CH624093A5 - Process for the preparation of biologically active polypeptides - Google Patents

Description

The present invention relates to a process for the preparation of new, low molecular weight polypeptides which are suitable as therapeutic agents in the treatment of allergic diseases or the allergic syndrome.

The present method enables the production of polypeptides with 3-10 amino acid residues that can block an allergic response in humans. These relatively short-chain polypeptides correspond to sequences (Se-5 sequences) which occur in the second (C-2), third (C-3) and fourth (C-4) domains of the constant (Fe) region of the (s) -peptide- chain of the IgE molecule.

The amino acid sequence of the entire e-chain has recently been determined by Bennich et al and in «Progress in Im-io munology II-Bd. 1: Immunochemical Aspects », July 1974, pages 49-58, North Holland Publishing Company, Amsterdam, (1974). The sequence of the Fe region in which the amino acid sequences obtainable according to the invention occur is as follows, the numbers on the edge indicating the 15 numerical position in the sequence of the amino acid to the right of it:

265 - (Met) -Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu-Ser-Glu-Gly-Glu-Leu-Ala-Ser-Thr-Glu-Ser-Glu-Leu- Thr-

20 289 - Leu-Ser-GIn-Lys-His-Trp-Leu-Ser-Asp-Arg-Thr-Tyr-Thr-Cys-Val-Thr-Tyr-Glx-Gly-His-Thr-Phen-Glx -

313 - Asx-Ser-Thr-Lys-Lys-Cys-Ala-Asp-Ser-Asp-Pro-Arg-Gly-Val-Ser-Ala-Tyr-Leu-Ser-Arg-Pro-Ser-Pro-Phe-

25 337 - Asp-Leu-Phe-Ile-Arg-Lys-Ser-Pro-Thr-Ile-Thr-Cys-Leu-Val-Val-Asx-Leu-Ala-Pro-Ser-Lys-Gly-Thr-Val -

361 - Asn-Leu-Thr-Trp-Ser-Arg-Ala-Ser-Gly-Lys-Pro-Val-Asx-His-Ser-Thr-Arg-Lys-Glu-Glu-Lys-Gln-Arg-Asn-

in

385 - Gly-Thr-Leu-Thr-Val-Thr-Ser-Thr-Leu-Pro-Val-Gly-Thr-Arg-Asx-Trp-IIe-Glu-Gly-Glu-Thr-Tyr-Glx-Cys-

409 - Arg-Val-Thr-His-Pro-His-Leu-Pro-Arg-Ala-Leu-Met-Arg-Ser-Thr-Thr-Lys-Thr-Ser-Gly-Pro-Arg-Ala-Ala-

433 - Pro-Glu-Val-Tyr-Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-Gly-Ser-Arg-Asp-Lys-Arg-Thr-Leu-AIa-Cys-Leu-Ile-

457 - Gln-Asn-Phe-Met-Pro-GIu-Asp-Ile-Ser-Val-GIn-Trp-Leu-His-Asn-Glu-Val-Gln-Leu-Pro-Asp-Ala-Arg-His-

40

481 - Ser-Thr-Thr-Glu-Pro-Arg-Lys-Thr-Lys-Gly-Ser-Gly-Phe-Phe-Val-Phe-Ser-Arg-Leu-Glu-Val-Thr-Arg-Ala-

505 - Glu-Trp-Gln-Glu-Lys-Asp-Glu-Phe-Ile-Cys-Arg-Ala-Val-His-Glu-Ala-AIa-Ser-Pro-Ser-GIn-Thr-Val-GIn-

45

529 - Arg-Ala-Val-Ser-Val-Asn-Pro-Gly-Lys.

The new compounds obtainable according to the invention are polypeptides of 3-10 amino acids in a sequence which is selected from a part of the above amino acid sequence, as well as their salts, esters, amides, N-acyl and O-acyl derivatives.

As already stated, the usual abbreviations 55 for the various amino acids, which are known to the person skilled in the art, are used to simplify the description of the present invention. For better clarity, however, the compounds in question are listed below. All chiral amino acid residues mentioned here have the natural or 60 L configuration, unless stated otherwise. All peptide sequences listed here are written in the usual way, whereby the N-terminal amino acid is on the left and the C-terminal amino acid on the right.

65 Asp - Aspartic Acid Ala - Alanine Arg - Arginine

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Asn - asparagine

Asx - aspartic acid or asparagine

(shows uncertainty in the decomposition analysis)

Cys - cysteine

Gly - glycine

Gin - glutamine

Glx - glutamic acid or glutamine

(shows uncertainty in the decomposition analysis)

His - histidine

Ile - isoleucine

Leu - leucine

Lys - lysine

Met - methionine

Phe - phenylalanine

Pro - proline

Ser - serine

Thr - threonine

Trp - tryptophan

Tyr - tyrosine

Val - valine

The term "salts" used here refers both to salts of a carboxyl group of the polypeptide chain and to acid addition salts of an amino group of the polypeptide chain. Salts of a carboxyl group can be formed with inorganic or organic bases. Inorganic salts include e.g. the alkali metal salts such as the sodium, potassium and lithium salt; Alkaline earth metal salts such as the calcium, barium and magnesium salt; iKid the ammonium, ferro, ferric, zinc, mangano, aluminum, manganese salt, etc. salts with organic amines include those e.g. with trimethylamine, triethylamine, tri- (n-propyl) amine, dicyclohexylamine, ß- (dimethylamino) ethanol, tris - (hydroxymethyl) aminomethane, triethanolamine, ß- (diethylamino) ethanol, arginine, lysine , Histidine, N-ethylpiperidine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazines, piperidines, caffeine, procain etc.

Acid addition salts include e.g. Salts with mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc .; and salts with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, etc.

The term "ester" used here refers to esters of a carboxyl group of the polypeptide, formed with straight or branched chain saturated aliphatic alcohols with 1-12 C atoms, such as the methyl, ethyl, n-propyl, isopropyl, n- Butyl, tert-butyl, n-amyl, n-hexyl, octyl, decyl and dodecyl esters.

The term “amides” used here refers to amides of a carboxyl group of the polypeptide, formed with ammonia or with primary or secondary amines with up to 12 C atoms, such as dimethylamiri, di-ethylamine, di- (n-butyl) amine, n-hexylamine, piperidine, pyrrolidine, morpholine, di- (n-hexyl) amine, N-methylpiperazine, etc.

The «N-acyl derivatives» refer to derivatives of an amino group of the polypeptide, formed with acyl parts (e.g. an alkanoyl or carbocyclic aroyl group) with up to 12 C atoms, such as formamides, acetamides, benzamides, etc.

The «O-acyl derivatives» refer to derivatives of a hydroxyl group of the polypeptide chain, formed with acyl parts (e.g. alkanoyl or carboxycyclic aroyl groups) with up to 12 C atoms, such as formates, acetates, propionates, benzoates etc.

