The peptides and compositions on their basis, is able to regulate immune function mammals

 

(57) Abstract:

Usage: in biology and medicine to regulate immune function mammals. The inventive peptides F.-ly (I): NR1R2where NR1R2methylamide, 2-phenylhydrazine, piperidine, cyclohexylamine and isopropylated, and a composition comprising a compound of f-crystals I and pharmaceutically acceptable carrier, and connections f-ly I - in the amount of 0.001 - 100 mg/kg of body weight. 2 S. and 2 C.p. f-crystals, 1 table.

The invention relates to peptides and compositions based on them, is able to regulate the immune function in mammals, which may find application in medicine.

Known immunomodulatory proteins, thymopoietin and displayn (or simply "splenin"), isolated from thymus and spleen, respectively bull and man. In addition, the known synthetic peptides which have biological activity thymopoietin and replenine was further modified with the acquisition of additional qualitative traits, such as resistance to enzymatic action.

Known incapatibility, which is the active site thymopoietin and has the sequence Arp-Lys-sp-Val-Tight. Also known PE deputies. As thymopoietin and thymopentin induce biological changes in two human T cell lines CEM and MOLT-4, indicating their participation in stimulating the activity of T cells as suppressor and as cell-attendant. The disadvantage of these peptides containing peptide chain of at least 5 amino acid residues, is their inaccessibility, in addition, they do not possess the ability to stimulate the activity of suppressor and helper of the human immune system in different T-cell disorders.

First, it was found that the number of tetrapeptides containing amides with limit substitution of carboxyl groups, has the activity of T-cell suppressor characteristic thymopentin in cyclic GMP in cells SEM. This discovery is surprising in view of the fact that the same tetrapeptide with a free carboxy end group or amidarone different C-terminal amide groups, do not cause such activity T-cell suppressor.

The purpose of the invention, the search range derived peptides containing a small number of amino acid residues, of new compounds with the ability to regulate insufficient or excessive T-cell function in the body is the soup of the formula I:

Arp-Lys-sp-ValNR1R2where NR1R2selected from the group including aniline, methylamide, 2-phenylhydrazine, piperidine, cyclohexylamine and Isopropylamine; preferred are peptides having the ability to regulate insufficient or excessive T-cell function of the body. And a composition capable of regulating immune function in mammals containing peptide and a pharmaceutically acceptable carrier, in which the said peptide is a compound of formula I:

Arp-Lys-sp-ValNR1R2where NR1R2selected from the group including aniline, methylamide, 2-phenylhydrazine, piperidine, cyclohexylamine and isopropylated, in the amount of 0.001-100 mg/kg of body weight; preferred is a composition suitable for oral administration.

Obtaining a peptide corresponding to the invention can be carried out by way of solid-phase synthesis or by standard methods of synthesis in solution (method of solid-phase synthesis is well known and straightforward way to obtain peptides). Solid-phase method for the synthesis include the phased introduction of protected amino acids to a growing peptide chain, which is linked by covalent bonds with Toluca thus the need for purification of intermediate products. The generally accepted concept of this method depends on the connection of the first amino acid chain with non-solid polymer by covalent bonds. Subsequent protected amino acids are introduced singly or jointly in a phased manner until, until it forms the desired sequence. And, finally, the protected peptide is removed from the solid resin base and protecting groups are split. Amino acids may be introduced in any suitable polymer in the form of a resin. This resin must contain a functional group which can be firmly connected by covalent linkage of the first protected amino acid. For this purpose various suitable polymers, such as cellulose, polyvinyl alcohol, polymethyl methacrylate, and N-alkylenediamines, N-alkylbenzenesulfonate and polystyrene resin. Suitable protective groups used in such synthesis processes include tert-butyloxycarbonyl (BOC), benzyl (Bzl), t-aryloxyalkyl (AOC), tosyl (s), ortho-bromophenylacetate (BrZ) and phenylmethanesulfonyl (Z or CZ).

In General terms, the procedure for obtaining the peptides according to the invention involves the initial binding of the protected C-terminal amino acids from the resin. After this binding is the notes removed. The removal of this protecting group should be carried out without breaking the connection between the amino acid and resin. The obtained resin peptide then a conjugate binds to protected-end penultimate amino acid. It paired binding occurs with the formation of amide bond between the free carboxyl group of the second amino acid and the amino group of the first amino acid linked to the resin. This sequence of procedures is repeated with the subsequent amino acids to until all amino acids are not bound with resin. And, finally, the protected peptide is cleaved from the resin in the usual way known to specialists, such as how aminolysis using cyclohexylamine, and protecting groups are removed with the formation of the desired peptide. Ways of splitting, used to separate the peptide from the resin and removal of protecting groups, depend on the choice of the resin and protecting groups and well-known to specialists involved in the synthesis of peptides.

The peptides can also be obtained by standard methods of synthesis in solution, including manual or bulk conjugate binding amino acids or peptide fragments with the use of chemical or Germnay area.

Peptides corresponding to the invention, showing biological effects similar to those of human thymopentin. This biological activity is determined primarily by analysis with measurement of inducing the formation of cyclic GM in human T-cell lines in comparison with good human thymopentin. The induction of education IDA peptide, corresponding to the invention, this analysis indicates the ability of this peptide to contact with the receptor site of human thymopentin in the cell and to induce biological activity similar to human thymopentin.

