JPH08119991A - Peptide derivative and il-1 production inhibitor - Google Patents

Abstract

PURPOSE: To obtain a peptide derivative useful for treating inflammations, allergies, autoimmune diseases (chronic rheumatoid arthritis and systemic lupus erythematosus), endotoxin shock, bone cacochymia, connective-tissue disorder and ulcerative colitis, improving, alleviating and preventing symptoms, having inhibitory action on IL-1 production, low adverse effects and toxicity. CONSTITUTION: This peptide derivative, for example, is represented by a peptide of the formula, H-Met-Ala-Arg-Gly-Ser-Val-Ser-Asp-Glu-Glu-Met-Met-NH2 and has a series of inhibitory actions on IL-1 production. Its acid addition salt or a complex. An IL-1 production inhibitor contains the peptide derivative, etc., as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel peptide derivative, an acid addition salt and complex thereof, and an IL-1 production inhibitor containing the above as an active ingredient.

[0002]

IL-1 is known as an important biological substance that induces and transmits a systemic biological reaction to, for example, infection and inflammation, and has a strong antitumor activity itself [Hirai Y . et al., "Gann Monograph on Can
cer Research ", Japan Scientific Societies Press, To
(kyo (1988)] at the same time, it is recognized that it induces reactions that occur in the body during inflammation, such as fever, an increase in white blood cell count, activation of lymphocytes, and induction of acute phase protein biosynthesis in the liver. [Dinarello CA: Interleukin-1; Rev. Infect.
Dis., 6 , 51-95 (1984), Kluger, MJ, Oppenheim, J.
J. & Powanda, MC The Physiologic, Metabolic an
d Immunologic Actions of interleukin-1, Alan R. Li
ss, lnc, New York (1985)].

The biological effects of IL-1 are diverse,
Although it is considered to be an important biological substance for maintaining homeostasis in the living body, an abnormality occurs in the IL-1 production regulation function, and IL-1
When the production of
It is considered to cause various diseases. For example, in rheumatoid arthritis, which is one of the autoimmune diseases, it has been reported that there is a strong correlation between the degree of inflammation of joint synovium, the degree of bone destruction, and the expression level of HLA-DR antigen in synovial tissues. [Miyasaka, N., Sato. K., Goto, M., Sasano,
M., Natsuyama, M., Inoue, K. and Nishioka, K., Aug
mented interleukin-1 production and HLA- DR expres
sion in the synovium of rheumatoid arthritis patie
nt Arthritis Rheum, 31 , (4), 480-486 (1988)]. Further, it is said that excessive production of IL-1 causes inflammation, allergy, endotoxin shock, bone metabolism disorder, connective tissue disorder, ulcerative colitis and the like.

Therefore, it is considered that various physiological actions of IL-1 can be blocked by inhibiting excessive IL-1 release from cells.

Currently, glucocorticoid hormone is used as a therapeutic agent for chronic inflammatory diseases, and it is known that a part of its action is suppression of IL-1 production [Lew, W., Oppenheim. , JJ & Matsushima, K., Anal
ysis of the suppression of IL- 1 α and IL- 1 β prod
auction in human peripheral blood mononuclear adher
ent cells by a glucocorticoid hormone, J. Immunol.,
140 (6), 1895-1902 (1988)], but the glucocorticoid has a disadvantage of causing various serious side effects due to its various physiological actions.

[0006] Therefore, in the pharmaceutical industry, a new substance which has no side effects as seen with glucocorticoids, is highly safe in terms of other toxicity and side effects, and has high selectivity,
In particular, the present situation is desired in the field of treatment of chronic inflammatory diseases.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a new IL-1 production inhibitor which meets the above-mentioned needs in the pharmaceutical field and an active ingredient compound therefor.

[0008] In the process of earnestly researching the IL-1 production mechanism and substances involved in the IL-1 production mechanism from the above-mentioned object, the present inventor has identified a specific novel peptide derivative (acid addition salt and acid addition salt It has been found that (including a complex) has an excellent IL-1 production inhibitory action that meets the above-mentioned object, and thus completed the present invention.

[0009]

That is, according to the present invention, M
Provided are a peptide derivative having at least an amino acid sequence of et-Ala-Arg-Gly-Ser, an acid addition salt and a complex thereof, and an IL-1 production inhibitor containing at least one selected from the above. .