Preferred polypeptides obtainable according to the invention have amino acid sequences which are not analogous to comparable regions in other immunoglobulins. In this regard, the following peptides can be specifically mentioned:

266 - Asp-Val-Asp-Leu-Ser

271 - Thr-Ala-Ser-Thr-Glu

266 - Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu

289 - Leu-Ser-Gln-Lys-His

319 - Ala-Asp-Ser-Asp-Pro-Arg

320 - Asp-Ser-Asp-Pro-Arg

321 - Ser-Asp-Pro-Arg

322 - Asp-Pro-Arg

354 - Ala-Pro-Ser-Lys-Gly-Thr

367-Ala-Ser-Gly-Lys-Pro

437 - Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-Gly-Ser

437 - Ala-Phe-Ala-Thr-Pro

442 - GIu-Trp-Pro-Gly-Ser

476 - Pro-Asp-Ala-Arg-His-Ser

521 - Ala-Ser-Pro-Ser-Gln and their salts, esters, amides, N-acyl and O-acyl derivatives.

A particularly preferred polypeptide is Asp-Ser-Asp-Pro-Arg.

The above list is not intended to be exhaustive, and other peptides with a shorter sequence than above or with sequences that have additional amino acids or sequences from other regions of the C-2, C-3 or C-4 s domain are important.

Such a polypeptide can thus be used to prevent or relieve symptoms associated with allergic manifestations, such as e.g. caused by antigen-antibody (allergic) reactions. It blocks (i.e. inhibits or prevents) the effects of the allergic reaction when administered in an effective amount.

As described above, the compounds presumably act by “blocking” the IgE binding sites.

The polypeptides are advantageously used in the form of pharmaceutical preparations for blocking (i.e. preventing or inhibiting) the effects of allergic reactions; the preparations contain an effective amount of a polypeptide or a derivative thereof described above in admixture with a pharmaceutically acceptable, non-toxic carrier.

For prophylaxis or treatment, an effective amount of a polypeptide or a derivative thereof or a pharmaceutical preparation containing it should be given in

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Customary, recognized way to be administered individually or in combination with other pharmaceutical agents such as antihistamines, corticosteroids, etc. Thus, the compounds or preparations can be administered orally, sublingually, topically (ie on the skin or in the eyes), parenterally (e.g. intramuscularly, intravenously, subcutaneously or intradermally) or by inhalation and in the form of solid, liquid or gaseous doses including tablets, suspensions and aerosols are administered. The administration can take place in continuous therapy with single dosage forms or ad libitum in single dose therapy.

Based on the above information and taking into account the extent or severity of the disease to be treated, the age of the patient, etc. - factors that can be determined by the expert through routine experiments - the effective dose can vary over a wide range. Since individual patients vary in their IgE content, an effective systemic dose is considered to be between the 2 X 10 :! up to 2 X 106 times the IgE content, on a molar basis. For an average patient, this would be between about 0.5-500 mg / kg / day depending on the strength of the compound. Of course, for localized treatment, e.g. of the respiratory system, proportionally less material needed.

Suitable pharmaceutical carriers for the preparation of the preparations can be solids, liquids or gases; The preparations can take the form of tablets, pills, capsules, powders, enterai-coated or other protected formulations (for example by binding to ion exchange resins or other carriers, by packaging in lipid-protein vesicles or by adding additional terminal amino acids or replacing a terminal amino acid in the L-form by one in the D-form), depot formulations, solutions (e.g. eye drops), suspensions, elixirs, aerosols etc. The carrier can be made from various oils including those of petroleum, as well as of animal, vegetable or synthetic origin, e.g. Peanut oil, soybean oil, mineral oil, sesame oil, etc. can be selected. Water, saline, aqueous dextrose and glycols are preferred as liquid carriers, especially (if isotonic) for injectable solutions. Suitable pharmaceutical extenders include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dry skimmed milk, glycerin, propylene glycol, water, ethanol, etc. Die Preparations can be subjected to the usual pharmaceutical measures, such as sterilization, and contain the usual pharmaceutical additives, such as preservatives, stabilizers, wetting agents or emulsifiers, salts for adjusting the osmotic pressure, buffering agents, etc. Suitable pharmaceutical carriers and their formulation are described in "Remington's Pharmaceutical Sciences" by E.W. Martin described. In any case, the preparations contain an effective amount of the active compound together with a suitable carrier amount to produce the correct dosage form for the appropriate administration to the patient.

In order to be effective in preventing or treating allergic reactions, it is important that the therapeutic agents be relatively non-toxic, non-antigenic and non-irritating at the concentrations of their actual use. As has been found, this is the case for all compounds whose preparation is described below.

The method according to the invention is defined in claim 1.

An excellent summary of the many methods available for condensation can be found in J. Meienhofer “Hormonal Proteins and Peptides”, Vol. 2, page 46, Academic Press, New York (1973) for peptide-5 synthesis in the solid phase, and in E. Schröder and K. Lübke "The Peptides", Vol. 1, Academic Press, New York (1965) for classical synthesis in solution.

These methods usually involve the sequential addition of one or more amino acids or correspondingly protected amino acids to the growing chain. Either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or converted amino acid can then either be bound to an inert solid support or in solution by adding the next amino acid in order with an appropriately protected, complementary (amino or carboxyl) group under suitable conditions to form the amide bond be used. Then the 2o protecting group is removed from this newly added amino acid residue, whereupon the next (appropriately protected) amino acid is added, etc. After all the desired amino acids have been bound in the correct order, any remaining protecting groups (and all 25 solid supports) become sequentially or simultaneously Removal of the final polypeptide. By simply modifying this general procedure, more than one amino acid can be added to a growing chain at the same time, e.g. by coupling (under conditions that do not racemize chiral centers 30) a protected tripeptide with an appropriately protected dipeptide to obtain a pentapeptide after removal of the protecting groups.

The protecting groups should be stable against the conditions of peptide bond formation and at the same time be easily removable without destroying the growing peptide chain or racemizing one of the chiral centers contained therein.

The classes of protective groups for amino groups in the step-by-step synthesis of polypeptides include (1) 40 acyl-like protective groups, such as formyl, trifluoroacetyl, phthalyl, toluenesulfonyl (tosyl), benzenesulfonyl, o-nitrophenylsulfenyl, tritylsulfenyl, o-chlorophenoxyacetyl tyl, acetyl, y-chlorobutyryl, etc .; (2) aromatic urethane-like protecting groups such as benzyloxycarbonyl and sub-45 substituted benzyloxycarbonyl, e.g. p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, 2- (p-biphenylyI) isopropyloxycarbonyl, 2-benzoyl-l-methylvinyl; (3) aliphatic urethane-like protective groups such as tert-butyloxycarbonyl, tert-50 amyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, allyloxycarbonyl; (4) cycloalkyl urethane type protecting groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl; (5) thiourethane-like protecting groups such as phenylthio-55 carbonyl; (6) alkyl type protecting groups such as triphenylmethyl (trityl) and benzyl; and (7) trialkylsilyl groups such as trimethylsilyl.