Many of the described peptides have other very important advantages. Many peptides, corresponding to the invention, can be characterized by resistance to enzymatic degradation as splitting and serum enzymes. Thus, they exhibit long half-life when the input is injected into a biological subject. Another advantage of many of these peptides is their ability to oral input. Due to the immunomodulatory characteristics of the claimed peptides they find therapeutic application for the treatment of humans and possibly animals, because they have the FPIC of the body. In the result of the claimed peptides can be considered as peptides multipurpose therapeutic use.

The proposed peptides are able to inform the overall immunity of the body in the sense that they increase, or give the ability to therapeutic stimulation of cellular immunity. The claimed peptides or containing pharmaceutical compositions are useful in applications where lost cellular immunity, especially where there is immunological failure. Thus, they are used to treat diseases associated with allergic reactions and autoimmune reactions. Where there is excess production of antibodies or cell-mediated immunity caused by unbalanced T-cells, the claimed peptides can correct this condition by stimulating the activity of T-cell suppressor. They are useful therapeutic use for some autoimmune diseases, in which produced damaging antibodies such as lupus erythematosus, rheumatoid arthritis and others. In the most broad application of the described peptides or containing pharmaceutical compounds are used for regulation, and that these connections lead to the return of the immune system from the abnormal state, i.e. the state of the disease, to a normal equilibrium. Although such regulation can find wide application in the correction of immunological abnormalities, such as the syndrome of DiGeorge, it can also be used to adjust a state of heightened immunological activity, such as autoimmune diseases. Thus, the invention encompasses methods of regulating the immune system of a human or animal in need of such regulation, which (way) enclose the entry in the human or animal immunoregulator-effective amount of at least one of the peptides, but also encompasses pharmaceutical compositions for the implementation of these methods. The invention encompasses a method of treating diseases caused by absolute or complete lack of the body's immune system, especially in the function of T-cell suppressor. The method includes entering into the organism a therapeutically effective amount of at least one of the peptides corresponding to this invention.

The term "therapeutically effective amount" means that amount which is effective for treatment of the above diseases. The invention also covers a method of endocervically at least one of the peptides, corresponding to this invention. In addition, the invention encompasses pharmaceutical compositions for the implementation of these methods. To obtain pharmaceutical compositions conforming to the invention, the proposed peptide is mixed as an active ingredient with a pharmaceutical carrier according to accepted pharmaceutical methods of compounding. The carrier may take various forms, which depend on the form of preparation desired for entry into the body, for example, for oral, sublingual, rectal, nasal or parenteral input. In the preparation of compositions in the preferred dosage form for oral input may be any of the commonly used pharmaceutical environment. For the preparation of liquid preparations for oral input, such as suspensions, elixirs and solutions, can be used a medium containing, for example, water, oils, alcohols, flavouring means of protection tools, coloring agents, etc. To prepare solid preparations for oral input, such as powders, capsules, tablets can be used in devices such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and so on Molotkovsky are the most preferred dosage form oral input and used in this case solid pharmaceutical carriers. If desired, tablets may be sugar coated or intersolubility coating standard ways.

For parenteral products entering the media typically comprises sterile water, although there may be included other ingredients that promote, for example, solubility or for defensive purposes. Can be prepared also injecting a suspension, in which case it may be a suitable liquid, suspendiruemye agents, etc.

Peptides corresponding to the invention, usually active when you enter them in amounts of about 1 g/kg body weight, and preferably in an amount of about 0.001 to 10 mg/kg of body weight, although there may be used lower doses. Usually the same range of measured quantities can be used to treat diseases or conditions listed above that are associated with immune deficiency. To suppress excessive immune activity are higher dosed quantity, for example, about 10-100 mg/kg of body weight.

In the examples the following abbreviations are used: FA triperoxonane acid; SPLA acetic acid; CH2Cl2methylene chloride; CH3CN acetonitrile; DMF is dimethylformamide; NH3sodium bicarbonate; MgSO4magnesium sulfate; NMM is N-methylmorpholine; Et2About simple diethyl ether; HPLC liquid chromatography high resolution; Pd/C palladium on coal; the DIEA diisopropylethylamine; iBuOCOCl isobutylparaben; i-Pr2O diisopropyl simple ether; ONp paranitrophenyl ester; the RPMI tissue culture medium; PBS, phosphate buffer saline.

P R I m e R 1. Synthesis of Arg-Pro-Asp-Val-Isopropylamine.