In the present specification, the abbreviations of amino acids, peptides and the like follow the conventions of IUPAC and IUB and symbols conventionally used in the field.

Representatives belonging to the above peptide derivatives according to the present invention include those represented by the following general formula (1).

R-Met-Ala-Arg-Gly-Ser- (X) n-R '(1) [wherein R is a hydrogen atom or an N-terminal protecting group, and R'is an amino group, a hydroxyl group or a C-terminal. A protecting group, X represents an amino acid residue or a peptide residue, and n is 0 or 1. In the above general formula (1), both the N-terminal protecting group defined by R and the C-terminal protecting group defined by R ′ are commonly used for protecting the N-terminal and C-terminal of a peptide or protein of this kind. It can be of various types. Typical examples thereof include N-terminal protecting groups such as methyl, ethyl, propyl, isopropyl,
Butyl, isobutyl, tert-butyl, pentyl, linear or branched lower alkyl groups such as hexyl group; methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl, tert-butoxycarbonyl,
Lower alkoxycarbonyl groups such as pentyloxycarbonyl and hexyloxycarbonyl groups; cyclopropyl,
Cycloalkyl groups such as cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl groups;
Lower alkanoyl groups such as acetyl, propionyl, butyryl, valeryl, pivaloyl, hexanoyl, heptanoyl groups; benzyl, 1-phenylethyl, 2-phenylethyl, 3-phenylpropyl, 4-phenylbutyl,
Phenyl lower alkyl groups such as 5-phenylpentyl and 6-phenylhexyl groups; halogen-substituted lower alkyl groups such as trifluoromethyl, pentafluoroethyl, heptafluoropropyl, nonafluorobutyl, undecafluoropentyl and tridecafluorohexyl groups Etc. can be illustrated.

The C-terminal protecting group is, for example, methoxy,
Lower alkoxy groups such as ethoxy, propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy;
Cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, cycloheptyloxy,
A cycloalkoxy group such as a cyclooctyloxy group; a linear or branched lower alkyl group described above; acetoxy, propionyloxy, butyryloxy, valeryloxy,
Lower alkanoyloxy groups such as pivaloyloxy, hexanoyloxy and heptanoyloxy groups;
Examples thereof include pyrrolidinyl group, piperidino group, morpholino group and the like.

In the general formula (1), the amino acid residue or peptide residue represented by X is, for example, Va.
1, Val-Ser, Val-Ser-Asp, Val
-Ser-Asp-Glu, Val-Ser-Asp-
Glu-Glu, Val-Ser-Asp-Glu-G
lu-Met or Val-Ser-Asp-Glu-G
lu-Met-Met and the like can be exemplified, and among them, in particular, Val-Ser-Asp-Glu-Glu-Met-M.
et is preferred.

Specific examples of the above-mentioned particularly preferred peptide derivative of the present invention include H-Met-Ala-Arg-Gly.
-Ser-Val-Ser-Asp-Glu-Glu-
Mention may be made of Met-Met-NH ₂ .

All of the derivatives of the present invention have an IL-1 production inhibitory action and are useful as an IL-1 production inhibitor. More specifically, the derivative of the present invention is the above-mentioned IL-
1. Treatment of various diseases caused by abnormal production of 1, such as inflammation, allergy, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, etc.), endotoxin shock, bone metabolism disorder, connective tissue disorder, ulcerative colitis, etc. It is useful for improvement, alleviation and prevention. In particular, a drug containing the derivative of the present invention as an active ingredient has the advantages of few side effects, extremely low toxicity, and excellent safety.

Further, the derivative of the present invention can be converted into a pharmaceutically acceptable acid addition salt by subjecting the derivative of the present invention to an addition reaction with an acid which is usually used according to a conventional method, and the acid addition salt is also in a free form. It has an excellent pharmacological action equivalent to that of the derivative, and is also useful as an active ingredient of the medicament of the present invention.

Specific examples of the acid capable of forming the pharmaceutically acceptable acid addition salt include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, formic acid, acetic acid, propionic acid and glycolic acid. , Lactic acid, pyruvic acid, oxalic acid, succinic acid,
Malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid,
Examples thereof include organic acids such as lower alkane sulfonic acid, benzene sulfonic acid, and toluene sulfonic acid.

