The method of producing peptides, peptides, immunomodulatory composition and method of regulation is insufficient or excessive functions of t cells in a patient

 

(57) Abstract:

Usage: in medicine. The essence of peptides f-ly 1: R - Arg - X - Y - Z - R1where R= hydrogen, acetyl, formyl; X= Proline; Y= L - Ala, L - Asp, L - Val; Z= L - Val, L - Ala; R1= H, OH, NH2, NH(CH3), NHCH(CH3)2under certain literal values and methods for their production or solid-phase method using the substrate polymer or copolymer comprising cellulose, polyvinyl alcohol, polymethylmethacrylate, polystyrene and politicalgovernmental, or in solution by sequential or block the attachment of amino acids or peptide fragments contained in f-Le 1 and immunomodulatory composition comprising as active principle at least one peptide f-crystals of 1 in an effective amount. 5 s and 5 C. p. F.-ly, 8 Il, 5 PL.

The invention relates to synthetic peptides capable of stimulating the activity of helper T cells. More specifically, the peptides obtained by the proposed method are tetrapeptide based on the molecule replenine.

Natural immunomodulatory proteins, thymopoietin and displayn (formerly known as splenin") were isolated from thymus and spleen (is devoted to a similar proteins and synthetic peptides. In U.S. patent N 4190646 described Pentapeptide thymopentin, which represents the area of activity (website) thymopoietin and contains the amino acid sequence arginine-lysine-aspartic acid-valine-tyrosine and peptide compositions in which various groups substituted on the amino and/or carboxyl end of the specified Pentapeptide. As thymopoietin and thymopentin cause biological changes in two lines of T-cells, GEM MOL and T-4, which indicates that their role in stimulating the biological activity of T cells. It was found that none of the analogues thymopentin, shorter than Pentapeptide (i.e., peptides, containing 5 amino acids in the sequence), it does not have activity against GEM cells.

In U.S. patent N 4428938 describes some of the peptides that affect immunoregulatory. Among these peptides are listed below tetrapeptide:

the arginine-lysine-aspartic acid-valine,

the arginine-lysine-asparagine-valine,

the arginine-lysine-alanine-valine,

the arginine-lysine-aspartic acid-alanine,

the arginine-lysine-aspartic acid-isoleucine,

the arginine-lysine-glutamic acid-valine,

pyroglutamyl-arginine-lysine-asparigine derivatives of these sequences, as well as methods of using these sequences for the treatment of immunological disorders caused by nedostatocnosti associated with the functioning of the thymus (thymus gland). According to this patent, these peptides were tested for the presence of activity carried out in vitro E-rosette test.

The same authors reported the synthesis of such tetrapeptides in Kisfaludi with TCS. Noppe-Seyler's L. Physid. hem. B. D. 364, S. 933-940 (1983). In this work it is reported that the sequence arginine-alanine-aspartic acid-valine is a very active analogue in the ongoing in vitro E-rosette test and that the sequence arginine-alanine-aspartic acid-valine and arginine-lysine-alanine-valine significantly reduce the activity.

According to the available prior art there is a need for additional peptides which have the ability to stimulate the immune system in humans under different cases of failure of T-cells.

The present invention describes a number of analogues of peptide replenine possess biological activity in relation to MOL T-4 cell line. Unlike the above-described thymopentin and its analogues these analogues want to make in relation to helper T cells MOL T-4, as is the case pentapeptidnogo analogues replenine.

One aspect of the present invention relates to new tetrapeptides that meet the following General formula

R-arginine-X-Y-Z-R1where R is hydrogen, acetyl, formyl;

X Proline;

Y L-amino acid belonging to the set consisting of alanine, aspartic acid;

Z L-amino acid belonging to a row consisting of valine or alanine,

R1hydrogen, hydroxyl or the group NH2, NH(CH3), NHCH(CH3)2.

These peptides and compositions containing them unexpectedly exhibit a biological activity characteristic of human replenine. A large number of these peptides also features an improved ability to resist the effects of endo - and ectopeptidases and trypsin-like enzymes in the digestive tract and serum. Thus, these peptides have significant advantages in the treatment of immune deficiency. In particular, tetrapeptide according to the present invention, for which X represents a Proline or methylalanine have the unexpected ability to resist degradation by enzymes such as peptidases serum.

Other aspects and advantages of the present invention are listed below.

In Fig.1 is an illustration of a circular formation of GMP MOL of T-4 in the form of a graph in a coordinate axis: concentration of peptide (μg/ml)-the level of cyclic GMP (PG/ml); it also provides a comparison of the activity thymopentin (TR-5) and peptides according to the invention in comparison to the control samples; Fig.2 shows a graphical illustration for cyclic GMP coordinates: concentration of peptide (ál/ml) levels of cyclic GMP (PG/ml); it also provides a comparison of the activity thymopentin (TR-5) and peptide acetyl-arginine-Proline-valine-alanine-NH2according to the present invention.

The peptides according to the present invention in the General case can be obtained according to known methods. Convenient to carry out the synthesis of the polypeptides according to the invention in accordance with the method of synthesis in the solid phase, as described by Merrifield, in J. A. C. S. 85, 2149-2154 (1963). This method is widespread and is a common method of producing peptides.

Amino acids can be attached to any suitable polymer resin. The resin shall contain a functional group through which the first protected amino acid can be firmly attached by covalent bonds. For this purpose, a suitable time is their protective groups, which can be used in such syntheses are tert-butoxycarbonyl (BOC), benzyl (BZL), t-meloxicamviagra (AOC), Casilina (TOZ), brompheniramineodeine (BrBz), 2,6-dichloraniline (BzLCl2and phenylmethanesulfonyl (or SV). Additional examples of protective groups described in the above source, as well as in the book. F. C. Mac-Omi Protective groups in organic synthesis. New York, Plenum Press, 1973.

A common method of obtaining a peptide according to the present invention includes the initial connection protected C-terminal amino acid to the resin. After this attach the resin is filtered, washed and remove the protective group from the alpha-amino group of the C-terminal amino acids. Deletes the specified protective group must of course take place without breaking the connection between the specified amino acid and resin. To the resulting resin peptide attach the penultimate C-terminal protected amino acid. Accession shall be effected by the formation of a connection between the free carboxyl group of the second amino acid and the amino group of the first amino acid attached to the resin. A similar sequence of operations is repeated with poledouris protected peptide otscheplaut from the resin and remove the protective group, receiving the target peptide. The method of removal used to separate the peptide from the resin and removal of the protective groups known to experts in the field of peptide synthesis technique.

In addition, the peptides according to the present invention can also be synthesized using the standard method for the synthesis of peptides in solution, which includes either Paladino or block the accession of amino acids or peptide fragments using chemical or enzymatic methods of amide bond formation.

It was found that the peptides according to the present invention have biological activity similar to human replenine. This biological activity is primarily manifested in the measurement of the degree of induction of the formation of cyclic GMP in MOL T-4-line of human T cells in comparison with human displaynum and human thymopentin. Inducing the formation of cyclic GMP peptide according to the present invention in these experiments indicates the presence of peptide ability to bind with the receptor area (site) of the human replenine in the cell and cause a biological activity equivalent to Akti is additional and significant advantages compared with human displaynum. Many of the peptides according to the present invention are resistant to enzymatic degradation by enzymes in the digestive tract or in the serum. As a result, they have a long half life in vivo when introduced through the injection of a biological object. Another advantage of many of these peptides is the possibility of oral administration. By itself, the human displayn is too large molecule and must be broken down in the digestive and intestinal tract.

Prior to the test peptides according to the present invention it was impossible to foresee what may be the analogs of human replenine that have the same biological specificity, because the sequence arginine-lysine-alanine-valentinois, which is the human analogue of bovine Pentapeptide SP=S (arginine-lysine-glutamic acid-valine-tyrosine), is inactive against cells MOL T-4.

Thus, the opening of tetrapeptide, which is similar to human replenine and active, similar to the biological activity of the whole molecule human replenine is neojidannaya useful from a therapeutic point of view means to ensure human and perhaps animals, because they have the ability to induce differencirovanie and maturation of T-cells that are able to engage in processes of the immune response of the body. As a result, the peptides according to the present invention can be considered as a means for many variants of therapeutic use.