In 40 ml of CH2CL2dissolving 2.25 g of BOC-Pro-(Bzl) - Asp-Val. The solution is cooled in an ice bath and add it to 0.67 g of HOBt and 0.90 g of DCC in 4 ml of DMF. After stirring for 10 min introduce 0,30 g Isopropylamine in 5 ml of CH2Cl2. The mixture is stirred in an ice bath for 15 min, then warmed to room temperature. After 3 h, the precipitate filtered. The filtrate is extracted with 10% citric acid solution, water and a saturated solution of NaHCO3. The organic layer is dried over MgSO4and the solvent is distilled off in vacuum. The resulting product is a whitish solid, yield 2,32, After crystallization from EtOAc-iPr2O poluchaets 40% TFA in CH2CL2. The solution is stirred for 30 min, then the solvent is distilled off in vacuum. After entering the simple ether in the residual product of the distillation, a solid Pro(Bzl) - Asp-Val-isopropylpiperazine. In 10 ml of DMF is dissolved 0,93 g BOC-arginine. The solution is quickly cooled to a temperature of -20aboutWith and enter 18 ml of NMM, then enter 0,22 g iBu OCOCl. The mixture is stirred at a temperature of -15oC for 20 min, then injected to 0.18 ml of NMM and chilled solution of triptoreline in 10 ml of DMF. After 30 min the cooling bath is removed and stirring is continued for 2 hours the Largest part of the solvent is removed in vacuum. In the residual product is injected EtOAc and water. These layers are separated and the organic layer extracted with citric acid solution and twice with saturated solution of NaHCO3. After removal of the solvent remains colorless glassy substance in the amount of 1.44 G. of This product was then purified by evaporative chromatography on silica gel 60 with elution CH2Cl2:MeOH in a ratio of 98: 2. First eluruumis component is the desired product. Pure fractions yield of 0.53 g of the product. Then obtain 0.32 g of product containing a small amount of impurities.

of 0.53 g of peeled protected t is rochestie one hour the mixture is filtered through Celite and washed with water. Meon is distilled off in vacuum. The residual product distillation diluted with water, lyophilizer and the result is 280 g of the product. This product is purified by chromatographic separation on R-Sephadex with elution 0.30 M NH4SLA, pH 5. Fractions containing the center of the main peak are pooled and lyophilizers and obtain 190 mg of an amorphous solid Arg-Pro-Asp-Val-Isopropylamine.

Amino acid analysis: Asp 1,01; PrO 1,03; Val 097; Arg 1,00.

The content of peptide 96%

Thin layer chromatography (Silica gel 60).

Rf(I) of 0.44 (n-BuOH HOAc H2O z 15:3:12:10)

Rf(II) 0,65 (tO Rog SPLA H2O 5:5:1:3)

Rf(III) 0,16 (n-BuOH HOAc H2O 3:1:1).

P R I m m e R 2. Synthesis of Arg-Lys-Asp-Val-cyclohexylamine.

To a solution of BOC-(Bzl) - Asp-Val-ONp (1,09 g, 2.0 mmol) and cyclohexylamine (0,27 ml, 1.2 EQ) in HF (10 ml) was injected HOBt (0.27 g). The obtained yellow solution is stirred for 24 h and evaporated in vacuum. The residual oil was diluted with EtOAc and washed with saturated NaHCO3H2O, 10% citric acid and saturated saline. The organic phase is dried over MgSO4, filtered, evaporated, and the result is protected dipeptide.

In a saturated solution of 4.2 M Hcl dioxane (10 ml) was injected protected dipap the every product in the form of muriate (HCl) of the dipeptide. In the mixed solution of HCl salt of the dipeptide, (Z)3Ary- (CBZ-Lys-ONp (1,34 g, 1.4 mmol) and t (0,19 g, 1.0 EQ) in DMF (10 ml) was injected NMM (0,18 ml). After 16 h is formed gelatinous precipitate. The reaction mixture is mixed with a saturated solution of NaHCO3, filtered and washed with H2O, 10% citric acid and H2O and E2O. After recrystallization from 4% SPLA/tOAc get (BZ)3-Arg-(CBZ)-Lys-(Bzl)-Asp-Val-cyclohexylamin in the form of a colorless solid of 1.36 g, 73%

This tetrapeptide (1,33 g) deprived of protection in HF/anisole (30 ml/8 ml) for 60 min at a temperature of 0oC. the Residual product is mixed with Et2O and filtered. United solids dissolved in 10% SPLA and lyophilizer and the result is Arg-Lys-Asp-Val-cyclohexylamin 460 mg.

This peptide is purified by liquid chromatography high resolution (LC): column Whatman M-20 10/50 ODS-3, the flow rate of 10.0 ml/min, 10% SN2JV, 0.01 mol, pH 5 NH4The OAc. Is three injection input 150 mg in 3 ml of buffer solution and faction, eluruume between 35,0 and 51.6 min, collected. After partial evaporation in vacuo and lyophilization obtained tetrapeptide Arg-Lys-Asp-Val-cyclohexylamin in the form of a colourless solid is Silica gel G): Rf(I) to 0.39 (n-BuOH: HOAc:H2O:EtOAc 1:1:1:1) Rf (II) 0,61 (n-BuOH:HOAc:H2O:Pyr 15:3:12:10)

Rf (III) 0,90 (tOAc:HOAc:H2O:Pyr 5:1:3:5).

P R I m e R 3. Synthesis Ary-Lys-Asp-Val-piperidylamine.