Further, the derivative of the present invention can be made into a complex by reacting it with a certain inorganic compound or organic substance according to a usual method, and the complex is also the above-mentioned derivative (free form) or its acid addition salt. Similarly to, it can be used as an active ingredient of the medicament of the present invention. Examples of the inorganic compound capable of forming the complex include compounds derived from metals such as calcium, magnesium, cobalt and zinc, and more specifically, phosphates, pyrophosphates and polyphosphates of various metals. In addition to slightly soluble salts and hydroxides, etc., alkali metal polyphosphates and the like can be exemplified. Examples of the organic substance capable of forming the same complex include non-antigenic gelatin, carboxymethyl cellulose (CMC), sulfonic acid ester or phosphoric acid ester of alginic acid, dextran, polyalcohol phytic acid, polyglutamic acid, protamine and the like. .

The production of the derivative of the present invention will be described in detail below. Since the derivative is an oligopeptide, the derivative can be easily produced by various means commonly used for peptide synthesis. Most commonly, it can be produced by a so-called stepwise method in which individual amino acids are sequentially amide-bonded from their terminal amino acids according to the amino acid sequence of the derivative of the present invention. It can be carried out by any of the synthetic methods. In particular, it is convenient and preferable to use an automatic synthesizer that employs a solid phase synthesis method. Specific examples thereof are as described in detail in Examples below.

As the reagents and raw materials used in the above-mentioned production, those already on the market can be used, and if necessary, commercially available reagents can be appropriately derivatized according to a conventional method.

The peptide derivative of the present invention thus obtained can be easily isolated and purified from the reaction system by a conventional separation means. Examples of such means include means such as column chromatography, solvent extraction and salting out.

Further, according to the present invention, there is provided an IL-1 production inhibitor containing at least one selected from the above peptide derivatives, acid addition salts and complexes thereof as an active ingredient.

The IL-1 production inhibitor can be prepared and put into practical use in the same manner as other ordinary pharmaceuticals, except that it contains the above-mentioned active ingredient.

Preparation of the inhibitor (dosage unit form of pharmaceutical preparation) is carried out, for example, by a commonly used filler, bulking agent, binder,
It can be carried out according to a general method using a diluent or excipient such as a moisturizer, a disintegrant, a surface active agent, a lubricant and the like.

As the dosage unit form of the above pharmaceutical preparation of the drug of the present invention, various forms can be appropriately selected according to the purpose of treatment, and typical examples thereof include tablets, pills, powders, solutions, suspensions, Examples thereof include emulsions, granules, capsules, suppositories, injections (solutions, suspensions, etc.), ointments and the like.

In the case of molding in the form of tablets, as the above-mentioned pharmaceutical carrier, excipients such as lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, potassium phosphate, etc.,
Binders such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, methylcellulose, polyvinylpyrrolidone, sodium carboxymethylcellulose, carboxymethylcellulose calcium, low-substituted hydroxypropylcellulose, dried Starch, sodium alginate, agar powder, laminaran powder, sodium hydrogen carbonate, calcium carbonate and other disintegrants, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, surfactants such as stearic acid monoglyceride, sucrose, stearin, cocoa butter, Disintegration inhibitors such as hydrogenated oil, quaternary ammonium bases, absorption enhancers such as sodium lauryl sulfate, glycerin, starch, etc. Moisturizers, starch, lactose, kaolin, bentonite, adsorbent such as colloidal silicic acid, purified talc, stearates, boric acid powder, a lubricant such as polyethylene glycol can be used. Further, the tablet may be a tablet coated with a usual coating as necessary, for example, a sugar-coated tablet, a gelatin-coated tablet, an enteric-coated tablet, a film-coated tablet, a double tablet or a multi-layer tablet.

In the case of molding in the form of pills, pharmaceutical carriers such as glucose, lactose, starch, cocoa butter,
Excipients such as hardened vegetable oil, kaolin and talc, gum arabic powder, tragacanth powder, binders such as gelatin and ethanol, and disintegrants such as laminaran and agar can be used.

In the case of molding into a suppository, polyethylene glycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like can be used as a pharmaceutical carrier.

Capsules are usually prepared by mixing the active ingredient compound of the present invention with the various pharmaceutical carriers exemplified above and filling hard gelatin capsules, soft capsules and the like.