The peptides according to the present invention can be considered as a means of facilitating the establishment of immunity of the patient's body because they increase or promote therapeutic stimulation of cellular immunity. Therefore, they can be useful in the treatment of diseases, which are characterized by chronic infection such as infectious fungal diseases or infections caused by Mycoplasma, tuberculosis, leprosy, acute and chronic viral infections and other diseases.

The peptides according to the present invention or compositions containing these peptides or their salts of acids or bases, can be used in all areas that require the achievement of cellular immunity, especially in those cases when there is immunodeficiency. So, in those cases where the merits of the Oia can correct this condition by stimulating the functions of T cells. Thus, we should expect that they may find use in the treatment of some autoimmune diseases, in which the formation of undesired antibodies, for example in the case of lupus erythematosus (erythema), rheumatoid arthritis and similar diseases.

In such a wide variation in the use of the peptides according to the present invention or pharmaceutical agents containing them can be used to regulate the immune system of the patient, human or animal in need of such regulation. The term "regulation" as used in the description of the present invention, means that the compounds according to the invention cause the immune system to return from the abnormal, painful condition in normal, balanced state. Although the regulation of this type can be widely used for the correction of immunological deficits (e.g syndrome Di George), other options are also possible applications for adjustment conditions corresponding to the excess of immunological activity (for example, in the case of autoimmune diseases).

Therefore, the present invention includes methods of regulating the immune system caloosa at least one of these peptides in number, effective from the point of view of the regulation of the immune system, as well as pharmaceutical compositions for the implementation of these methods. Preferably the effective amount is from 1 μg to 100 mg/kg of body weight.

The invention also provides a method of treating conditions that occur when the relative or full immune deficiency of the immune system of the patient, particularly from the point of view of the functioning of helper T cells; this method includes the introduction of the specified effective patient with a therapeutic standpoint amount of at least one of the peptides according to the present invention.

In the sense in which it is used in the present description, the term "effective from a therapeutic point of view, the amount" means that amount of a drug is effective in treating the above conditions. The present invention also provides a method of inducing the differentiation and maturation of T cells, including introduction to patient effective from the point of view of the specified inducing amount of at least one of the peptides according to the present invention.

In addition, the invention provides the pharmaceutical according to the present invention the peptide according to the present invention combine as an active ingredient with suitable for pharmaceutical use of the media by thorough mixing in accordance with the usual methods of making pharmaceutical compositions. The media can be in various forms, depending on the method, which provides for the introduction of the drug, for example, oral, rectal, parenteral through the nose or under the tongue.

Upon receipt of the compositions in the preferred form for oral administration, you can use any of the usual pharmaceutical environment. In the case of liquid compositions for oral administration (for example, suspensions, elixirs and solutions) you can use environment containing, for example water, oils, alcohols, flavouring agents, preservatives, dyes and the like agents. Such carriers such as starch, sugars, diluents, granulating agents, lubricating agents, binding agents, dezintegriruetsja agents and similar components can be used for solid compositions for oral administration (for example, powders, capsules and tablets). You can also use the recipe for the managed introduction (formulation prolongirovannogo actions). Due to ease their introduction of tablets and capsules represent the most preferred dosage forms for oral method of administration; obviously, in this case, use a solid pharmaceutical carriers. If desired, tablets Magi.

In the case of products for parenteral route of administration, the media is usually a sterile water, although such compositions may also comprise other components, for example, contributing to the dissolution or having the properties of preservatives. Can be obtained suspension for injection; in this case, you can use the appropriate liquid carriers, suspendresume agents and similar tools.

As already mentioned, tetrapeptide according to the present invention is usually active when you enter them in quantities of more than 1 µg/kg body weight, preferably from about 0.001 to about 10 mg/kg of body weight. In General, dosages in the same range can be used for the treatment of diseases in which there is an immune deficiency. Large quantities (for example, in the range from about 10 to 100 mg/kg of body weight of the patient) can be used to suppress excessive immune activity.

The following examples illustrate the invention, without restricting the limits of its application. In the examples and in the description, parts are understood mass parts, unless otherwise specified. In the examples used the following abbreviations: FA (TFUC) Tril, (DMF) dimethyl formamide, N4OAc ammonium acetate, NH4HE hydroxide ammonium, n-Gon n-propanol, n-VION n-butanol, pyr is pyridine, DCC (DCC) dicyclohexylcarbodiimide, OBt (MBT) 1-oxybisethanol, DMAP (DMAP) dimethylaminopyridine, HF hydrogen fluoride, TCA (THUK)-trichloroacetic acid, BHA (BHA) benzhydrylamine and Meon methanol.

P R I m e R 1. Synthesis of tetrapeptide: acetyl-arginine-Proline-alanine-valine-NH2.

The above tetrapeptide synthesized using solid-phase synthesis method with sequential binding. All amino acids protected at the amino group by tert-butyloxycarbonyl group. Tosyl (TOZ) are used to protect the side chain of arginine. Protected amino acids are used in excess, corresponding to 2.5 equivalents of I equivalent substitution resin. All the bind operation is performed with the use of dicyclohexylcarbodiimide and 1-oxybisethanol equal the equivalent amount in relation to a protected amino acid. All amino acids are dissolved in methylene chloride, with the exception of arginine, which is dissolved in dimethylformamide.

Wash, remove protection and binding spend Paladino, in accordance with the following scheme, min:th 1 methylene 30 4. Methylene chloride 1 3 5. 40% Isopropanol chlorine - risty methylene 2 3 6. Methylene chloride 2 3 7. 10% of Diisopropylamine - Mina 2 10 8. Methylene chloride 1 3 9. Amino acid 1 3 10. 1-Oxybisethanol 1 3 11. Dicyclohexylcarbodimide MFA - 1 120 12. Methylene chloride 2 3 13. 40% isopropanol chlorine - risty methylene 2 3 14. Dimethylformamide 1 3 15. Methylene chloride 2 3

a) Synthesis.

The protected peptide is synthesized using a peptide synthesizer company "Beckman model 990. The resulting resin with tetrapeptides corresponds to 0.67 Meg/g; synthesis starting with 5.0 g (3.35 mmol) of resin. The resulting tetrapeptide will acetimidoyl as a result of processing of the resin in accordance with the following procedure, min: 1. Methylene chloride 2 3 2. 50% triperoxonane acid methylene chloride 1 3 3. 50% triperoxonane KIS lots of methylene chloride 1 30 4. Methylene chloride 1 3 5. 40% isopropanol chlorine - risty methylene 2 3 6. Methylene chloride 2 3 7. 10% diisopropylethylamine - 2 10 8. Methylene chloride 1 3 9. 50% acetic anhydride methylene chloride, with rolled - cal number of dimethy - aminopyridine 1 120 10. Methylene chloride 2 3 11. 40% isopropanol chlorine - risty methylene 2 3 12. Dimethylformamide 1 3 13. Methylene chloride 2 3

Containing the peptide resin was dried under reduced pressure get in the breakdown of communication resin peptide by HF (25 ml) and anisole (25 ml) for 4 h at 0aboutC. HF is removed under reduced pressure. To the mixture is added 150 ml of diethyl ether, the solution is stirred and transferred to a glass filter and then washed with 3 portions of 100 ml of diethyl ether. The peptide is extracted from the resin by 10% acetic acid (3 100 ml), and aqueous solution lyophilizer, receiving 1.20 g of the product.

C) Cleaning.

The product was then purified by the method of preparative high-performance liquid chromatography (PUGH) under the following conditions: Column "Whatman", Partial M-20 10/50 ODS-3, samples of 300 mg used socrata system 15% acetonitrile/0.01 M ammonium acetate (pH is brought to 5 by means of acetic acid), flow rate 15 ml/min, wavelength used for detection, 2120 nm. All fractions containing the pure product are collected. The organic solvent is distilled off and lyophilizers aqueous solution, resulting in the 258 mg of a white solid product.

Amino acid analysis: Proline 1,00, alanine 1,00, valine 1,00, arginine 1,00, 83% of the peptide. Thin layer chromatography (silica 60/Analtech): Rf (I) 0,50 (butanol: acetic acid: water 3 1 1) Rf (II) 0,59 (butanol: pyridine: acetic acid-water 4:1:1:2) Rf (III) 0,29 (butanol: acetic acid: water: ethyl the ID is obtained using a peptide synthesizer "Applid of Biosystems-A". Use for this purpose the standard cyclic procedure I (production version 1.40), through which the binding of one amino acid at a time, with the exception of a fragment of tert-butyloxycarbonyl-tosyl-arginine, which is bound twice. For synthesis use the original products, are given in table.1.