In the mixed solution of BOC-(Bzl)-Asp-Val-ONp (2,72 g, 5.00 mmol) and piperidine (0.6 ml, 1.2 EQ) in HF (10 ml) was injected HOEt (0.68 g, 1.0 EQ.). After 48 h, the yellow solution is evaporated in vacuum. Oily residual product is dissolved in EtOAc and washed once with saturated solution of NaHCO3cold 2 M NaOH, twice H2O, once with 10% citric acid and saturated saline. The organic phase is dried MgSO4and it turns colorless oil BOC-(BZl)-Asp-Val-piperidine, 2,36,

After purification using the evaporative chromatography with elution Meon/CH2Cl2get protected dipeptide in the form of a colorless solid substance, of 1.65 g, 67%

4.2 M HCl-dioxane (10 ml) is introduced protected dipeptide (1.06 g, 2,17 mmol). After 1 h, the solution is evaporated in vacuo, dissolved in CH2CL2and again evaporated, and the resulting HCl salt as a colourless oil.

In the mixed solution (CBZ3-Arg-(CBZ)-Lys-OWp (2,08 g, 2,17 mmol), colorless oil, described above, and HOBt (0.29 grams) in DMF (10 ml) is introduced N (0,29 ml). Later tOAc and washed with saturated solution of NaHCO3H2O, 10% citric acid, H2O and E2O. After recrystallization from HOAc/EtOAc/Et2O get (BZ)3-Arg-(CBZ)-Lys-(Bzl)-Asp-Val-piperidine in the form of a colorless solid, 2,40 g, 91%

Protected tetrapeptide (1,87 g, 1.55 mmol) is mixed in a nitrogen purged 10% SPLA (100 ml), to which is added 10% PdC (0.8 g). The mixture was stirred at hydrogenator Parra (500 ml vessel, the pressure of 43.5 psi or 3 bar). After 48 h, the reaction mixture was filtered through a nylon filter of 0.45, partially evaporated, diluted with H2O and liabilitiesa, and the result is a crude tetrapeptide in the form of a colorless solid, 890 mg of This peptide is purified by a method LC (chromatography) column Whatman-M 10/50 ODS-3, the flow rate of 10.0 ml/min, 10% SN3JV, 0.01 mol, pH 5 NH4The OAc. Is three injection input 300 mg in 3 ml of buffer solution, and faction, erwerbende in the time interval between min 23,0 and 44.0 min, collected. After partial evaporation in a vacuum and freeze-drying is obtained the desired tetrapeptide Arg-Lys-Asp-Val-piperidine in the form of colorless solids, 510 mg.

Amino acid analysis: Arg 0,95; Lys 1,00; Asp 1,02; Val 1,00. The content of peptide 54,2%

Thin layer of chromous:H2O:Pyr 5:1:3:5).

P R I m e R 4. Synthesis of Arg-Pro-Asp-Val-N-methylamide.

In the mixed solution of BOC-Val (10,86 g, 50.0 mmol) and DIFA (to 8.70 ml, 1.0 EQ), methylamine HCl (3,38 g, 1.0 EQ) and NOT (7,66 g, 1.0 EQ) in CH2CL2and DMF at a ratio of 12:5 (85 ml) was injected DCC (10,32 g, 50.0 mmol). A precipitate. After 2 h, the reaction mixture is filtered and the filtrate evaporated to dryness. The oily residue is dissolved in Eoas and washed three times with saturated solution of NaHCO3H2O, 10% citric acid and saturated saline. After drying over Na2SO4the organic phase is filtered and evaporated, and the result is a colorless solid product. Grinding it in hot hexane gives the desired amide, BOC-Val-methylamide, 7.98 g, 69% melting point 120-122aboutC.

This amide (4,60 g, 20.0 mmol) is added (50) FA/CH2Cl2(20 ml). After stirring for 30 minutes the solution is evaporated at a temperature of less than 30aboutWith that oily evaporation residue is dissolved in CH2CL2, washed twice with saturated solution of NaHCO3. The organic phase is dried over Na2SO4, filtered and evaporated, and the result is a Val-methylamide, 1,90 g, 73%

To p the CC (3,01 g, 14.6 mmol). After 5 min, a precipitate may form. After 16 h, the reaction mixture is filtered and the filtrate is mixed with polysystem solution of NaHCO3.

The obtained solids are joined, washed with H2O, 10% citric acid, H2O and Et2O, dried by air and turns colorless solid. After recrystallization from EtOAc get the desired dipeptide, of 5.85 g, 90% BOC-(Bzl)-Asp-Val-methyl - amide.

This dipeptidase (3.94 g, 9,05 mmol) is added 50% TFA/CH2Cl2(20 ml). After stirring for 30 minutes the solution is evaporated at a temperature of less than 30aboutWith and mixed with Et2Oh, the result is a dipeptide TFA, 4,10 g, 100% in the form of a colorless glassy substance.

To a stirred solution of BOC-Pro (1,95 g, 0.95 mmol), FA of the dipeptide (4,10 g, 9,05 mmol), DIEA (1.73 ml, 1.1 EQ), DMA (0.12 g, 0.1 EQ) is added DCC (1,87 g, 9,05 mmol). After 5 min, a precipitate may form. After 16 h, the reaction mixture is filtered and the filtrate is mixed with polysystem solution of NaHCO3. The obtained solid product is extracted, washed with H2Oh, 10% citric acid, H2Oh and Et2Oh, dried air and the result is the desired Tripeptide, 4,15 Cl2(15 ml). After stirring for 30 minutes the solution is evaporated at a temperature lower than the 30oC, mixed with Et2And the result is a Tripeptide TFA, 4.26 deaths g, 100% in the form of a colorless solid.