Preferred protecting groups are tert-butyloxycarbonyl (t-BOC) and tert-amyloxycarbonyl (AOC).

S3 The classes of carboxy protecting groups suitable for the step-wise synthesis of polypeptides include (1) substituted and unsubstituted aliphatic ester groups, such as methyl, ethyl, tert-butyl, 2,2,2-trichloroethyl and tert-butyl esters; (2) aralkyl ester groups such as benzyl, p-Ni-65 trobenzyl, p-methoxybenzyl, diphenylmethyl or tri-pheny! Methyl (trityl) esters; (3) N-substituted hydrazides such as tert-butyloxycarbonylhydrazide and carbobenzyloxycarbonylhydrazide; (4) amide protecting groups formed by

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Condensation of a carboxyl part with e.g. Ammonia, methylamine, ethylamine, diphenylmethylamine, etc.

Hydroxyl groups in amino acids such as in serine, threonine and hydroxyproline can be used as aralkyl ethers, e.g. Ben-cyl ether, protected.

Suitable as solid supports for the above synthesis are materials which are inert to the reactants and reaction conditions of the stepwise condensation and deprotection reactions and are insoluble in the media used. Usable materials include e.g. crosslinked polystyrene divinylbenzene resins, crosslinked polyamide resins, polyethylene glycol resins, correspondingly functionalized glass beads, etc.

The first amino acid residue is bound to the solid support by forming a covalent bond with an active group on the resin. Active groups suitable for this purpose include e.g. Chloromethyl, benzhydril-amino, hydroxymethyl, phenacyl halide, dehydroaia-nin, etc., with chloromethyl being preferred as the active group. The first amino acid is bound to the preferred chloromethyl resin by a base catalyzed process in which the triethylamine, tetramethylammonium or cesium salt (or a similar salt) of the carboxylic acid with the resin in a solvent such as ethanol, dioxane, dimethylformamide etc., is heated.

Suitable reactants for amide formation between carboxyl and amino groups are known and include e.g. (1) carbodiimides such as dicyclohexylcarbodiimide (DCC); (2) a carbodiimide plus an additive such as 1-hydroxybenzotriazole or ethyl 2-hydroximino-2-cyanoacetate; (3) alkyl chloroformates such as isobutyl chloroformate or ethyl chloroformate; (4) N-protected amino acids activated by the formation of a suitable ester, e.g. substituted phenyl esters, aryl or alkyl thioesters, substituted 8-hydroxyoxyquinoline esters, 2-thiopyridyl esters and similar known esters.

A preferred method for producing the peptides is the so-called “Merrifield” method, which is known and is described in detail by R.B. Merrifield in "Synthesis of a Tetrapepti-de", J. Am. Chem. Soc., Vol. 85, pages 2149-2154 (1963), and by the above-mentioned Meienhofer publication.

In this preferred method, a peptide of any desired length and sequence is made by the stepwise addition of amino acids to a growing peptide chain which is covalently bound to a solid resin particle.

In the preferred application of this method, the C-terminus of the growing peptide chain is covalently bound to a resin particle, and amino acids with protected amino groups are gradually added in the manner outlined above. A preferred group protecting the amino group is the t-BOC group, which is stable under the condensation conditions and yet is easily removable without destroying the peptide bonds or racemization of the chiral centers in the peptide chain. At the end of the process the final peptide is cleaved from the resin and any remaining protective groups are removed by treatment under acidic conditions e.g. removed with a mixture of hydrobromic acid and tri-fluoroacetic acid or with hydrofluoric acid;

or the cleavage from the resin can be carried out under basic conditions, e.g. with triethylamine, the protective groups then being removed under acidic conditions.

The cleaved peptides can be isolated and purified in a known manner, e.g. by lyophilization and subsequent exclusion or division chromatography on polysaccharide gel media, such as «Sephadex G-25», or countercurrent distribution. The composition of the final peptide can be confirmed by amino acid analysis after degradation of the peptide by standard measures.

Salts of the carboxyl groups of the peptide can be conveniently prepared by contacting the peptide with one or more equivalents of a desired base, such as a basic metal hydroxide, e.g. Sodium hydroxide, a metal carbonate or bicarbonate, such as sodium carbonate or sodium bicarbonate, or an amine base, such as triethylamine, triethanolamine, etc., can be produced.

Acid addition salts of the polypeptides can be obtained by contacting the polypeptide with one or more equivalents of the desired inorganic or organic acid, e.g. Hydrochloric acid.

Esters of carboxyl groups of the polypeptides are prepared in the usual way by converting a carboxylic acid or a precursor into an ester. A preferred method of making esters of the polypeptides using the Merrifield synthetic method described above is to cleave the complete polypeptide from the resin in the presence of the desired alcohol under basic or acidic conditions, depending on the resin. Thus, the C-terminal end of the peptide is esterified directly, without isolation of the free acid, after being freed from the resin.

Amides of the polypeptides are also prepared according to the usual manner for converting a carboxylic acid group or a precursor into an amine. A preferred method for amide formation at the C-terminal carboxyl group is cleavage of the polypeptide from a solid support with a corresponding amine or cleavage in the presence of an alcohol, which leads to an ester, and the subsequent aminolysis with the desired amine.

The N-acyl derivatives of an amino group of the polypeptides are prepared using an N-acyl protected amino acid for the final condensation or by acylation of a protected or unprotected peptide. The O-acyl derivatives are produced by O-acylation of a free hydroxypeptide or peptide resin. Any acylation can be carried out using conventional acylating agents, such as acyl halides, anhydrides, acylimidazoles, etc. If necessary, N- and O-acylation can be carried out together.

The coupling, deprotection / cleavage reactions and the preparation of derivatives of the polypeptides are conveniently carried out at temperatures between about -10 ° C and + 50 ° C, preferably about 20-25 ° C. Of course, the exact temperature for each particular reaction will depend on the substrates, reactants, solvents, etc., as is known to those skilled in the art. Examples of reaction conditions for these processes can be found in the examples.

The following examples illustrate the present invention.

