column: YMC-D-ODS 20mmX250mm
(Manufactured by Yamamura Kagaku Kenkyushin) Solvent: 0.1% TFA and acetonitrile from 0% to 7%
Solvent flow rate with linear gradient to 0% mixture: 1. 28 mg of peptide with a purity of 98% was obtained by Oml/min preparative high performance liquid chromatography. The analysis results of this purified peptide are as follows.

Analytical high-performance liquid chromatography: Figure 1 Amino acid analysis values after acid decomposition = (molar ratio) Alanine 1.0 Serine 0.8 Glycine 1.0 Lysine 1.1 In addition, analytical high-performance liquid chromatography is performed using columns and KASEISORB (KASEISORB). ) C8-300X
5 (manufactured by Tokyo Kasei Kogyo, 4.6 x 250 mm), the solvent was an aqueous solution containing 0.1% TFA and acetonitrile containing 0.1% TFA from 100:0 (volume ratio) to 70:3.
0 (volume ratio) Using a solution with a linear gradient, the flow rate was 1, 0 ml/min, and the detection wavelength was 210 nm.
The acid decomposition of the peptide was performed in 6N hydrochloric acid (containing 0.1 wt% phenol) at 120°C for 15 hours, and the amino acids were analyzed using a Hitachi amino acid analyzer Model 835.

Next, the tetrapeptide H-Ala-S represented by formula [I]
The results of a pharmacological activity test of er-G 1y-Lys-OH are shown.

Male Wistar rats weighing 300 to 350 g were passively sensitized, and their intraperitoneal mast cells were used for testing. The rat antiserum used for passive sensitization was prepared according to the method of Mota [Imm
unology, 2 L P, 681 (1964)] and Hamaoka's method [J, Immunolog
y, 113. 958 (1974)]. That is, 5 ml/kg of ovalbumin (10 mg/kg) was injected into both thigh muscles of male Wistar rats (body weight 200-250 g), and at the same time 2 x 101
0 Killed Bordetell
aPertussis) was administered intraperitoneally for immunization.

On the 12th day after the first sensitization, blood was collected from the abdominal aorta under ether anesthesia, and the antiserum was separated. Antiserum was stored frozen at -20°C. The titer of the antiserum was measured by a 48-hour rat PCA reaction, and those with a titer of 128 to 256 times were used for the experiment. The obtained ovalbumin rat IgE serum was diluted 2 times, and 1 ml thereof was intraperitoneally administered for sensitization.

After 48 hours of sensitization, the rats were sacrificed by bleeding and intraperitoneally injected with phosphate buffered solution (18 g of NaC, 0.2 g of KCI, 0.2 g of Na
zHPO442Hz02.88g, KH2PO40,
2g, EDTA・2NaO02g and bovine serum albumin 1g dissolved in purified water to make 1 liter solution, p
15 ml of H7,4 (hereinafter abbreviated as PBS (-)) was injected, the abdomen was lightly massaged for about 2 minutes, and the abdomen was opened to collect intraperitoneal cells.

This cell suspension was centrifuged (1,00 rpm, 10
1 minute), resuspended in PBS (-), layered with gum arabic specific gravity solution (specific gravity 1.075), and centrifuged (2.5 minutes).
0 rpm, 10 minutes). PBS the precipitated cells.
Wash twice with PBS (-) and add new PBS (+) [PBS (-).
), CaC1zO,1 was used instead of EDTA-2Na.
After suspending the cell suspension in a solution abbreviated as PBS (+) and adjusting the cell density to 1 x 10 cells/ml, 0.8 ml of the cell suspension was dispensed into test tubes treated with silicone.
Breincubate for 10 minutes at 37°C. Add 0.1 ml of various sample solutions diluted with PBS (+) to a test tube containing cell suspension, incubate at 37°C for 15 minutes, and add egg white, an antigen, to suspend histamine from mast cells. Albumin (final concentration 1mg/ml)
and phosphoadityl-cy-serine (final concentration 100 μg
0.1 ml of a mixed solution of 0.2 mg/mg) was added, and the mixture was further incubated for 15 minutes to release histamine. However, DSCG, one of the comparative drugs, was added 30 seconds before antigen addition, and incubated for an additional 15 minutes after antigen addition. Add 1 ml of ice-cold PBS (+) to stop the reaction, and
Centrifugation was performed at 0 rpm for 10 minutes. 2 ml of the supernatant was taken, and 1 ml of 4 wt % perchloric acid solution was added thereto to prepare a sample for quantifying the amount of free histamine. The sample for quantifying the total histamine amount was an untreated mast cell suspension (IX 105 cells/ml).
0.8ml was placed in boiling water for 10 minutes, then 4wt%
Perchloric acid was added to prepare a sample.