After tert-butoxycarbonyl protective group of the arginine removed and the resin peptide was subjected to neutralization and washing, the connection will acetimidoyl using 10% acetic anhydride in methylene chloride (15 ml) in the presence of 4-dimethylaminopyridine (20 mg). After 60 min, the peptide is subjected to the test ninhydrin test to ensure the completeness of acetylation. The resin-peptide is washed with methylene chloride (3 servings 15 ml) and dried in vacuo (room temperature) to give 1.0 g of product.

b) Splitting HF:

Peptide otscheplaut from the resin with 10 ml of HF in the presence of 1.5 ml of anisole. The mixture was stirred at 0aboutC for 1.5 h, after which the HF is removed under reduced pressure. The mixture was washed with diethyl ether (3 portions of 50 ml) and dried in air. The mixture of the resin and the peptide extracted with a 25% aqueous solution of the UKS crude product. This product is passed through an ion-exchange resin "Amberlite IRA-68 (acetate form) in water. The fractions containing the peptide, unite and lyophilizers, receiving 200 mg of product.

The peptide was subjected to purification on a column of companies "Whatman", Particel 20-M 10 ODS-3 (22 mm x 50 cm) using as solvents for elution of 15% acetonitrile (0.1% of triperoxonane acid) and 0.1% aqueous solution triperoxonane acid (speed of 10.0 ml/min). For the elution process is supervised by high performance liquid chromatography, the appropriate fractions are collected, combined and evaporated from their solvent under reduced pressure. The residue is dissolved in water and lyophilizers, repeating the process twice, receiving the 167 mg of product with a total yield of 50%

Amino acid analysis: arginine 1,04, Proline 1,02, valine 1,00, analin 1,01, 72% of the peptide.

Thin layer chromatography (plate with silica gel CF-250 microns):

Rf (I) 0,37 (n-butanol: acetic acid: water 3:1:1)

Rf (II) to 0.15 (chloroform: methanol: acetic acid 60:35:5)

Rf (III) of 0.48 (n-butanol: acetic acid: water: ethyl acetate 1:1:1:1).

P R I m e R 3. Synthesis of tetrapeptide acetyl-arginine-Proline-aspartic acid-valine-NH2.

a) Synthesis. using an automatic synthesizer company "Beckman", the model 990. Synthesis starting with 5.0 g benzhydrylamine resin (0,67 Meg/g). After binding of tert-butoxycarbonyl-arginine (Totila) tert-butoxycarbonyl protective group is removed by 50% triperoxonane acid in methylene chloride and acetimidoyl the resin with the peptide by 50% acetic anhydride in methylene chloride in the presence of catalytic amounts of dimethylaminopyridine. The resin-peptide is washed with ethanol (2 50 ml) and dried under reduced pressure over night before making cleavage by HF. The dry mixture of the resin and the peptide weighs 6,5,

b) Splitting HF.

The crude tetrapeptide get through cleavage of 6.5 g of a mixture of resin with the peptide using 25 ml of HF and 25 ml of anisole at 0aboutC for 4 h, HF is distilled off under reduced pressure and add diethyl ether (150 ml) to the obtained reaction mixture. The crude peptide in a mixture with the resin was transferred to a glass filter and washed with 3 portions of diethyl ether, 100 ml of the Peptide extracted with 10% acetic acid (2 100 ml) and the resulting aqueous solution lyophilizer, receiving 900 mg of a white solid.

C) Cleaning.

The crude product (degree Chistota, 2,5 89 cm) using 10% acetic acid as eluent. After lyophilization the product is snegopady white solid product weighing 800 mg.

Amino acid analysis: arginine f 0,99, valine f 1,00, Proline 1,05, aspartic acid 1,01.

Thin layer chromatography (silica-60/Merck, 250 g):

RF (I) of 0.51 (n-butanol: acetic acid: water 3:1:1)

RF (II) 0,60 (n-butanol: acetic acid: water: ethyl acetate 1:1:1:1)

Rf (III) 0,59 (n-butanol: pyridine: acetic acid: water 4:1:1:2).

P R I m e R 4. Synthesis of N-formyl-arginine-Proline-aspartic acid-valine

a) Benzyl ester of N-tert-butyloxycarbonyl- -benzyl-aspartyl-valine.

3,23 g of N-tert-butyloxycarbonyl- -benzyl-asparagine and 3.97 g valine benzyl ester are dissolved in 80 ml of methylene chloride. To the solution add 1,74 ml diisopropylethylamine and maintain the solution at the 5aboutC. Then added to the mixture of 2.06 g dicyclohexylcarbodiimide. The reaction mixture is stirred at 5aboutC for 30 min and then 2 h at room temperature. The precipitate is filtered off and the filtrate is extracted with water, 10% citric acid solution and saturated aqueous sodium bicarbonate. After removal of solvent the initial methylene:chloroform. The product in the form of butter weighs 3.46 in,

b) Benzyl ester of N-tert-butyloxycarbonyl-shed-benzyl-aspartyl-valine.

After removing protection from 3,41 g of the above product by 60 ml of a mixture of methylene chloride-triperoxonane acid for 30 min are salt (triptorelin) in the form of oil. The free amine is obtained by neutralization with a saturated aqueous solution of sodium carbonate with the extraction of methylene chloride. The volume of extract to reduce by evaporation; then added to 1.05 g of N-tert-butyloxycarbonyl-Proline. To the mixture then add a solution of 0.73 g of 1-oxibendazole in 2 ml of dimethylformamide and 0.99 g dicyclohexylcarbodiimide. The mixture is stirred for 2.5 h, and then filtered. The filtrate is extracted with water, 10% citric acid solution and twice with saturated sodium bicarbonate solution. After drying, the solvent is removed, getting the oil. The product is purified by chromatographytandem on silica gel-60 using a mixture of 9:1 methylene chloride:acetonitrile. Fractions containing a major component, give 2,31 g of colorless oil. The IR spectrum of 1740, 1695, and 1675 cm-1.

C) Benzyl ester of N-formyl-N2-nitroarginine- -benzyl-aspartyl-VA is methylene 1:2. The solution is stirred for 30 min, after which the solvent is removed under reduced pressure, resulting in residue waxy solid product, which is washed with petroleum ether. In 15 ml of dimethylformamide is dissolved to 0.72 g of N-formyl-N2-nitroarginine and 0.33 ml of N-methylmorpholine. The solution is cooled to -20aboutWith and add to it dropwise 0.40 g of isobutylacetate. The reaction mixture is stirred at a temperature of from -20 to -10aboutC for 20 min, then add the cooled solution of benzyl ether prolyl- -benzyl-aspartyl-valine (triptorelin) in 20 ml of dimethylformamide and 0.33 ml of N-methylmorpholine. The mixture was stirred at -15aboutC for 20 min, then bath for cooling is removed and continue stirring at room temperature for 2 hours the Greater part of the solvent is removed under reduced pressure. The residue is dissolved in 50 ml of methylene chloride and extracted with water, 10% citric acid solution and saturated sodium bicarbonate solution. The organic layer is dried and the solvent is distilled off, the best result of 2.08 g of the product.

Thin layer chromatography (silica gel GF):

Rf (I) 0,56 (methylene chloride: methanol= 9:1).

Rf (II) to 0.72 (chloroform: methanol: watom hydrogenation transmission using 30 ml of 5% formic acid-ammonium formate at the palladium. The reaction mixture was stirred in the flask, placed in the container, within 18 hours After filtering the mixture through Celite, the solvent is removed under reduced pressure. The residue is dissolved in water and lyophilizers. The product is purified on a column with DEAE-Sephadex, 1,6 60 see the Elution start using a 0.02 M solution of ammonium bicarbonate (pH 8.5), collecting fractions of 150 drops. After selection 52 factions begin to use gradient solution from 0.02 to 0.2 M ammonium bicarbonate (>2 l). The largest peak on the chromatogram lirovannomu product corresponds to the fraction of 88. Faction 75-100 unite and lyophilizers, receiving an amorphous solid. Data high-performance liquid chromatography: retention time of 9.4 minutes in 10% methanol in 0.01 n solution of ammonium acetate, pH 5 at a speed of 1.5 ml/min column Bendpak-C18.

Thin layer chromatography (silica gel-60):

Rf (I) 0,16 (n-butanol: acetic acid: water: 3:1:1).