In the mixed solution of 86.9 AOC-TOS-Arg (39,7 g, 7,78 mmol), Tripeptide (4,25 g, 7,78 mmol), NMM (0,86 ml, 1.1 EQ) and HOBt (1.19 g, 1.0 EQ) in DMF (15 ml) is injected DCC (1,61 g, for 7.78 mmol). After 10 min, a precipitate may form. After 16 h, the reaction mixture is filtered and the filtrate is mixed with polysystem solution of NaHCO3. The obtained solid product is extracted, washed with H2O, 10% citric acid, H2About simple ether, dried by air and the result is a protected tetrapeptide, 4,82 g, 72% Half of this solid product is deprived of protection in 50% TFA/CH2Cl2(10 ml) for 30 min, evaporated in vacuo and mixed with Et2O. the Obtained solid product is cleaved with HF /anisole (30 ml/8 ml) for 60 min at a temperature of 0aboutC. the Residual product was stirred in EtOAc and extracted twice with 10% SPLA (50 ml). Water extracts lyophilizers and the result is a raw Arg-Pro-Asp-Val-methylamide.

This crude product is purified on SPC-25 Sephade (column razmera going and lyophilizers, and the result is 925 mg tetrapeptide, Arg-Pro-Asp-Val-methylamide.

Amino acid analysis: Arg 1,06; Pro 0,99; Asp 0,95; Val 1,00.

The content of peptide 54%

Thin layer chromatography (250 MK, Silica gel G). Rf (I) 0,20 (n-BuOH: HOAc: H2O: Upper phase 4:1:5) Rf (II) 0,26 (n-BuOH:HOAc:H2O:Rog 15:3:12:10) Rf (III) 0,26 (tOAc:HOAC:H2O:Pyr 5:1:3:5).

P R I m e R 5. Synthesis of N-formyl-Arg-Pro-Asp-Val-methylamide.

In AOC-(TOS)-Arg-Pro-(Bzl)-Asp-Val-methylamide (1,00 g at 1.17 mmol) introduces a 4.5 M HCl Dioxane (10 ml). After 60 min reaction mass is evaporated, diluted with H2O and liabilitiesa, and the result is HCl Tetrapeptide in the form of a colorless solid powdery substance to 0.92 g, 100%

This solid product is dissolved in DMF (10 ml) and injected DIFA (0,93 ml, 4.0 EQ), DMAP (0.08 g) and para-nitrophenylphosphate (200 mg, of 1.02 EQ). After 2 h, the reaction mixture is treated with 10% citric acid. The obtained solid product is filtered, washed with N2Oh, dried air and the result is formirovanii peptidome, 0,41,

This solid product is cleaved with HF/anisole (30 ml/8 ml) for 60 min at 0aboutC. the Residual product is mixed in tOAc and extracted twice with 1% NH4OH (50 ml). Aqueous extracts of leaf is on DEAE Sephadex (column 2.6 x 90 cm 0.1 M H4HCO3, non-buffer solution; flow rate 100 ml/h 10 ml/fraction, 206 nm detector). Faction 38-43 going lyophilizers and the result is aminirovanie peptide, 80 mg.

Amino acid analysis: Arg 1,00; Pro 1,00; Asp 1,00; Val 1,00.

The content of peptide 50%

Thin layer chromatography 250 micron Silica gel G). Rf (I) 0,32 (n-VION:HOAc:H2O:Upper phase 4:1:5)

Rf (II) 0,38 (tOAc:SPLA:H2O:Pyr 5:1:3:5) Rf (III) 0,34 (triptoreline: NH4OH 1:1).

P R I m e R 6. Synthesis of Arg-Lys-Asp-Val-2-phenylhydrazine.

In the mixed solution of BOC-Val (4,34 g, 20.0 mmol) in THF (40 ml) at -15aboutTo enter NMM (2.30 ml), and then enter i-BuOCOCl (2,64 ml). The resulting suspension stirred for 15 min and then added dropwise introduced phenylhydrazine (1,98 ml). The suspension is stirred at a temperature of from -10 to 0aboutC for 1 h and then warmed to room temperature. The reaction mixture is filtered and the filtrate evaporated under reduced pressure. The residual product is dissolved in EtOAc and washed with saturated solution of NaHCO3H2O and saturated salt solution.