The amount of histamine in each sample was measured by a fluorescence method, and the histamine release rate (%) was calculated using the following formula.

Histamine release rate (%) = (Free histamine amount/Total histamine amount) X100 formula [
Figure 2 shows the histamine release rate (%) of the peptide represented by I] and the comparative drug. As is clear from FIG. 2, the peptide represented by formula [I] exhibits an inhibitory effect on histamine release at a concentration of 10-6 M or higher, and this effect is similar to that of the comparative drug H-Asp-3er-ASp-PrO-Arg- O
It was stronger than H, and comparable or stronger than DSCG.

■E production of H-3er-As -Gl -L 5-0H ([I peptide)] The immunized animals were 5 BALB/C male mice (6 weeks old) per group, and the antigen dinitrophenyl aska Squirrel (DNP-
Ascaris) (10 μg) was adsorbed onto 4 mg of aluminum hydroxide gel, which is an immune enhancer, and the following two experiments were conducted.

In one experiment, 1 mg of the peptide of formula [I] was administered intraperitoneally, 30 minutes later DNP-Ascaris and aluminum hydroxide gel were intraperitoneally administered, and then blood was collected on the 14th day to obtain serum.

In the other experiment, DNP-Ascaris and aluminum hydroxide gel were administered intraperitoneally on days 7, 14, and 2.
On the 1st day, 1 mg of the peptide of formula CI was intraperitoneally administered three times in total, and on the 28th day, blood was collected to obtain serum.

The serum obtained in both experiments was subjected to a 48-hour rat PCA reaction, and the antibody titer was measured.

That is, Wistar male rats (200-250g
) was sensitized intradermally with serum on the back of the body, and 48 hours later,
A DNP-Ascaris solution containing wt% Evans blue was injected into the tail vein, and the pigment spots that appeared were measured 30 minutes later to determine the IgE antibody titer. In addition, in order to confirm that the antibody titer obtained by the PCA reaction is an IgE antibody,
Sensitization was performed using serum that had been previously heat-treated at 56° C. for 3 hours, and the antibody titer was measured by PCA reaction using the same procedure.

Ig when 1 mg/kg of the peptide of formula [I] was administered
Figures 3 and 4 show the amount of E antibody produced expressed by the antibody titer determined by PCA reaction. As is clear from FIGS. 3 and 4, the peptide of formula CI strongly suppressed IgE antibody production. The antibody titer of the heat-treated serum was almost 0 in one experiment (the shaded area in Figure 3), but it showed a slight antibody titer in the other experiment (the shaded area in Figure 4). .

[Effects of the Invention] The peptide represented by formula (1) of the present invention exhibits an inhibitory effect on histamine release and an IgE antibody production inhibitory effect. ADVANTAGE OF THE INVENTION According to the present invention, a novel peptide with excellent properties as an antiallergic agent could be provided.

[Brief explanation of the drawing]

Figure 1 shows H-Ala-3er-Gly-Lys of the present invention.
A high performance liquid chromatogram of -OH is shown. The vertical axis is 220
The intensity of ultraviolet absorption in nm, and the horizontal axis is the elution time (minutes). FIG. 2 shows H-Ala-3er-Gly-Ly of the present invention.
s-OH and comparative drug (H-Asp-Ser-Asp-
It is a graph showing the histamine release rate (%) of Pro-Arg-OH and DSCG). The vertical axis is histamine release rate (%), and the horizontal axis is compound and concentration (M). FIG. 3 shows H-Ala-3er-Gly-Ly of the present invention.
This is a graph showing the IgE antibody titer produced by pre-administration of s-OH, and the shaded area is the antibody titer of the heat-treated serum. FIG. 4 shows that H-Al of the present invention was used during the sustained period of IgE antibody production.
This is a graph showing the IgE antibody titer produced when a-Ser-Gly-Lys-OH was administered, and the shaded area is the antibody titer of the heat-treated serum. In Figures 3 and 4, the vertical axis represents the antibody titer, and the horizontal axis represents the compound and its dosage (
mg/kg).

Claims (1)

[Claims] 1. The following formula [ I ] H-Ala-Ser-Gly-Lys-OH [ I ] (
(Ala is an L-alanine residue, Ser is an L-serine residue, Gly is a glycine residue, and Lys is an L-lysine residue) or a pharmaceutically acceptable salt thereof. 2. An anti-allergic agent containing the peptide according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.