Rf (II) a 0.27 (n-butanol: acetic acid: water: pyridine: 15:3:12:10).

Rf (III) of 0.42 (ethyl acetate: pyridine: acetic acid: water 5:5:1:3).

Amino acid analysis: asparagine 0,99, Proline 0,97, valine 1,05, arginine 1,00, 38% of the peptide.

P R I m e R 5. Synthesis of arginine-Strait is oxycarbonyl-Proline-(benzyl) aspartic acid-valine. The solution is cooled in an ice bath and add to 0.67 g of 1-oxibendazole and 0.90 g dicyclohexylcarbodiimide in 4 ml of dimethylformamide. After stirring for 10 minutes add a solution of 0.30 g of Isopropylamine in 5 ml of methylene chloride. The mixture is stirred in an ice bath for 15 min, and then leave to heat to room temperature. After 3 hours the precipitate is filtered off. The filtrate is extracted with 10% citric acid solution, water and saturated sodium bicarbonate solution. The organic layer is dried on magnesium sulfate and remove the solvent under reduced pressure. The result: an off-white solid weighing 2,32, During crystallization from ethyl acetate-i - Pr2O obtain 1.64 g of a substance having a melting point 179,5-181,5aboutC.

To 0.84 g of the above substances are added 50 ml of 40% aqueous solution triperoxonane acid in methylene chloride. The solution is stirred for 30 min, then the solvent is removed under reduced pressure. The remaining substance is added diethyl ether, and get a solid substance triptorelin Proline-(benzyl) aspartic acid-valine-Isopropylamine.

In 10 ml of dimethylformamide is dissolved 0,93 g of tert-butyloxycarbonyl-and is Miata. The mixture was stirred at -15aboutC for 20 min, then add to 0.18 ml of N-methylmorpholine and cooled solution of the above salt trifenatate in 10 ml of dimethylformamide. After 30 minutes remove the cooling bath and continue stirring for 2 hours the Greater part of the solvent is removed under reduced pressure. The remaining substance is added ethyl acetate and water. The layers are separated and the extracted organic layer with citric acid solution and twice with saturated sodium bicarbonate solution. After removal of the solvent remains colorless glassy substance weight of 1.44, Cleaning substances are produced using a mixture of 98:2 methylene chloride: methanol. When the first elution allocate specified in the header of the substance; the yield of pure fraction of 0.53 g, as well as obtain 0.32 g of a substance with a low content of impurities.

of 0.53 g of peeled protected tetrapeptide hydrogenizing 1 ml of formic acid in 20 ml of methanol, palladium on mobiles. After 1 h the mixture is filtered through Celite and washed with water. The methanol is removed under reduced pressure. The precipitate is dissolved in water and lyophilizer to obtain 280 g of substance. Purification of the obtained substances produced by the method of chromatography was carried out on S is eraut and lyophilizer to obtain 190 mg of amorphous solids arginine-Proline-aspartic acid-valine-Isopropylamine.

Amino acid analysis: aspartic acid 1,01; Proline 1,03; valine 0,97; arginine 1,00; 96% of the peptide.

Thin layer chromatography (silica gel-60):

Rf (I) of 0.44 (n-butanol:acetic acid: water: pyridine 15:3:12:10).

Rf (II) of 0.65 (ethyl acetate, pyridine, acetic acid water 5:5:1:3).

Rf (III) 0,15 (n-butanol acetic acid-water 3:1:1).

P R I m e R 6. Synthesis of arginine-Proline-aspartic acid-valine-N-methylamide.

To a stirred solution of tert-butoxycarbonyl-valine (10,86 g, 50.0 mmol) and diisopropylethylamine (to 8.70 ml, 1.0 EQ.), of methylamine Hcl (3,38 g, 1.0 EQ.) 1-oxybisethanol (7,66 g, 1.0 EQ.) in 85 ml of methylene chloride and dimethylformamide, taken in the ratio of 12:5, add dicyclohexylcarbodiimide (10.3 g, 50.0 mmol). A precipitate. After 2 h the precipitate was filtered and the filtrate is evaporated to the dry state. The oily residue is dissolved in ethyl acetate and washed three times with saturated sodium bicarbonate solution, water, 10% citric acid and saturated saline. After drying on sodium sulfate the organic phase is filtered and evaporated to obtain a colorless solid. It dissolved into powder in hot hexano and get 7.98 g 69% of the desired amide: the (4,60 g, 20.0 mmol) is added 20 ml of a 50% aqueous solution triperoxonane acid in methylene chloride. After stirring for 30 minutes the solution is evaporated at a temperature of less than 30aboutWith, dissolve the oily residue in methylene chloride, washed twice with a saturated solution of sodium bicarbonate. The organic phase is dried on sodium sulfate, filtered and evaporated to obtain 1.90 g 73% valine-methylamide.

To a stirred solution of tert-butoxycarbonyl-benzyl-aspartic acid (4.72 in g, 14.6 mmol), valine-methylamide (1.90 g, 14.6 mmol), 1-oxybisethanol (2.24 g, 1.0 EQ.) in dimethylformamide (20 ml) add dicyclohexylcarbodiimide (3,01 g, 14.6 mmol). After 5 min, a precipitate may form. After 16 h, the precipitate is filtered off and the filtrate extinguish polysystem solution of sodium bicarbonate.

The obtained solid is collected, washed with water, 10% citric acid, water and diethyl ether and dried in air to obtain a colorless solid. Recrystallization from ethyl acetate receive 5,85 g 92% of the desired dipeptide: tert-butoxycarbonyl-(benzyl)aspartic acid-valine-methylamide.

This dipeptidase (3.94 g, 9,05 mmol) add 20 ml of 50% restorenatural less than 30aboutC and ground to powder with diethyl ether, receiving 4,10 g 100% of the dipeptide triperoxonane acid in the form of a colorless glassy substance.

To a stirred solution of tert-butoxycarbonyl-Proline (1,95 g, 9,05 mmol), dipeptide triperoxonane acid (4,10 g, 9,05 mmol), diisopropylethylamine (1.73 ml, 1.1 equiv.) dimethylaminopyridine (0.12 g, 0.1 EQ.) in dimethylformamide (20 ml) add dicyclohexylcarbodiimide (1,87 g, 9,05 mmol). After 5 min, a precipitate may form. After 16 h the reaction products is filtered and the filtrate extinguish polysystem solution of sodium bicarbonate. The obtained solid is collected, washed with water, 10% citric acid, water and diethyl ether and air-dried to obtain 4.15 g of 86% Tripeptide. It is used without purification.

This amide (4.15 g, 7,78 mmol) is added 15 ml of a 50% aqueous solution triperoxonane acid in methylene chloride. After stirring for 30 minutes the solution is evaporated at a temperature of less than 30aboutC and ground to powder with diethyl ether, the output is of 4.25 g 100% Tripeptide triperoxonane acid as colorless solid.

To mixed 86.9% of the resultant solution AOC-tosyl-arginine (39,7 g, 7,78 mmol), this triple (15 ml) add dicyclohexylcarbodiimide (1,61 g, for 7.78 mmol). After 10 min, a precipitate may form. After 16 h the reaction products are filtered out and the filtrate extinguish polysystem solution of sodium bicarbonate.

The obtained solid is collected, washed with water, 10% citric acid, water, ether and dried in air to obtain 4,82 g 72% protected tetrapeptide.

Half of this solid matter is removed protection in 10 ml of 50% aqueous solution triperoxonane acid in methylene chloride for 30 min, evaporated under reduced pressure and ground to powder with diethyl ether. The obtained solid is digested using a mixture of 30 ml of HF and 8 ml of anisole for 60 min at 0aboutS. Of sediment prepare a slurry in ethyl acetate and extracted twice with 50 ml of 10% acetic acid. Water extracts lyophilizer with obtaining the crude arginine-Proline-aspartic acid-valine-methylamide.

Purification of the crude peptide is produced on Sephadex SPO-25 (column 2,6 83 cm, from 0.05 to 0.3 M ammonium acetate, pH 6.5, gradient: flow rate 100 ml/h, fraction of 9.5 ml, detection at a wavelength of 206 nm). Faction 113-128 unite and lyophilizers obtaining 925 mg tetrapeptide arginine-Proline-aspartic acid-shaft is holding peptide 54%

Thin layer chromatography (silica gel G, 250 microns):

Rf (I) 0,20 (n-butanol acetic acid-water 4:1:5).