The organic phase is dried over Na2SO4, filtered, evaporated and the resulting solid is hydrazine in the form of a colorless solid, melting point 140-141aboutWith, 4,96 g, 74%

In the mixed solution of 4.2 M HCl dioxane (10 ml) is introduced BOC-Val-2-phenylhydrazine (1.13 g, 3,68 mmol). After 1 h the reaction mixture was evaporated in vacuo, stirred Et2O and filtered, and the result is a Val-2-phenylhydrazine in the form of a di-HCl salt as a colourless solid product of 1.03 g

In the mixed solution of this di-HCl salt (1,03 g, 3,68 mmol). BOC-(Bzl)-Asp (0.88 g) and HOBt (0,37 g) in THF (10 ml) is introduced NMM (0,60 ml) and then DCC (0.56 g). Almost immediately a precipitate. After 16 h, the reaction mixture is filtered and the filtrate evaporated in vacuum. This residual product evaporation is dissolved in tOAc, washed with saturated solution of NaHCO3and a salt solution. The organic phase is dried over Na2SO4, filtered and evaporated in vacuum. After recrystallization from EtOAc/hexane obtained BOC-(Bzl)-Asp-Val-2-phenylhydrazine in the form of a colorless solid, 1.07 g, 64%

In the mixed solution of 4.2 M HCl dioxane (10 ml) is introduced BOC dipeptide phenylhydrazide (1.07 g, a 1.75 mmol). After 60 minutes the solution is evaporated in vacuum and the obtained colorless oil (Bzl)-Asp-Val-2-phenylhydrazide.

In the mixed solution (CBZ)3-Arg-(CBZ)Lys-0 Np (1.68 g) mA who is with a saturated solution of NaHCO3and solids are extracted, washed with H2O, 10% citric acid, H2O and Et2O. After recrystallization from 2% HOAc/EtOAc obtained (CBZ)3Ary-(CBZ)-Lys-(Bzl)-Asp-Val-2-phenylhydrazide in the form of a colorless solid, 2.15 g, 92% of This protected peptide (1.88 g) is cleaved in HF/anisole (30 ml/8 ml) for 60 min at 0aboutC. the Residual product splitting mixed with Et2O and filtered. Solid products are extracted with 10% HOAc (100 ml) for 1 h, filtered extract liabilitiesa and the result is Arg-Lys-Asp-Val-2-phenylhydrazide in the form of a colorless solid, 890 mg of This crude peptide is purified on CM-Sephadex (column 2.6 x 83 cm) 0.3 M NH4SLA, non-buffer solution; flow rate 10 ml/h, 12.5 ml/fraction, 225 nm detector).

Faction 270-310 going lyophilizers and the result is 390 mg of the final product, Arg-Lys-Asp-Val-2-phenylhydrazide.

Amino acid analysis: Arg 0,98; Lys 1,03; sp 1,00; Val 0,99. The content of peptide 83%

Thin layer chromatography: (250 micron Silica gel G) Rf (I) to 0.39 (n-BuOH: HOAc: H2O: tOAc 1: 1:1:1) Rf (II) of 0.51 (n-BuOH:HOAc:H2O:Pyr 15:3:12:10) Rf (III) 0,59 (tOAc:HOAc:H2O:Pyr 5:1:3:5).

P R I m e R 7. Synthesis of Arg-Lys-Asp-Val-onlinemed.

In the mixed solution of BOC-V). Within 5 min formed a thick precipitate. After 4 h, the reaction mixture is filtered and the filtrate poured into Polynesians NaHCO3(50 ml). The resulting precipitate is extracted. The solids are washed with water (H2O (1: 1) and dried in air. After recrystallization from hexane-isopropanol obtained colorless solid product anilide BOC-Val-aniline, 3,76 g, 64%

This BOC-Val-aniline (2.50 g, 8,55 mmol) was dissolved in 50% FA/CH2Cl2(10 ml) and stirred for 30 minutes, the Reaction mixture was evaporated in vacuum at room temperature, and the residual product evaporation is dissolved in CH2CL2. This solution is washed twice with saturated solution of NaHCO3dried over MgSO4, filtered and evaporated, and the result is a Val-aniline in the form of a pale yellow oil, 1.42 g, 88%

To a solution of BOC-(Bzl)-Asp (2.38 g, 1.0 EQ), Val-aniline and HOBt (1.13 g) in DMF (7.5 ml) was injected DCC (1.52 g, 1.0 EQ). A precipitate, and the reaction mixture is stirred for 3 hours the Mixture is filtered, the filtrate is mixed with a saturated solution of NaHCO3. The obtained solids are extracted, washed with H2O and air dried. After recrystallization from EtOAc/petroleum ether recip/P> BOC dipeptide (1,71 g, 3,44 mmol) was dissolved in 50 TFA/CH2Cl2(10 ml) and stirred for 30 minutes, the Reaction mixture was evaporated in vacuum at room temperature and the residual product is mixed in a simple ether, filtered and dried air.

In the mixed solution of BOC-(Cbz)-Lys (1,33 g, 3.50 mmol) in DMF (10 ml) at -20aboutTo enter NMM (and 0.46 ml, 1.2 EQ) and then i BuOCOCI ones (0.46 ml, 1,01 EQ). The resulting suspension stirred for 30 min and then introduced TFA salt of the dipeptide, and then NMM (0,46 ml). The reaction mixture was stirred and heated to room temperature. After 1 h the reaction mixture was poured into Polynesians solution of NaHCO3(20 ml). Formed colorless solid. This substance is extracted and dried by air. After recrystallization from EtOAc/i-Pr2O is obtained BOC Tripeptide, 2,05 g, 78% melting point 164-167aboutC.