Example 1 Preparation of the tripeptide Asp-Pro-Arg

1.6 g (5 millimoles) of t-BOC-nitroarginine was mixed with 10 g of chloromethyl resin (beads of copolystyrene -2% divinyl-benzene with 0.5-1 meq of chloromethyl groups per g of resin) in a mixture of 1.4 ccm (10 Millimoles) of triethylamine and 100 cc of ethanol for 24 hours at 22 ° C with constant stirring. The arginimene resin was then thoroughly washed in succession with acetic acid, abs. Washed ethanol, water with increasing amounts of ethanol, methanol and finally with methylene chloride. Then that became

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Resin thoroughly dried under vacuum. Analysis showed 0.05 millimoles Arg / g resin. 2.5 g of the resin so prepared was placed in a Merrifield solid phase reaction vessel equipped with stirring means and subjected to the following deprotection cycle:

(a) With stirring, the t-BOC group was cleaved with 10 ccm 4N HCl in dioxane at 22 ° C. for 30 minutes

(b) Washed twice with 10 cc dioxane

(c) Washed twice with 10 cc methylene chloride

(d) Washed twice with 10 cc chloroform

(e) the hydrochloride was neutralized with 10 cc 5:95 triethylamine / chloroform

(f) washed twice with 10 cc methylene chloride

(g) washed twice with 10 cc chloroform.

The resin was then subjected to the following synthetic cycle: a 1-fold excess of t-BOC-proline (1.25 millimoles) in methylene chloride solution was added, then 258 mg (1.25 millimoles) of dicyclohexylcarbodiimide (DCC) was added and the mixture Shaken for 2 hours at 22 ° C. The resin was then washed three times with 10 cc of dioxane, chloroform or methylene chloride.

The dipeptide resin was then subjected to the deprotection cycle and reacted with a 4-fold excess of t-BOC-β-benzyl aspartate (0.5 millimoles) as in the synthesis cycle described above. Then a 0.5 g portion of the resin was removed from the reaction vessel and subjected to a cleavage process as follows:

0.5 g of the tripeptide resin was suspended in 5 cc of dry trifluoroacetic acid, then a slow stream of anhydrous HBr was passed through the solution for 90 minutes. The resin was filtered off and washed twice with 5 cc trifluoroacetic acid. The combined filtrates were concentrated under vacuum and the excess HBr was removed from the peptide by repeated evaporation of 1: 1 methanol / water solutions. The peptide was finally dissolved in water and, after lyophilization, gave aspartyl-prolyl-e-nitroarginine. Then the nitro group was removed by hydrogenation in a Parr low pressure shake hydrogenator as follows: The nitro protected tripeptide was dissolved in a 10: 1: 1 methanol / acetic acid / water mixture (about 10-20 mg / ccm) and the like The weight of a 5% palladium-on-BaS04 catalyst was added and the mixture was shaken overnight at a hydrogen pressure of about 3.5 at. The catalyst was filtered off and the filtrates were concentrated under vacuum. The peptide residue was chromatographed on a Sephadex G-25 column. The yield of purified tripeptide determined by conventional amino acid analysis was approximately 24%, based on the arginine incorporated into the resin. Part of the product was hydrolyzed with 5.7N HCl in water and examined in an amino acid analyzer; a ratio of Asp 1.05, Pro 0.95, Arg 1.00 was found.

Purity was determined in the usual way by paper electrophoresis at a number of pH values.

Example 2

Production of the tetrapeptide Ser-Asp-Pro-Arg

The tri-peptide resin of Example 1 not used for the synthesis of the tripeptide was subjected to the protective group removal cycle (see Example 1) and then with 0.111 g of t-BOC-O-benzylserine and 0.13 g of dicyclohexylcarbo-

diimide reacted in 20 cc methylene chloride as in the synthesis cycle of Example 1.

A portion of the resin was then subjected to the cleavage and hydrogenation procedures described in Example 1 and recovered as in Example 1, yielding Ser-Asp-Pro-Arg in 20% yield based on the arginine esterified on the resin. After hydrolysis with HCl, a sample of the tetrapeptide obtained was examined in an amino acid analyzer and showed a ratio of Ser 0.79, Asp 1.18, Pro 1.02 and Arg 1.01 (serine was partially destroyed during acid hydrolysis).

The purity was determined in the usual way at a number of pH values by paper electrophoresis.

Example 3

Preparation of the pentapeptide Asp-Ser-Asp-Pro-Arg

A.) The non-cleaved tetrapeptide resin from Example 2 was subjected to the deprotection cycle of Example 1 and treated in the synthesis cycle using 0.152 g of t-BOC-β-benzyl aspartate.

The pentapeptide was cleaved from the resin part with HBr in trifluoroacetic acid as above. The obtained polypeptide was dried under vacuum, washed thoroughly with water and then lyophilized. The analysis showed a yield of 16% based on the arginine.

The pentapeptide product was hydrolyzed with HCl and examined in the amino acid analyzer; a ratio of Asp 2.12, Ser 0.74, Pro 1.12 and Arg 1.01 was found.

B.) The pentapeptide can also be prepared by modifying the procedures of Example 1-3A:

To a solution of 3.02 g (6.82 millimoles) of at-amyloxy-carbonyl-ne-tosyl-L-arginine (t-Aoc-Tosyl-Arg) in 15 ccm of ethanol and 6 ccm of water was added a solution of cesium Bicarbonate (1.4 g in 3 cc HaO) was added dropwise until the pH of the solution was 7.0. The solution was concentrated in vacuo to a foam which was dried thoroughly under high vacuum over P2On. 25 cc of dry dimethylformamide (DMF) and 4.5 g of chloromethylated resin (beads of copolystyrene-1% divinylbenzene with 1.10 meq of chloromethyl groups / g of resin) were added to this residue and the mixture was shaken at 50 ° C. for 3 days. The resin was filtered and washed 5 times with 20 cc of dimethylformamide, 3 times with 20 ccm of 90% dimethylformamide / water, 2 times with 20 ccm of dimethylformamide and 2 times with 20 ccm of ethanol and then under vacuum over P2Or , dried; 5.54 g of argininated resin were thus obtained (incorporation of about 50%).

The resin was then subjected to 4 deprotection and synthesis cycles using 4 equivalents of the corresponding t-BOC amino acid in each chain extension step to give a protected pentapeptide resin material.

This resin material was then placed in an HF-resistant reaction vessel, 8 cc of anisole was added and the vessel was connected to an HF line. About 70 cc of HF was distilled into the reaction vessel at 0 ° C. and the mixture was stirred at 0 ° C. for a further 30 minutes. The HF was pumped out and the resin was washed 5 times with 30 cc ether and then extracted 5 times with 30 cc water. The aqueous layer was lyophilized to a yellow glassy powder which, after purification according to Example 1, gave the pentapeptide Asp-Ser-Asp-Pro-Arg.

The pentapeptide prepared above shows an [a] n20 ° = -78.6 ° (c = 1, H20). The purity was in the usual way

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Example 4

Preparation of the hexapeptide Ala-Asp-Ser-Asp-Pro-Arg

Another batch of the argininated resin (0.20 millimoles) was subjected to the procedures of Example 1-3A, but after binding the second aspartic acid residue and deprotection, an equivalent amount of t-BOC-alanine was coupled with dicyclohexylcarbodiimide in the usual manner.