Rf (II) 0,26 (n-butanol acetic acid water pyridine 15:3:12:10).

Rf (III) of 0.26 (ethyl acetate acetic acid water pyridine 5:1:3:5).

P R I m e R 7. Synthesis of N-formyl-arginine-Proline-aspartic acid-valine-methylamide.

To AOS-(tosyl)-arginine-Proline-(benzyl)-aspartic acid-valine-methylamide (1,00 g at 1.17 mmol) is added 4.5 M Hcl dioxane (10 ml). After 60 min the reaction product is evaporated, dissolved in water and lyophilizers obtaining 0,92 g 100% tetrapeptide Hcl in the form of colorless powdery solid.

This solid is dissolved in dimethylformamide (10 ml) and diisopropylethylamine (0,93 ml, 4.0 EQ.), add dimethylaminopyridine (0.08 g) and p-nitrophenylphosphate (200 mg, of 1.02 EQ.). After 2 h produce acid purification of reaction mixture with 10% citric acid. The resulting reaction, the solid is filtered off, washed with water and dried in air to obtain 0,41 g formirovala peptided.

This solid is digested with a mixture of 30 ml of HF and 8 ml of anisole for 60 min at 0aboutS. Of sediment prepare a slurry in ethyl acetate and DV is N-formyl-arginine-Proline-aspartic acid-methylamide.

This crude peptide was subjected to purification on DEAE-Sephadex (column 2,690 cm, 0.1 M hydroxide ammonium without buffer solution; flow rate 100 ml/h and fractions of 10 ml, the detection at a wavelength of 206 nm). Faction 38-43 unite and lyophilizer to obtain 80 mg of the amide peptide.

Amino acid analysis: arginine 1.00; Proline 1,00; aspartic acid 1,00; valine 1.00; peptide content 50%

Thin layer chromatography:

Rf (I) 0,32 (n-butanol acetic acid-water 4:1:5).

Rf (II) of 0.38 (ethyl acetate acetic acid water pyridine 5:1:3:5).

Rf (III) 0,34 (triptoreline: hydroxide ammonium 1:1).

P R I m e R 8. Synthesis of tetrapeptide: acetyl-arginine-Proline-valine - alanine-NH(CH3).

a) Synthesis.

Tetrapeptide get by using a peptide synthesizer company "Applid of Biosystems" model A. To do this, use the standard cyclic procedure I (variant manufacturer 1.40) which produce the binding of one acid at a time, except tert-butyloxycarbonyl-arginine, which bind twice. For synthesis use of source materials, are given in table.2.

After tert-butoxycarbonyl group of arginine UD%-aqueous solution of acetic anhydride in methylene chloride with 4-dimethylaminopyridine (15 mg). The resin-peptide is washed with dimethylformamide (3 portions of 10 ml), then methylene chloride (3 portions of 10 ml) and dried in vacuo, yield 1.6 g of substance.

b) Splitting using HF.

Peptide otscheplaut from the resin using 30 ml of HF in the presence of 1.5 ml of anisole. The mixture was stirred at 0aboutC for 1 h and for 4 h at a temperature of 23aboutWith, then HF is removed under reduced pressure. The mixture was washed with diethyl ether (3 portions of 50 ml) and dried in air. The peptide is extracted from the resin of 20% aqueous solution of acetic acid (3 portions of 50 ml).

C) Cleaning.

Aqueous extract lyophilizer and get crude solid. This substance is passed through an ion-exchange resin "Amberlite IR A 68" (acetate form) in water. The fractions containing the peptide, unite and lyophilizers.

This peptide was subjected to purification on preparative column reverse phase of the firm "video recorder" model TR C-18 using the gradient of the solution from 0 to 3.75% acetonitrile in 0.1% aqueous solution triperoxonane acid over 120 min at a flow rate of 14 ml/min Elution control by high-performance liquid chromatography, Eaut in 10 ml of water and lyophilizers, the result 218 mg of the pure product.

Amino acid analysis: arginine 1.00; Proline 0,98; valine 0,99; alanine 1,02; 68% of the peptide.

Thin layer chromatography (plate of silica gel Merck):

Rf (I) 0,31 (n-butanol acetic acid-water 4:1:1).

Rf (II) 0,41 (n-butanol acetic acid in ethyl acetate water 1:1:1:1).

Rf (III) of 0.71 (n-butanol pyridine acetic acid water 5:4:4:2).

P R I m e R 9. Biological activity: the experience of cyclic GMP.

As a result of this experience reveals the activity of the peptide according to the present invention in relation to the binding of receptors in the cell membranes of intact cells OL T-4 and in relation to selective stimulation of the production of cyclic GMP, which corresponds to the activity of the human replenine and human thymopentin.

Line MOL of T-4 cells obtained from the American type culture collection, Rockville, Maryland. Cell MOL T-4 re-sown and grown for 3 days using the method of cultivation described So Audia with TCS. Arch. Biochem. Biophys. 234, 167-177 (1984). Cells are washed three times in phosphate buffer solution and again transferred in suspension in RPMI-1640 at a concentration of 1.0 to 107cells/ml, after which the balance is stalnye peptides. Incubation is carried out in a water bath with shaking for 4-5 minutes and finish by injection of 1 ml ice trichloroacetic acid (10% solution).

Cells in trichloroacetic acid homogenized and treated with ultrasound for the removal of cyclic nucleotides. The suspension is centrifuged at 3000 g for 20 min and a temperature of 4aboutC. the Resulting precipitate was dissolved in 0.1 N. the solution of hydrate of sodium oxide to determine protein content. Trichloroacetic acid is removed from the top of the fraction by 4-fold extraction portions 5 ml of water-saturated diethyl ether. After extraction of the remaining traces of ether are removed by heating for 10 min in a water bath at 50aboutC. After liofilizirovanny the sample is again dissolved in 50 mm acetate buffer (pH 6,2) for radioimmunoassay studies on cyclic GMP.

In Fig.1 shows typical curves in the coordinates of the dose response defined in the range from 1 to 1000 μg/ml obtained for active peptides acetyl-arginine-Proline - D-aspartic acid-valine-NH2arginine-Proline-alanine-valine-tyrosine and acetyl-arginine-Proline-alanine-valine-NH2in crawlin-lysine-aspartic acid-valine and lysine-lysine-aspartic acid-valine-NH2for cell MOL T-4.

In Fig. 2 shows the curves in the coordinates of the dose-response defined in the range from 1 to 100 µg/ml for the active peptide acetyl-arginine-Proline-valine-alanine-NH2in comparison with thymopentin (cell MOL T-4).

Threshold activity determined for each of the investigated peptides. Threshold activity is defined as the lowest concentration of the test peptide, which causes intracellular level of cyclic GMP, greater than 2 standard deviation values for the control sample. Control samples have values of intracellular cyclic GMP is less than 0.5 g mol/ml (mean standard deviation). The test results are considered positive if the level of cyclic GMP in more than 2 times (more than 2 standard deviations) level defined for the parallel negative control sample.

The results from the conducted experiments on cyclic GMB shown in Fig.1 and in the corresponding table 3 and Fig.2 and in the corresponding table.4, in which the representatives of the peptides according to the present invention was tested in comparison with thiopentone and control peptides. Mon-tyrosine) cell MOL T-4, because Pentapeptide of human replenine SP-5 (arginine-lysine-alanine-valine-tyrosine) is inactive against cells MOL T-4. These results indicate that the peptides according to the present invention show a biological activity in stimulating the activity of helper T-cells if cells MOL F-4.

Values for the zero control peptide sample are in the range from 0 to 0.3 PG/ml

P R I m e R 10. Stability with respect to the action of enzymes.

To illustrate the stability of the peptides according to the present invention in relation to degradation by enzymes in the serum and digestive tract, taken as example, the peptide acetyl-arginine-Proline-aspartic acid-valine-NH2incubated at 37aboutWith the gastric juice of the rat and human serum for 120 minutes For comparison, similar processing is subjected to tetrapeptide of arginin-lysine-aspartic acid-valine and thymopentin. Analysis by high performance liquid chromatography shows that the peptide arginine-lysine-aspartic acid-valine completely decompose after 25 C. Thymopentin subjected to a 50% increase decay within about 20 C. the Peptide according to at least 120 C.

P R I m e R 11. Biological activity: analysis of tumor suppression.