When the drug of the present invention is prepared as an injection such as a solution, emulsion or suspension, it is preferable that the drug is sterilized and isotonic with blood. As the diluent, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid ester, etc. can be used. In this case, a sufficient amount of sodium chloride, glucose or glycerin to prepare an isotonic solution may be contained in the drug of the present invention, and a usual solubilizing agent, buffer, soothing agent, etc. are added. You may.

Further, the drug of the present invention may contain a colorant, a preservative, a flavoring agent, a flavoring agent, a sweetening agent, and other pharmaceuticals, if necessary.

When the paste, cream, gel or the like is formed into an ointment, white diluents such as white petrolatum, paraffin, glycerin, cellulose derivatives, polyethylene glycol, silicone and bentonite can be used.

The amount of the active ingredient compound to be contained in the drug of the present invention is not particularly limited and is appropriately selected from a wide range, but it is usually preferable to contain about 1 μg to 100 mg in a pharmaceutical preparation. .

The administration method of the above-mentioned pharmaceutical preparation is not particularly limited, and it is determined according to various preparation forms, age of the patient, sex and other conditions, degree of disease and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules, and capsules are orally administered, and injections are intravenously administered alone or in a mixture with a normal replenishing solution such as glucose and amino acid. Independently, it is intramuscularly, intradermally, subcutaneously or intraperitoneally administered, and the suppository is intrarectally administered.

The dose of the above-mentioned pharmaceutical preparation is appropriately selected depending on the usage, age of the patient, sex and other conditions, degree of disease and the like, but usually the amount of the compound of the present invention as an active ingredient is 1.
The amount is preferably about 1 μg to 1000 mg per 1 kg of body weight per day, and the preparation can be administered in 1 to 4 divided doses per day.

The active ingredient selected from the peptide derivative of the present invention, its acid addition salt, and its complex is effective as the above-mentioned drug by utilizing it alone, and in addition to this, it further promotes usual protein absorption. It can be prepared into a pharmaceutical preparation form in which it is mixed with an agent, and can be administered to a patient in need of treatment therewith, and can also be administered in combination with a separately prepared protein absorption enhancer. It is possible to increase the absorption rate of the active ingredient and enhance the pharmacological effect.

Examples of such protein absorption promoters are preferably soybean trypsin inhibitor, leupeptin, antipain, aprotinin, nafamstat mesylate, gabexate mesylate, ulinastatin, N-α-paratosyl-L-lysine-chloromethyl. Trypsin inhibitors such as ketones [Life Science, 31 , 2837
(1982); Biochemical Pharmacology (Bioche
mical Pharmacology), 36 , 1035 (1987); History of Medicine, 138, 59 (1986), etc.] and chymotrypsin inhibitors such as chymostatin [JP-A-58-225080; The Journal of Biochemistry (J. Bioche).
m.), 95 , 319 (1984); The Journal of Biochemistry (J. Biochem.), 2 252 (1963); Journal
Of the American Chemical Society (J.Am.C
hem.Soc.), 93 , 2351 (1971); Journal of Pharmacy and Pharmacology (J.Pharm.Pharmc
ol.), 32 , 182 (1980); The Journal of Antibiotocs, 23 , 425 (197
0)] and the like.

[0039]

EXAMPLES In order to explain the present invention in more detail, examples of production of the peptide derivative of the present invention will be given below as examples, and then pharmacological test examples for the IL-1 production inhibitory action performed on the derivatives will be given. Invention Formulation examples of the IL-1 production inhibitor are given below.

The following examples show one embodiment of the present invention, and the present invention is not limited to these examples. Various modifications are possible without departing from the gist of the present invention. Of course, it can be implemented in the form of

Example 1 Automatic synthesizer manufactured by Pharmacia LKB (4175 BIOL
YNX Peptide Synthesizer), set the appropriate reagents and operate according to the program, take out the solid phase resin from the synthesizer, put it in the HF reaction tube and extract the product. It was freeze-dried and the resulting crude product was purified by reverse phase HPLC to give the peptide of the formula below.

H-Met-Ala-Arg-Gly-S
er-Val-Ser-Asp-Glu-Glu-Me
t-Met-NH _{2 The} obtained peptide was found to have a molecular ion peak M / Z = 1341.4 (calculated value = 134) as a result of mass spectrum analysis.
1.6).