The resin was then subjected to the cleavage and hydrogenation procedures as in Example 1 and recovered there. This gave 0.026 millimoles of Ala-Asp-Ser-Asp-Pro-Arg in 13 9r yield.

The obtained polypeptide was examined in an amino acid analyzer and showed an amino acid ratio of Ala 0.95, Asp 2.05, Ser 0.80, Pro 0.98 and Arg 1.00.

The purity was determined in the usual way at a number of pH values by paper electrophoresis.

Example 5

The following polypeptides can be prepared according to the synthetic procedures of Example 1-4:

Asp-Val-Asp-Leu-Ser Thr-Ala-Ser-Thr-GIu

Asp-Val-Asp-Leu-Ser-Thr-Ala-Ser-Thr-Glu Leu-Ser-Glu-Lys-His Ala-Pro-Ser-Lys-Gly-Thr Ala-Ser-Gly-Lys-Pro

Ala-Phe-Ala-Thr-Pro-Glu-Trp-Pro-GIy-Ser

Ala-Phe-Ala-Thr-Pro

Glu-Trp-Pro-GIy-Ser

Pro-Asp-Ala-Arg-His-Ser

Ala-Ser-Pro-Ser-Glu

Example 6 Preparation of metal and amine salts

A.) The pentapeptide Asp-Ser-Asp-Pro-Arg was converted to its sodium salt as follows:

A solution of 0.05 millimole of the pentapeptide in water was carefully treated with exactly 1 equivalent of 0.1N NaOH and the monosodium salt of the peptide was isolated by lyophilization. By using exactly 2 or 3 equivalents of 0.1N NaOH, the corresponding di- or. Trisodium salts obtained.

Similarly, by using the appropriate base, this peptide could be converted into other metal salts, e.g. the potassium, lithium, calcium, barium, magnesium, ammonium, ferro, ferric, zinc, mangano, mangani and aluminum salts are converted.

B.) The pentapeptide Asp-Ser-Asp-Pro-Arg was converted to its triethylamine salt as follows:

The careful addition of 1, 2 or 3 equivalents of triethylamine to the solution of the peptide in methanol and subsequent careful evaporation of the solvent gave the mono-, bis- or tristriethylammonium salt. Similarly, this pentapeptide can be converted to other amine salts using the appropriate amine, e.g. that of trimethylamine, tri- (n-propyl) amine, dicyclohexylamine, ß- (dimethylamino) ethanol, ß- (diethyIamino) ethanol, triethanolamine, tris- (hydroxymethyl) aminomethane, arginine, lysine, Histidine, N-ethylpiperidine, hydrabamine, choline, betaine, ethylene diamine, glucose amine, methyl glucamine, theobromine, purine, piperazine, piperidine, caffeine and procyne.

C.) Similarly, the other peptides of Examples 1, 2, 4 and 5 can be converted to their corresponding metal and amine salts.

Example 7

The pentapeptide Asp-Ser-Asp-Pro-Arg can be converted into its hydrochloric acid addition salt as follows:

Careful neutralization of a solution of the peptide in water or methanol with exactly 1 or 2 equivalents of hydrochloric acid provided the mono- or dihydrochloride salt. The salts were isolated by lyophilization of an aqueous solution or by precipitation with ether from a methanolic solution.

Similarly, the peptide can be converted to other acid addition salts, e.g. in the hydro-bromide, sulfate, phosphate, nitrate, acetate, oxalate, tartrate, succinate, maleate, fumarate, gluconate, citrate, malate, Ascor-bat and benzoate.

Similarly, the other peptides of Examples 1, 2, 4 and 5 can be converted to their corresponding acid addition salts.

Example 8 Preparation of Esters

A.) 1.0 g of the corresponding peptide resin from Example 5 was suspended in 40 cc of anhydrous methanol per g of resin, 50 millimoles of triethylamine were added and the mixture was stirred at 22 ° C. for 20 hours. The resin was filtered off and the combined filtrates were concentrated under vacuum. The residue was dissolved in ethyl acetate, saturated with 5 cc of hydrogen chloride and the solution was stirred at 22 ° C. for 30 minutes. The product was precipitated by the addition of ether and gave a hydrochloride salt of the peptide. The protective O-benzyl ether groups of Ser or Thr were removed by hydrogenolysis using Pd / BaS04 in the manner described in Example 1 to remove the nitro group in nitroarginine derivatives; so you got

Ala-Pro-Ser-Lys-Gly-Thr-OMe Ala-Ser-Gly-Lys-Pro-OMe or

Ala-Phe-Ala-Thr-Pro-OMe.

By using other alcohols instead of methanol and increasing the reaction temperature to 45-80 ° C and the reaction time to 45-90 hours, the corresponding ethyl, propyl, butyl, hexyl, octyl, decyl or dodecyl ester was obtained .

B.) This method used another type of anchoring bond to bind the arginine residue, namely that of S. Wang and R.B. Merrifield in J. Am. Chem. Soc., 91 6488 (1969) described Resin-0-CH2-CH2-C (CHa) 2-OCONHNH2 bond. Furthermore, Na-2- (p-biphenylyl) isopropyloxycarbonyl protecting groups (Bpoc) were used in the process instead of t-BOC for protecting the a-amino group, since the Bpoc group with a very mild acid un -

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conditions in which the anchoring bond is stable. The Bpoc-Ns-Nitro-Arg is bound to the resin by the DCC method, and the synthesis is carried out essentially according to Example 1-3, but using 1% trifluoroacetic acid (TFA) / CH2Cl2 for the protective group removal cycle in order to split off the Bpoc group . The final amino acid incorporated is protected as an N «-benzyloxycarbonyl derivative (Z), so that the N-terminal group remains protected from the resin during the cleavage of the protected peptide. The cleavage takes place as follows: 500 mg of the peptide resin were suspended in 12 cc 50% trifluoroacetic acid in CH2Cl2 and the mixture was shaken for 30 minutes at room temperature. The resin was filtered off, washed twice with 10 cc of CH2C12 each time and the combined filtrates were concentrated under vacuum; this gave Z-ß-benzyl-Asp-O-benzyl - Ser-ß-benzyl-Asp-Pro-NE-Nitro-Arg-NHNH2 as a white powder.