BOC Tripeptide (2.00 g, 2,63 mmol) was dissolved in 50% FA/CH2Cl2(6 ml) and stirred for 30 minutes, the Reaction mixture was evaporated at room temperature in a vacuum and the evaporation residue is pulverized in a simple ether, filtered and dried air, and the result is salt FA.

In lane is BuOCOCl (0.35 ml, a 1.01 EQ). The resulting suspension is stirred for 30 minutes Then injected NMM (0.35 ml), and then salt FA. The reaction mixture is heated to room temperature within 1 h the Mixture was poured into a saturated solution of NaHCO3and the resulting solids filtered, washed with N2O and air dried. After stirring for tOH have protected tetrapeptide, BOC-(CBZ)-Lys-(BZl)-Asp-Val-aniline, 2.55 g, 78%

Variable suspension of tetrapeptide (2.30 g, 1,89 mmol) in triptoreline (50 ml) and formic acid (97% 5 ml) was injected palladium black (0.95 g). The reaction mixture is intensively stirred. After 1 h, the solution was filtered through Celite and washed with H2O. the Filtrate is partially evaporated in vacuo, the residual product lyophilizer, the result is colourless solid, 0,89,

The crude peptide is purified on SPC-25 Sephadex (column 2,6 x 89 cm, about 0.1-0.3 mol NH4OAc, pH 5 gradient; flow rate 100 ml/h, 15 ml/fraction, 206 nm detector). Fraction 253-285 collect, lyophilizer and the result is 680 mg Arg-Leu-Asp-Val-aniline.

Amino acid analysis: Arg 1,00; Lys 0,98; Asp 1,00; Val 1,01.

The content of peptide 100%

Thin layer chromatography (250 micron Silica gel G) Rf (I) of 0.42 (EtOAc: HOAc: H2O: Pyr) (5: 1:3:5) Rf (II) 0,32 (nBuOH:HOAc:H. a-tert.-butyloxycarbonyl-(N-benzyloxycarbonyl)-lysyl-(- benzyl)aspartyl)-poured-pencilwork of ester. Introducing zinc powder (5.2 g) and the mixture is stirred intensively for one hour. The reaction mixture is filtered, the filtrate is diluted with water and extracted three times with ethyl acetate. The combined organic layers are extracted with water and saturated aqueous sodium chloride, then dried over anhydrous magnesium sulfate. After removal of the solvent remains in the oil. Introduces a simple ether, then hexane and forms a resinous precipitate. This product is mixed with rubbing with hexane until then, until it turns dry white solid in the amount of 1.75 G. of This product is dissolved in 14 ml of ethyl acetate and 1 ml of dimethylformamide. After cooling the solution to -20aboutTo enter to 0.30 ml of N-methylmorpholine, then introduced dropwise 0.36 g of isobutylacetate. The reaction mixture was stirred at -15aboutC for 20 min, then introduced to 0.29 ml of N-methylmorpholine and chilled solution of 0.18 g of the hydrochloride of methylamine in 10 ml of 95% ethanol. Forms a thick sludge. To accelerate the mixing of injected 5 ml of dimethylformamide and the mixture is stirred at 0aboutWith over 90 mi the ri stirring the mixture turns into a globule waxy solids. This solid is filtered and the filtrate is extracted with ethyl acetate. The residual product evaporation of the organic phase is connected with this solid product is crystallized from a mixture of ethyl acetate-isopropyl ether. The product yield is 1,53 g, melting point 183,5-184,5aboutC.

The data of elementary analysis: C 61,67 (61,96); N 7,21 (7,37); 9,81 N (10,04).

C. Three-benzyloxycarbonyl-arginyl-(N-benzyloxycarbonyl)-lysyl-(-BOC-(CBZ)-Lys-(-Bzl)-Asp-Val

In a rapidly stirred solution of BOC-(CBZ)-Lys-(-Bzl)-Asp-Val-final - business complex ether (8,02 g, 10.0 mmol) in 85% of the SPLA (100 ml) at room temperature is injected subjected to etching by acid zinc dust (12.4 g). After 1 h the reaction mixture was filtered and the filtrate is evaporated in vacuum. Oily residual product mix with rubbing at E2O. the Resulting solid product is extracted, washed in Et2O and dried in vacuum at 40aboutWith, the result of 4.95 g, 72%

Century-VOS-(Cbz)Lys-(-Bzl)-Asp-Val-NHCH(CH3)2.

In the mixed solution of BOC Tripeptide (1,00 g of 1.46 mmol) in THF (5 ml) at -15aboutTo enter NMM (170 μl, 1.06 EQ) and l (195 μl, 1.03 EQ). The resulting weakly turbid solution is stirred for 15 min and then injected 2-p and the reaction mixture is heated to room temperature. After 16 h, the reaction mixture is evaporated in vacuum at 30aboutC and the residual solid product is washed with 10% citric acid, H2O, a saturated solution of NaHCO3and H2O. After drying in the air get colorless BOC Tripeptide, 2.58 g, 55%