Further, the above-mentioned HPL carried out under the following conditions:
The elution pattern of C is shown in FIG.

Column: manufactured by Yamamura Chemical Co., Ltd. (YMC-PACK
ODS-A302) Sample: 20 μl of an aqueous solution with a concentration of 1 mg / ml Eluent: A solution: 10% trifluoroacetic acid (TFA): water = 1: 100 Solution B: 10% TFA: acetonitrile: water = 1:60:
40 Concentration gradient: The mixing ratio of solution B to solution A was 0 in 30 minutes
It was changed linearly from 100% to 100%.

Flow rate: 1 ml / min Chart speed: 12 cm / min Detection: UV220 nm Further, Table 1 shows the results of amino acid analysis of the obtained peptides (average value of 3 times based on Ala).

[0046]

[Table 1]

Pharmacological Test Example 1 IL-1 Production Inhibition Test Female C3H / He mice were intraperitoneally administered with 2 ml of thioglycollate medium, and 4 days later, peritoneal exudate cells were treated with RPMI.
Recovered with 1640 medium (Nissui Pharmaceutical Co., Ltd.) and RPM
It was washed twice with I1640 medium (manufactured by the same company).

1 × 10 ⁵ cells of the above were added to a culture medium [RPM
I1640 medium (manufactured by the same company) + 10% FBS (manufactured by Hyclone)] at 37 ° C., and 2 hours later, a predetermined amount of the peptide aqueous solution obtained in Example 1 and 1 μg / ml of lipopolysaccharide ( Difco), and add 3 more
It was cultured at 7 ° C. for 20 hours.

Thereafter, the amount of IL-1 in the culture solution was measured using a measurement kit (IL-1 ELISA Kit, manufactured by Genzyme), and the amount of IL-1 when only lipopolysaccharide was added to the culture was controlled. As a value, I
The L-1 production suppression rate was calculated by the following formula.

IL-1 production inhibition rate (%) = [1- (measured value) / (control value)] × 100 The results are shown in Table 2.

[0051]

[Table 2]

Formulation Example 1 Peptide 1 g obtained in Example 1 and purified gelatin 200
After dissolving mg, mannitol 1 g, sodium chloride 70 mg and sodium phosphate 150 mg in 20 ml of distilled water for injection and dispensing into 10 vials, a freeze-drying operation consisting of conventional pre-freezing, primary drying and secondary drying Then, a freeze-dried preparation for intravenous injection was obtained.

[Brief description of drawings]

FIG. 1 HPL of the peptide derivative of the present invention according to Example 1.
The elution pattern by C is shown.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication location A61K 38/00 ABE ABF ABG ABN ADD AED C07K 7/08 8318-4H A61K 37/02 ABE ABF ABG ABN ADD AED

Claims (6)

[Claims] 1. Met-Ala-Arg-Gly-Se
a peptide derivative having at least the amino acid sequence of r,
An acid addition salt or complex thereof. 2. The general formula R-Met-Ala-Arg-Gly-Ser- (X).
n-R '[wherein R is a hydrogen atom or an N-terminal protecting group, R'is an amino group, a hydroxyl group or a C-terminal protecting group, X is an amino acid residue or a peptide residue, and n is 0 or It is 1. ] The peptide derivative of Claim 1 represented by these, its acid addition salt or complex. 3. n is 1, X is Val, Val-Se.
r, Val-Ser-Asp, Val-Ser-Asp
-Glu, Val-Ser-Asp-Glu-Glu,
Val-Ser-Asp-Glu-Glu-Met and Val-Ser-Asp-Glu-Glu-Met-M
The peptide derivative according to claim 2, which is any of et, or an acid addition salt or complex thereof. 4. n is 1 and X is Val-Ser-Asp.
-Glu-Glu-Met-Met, The peptide derivative according to claim 3, an acid addition salt or complex thereof. 5. H-Met-Ala-Arg-Gly-
Ser-Val-Ser-Asp-Glu-Glu-M
The peptide derivative according to claim 4, which is et-Met-NH ₂ , its acid addition salt or complex. 6. An IL comprising at least one selected from the peptide derivative according to any one of claims 1 to 5, an acid addition salt and a complex thereof as an active ingredient.
-1 production inhibitor.