A solution of 0.2 millimoles of the protected peptide hvdrazide in 1 cc of dimethylformamide was cooled to -20 ° C, then 3.35N HCl in dioxane (0.5 millimoles) was added. The bath was warmed to -15 ° C, 0.03 cc of tert-butyl nitrite was added and the mixture was allowed to stand at -10 ° C for 10 minutes, thereby obtaining the peptide azide derivative. Excess methanol was then added at -10 ° C. and 0.5 millimole of ethyldiisopropylamine and the mixture was kept at 0 ° C. for 24 hours. For the first 6 hours, 5 μl of the base was added every hour. Then the protected peptide was precipitated by pouring the mixture into 15 ccm cold, 1% acetic acid and the collected precipitate was collected by filtration and washed. Then the protective groups based on benzyl were removed by hydrogenolysis according to Example 1 and the product was purified by division chromatography on “Sephadex G-25” or by countercurrent distribution, giving Asp-Ser-Asp-Pro-Arg-OMe.

If the methanol was replaced by other alcohols in this process, the corresponding ethyl, propyl, butyl, hexyl, octyl, decyl and dodecyl esters were obtained.

C.) By using the methods described in A.) and B.), the corresponding esters of the polypeptides of Examples 1, 2, 4 and 5 can be prepared.

Example 9 Preparation of Amides

A.) The products of Examples 8A and 8B were treated with a saturated solution of ammonia in methanol for 2 days at room temperature; after removal of the solvent under vacuum, the following was obtained:

AIa-Pro-Ser-Lys-Gly-Thr-NH2

Ala-Ser-Gly-Lys-Pro-NH2

AIa-Phe-Ala-Thr-Pro-NH2 and

Asp-Ser-Asp-Pro-Arg-N H2

B.) The peptide azide of Example 8B was reacted with ammonia in dimethylformamide solution under the conditions described in Example 8B for reaction with methanol. The protected peptide amide was isolated and the protecting groups removed as described, giving Asp-Ser-Asp-Pro-Arg-NH2.

C.) The protected peptide resin product of Example 3A was suspended in a saturated solution of ammonia in methanol and the mixture was stirred at room temperature for 2 days. The resin was filtered off, washed with methanol and the combined filtrates concentrated under vacuum; this gave t-BOC-Asn-O-Benzyl-Ser-Asn-Pro - NE-Nitro-Arg-NH2-. The t-BOC group and the Ne-nitro group were then removed by acid hydrolysis or hydrogenolysis as above and gave Asn-Ser-Asn-5 -Pro-Arg-NH2.

By using other amines instead of ammonia and dimethylformamide as solvents (if appropriate) and by increasing the reaction temperature and time (if necessary), e.g. the corresponding io de dimethyl, diethyl, di (n-butyl), n-hexyl, piperidyl, pyrrolidinyl, morpholinyl, di (n-hexyl) and N-methylpiper-azinylamine.

D.) The corresponding amides of the other polypeptides from Examples 15, 1, 2, 4 and 5 can be prepared by the process according to A.) B.) and C.)

Example 10 Preparation of N-acyl derivatives

20 Na-acyl derivatives of Asp-Ser-Asp-Pro-Arg were prepared by replacing the terminal t-BOC amino acid (t-BOC-ß-benzyl aspartate) with the corresponding Na-acyl-amino acid (eg Na-acetyl-ß -benzylaspartate), with all other stages of deprotection, synthesis 25 se and cleavage cycles remaining the same.

The following can be prepared: N n-acetyl-Asp-Ser-Asp-Pro-Arg Nre-butyryl-Asp-Ser-Asp-Pro-Arg N «-hexanoyl-Asp-Ser-Asp-Pro-Arg Na-octanoyl- Asp-Ser-Asp-Pro-Arg Na-Decanoyl-Asp-Ser-Asp-Pro-Arg

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Na-DodecanoyI-Asp-Ser-Asp-Pro-Arg.

The corresponding N "-acyl derivatives of the other 40 peptides mentioned in Examples 1, 2, 4 and 5 can be prepared in a similar manner.

Example 11 Preparation of O-Acyl Derivatives

45 To produce the protected pentapeptide resin material, in which the hydroxyl group of serine is unprotected, the following modification of the synthesis process was applied in the solid phase:

The tripeptide resin material of Example 1 was subjected to the 50 deprotection cycle and then allowed to react with the t-BOC-serine-N-hydrosuccinimide ester to give a t-BOC-Ser-β-benzyl-Asp-Pro-NE - nitro-Arg- Resin was obtained which was coupled with p-nitrophenyl-t-BOC-ß-55-benzyl aspartate after deprotection under usual conditions, whereby a t-BOC - ß-BenzyI-Asp-Ser-ß-Benzyl-Asp-Pro Ns-Nitro-Arg resin was obtained.

0.5 millimoles of this protected peptide resin material was replaced with octanoic acid, decanoic acid and dodecanoic acid, then 60 1.5 millimoles of hexanoic acid dissolved in 1: 1 dimethylformamide / CHCls and 1.5 millimoles of carbonyldiimidazole dissolved in the same solvents were added. The mixture was shaken in a Merrifield reaction vessel at room temperature for 2 hours and the peptide was then cleaved from the resin in the manner described above. The NE nitro group was removed by hydrogenolysis and the peptide purified as above to give Asp-O-Hexanoyl-Ser-Asp-Pro - Arg.

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10th

If the hexanoic acid was replaced by acetic acid, butyric acid, octanoic acid, decanoic acid and dodecanoic acid, the corresponding O-acetyl, butyryl, octanoyl, decanoyl and dodecanoyl compounds could be prepared.

Similarly, the corresponding O-acyl derivatives of the other peptides with side chain hydroxyl groups of Examples 2, 4 and 5 can be prepared.

The “blocking” activity of the polypeptides obtainable according to the invention can be investigated using the classic Prausnitz-Kuster (P-K) reaction. A known allergic serum, i.e. one containing an IgE specific for a known antigen or allergen is injected intradermally. After a certain time, e.g. For 20 hours or more, the injection sites are irritated by a prick or injection of a solution of the antigen specific for the IgE in the injected serum. Within the next 10-30 minutes there will be a positive reaction from swelling (and redness) at the injection site. The larger the swelling diameter, the more intense the allergic reaction. This means that a stronger swelling means a greater release of histamine into the tissue at the injection site. Conversely, the formation of a smaller diameter swelling or the absence of any swelling shows a reduced and / or no allergic reaction. The P-K reaction shown is a classic, well-known test used by allergy researchers.

As mentioned, the classic P-K reaction is used to investigate the "blocking" abilities of the polypeptides.

The following studies of various suitable polypeptides, the synthesis of which has been described, were carried out using a single, recognized as safe P-K donor serum containing an IgE specific for guinea pig allergens.

The peptide solutions were either injected intradermally 1-24 hours before the P-K serum or mixed with dilutions of the P-K serum for simultaneous injection. The initial tests were done with a P-K serum at 1: 4 to 1: 200 dilutions. Further experiments were carried out at a fixed P-K dilution of 1:32, the peptide solutions being varied so that they contained about 1 millimole to 2 mol of the peptide to be tested. The injection sites of the test subjects were determined by guiding pigs of guinea pigs BCA 1:40 w / v. (Purchased by Berkeley Biologicals, Inc.) irritated.