The analysis is performed using a normal healthy mice of experimental animals. In the mice (groups of 12-20 units) is administered by subcutaneous injection either 30 mg or 30 mg of peptide, corresponding to this invention, once a week. This peptide (acetyl-Arg-Pro-Val-Al-NH2) called splenorenal, enter in citrate buffer containing 1% glycine and 2% pentahydrate-raffinose. In the mice of the control group is administered by a similar injection of 30 μg of glycine-raffinose medium without peptide.

4 weeks after the start of these weekly injections each mouse inoculant intradermally tumor cells of mice 510 WR, WR is a regular benzo (a) pyrene-induced tumor mice. Then once in every week in the body of each mouse is injected by the injection of the same number or peptide compositions, either alone media.

In Fig. 3 shows the percentage of mice in each group who show a negative reaction to the presence WR tumors, in the days after insulinopenia tumor cells. In Fig.4 shows for the same experiment, the maximum size of tumors in those mysod peptide, meets the present invention provides mice the ability to resist the spread and growth of tumor cells. It was shown that in mice treated with placebo, found developed VR tumors. Thus, it is found that the peptide activates the immune system of an animal, inhibiting tumor growth, showing thus an increased activity of T-cell phage helper.

P R I m e R 12. Biological activity: analysis of viremia.

Carrying out an analysis using normal healthy mice without family joining CDI), in mice groups of 30 pieces first injected by subcutaneous injection of lethal dose 50 adapted mice influenza virus A/HK/68. 2 days after infection infection in the body of each mouse is injected 0,03 µg peptide corresponding to this invention, by subcutaneous injection. This peptide (acetyl-Arg-Pro-Val-Al-NH2) called splenorenal, enter in the formulation described above, containing glycine and raffinose. In the control group mice injected through the same injection of 0.03 µg of glycine-raffinose medium without peptide.

In the body of each mouse injected with the same dose of either peptide, either alone media petemote the overall proportion of deaths from the number of days after viral infection. This graph shows that those mice treated with the peptide corresponding to the present invention have a lower risk of death after 12 days, than mice treated with a placebo. Thus, this peptide exhibits a therapeutic or activating effect on the immune system of the treated mice, which enables a greater number of mice to resist viral infection.

P R I m e p 13. Biological activity: testing in diabetic mice.

Put strain noirish (NOD), diabetic mice, in Nopital Necker, Paris, France. Females spontaneously developed autoimmune disease of the pancreas. In the normal course of the disease mice cells of the pancreas were destroyed that had resulted in the loss of production of insulin and diabetes. Thus, this mouse strain is a model similar to the juvenile diabetes type I, which is usually treated by insulin replacement, but which can usually slow down by immunosuppressive drugs such as cyclosporine.

In this analysis in mice NOD females in groups of 1-12 units administered by injection 2 mg/mg mA is, the so-called splenorenal injected into the above formula, containing glycine and raffinose. In the mice of the control group injected through the same injection 1 mg glycine-raffinose medium without peptide once a week.

In Fig.6 shows the results of this treatment on the curves decrease diabetes (in percent) age (in weeks) of the studied mice. Strikingly, treatment with a peptide corresponding to this invention, maintains immune function and ensures the survival of a larger number of animals than treatment with placebo, indicating positive effects, such as stimulation or recovery incomplete cellular function of suppressor T these animals.

P R I m e R 14. Neuromuscular analysis.

This analysis measures the ability of the peptide to reduce holinoliticheskoy the transmission of neuromuscular function. It is known that thymopentin and thymopentin cause this decrease neuromuscular transmission.

Dissolve various doses of the test peptide in phosphate buffer saline and injected subcutaneously or orally to female mice, weighing 25-30 g (line CD). Spend electromyographic analysis, according to Goldstein with TCS. j. Neurosurg. Psychlat. 31, 453-459 (1968 the t Grasse S-48 stimulator and grass SIU-5A, isolating stimulus device (grass medical instruments, Quincy, MA), and record the electromyographic response to Tektronix nakaplivaya oscilloscope-SIII, United with 521N differential amplifier and 510N time counter (Tektronix, Beaverton, OR).

When supramaximal nerve stimulation-30 pulses per second, the height of the tenth potential muscle actions expressed as a percentage of the first. The p-values to determine the statistical difference between control and test compounds are calculated using duraderos t-test Dunnet. The results obtained in neuromuscular analysis with the test peptide, compared to the results with thymopoietin and thymopentin, which are listed in the table.5.

P R I m e R 15. Regulation of Anti-CD-3-stimulated T-suppressor cells.

Mouse: Female NZB x NZW FI mice purchased from Jackson laboratories (Bar Harbur, IU) and placed in cages the size of a Shoe box on the floor, five animals in a cage, in the IRI vivarium. Food and water provide plenty. Animals humanely kill CO2upon completion of the test period.

Tests. Mice treated subjects peptides before killing one of the two about the (Abbott) 1 mg/kg subcutaneously once a week for four weeks, starting from four weeks of age. In the Protocol In a seven-mice do four injections of 1 mg/kg subcutaneously consistently for four days. All animals kill to analyze the activity of T-suppressor cells the next day after the last injection.

Splenic drug.

Aseptically remove the spleen, preparing a suspension of single cells for each individual animal by passing through a sieve of 60 mesh (Sigma) and the suspension in the medium for tissue culture containing RPMI 1640 c 25 mm HEPES buffer, 10% fetal bovine serum, 2% sodium pyruvate, 1% NEN optional amino acids, and 1% glutamine (all from Flexible, Grand Island, N. Y.), 1% -mercaptoethanol (Sigma, St, Louis, MO) and 0.05% gentamicin (Flow, McLean, VA).

Anti-SRS-generated suppressor cells.

Suppressor cells for proliferative responses to the mitogen-stimulated T cells generated in cells in 35 mm tissue culture (Corning, Corning, N. Y.), coated with anti-SRS (Clone 145-S, Boehringer Mannheim, Indianapolis, IN) diluted in borate buffer saline pH 8.5 at a concentration of 0.5 ng/ml These plates are incubated at room temperature for 4 h and washed three times of balansera the data at 5106cells /ml and the culture incubated for 48 h in 5% CO2at 37aboutC. Collect stimulated culture and bring the number of cells to 1,25106cells/ml by adding culture analysis to suppress at 25% of the number of responding cells. Suppression of proliferation of T cells measured in a 48-hour mitogen-stimulated cultures (0.25 ng/ml anti-SRS, Boeringer Mannheim) containing 5105fresh singining of splenocytes and 1,25105the tested cells in triplicate cultures in plates of 96 wells. The cells shaken in the last 7 hours with 1 NCU (3H) - thymidine (NEN research products, Cadds, Ford, PA), collected on a filter plates (Pharmacia, Turku, Finland) and count on metaplot-liquid acquired scintillation counter USING 1205 (see Fig.7).

P R I m e R 16. Mouse NZB x NZW FI: Test for survival and the study of proteinuria.

Mouse: female NZB x NZW FI (BW) three weeks of age purchased from Jackson laboratories (Bar Harbur, IU). Animals kept on litter in cages the size of a Shoe box for 5 mice in a cage, give them water and drink plenty. After 1 week quarantine period, the animals placed on study in one of the three groups of subjects; 30 animals per group (6 cells). Control stereotactically water for injection (Abbott). The treatment is carried out once a week or three times a week, starting from 4 weeks of age.

Control. Animals check every day for survival.

Tests for proteinuria are conducted monthly during the first six months and every week after you have marked the initial changes.

In NZB x NZW FI mice spontaneously develop an autoimmune disease, which leads to their premature death from kidney disease. Because IRI-780 restores the deficit generation of suppressor cells, manifested in these mice, it is periodically injected to determine the impact of such treatment on their autoimmune disease.

Result. Although there is no statistically significant difference between the three groups regarding life expectancy, there is a significant (p 0,007) number of animals treated IRI-780 (AU-RPv-NH2) h week, have not developed proteinuria compared with the control group (see Fig. 8). This involves the suppression of the autoimmune process that causes the kidney disease, which is defined by proteinuria.

