JPH03123796A - Novel hexapeptide, its salt and anti-allergic agent - Google Patents

Abstract

NEW MATERIAL:The hexapeptide (salt) expressed by formula (Ala is L-alanine residue; Ser is L-serine residue : Asn is L-asparagine residue ; Pro is L-proline residue; Gly is glycine residue; Lys is L-lysine residue). USE:An antiallergic agent. PREPARATION:The objective hexapeptide of formula is produced e. g. by using chloromethyl resin, etc., as a solid-phase carrier of a solid-phase synthetic process, synthesizing a peptide chain by the dehydrative condensation of (A) a raw material having reactive carboxyl group and corresponding to one of two kinds of fragments obtained by dividing a hexapeptide into two at an arbitrary position of a peptide bond according to the amino acid sequence of the hexapeptide and (B) a raw material having reactive amino group and corresponding to another fragment, desorbing the peptide from the resin and removing the protecting group.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel hexapeptide, a salt thereof, 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 I 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 mediators) 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 an IgE 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, H-Ala-8er-Asn-Pro-Gly-Ly
The present invention was completed based on the discovery that a hexapeptide represented by s-OH suppresses histamine release and IgE antibody production.

That is, the present invention provides: (1) The following formula CI) H-Ala-Ser-Asn-Pro-Gly-Lys
-OH[I] (where A1a is an L-alanine residue,
ser is L-serine residue, Asn is L-asparagine residue, Pro is L-proline residue, Gly is glycine residue, Lys is L-lysine residue) or its pharmaceutical Salt allowed in.

(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.

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

The hexapeptide 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 hexapeptide 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 hexapeptide 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 hexapeptide 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, poultices, 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.

In the case of 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 10 mg per day.
/kg, intranasal dosage is 0.1-100mg
It is. For parenteral administration, the dose is 10 to 1 per day.
, 000 μg/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 G1y nigericine residue Asn: L-asparagine residue Pro: L-proline residue Lys: L-lysine residue Boc: t-butyloxycarbonyl group 2: benzyl Oxycarbonyl group Z (2CI) + 2-chlorobenzyloxycarbonyl group PAM: 4-(oxymethyl)phenylacetamidomethyl TFA nitrifluoroacetic acid DCC dicyclohexylcarbodiimide H-Ala-
The 8 peptide synthesizer for Ser-Asn-Pro-Gl-L 5-OH is a Beckman peptide synthesizer Mo.
de1990B was used. 1.50g of Boa in the reaction tank
-Lys[Z(2CI))-PMA resin CBoc-Ly
s (Z(2C1)) containing 0.7 mmol 7g,
(manufactured by Applied 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 with 20 ml of methanol for 2 minutes/3 times.

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

(4) Washed 71 times for 5 minutes with 45 wt% TFA and 20 ml of dichloromethane.

(5) 1 with 5 wt% TFA and 20 ml of dichloromethane
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 Jig 00 methane.

(10) Wash with 20 ml of dichloromethane 73 times for 2 minutes.

(11) A solution of 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 73 times for 2 minutes.

(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-F'ro-OH4, Z mmol Boc-Asn
-01(4, Z mmol Boc-8er (OBzl)-
0H4, 2 mmol Boc-Ala-OH4, 2 mmol or more When all the operations are completed, the next protected peptide is synthesized on the resin.

BoC-Ala-Ser(OBzl)-Asn-Pro
-Gly-Lys CZ(2CI))-resin The protected peptide conjugated onto this resin was prepared as described in the previous procedure (1).
After performing ~(14), it was filtered and dried in a desiccator overnight. 690 mg of dried protected peptide resin was obtained.

Take this, add 2 ml of anisole, dimethyl suberimidate dihydrochloride (dimethyl-suberimid),
ate dihydrochloride) 0.5m
The mixture was treated with 30 ml of hydrogen fluoride for 1 hour at 0°C in the presence of 1 hour. The fluorine hydride was distilled off and anhydrous ether/n-hexane (
After washing with a mixed solution (volume ratio 1:1) and then with anhydrous ether and thoroughly drying, the peptide was dissolved in 50 ml of 10 wt % acetic acid to remove the insoluble resin.

The resulting solution was Dowex LX-2
The mixture was applied to a column (1, OX15 cm, manufactured by Dow Chemicals), eluted with 2N acetic acid, filtered through a 0.22 μm Millipore filter, and lyophilized. 163 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 a linear gradient up to 0% mixture: l, Oml/min 17 mg of peptide with a purity of 98% was obtained by 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 0.9 Aspartic acid 1.1 Proline 1.1 Glycine 1.0 Lysine 1.2 In addition, high-speed analysis Liquid chromatography uses KASEISORB C8-300x as a column.
5 (manufactured by Tokyo Kasei Kogyo, 4.6 x 250 mm), and 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) A solution with a linear gradient was used, the flow rate was 1.0 ml/min, and the detection wavelength was 210 nm. After CI treatment for 5 hours, amino acids were analyzed using a Hitachi amino acid analyzer model 835.