Test subjects with serum IgE values below 100 U / ccm (242 ng / ccm) had; these values are known to ensure successful P-K responsiveness. Furthermore, people with a negative direct skin test for guinea pig antigen were selected for these tests. The P-K and skin tests were carried out on the back and / or on the forearm. Multiple test sites approximately 25 mm in diameter were identified by a marker and all injections were made within the framed skin areas.

A typical consequence was the intradermal injection of 0.1 cc of the peptide solution or a buffered control solution with saline dilution; In 1-24 hours this was followed by the intradermal injection of 0.05 cc P-K serum into every previous injection site. After 20-24 hours had elapsed, each site was punctured with the antigen solution, blotted dry in 5 minutes, and then, usually 15, 20 and 25 minutes after the puncture puncture, the swelling and redness were measured three times in their narrowest and widest diameters.

The blocking activity was tested for the following polypeptides: Asp-Pro-Arg; Ser-Asp-Pro - Arg; Asp-Ser-Asp-Pro-Arg; and Ala-Asp-Ser-Asp-Pro-Arg. For comparison tests, Asp-Thr-Glu-Ala-Arg and Tosyl-L-arginine sarcosine methyl ester (TASME) were also synthesized and investigated.

The polypeptides mentioned were examined as above in six different people with the following results:

For Asp-Pro-Arg, the average inhibitory effect was 15% with an individual range of only 0c / c up to 38%.

For Ser-Asp-Pro-Arg, the average inhibitory effect was 18% with an individual range of only 0% to 50%.

For Asp-Ser-Asp-Pro-Arg, the average inhibitory effect was 72% with an individual range of 60 to 89%.

For Ala-Asp-Ser-Asp-Pro-Arg, the average inhibitory effect was 46% with an individual range between 10 to 61%.

For Asp-Thr-Glu-Ala-Arg, the average inhibitory effect was 58% with an individual range between 30 to 80%.

For TASME, the average inhibitory effect was 24% with an individual range between 0 to 40%.

The results above are the average of duplicate measurements at three time intervals for each response of each subject, subtracted from the average control swell measurements and divided by the average control swell measurement for each subject. The control swellings in the various test subjects varied from 8-40 mm 2 with an average value of 17 mm 2. Each peptide was used in approximately 6 μg / ccm dilution and 0.1 ccm and then 0.05 ccm diluted P-K serum with an IgE content of 0.2 ng was injected into each site. Thus 10 ~ nM of the peptide competed with 10 ~ lr, M IgE for the binding sites of the mast cells; or the ratio was 1 IgE molecule per 10 "peptide molecule. From the above investigations it can be seen that the pentapeptide, ie Asp-Ser-Asp - Pro-Arg, shows the strongest" blocking "activity, the hexapeptide, ie Ala -Asp-Ser-Asp-Prop-Arg, shows a slightly lower activity, the tetrapeptide Ser-Asp-Pro-Arg and the tripeptide Asp-Pro-Arg showed the least effectiveness.

It should be noted that the pentapeptide Asp - Thr-Glu-Ala-Arg was prepared and studied together with the other peptides above. This polypeptide does not have the analogous sequence of the amino acids appearing in the C 2, C 3 or C 4 domains of the IgE molecule, but shows a high effectiveness in the above test.

Furthermore, it was found that the active polypeptides apparently have the ability to “remove” the IgE from the mast cell sites and to prevent the IgE from binding to these sites. In a single test on a subject with known, extreme sensitivity to guinea pig antigens, i.e. a person with a high natural concentration of IgE sensitive to guinea pig antigen, the polypeptides were injected and the subject's response to guinea pig antigen was determined.

In particular, about 2 nM Asp-Ser-Asp-Pro-Arg and Ala-Asp-Ser-Asp-Pro-Arg were injected intradermally into 3 marked positions. For comparison, TASME and a control of the buffer diluent were injected into 3 marked sites alone. 1, 5 and 24 hours after the polypeptide and control injection, a peptide was

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and diluent point irritated by puncture puncture with guinea pig antigen. At the 1 and 5 hour intervals, no inhibition of the swelling and redness response was observed at any of the sites. However, after 24 hours of irritation, the swelling at the Asp-Ser-Asp-Pro-Arg site was approximately 45% less, while at the Ala-Asp-Ser-Asp-Pro-Arg site the swelling was approximately 23% less. No reduction in swelling size was observed at the TASME site compared to the site of injection with buffered saline diluent.

Thus, at least the most active peptides apparently "remove" an IgE that is already bound to mast cell sites and thus suppress a natural allergic reaction. As already shown above, the same pentapeptide is extremely effective in inhibiting a passively transmitted (P-K) allergic reaction.

The acute toxicity was determined as follows:

White BBA mice with an average weight of 15 g were each injected with 1.4 cc of a solution of the peptide in phosphate-buffered saline at a pH of 7.4 as follows:

0.1 ccm X 3 0.1 ccm X 3 0.2 ccm 0.6 ccm intradermally subcutaneously intravenously intraperitoneally

24-72 hours after the injection, the mice that were still alive were sacrificed and examined.

The peptides used and their concentrations were as follows:

Ala-Asp-Ser-Asp-Pro-Arg

(Example 4) 5 (xg / ccm (375 mg / kg) -6 mice

Asp-Ser-Asp-Pro-Arg (Example 3)

10 (ig / ccm (1 mg / kg) -8 mice

Asp-Ser-Asp-Pro-Arg

(Example 3) 13 jx / ccm (1.3 mg / kg) -8 mice

20 Visual and microscopic examination of tissues and organs showed no local or systemic toxicological abnormalities.

v

Claims (3)