1. The method of producing peptides of General formula I

R Arg X Y Z R1,

where R is hydrogen, acetyl, formyl;

X Proline;
series: valine, alanine;

R1H, OH, NH2, NH(CH3), NHCH(CH3)2,

characterized in that a) the appropriate amino acid, which is a compound of formula I, protected by amino-hydroxy-, indomitable group; b) then carry out the etherification aminosidine patinage fragment containing the C-terminal amino acid, resin selected from the group consisting of a polymer or copolymer comprising cellulose, polyvinyl alcohol, polymethylmethacrylate, polystyrene and politicalgovernmental; (c) removing the protective group from the resulting aminosidine connection, d) to the resulting product, attach the following aminosidine C-terminal amino acid or the second peptide fragment; (e) remove the protective group from the resulting aminosidine peptide derived; (f) repeating steps (d) and (e) for the remaining amino acids of the compounds of General formula I; g) otscheplaut peptide from the received peptidylarginine and, if necessary, removing the protective group.

2. The method according to p. 1, characterized in that, as the protective groups using t-butyloxycarbonyl and toil.

3. The method according to p. 1, characterized in that the peptide is chosen from the group of: formyl-arginine-Proline aspartic acid-valine acetyl-arginine-Proline-alanine-valine-NH2arginine-Proline-aspartic acid-valine-NHCH(CH3)2, formyl-arginine-Proline-aspartic acid-valine-NH(CH3), acetyl-arginine-Proline-valine-alanine-NH(CH3)

4. The method according to p. 1, characterized in that the peptide represents acetyl-arginine-Proline-valine-alanine NH(CH3).

5. The method of producing peptides of General formula I

R Arg X Y Z R1,

where R is hydrogen, acetyl or formyl;

X Proline;

Y L-amino acid is selected from the range of: alanine, aspartic acid, valine;

Z L-amino acid is selected from the range of: valine, alanine;

R1H, OH, NH2, NH(H3)NHCH (CH3)2,

characterized in that the solution is carried out stepwise or block the accession of amino acids or peptide fragments contained in the above formula, with the formation of the corresponding amide bonds.

6. The peptides of General formula I

R Arg X Y Z R1,

where R is hydrogen, acetyl or formyl;

X Proline;

Y L-amino acid selected from alanine, asparginase acid or valine;

Z L-form amino acids selected from valine or alanine;

R1H, OH, NH2, NH(CH3), NHCH(CH3)2.

7. Peptides under item 6, characterized in that iSlate-valine-NH2acetyl-arginine-Proline-alanine - valine-NH2arginine-Proline-aspartic acid-valine-NACH(CH3)2arginine-Proline-aspartic acid-valine-NH(CH3), acetyl-arginine-Proline-aspartic acid-valine-NH(CH3), arginine-Proline - valine-alanine-NH2, formyl-arginine-Proline-aspartic acid - valine-NH(CH3), acetyl-arginine-Proline-valine-alanine-NH(CH3).

8. Immunomodulatory composition comprising the active principle and a pharmaceutically acceptable carrier, characterized in that it contains as active principle at least one peptide of formula I on p. 7 in an effective amount.

9. The composition according to p. 8, characterized in that it is intended for oral administration, and an effective amount of the peptide of formula I on p. 7 ranges from 1 μg to 100 mg/kg body weight.

10. The way the regulation is insufficient or excessive functions of T cells in a patient by introducing a peptide, characterized in that the injected peptide of General formula I on p. 7 at a dose of from 1 μg to 100 mg/kg body weight.

 

Same patents:

FIELD: medicine, immunology, peptides.

SUBSTANCE: invention relates to a new composition of biologically active substances. Invention proposes the composition comprising of peptides of the formula: Arg-Gly-Asp and H-Tyr-X-Y-Glu-OH wherein X means Gln and/or Glu; Y means Cys(acm) and/or Cys that elicits ability to inhibit the proliferative response for phytohemagglutinin, to induce the suppressive activity of mononuclear cells and ability of peptides to induce secretion of immunosuppressive cytokines of grouth-transforming factor-β1 and interleukin-10 (IL-10). The composition can be prepared by a simple procedure.

EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

FIELD: organic chemistry, medicine.

SUBSTANCE: invention represents ligands MC-4 and/or MC-3 of the formula (I): , wherein X means hydrogen atom, -OR1, -NR1R1' and -CHR1R1' wherein R1 and R1' are taken among the group: hydrogen atom, (C1-C6)-alkyl and acyl; (1) each R2 is taken independently among the group: hydrogen atom, (C1-C6)-alkyl; or (2) (a) R2 bound with carbon atom that is bound with X and Z1 and substitute R5 can be optionally bound to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; or (b) R2 bound with carbon atom that is bound with ring Ar can be bound with R7 to form ring condensed with ring Ar; each among Z1, Z2 and Z3 is taken independently from the following groups: -N(R3e)C(R3)(R3a)-, -C(R3)(R3a)N(R3e)-, -C(O)N(R3d)-, -N(R3d)C(O)-, -C(R3)(R3a)C(R3b)(R3c)-, -SO2N(R3d)- and -N(R3d)SO2- wherein each among R3, R3a, R3b and R3c, R3d, R3e when presents is taken independently among hydrogen atom and (C1-C6)-alkyl; p is a whole number from 0 to 5 wherein when p above 0 then R4 and R4' are taken among hydrogen atom, (C1-C6)-alkyl and aryl; R5 represents 5 substitutes in phenyl ring J wherein each R5 is taken among hydrogen atom, hydroxy-, halogen atom, thiol, -OR12, -N(R12)(R12'), (C1-C6)-alkyl, nitro-, aryl wherein R12 and R12' are taken among hydrogen atom and (C1-C6)-alkyl; or two substitutes R5 can be bound optionally to form carbocyclic or heterocyclic ring that is condensed with phenyl ring J; q = 0, 1, 2, 3, 4 or 5 wherein when q above 0 then R6 and R6' are taken among hydrogen atom and (C1-C6)-alkyl; Ar is taken among the group consisting of phenyl, thiophene, furan, oxazole, thiazole, pyrrole and pyridine; R7 are substitutes at ring Ar wherein each R7 is taken among hydrogen, halogen atom, -NR13R13', (C1-C6)-alkyl and nitro- wherein R13 and R13' are taken among hydrogen atom and (C1-C6)-alkyl; r is a whole number from 0 to 7 wherein when r is above 0 then R8 and R8' are taken among hydrogen atom and (C1-C6)-alkyl; B is taken among -N(R14)C(=NR15)NR16R17, -NR20R21, heteroaryl ring and heterocycloalkyl ring wherein R14-R17, R20 and R21 are taken independently among hydrogen atom and (C1-C6)-alkyl; s = 0, 1, 2, 3, 4 or 5 wherein when s is above 0 then R and R9' are taken among hydrogen atom and (C1-C6)-alkyl; R10 is taken among the group consisting of optionally substituted bicyclic aryl ring and optionally substituted bicyclic heteroaryl ring; D is taken among hydrogen atom, amino- and -C(O)R11 wherein R11 is taken among the following group: hydroxy-, alkoxy-, amino-, alkylamino-, -N(R19)CH2C(O)NH2 wherein R19 represents (C1-C6)-alkyl, -NHCH2CH2OH and -N(CH3)CH2CH2OH, or its isomers, salts, hydrates or biohydrolysable ester, amide or imide.

EFFECT: valuable medicinal properties of compounds.

18 cl, 107 ex

FIELD: medicine.

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EFFECT: higher efficiency and prophylaxis.

4 dwg, 5 ex

FIELD: medicine, chemistry of peptides, amino acids.

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EFFECT: simplified method for preparing composition, valuable medicinal properties of composition.

4 cl, 16 tbl, 9 ex

FIELD: medicine.

SUBSTANCE: method involves applying composition based on receptor antagonist P substance and magnesium salt.

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23 cl, 2 dwg, 3 tbl

FIELD: medicine, peptides.

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EFFECT: enhanced and valuable properties of oligopeptides.

34 cl, 2 tbl, 7 dwg, 4 ex

FIELD: medicine; pharmacology.

SUBSTANCE: releasing peptides of growth hormone are described with formula (I): R112345-R2, where:А1 designates Aib, Apc or Inp; А2 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-Ser(Bzl) or D-Тrp; А3 designates D-Bal, D-Bip, D-Bpa, D-Dip, D-1Nal, D-2Nal, D-2Ser(Bzl) or D-Trp; А4 designates 2Fua, Orn, 2Pal, 3Pal, 4Pal, Pff, Phe, Pim, Taz, 2Thi, 3Thi, Thr(Bzl); А5 designates Apc, Dab, Dap, Lys, Orn or deleted; R1 designates hydrogen; and R2 designates NH2; and their pharmaceutically acceptable salts.