Next, hexapeptide H-Ala-S represented by formula [1]
The results of a pharmacological activity test of er-Gly-Asn-Pro-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, NiLP, 681 (1964)
] and Hamaoka's method [J, Immunol
ogy, 113. ．． 958 (1974)]. That is, ovalbumin (10 mg/kg
) was injected at 5 ml/kg into both thigh muscles of male Wistar rats (body weight 200-250 g), and at the same time 2
loIO Killed Bordet
ellaPertussis) 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 is 120
Stored frozen at ℃. Antiserum titer was 48hr rat PC
It was measured by the A reaction, and those with a titer of 128 to 256 times were used for the experiment. Obtained ovalbumin rat Ig
E 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 NaCl, 0.2 KCI).
gNazHP0442HzO2, 88g, KH2PO
40.2 g, 1 liter solution of 92 g of EDTA/2NaO and 1 g of bovine serum albumin dissolved in purified water, pH 7.4, hereinafter abbreviated as PBS (-)) 15 m
1 was injected, the abdomen was gently massaged for about 2 minutes, the abdomen was opened, and intraperitoneal cells were collected.

This cell suspension was centrifuged (1,00 rpm, 10
minutes), 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 (-).
), CaC1z 0.1 was used instead of EDTA/2Na.
(abbreviated as PBS(+))] and adjusted to 1×105 cells/ml, then dispensed 0.8 ml each of the cell suspension into silicone-treated test tubes.
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 /mg) was added and incubated for an additional 15 minutes to release histamine. however,
DSCG, one of the comparative drugs, was added 30 seconds before antigen addition.
After antigen addition, incubation was continued for an additional 15 minutes. Add 1 ml of water-cooled PBS (+) to stop the reaction, and
Centrifuged at Orpm for 10 minutes. Take 2ml of the top and
1 ml of 4 wt % perchloric acid solution was added to prepare a sample for quantifying the amount of free histamine.

A sample for quantifying the total amount of histamine was obtained by placing 0.8 ml of an untreated mast cell suspension (IXIO' cells/ml) in boiling water for 10 minutes, and then adding 4 wt % perchloric acid.

The histamine content of 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 i) 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 histamine release inhibiting action at a concentration of 10-6 M or higher, and this action is similar to that of the comparative drug H-Asp-Ser-Asp-Pro-ARg- O
Stronger than H (, I) It was as strong as or stronger than SCG.

The immunized animals were BALB/c male mice (6 weeks old), 5 mice per group, and the antigen dinitrophenyl ascaris (DNP-
Ascaris) 10 μg was adsorbed onto 4 mg of aluminum hydroxide gel, an immunostimulant, and two experiments shown below were conducted.

In one experiment, 1 mg of the peptide of formula [1] 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.
1 mg of the peptide of formula [1] was intraperitoneally administered three times on the first day, and blood was collected on the 28th day 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 to 25 CI
g) Serum is sensitized intradermally on the back of the patient, and 48 hours later, 0.5
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 [I] strongly suppressed IgE antibody production. The antibody titer of the heat-treated serum was almost O in one experiment (shaded area in Figure 3), but it showed a small antibody titer in the other experiment (shaded area in Figure 4). .

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

[Brief Description of the Drawings] Figure 1 shows the H-Ala-Ser-Asn-Pro of the present invention.
An analytical high performance liquid chromatogram of -Gly-Lys-OH is shown. The vertical axis is the intensity of ultraviolet absorption at 220 nm, and the horizontal axis is the elution time (minutes). FIG. 2 shows H-Ala-5er-Asn-Pr of the present invention.
o-Gly-Lys-0) 1 and comparative drug (H-Asp
-Ser-Asp-Pro-Arg-OH and DSCG
) is a graph showing the histamine release rate (%). The vertical axis is histamine release rate (%), and the horizontal axis is compound and concentration (
M). FIG. 3 shows H-Ala-5er-Asn-Pr of the present invention.
This is a graph showing the IgE antibody titer produced by pre-administration of o-Gly-Lys-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-Asn-Pro-Gly-Lys-OH was administered, and the shaded area is the antibody titer of the heat-treated serum. In FIGS. 3 and 4, the vertical axis shows the antibody titer, and the horizontal axis shows the compound and its dosage (mg/kg).

Claims (1)

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