624093 1. A process for the preparation of a polypeptide from 3 to 10 amino acids in a sequence selected from a part of the amino acid sequence 265-537 of the Fe region of immunoglobulin E, or a pharmaceutically acceptable, non-toxic salt thereof, characterized in that (a) a first amino acid or a peptide, which may be covalently bound to a solid support, is condensed with a second amino acid or a peptide, the carboxyl or amino groups not intended for the condensation reaction being blocked by protective groups and the total number of amino acid residues in the the first and second amino acids and / or peptides are the same as those of the final polypeptide and (b) successively or simultaneously cleaves the protective groups and / or the solid support from the product from step (a). 2. The method according to claim 1, characterized in that converting the polypeptide obtained into a salt. 3. The method according to claim 1 or 2, characterized in that the polypeptide Asp-Ser-Asp-Pro-Arg or a pharmaceutically acceptable, non-toxic salt thereof is prepared. 4. The method according to any one of claims 1 to 3, characterized in that the protective group is an amino protecting group from acyl, aromatic urethane, aliphatic urethane, cycloalkyl urethane, thiourethane, aralkyl or trialkylsilyl groups or a carboxyl protecting group from substituted or unsubstituted aliphatic esters, aralkyl esters, N-substituted hydrazides and amides. 5. The method according to any one of claims 1 to 4, characterized in that the covalently bound solid support is a crosslinked polystyrene-divinyl resin, crosslinked polyamide resin, polyethylene glycol resin or functionalized glass beads. 6. A process for the preparation of esters of a polypeptide from 3 to 10 amino acids in a sequence which is selected from a part of the amino acid sequence 265-537 of the Fe region of immunoglobulin E, characterized in that said polypeptide is prepared by the process according to claim 1 and then subjected to esterification. 7. The method according to claim 6, characterized in that one produces esters of the polypeptide Asp-Ser-Asp-Pro-Arg. 8. A process for the preparation of amides of a polypeptide from 3 to 10 amino acids in a sequence which is selected from a part of the amino acid sequence 265-537 of the Fe region of immunoglobulin E, characterized in that said polypeptide is prepared by the process according to claim 1 and then converted to an amide. 9. The method according to claim 8, characterized in that one produces amides of the polypeptide Asp-Ser-Asp-Pro-Arg. 10. A process for the preparation of N-acyl derivatives of a polypeptide from 3 to 10 amino acids in a sequence which is selected from a part of the amino acid sequence 265-537 of the Fe region of immunoglobulin E, characterized in that said according to the process of claim 1 Prepares the polypeptide and then subjected to N-acylation. 11. The method according to claim 10, characterized in that one produces N-acyl derivatives of the polypeptide Asp-Ser-Asp-Pro-Arg. 12. A process for the preparation of O-acyl derivatives of a polypeptide from 3 to 10 amino acids in a sequence consisting of part of the amino acid sequence 265-537 the Fe region of immunoglobulin E is selected, characterized in that said polypeptide is prepared by the process according to claim 1 and then subjected to O-acylation. 13. The method according to claim 12, characterized in that one produces O-acyl derivatives of the polypeptide Asp-Ser-Asp-Pro-Arg. The symptoms of allergic diseases in humans, especially the allergic syndrome, are caused by the release of vasoactive amines, especially histamine, into the organism. Histamine is normally stored in special cells and basophil eucocytes known as mast cells, which are distributed throughout the organism. The mast cells are distributed over the entire tissue structure in humans, while the basophils with the blood in the body, i.e. circulate within the vascular system. The above cells produce and store histamine within their internal structures, in which the histamine remains, unless a particular sequence of events occur to trigger the release of the histamine from within the cell structures into the surrounding tissue and vascular system. The histamine is released in particular in response to the presence of specific antigens (allergens) that enter the organism or can be released by the organism in response to a traumatic event. However, the usual histamine release from the mast cells or basophils is triggered by a necessary sequence of chemical and immunological events that take place on and in the mast cell and basophil structures. The allergen mast cell (basophil) reaction is particularly favored by a group of proteins known as immunoglobulin E (IgE) that are produced within the body. The IgE formed by the human organism is a complex structure of polypeptide chains, the molecules of which can each have certain variations in the sequence of the amino acids in the polypeptide chain, but all of which are essentially characterized by the fact that they have a “Y” -shaped structure, the "foot" (ie the lower part of the "Y") in the form of the (Fe) polypeptide portion or fragment contains a fixed sequence (sequence) or "constant region" of peptides along the chain. The "head" (corresponding to the upward-pointing arms of the "Y" structure) can have regions in which the polypeptide chain varies (the variable region of the Fab), from molecule to molecule. The IgE molecules usually have identical "foot" peptide sequences, but can have a large number of different "head" peptide sequences. The allergic or immunological histamine release within the organism from the specialized mast cells and basophils can occur under the following circumstances: All mast cells or basophils have a number of receptor sites that are available for "coupling" the constant region or the Fe portion of the IgE molecules. These "binding sites" are specialized areas on the cell membrane in which a special geometric or spatial molecular arrangement takes place, whereby this "binding or receptor site" can "couple" to the Fe fragment or a site in the constant region of the IgE molecule. 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65 2nd PATENT CLAIMS 3rd 624093 If a migrating IgE molecule finds a free "binding receptor site" on a mast cell or a basophile, it couples its Fe part to the binding (receptor) site of the cell in order to attach the IgE molecule to the mast cell or the basophil. When the Fe portion of the IgE molecule is attached to the binding "receptor site", the upward-facing arms of the "Y" shaped molecule (the F (ab) portion) are free to expand above the cell surface. These extended or pointing upper peptide chains in turn act as receptors for allergens that may be present in the environment of the organism. If the polypeptide structure of the Fab components are compatible with a particular allergen, the allergen can bind to the outwardly extending Fab of the IgE polypeptide chain. In the event of binding, the mast cell or basophil is automatically stimulated to release the histamine from the internal cell structure into the local environment of the mast cell or basophil. Once the histamine is released, the known «allergic symptoms» appear. Current therapy for allergic diseases includes hyposensitization (repeated injections of harmful allergens to form “blocking antibodies”), systemic therapy with antihistamines (which compete with the histamines released during the allergic reaction) and disodium cromoglycate (the histamines released by allergic reactions) -ge can decrease). Corticosteroids, isoprenaline and theophylline and other medicines have also been used in allergy therapy. However, none of the above-mentioned drugs or procedures interfere with the basic response of the IgE mast cell (basophil), and all of them have significant restrictions on their suitability. Another therapeutic route proposed by the above analysis of the allergen-IgE mast cell (basophil) reaction would be the introduction of a drug into the organism that acts on the receptor or binding sites of mast cells (basophils) against the binding of the IgE molecule « block ». Of equal importance would be a drug that would not only "block" the binding sites, but would also remove the IgE from the binding sites to which it has already accumulated. Any replenishment or reduction of the binding sites available for IgE accumulation would of course reduce the number of Al-allergen-IgE mast cell (basophil) reactions, thereby reducing the histamine release into the organism and thus reducing or preventing allergic reactions. There are already several attempts to follow this therapeutic route. For example, published a study in "Lancet" of July 6, 1968, in which the entire Fe portion of the IgE and small proteolytic digestive fragments thereof were tested for their ability to suppress allergic reactions. This study suggested that only the entire Fe fragment of IgE was as effective as the intact IgE molecule in inhibiting allergic reactions, while the digestion fragments were ineffective. This means that all fractions of the FC peptide chain smaller than the entire polypeptide were unsuitable for preventing induced allergic reactions. The Fe fragment itself cannot be used as a therapeutic or medicinal product.