EFFECT: pharmaceutical compositions and the methods of their application are presented.

25 cl, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention can be used for medical treatment of secondary hypothyroid state accompanied by low synthesis of thyrotrophic hormone by hypophysis and of iodine hormone by thyroid gland. Substance of invention implies application of peptide Lys-Glu-Asp-Gly as a medicine stimulating synthesis of thyrotrophic hormone by hypophysis and of thyroid hormone by thyroid gland.

EFFECT: high specific activity of introduced peptide and decrease of side effect risk.

4 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: present invention refers to compounds of Formula II and to methods of immune response suppression, e.g. by inhibition of indirect MHC type II of T-cells activation. Compounds under invention can be applied to treatment or prevention of derangements, such as rheumatoid arthritis and/or multiple sclerosis.

EFFECT: production of compounds which can be used for immune response suppression.

25 cl, 19 dwg, 4 tbl, 22 ex

FIELD: medicine.

SUBSTANCE: invention refers to pharmaceutical industry, namely to medicated products normalising cardiac function, particularly being perspective for treatment of coronary heart disease (CHD). Peptide of general formula CH3CO-Lys-Lys-Arg-Arg-NH2 is disclosed as an anti-ischemic and antihypoxic agent.

EFFECT: declared invention ensures apparent anti-ischemic, antihypoxic and adaptive action and is being available in medical practice.

1 cl, 3 ex, 5 tbl

FIELD: organic chemistry, medicine, pharmacy.

SUBSTANCE: invention relates to compounds of the formula (I):

wherein r = 1, 2 or 3; s = 0; t = 0; R1 is taken among group including R11-CO and R12-SO2- wherein R11 is taken among group including (C6-C14)-aryl, (C1-C8)-alkyloxy-group wherein all given group are unsubstituted or substituted with a single or some similar or different substitutes R40; R12 means (C6-C14)-aryl wherein indicated group is unsubstituted or substituted with a single or some similar or different substituted R40; R2 means R21(R22)CH-, R23-Het-(CH2)k-, R23(R24)N-(CH2)m-D-(CH2)n- or R25(R26)N-CO-(CH2)p-D-(CH2)q- wherein D means bivalent residue -C(R31)(R32)-, bivalent (C6-C14)-arylene residue or bivalent residue obtained from aromatic group Het comprising 5 or 6 atoms in cycle among them 1 or 2 are similar or different cyclic heteroatoms taken among group including nitrogen and sulfur atoms; numbers k, m, n, p and q = 0, 1, 2; R21 and R22 that are independent of one another can be similar or different and taken among group including hydrogen atom, (C1-C12)-alkyl, (C6-C14)-aryl and so on; R23 means hydrogen atom, R27-SO2- or R28-CO-; R24, R25 and R26 mean hydrogen atom; R27 is taken among group including (C1-C8)-alkyl, (C6-C14)-aryl and so on; R28 is taken among group including R27, (C1-C8)-alkyloxy-group; R31 and R32 mean hydrogen atom; R40 is taken among group including halogen atom, hydroxy-, (C1-C8)-alkyloxy-group, (C1-C8)-alkyl, (C6-C14)-aryl and so on; R91, R92, R93 and R96 means hydrogen atom; R95 means amidino-group; R97 means R99-(C1-C8)-alkyl; R99 is taken among group including hydroxycarbonyl- and (C1-C8)-alkyloxycarbonyl-; Het means saturated, partially unsaturated or aromatic monocyclic structure comprising from 3 to 6 atoms in cycle among them 1 or 2 are similar or different heteroatoms taken among group comprising nitrogen and sulfur atoms; in all its stereoisomeric forms and also their mixtures in any ratios, and its physiologically acceptable salts. Invention proposes a method for preparing compound of the formula (I). Also, invention proposes a pharmaceutical preparation eliciting inhibitory activity with respect to factor VIIA and containing at least one compound of the formula (I) and/or its physiologically acceptable salts and pharmaceutically acceptable carrier. Invention provides preparing compounds of the formula (I) eliciting power anti-thrombosis effect and useful for treatment and prophylaxis of thrombosis-embolic diseases.

EFFECT: valuable medicinal properties of compounds and composition.

10 cl, 70 ex

FIELD: chemistry; medicine.

SUBSTANCE: invention relates to derivatives of 2-hydroxytetrahydrofurane , of general formula (I) , which possess ability to inhibit calpaines and/or ability to catch active oxygen forms and can be used to obtain medication, intended for inhibiting calpaines and/or lipid peroxidation.

EFFECT: medications possess higher efficiency.

9 cl, 64 ex

FIELD: medicine.

SUBSTANCE: invention refers to inhibitors of enzymes cleaving protein after proline, such as depeptidyl peptidase IV inhibitors, as well as to their pharmaceutical compositions, and methods of application of such inhibitors. Particularly, inhibitors under this invention are improved in comparison with those currently in use in the present art by selecting special classes of side chains in P1 and/or P2 positions of inhibitor which contains carboxylic acid grouping.

EFFECT: compounds of specified formulas I, II and III can have the improved therapeutic index, partially owing to reduced toxicity and improved specificity in relation to target protease.

15 cl, 2 dwg, 6 ex

FIELD: chemistry.

SUBSTANCE: lipodipeptide is 1-hexadecyl-5-(1-pyrenebutyl)-N-(L-ornitile)-L-glutamatebischloride, which can deliver nucleic acids into cells and contains in its structure a stable fluorescent mar in form of pyrene. The obtained compound is 10 times less toxic to Hela and HEP cells compared to Lipofectamine. The synthesised compound has more efficient transfection on neoplastic (HeLa, HEP, COS-7) and nonneoplastic cell lines (CHO, rat leucocyte) than special commercial transfection agents for HEP and CHO cells.

EFFECT: owing to presence of a stable fluorescent mark in the structure of the synthesised compound, the field of using such substances widens considerably and it becomes possible to study localisation of labelled liposomes and duration of the transfection process.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to compounds, which possess agonistic or antagonistic activity of NGF and BDNF neutrophins and represent monomer or dimmer substituted dipeptides, which are analogues of exposed outside parts of loops 1 and 4 of said neutrophins, close to beta-bends of said loops or coinciding with them. Effects in vivo are demonstrated by claimed compounds within dose interval 0.01-10 mg/kg in intraperitoneal introduction.

EFFECT: claimed compounds possess neuroprotective and differentiating activity on cell models, increase concentration of phosphorylated form of tyrosine kinase A and proteins of heat shock Hsp32 and Hsp70 in concentrations10-5-10-9 M, they also possess neuroprotective, antiparkinsonian, anti-stroke, antiischemic, antidepressant, antiamnestic activity on animal models and demonstrate activity on experimental models of Alzheimer's disease.

20 cl, 33 dwg, 23 tbl, 18 ex

FIELD: chemistry.

SUBSTANCE: synthesis is carried out using a liquid-phase technique via condensation of pentafluorophenyl ether of Nα-benzloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with methyl ether of O-tert-butyl-L-threonine in the presence of N-methylmorpholine in ethyl acetate medium and followed by hydrogenation of the obtained compound with hydrogen in methyl alcohol using palladium hydroxide as a catalyst. Purification of the desired protected dipeptide is carried out using a salt with oxalic acid. The initial pentafluorophenyl ether is obtained from reaction of Nα-benzyloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with pentafluorophenol and is then used without extraction. The disclosed method enables to obtain the desired product with output of over 96% and high degree of purity.

EFFECT: obtained protected dipeptide can be used to produce octreotide or analogues thereof, or other peptides containing a L-lysine-L-threonine fragment.

2 ex

FIELD: pharmacology.

SUBSTANCE: invention relates to a dipeptide prodrug of general formula (I)

,

which can be used to treat and/or prevent cardiovascular diseases.

EFFECT: increased application effeciency.

3 cl, 4 tbl, 7 ex

FIELD: pharmacology.

SUBSTANCE: invention relates to a germanium compound of the general formula (I): Ge[P]x[CA]y, where P is a peptide that is Lys-Glu; CA is a hydroxycarboxylic acid selected from malic acid or citric acid; x=1 for y=1, x=2 for y=0. A method for preparation of a formula (I) compound and a pharmaceutical composition having an immunomodulatory activity comprising a formula (I) compound are also provided.

EFFECT: increased activity of the composition.

15 cl, 6 dwg, 5 ex

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