C. (bz)3Arg-(Cbz)-Lys-( -Bzl)-Val-NHCH(CH3)2.

BOC Tripeptide (0.55 g, from 0.76 mmol) dissolved in 4.4 HCl dioxane (5 ml) and stirred for 45 minutes the Solution is evaporated in vacuum and the solid residue triturated in Et2O. After filtering receive colorless HCl Tripeptide 0.51 g, 100% In the mixed solution of Hcl Tripeptide, (bz)3-Arg-para-nitrophenylamino of ester (0,53 g, 1.0 EQ) and HOBT (0,12 h, 1.0 EQ) in DMF (5 ml) at room temperature is injected NMM (92 μl, 1.1 EQ). Slowly precipitate for 16 hours Gelatinous reaction mixture is stirred with a saturated solution of NaHCO3and filtered. The solid residue was washed with H2O, 10% citric acid, H2O and E2O. the Obtained protected tetrapeptide recrystallized from HOAc/tOAc, the result is colourless solid, 743 mg, 83%

D. Arg-Lys-Asp-Val-NHCH(CH3)2< / BR>
Protected tetrapeptide (0,70 g, 0.59 mmol) is suspended in 90% of the SPLA (50 ml). Smgd capacity of 250 ml, Po52,0 psi 3.64 ATM) for 64 hours, filtration through a nylon filter of 0.45 μm, partial evaporation and lyophilization get raw tetrapeptide, 385 mg.

The crude peptide is purified on CM-Sephadex (column 2,6 x 93 cm, 1.7 l, 0.2 mol, then 0.3 mol, NH4OAc, non-buffer solution; flow rate 100 ml/h 10 ml/fraction, 225 nm detector). Faction 335-370 merge and lyophilizers and it turns out 231 mg of product.

Amino acid analysis: Arg 1,00; Lys 1,02; sp 0,98; Val 1.00; peptide content of 80.3%

Thin layer chromatography (250 MK, Silica gel G) Rf (I) 0,24 (n-BuOH: HOAc:H2O:EtOAc 1:1:1:1) Rf (II) 0,52 (n-BuOH:HOAc:H2O:pyridine 15:3:12:10)

Rf (III) 0,61 (tOAc:HOAc:H2O:pyridine 5:1:3:5).

Biological activity analysis of cyclic GMP.

In this analysis, we determined the ability of a peptide corresponding to the invention, to contact with a cell membrane receptor CEM cells and selectively stimulate the formation of cyclic CMP, as human thymopentin.

Cell line CEM was obtained from the culture Collections of the American type, Rockville, MD., Md, CEM Cells were inoculated, were grown for 3 days and was going.

Cells was three times washed in PBS and re-suspensibility in RRM 1640 with concentric the e peptides (25 μl) and control peptides. Incubation was carried out in shake a water bath for 4-5 minutes and then the process continued input 1 ml ice TCA (10%).

Cells in TCA was gomogenizirovannykh and was exposed to ultrasound for the selection of the cyclic nucleotide. This suspension was centrifugals with acceleration 3000 x g for 20 min at 4aboutC. the precipitate was dissolved in 0.1 G. of NaOH to determine protein content. TCA was removed from the surface fraction by 4-fold extraction with 5 ml of water-saturated Et2O. After the final extraction, the remaining traces of ether were removed by heating for 10 min in a water bath with a temperature of 50aboutC. After lyophilization, the sample was injected in 50 mm acetate buffer solution (pH 6,2) for radioimmunoassay of cyclic CMP.

For example, for each compound was determined curve "dose response" using 1000 μg/ml of peptide. For each test peptide was determined threshold activity. It is defined as the lowest concentration of the test peptide, which induced the intracellular level of cyclic GMP is more than two standard deviations above the control. Control samples had a value of the test was considered positive, if the level of cyclic GMP in more than 2 times the level that was specified for the parallel negative control sample. Active peptides have activity thresholds in the range from 1 to 10 g/L.

The table illustrates the results of the analysis of cyclic GMP. Positive (+) results show that the level of action of cyclic GMP in more than 2 times higher than the level defined for the parallel negative control.

P R I m e R 9. Enzyme stability.

To illustrate the resistance of the peptide to degradation of the intestinal and serum enzymes, peptide Arg-Pro-Asp-Val-isopropylated can be incubated at a temperature of 37aboutWith intestinal fluids, and human serum over time up to 120 minutes To compare similarly processed such peptides, as Arg-Lys-Asp-Val-dimethylamide and thymopentin. Analysis by liquid chromatography high resolution showed that Arg-Lys-Asp-Val-dimethylamide fully vivariums a few seconds. Under these conditions thymopentin decomposes at 50% for about 20 C. the Proposed peptides remain almost nerazrushennymi as in serum and intestinal juice over a longer time is 2,

where-NR1R2selected from the group including aniline, methylamide, 2-phenylhydrazine, piperidine, cyclohexylamine and Isopropylamine.

2. Peptides under item 1, characterized in that they have the ability to regulate insufficient or excessive T-cell function of the body.

3. A composition capable of regulating immune function in mammals containing peptide and a pharmaceutically acceptable carrier, wherein the peptide compound represented by the General formula

Arg Lys Asp Val NR1R2< / BR>
where-NR1R2selected from the group including aniline, methylamide, 2-phenylhydrazine, piperidine, cyclohexylamine and isopropylated,

in the amount of 0.001 to 100 mg/kg of body weight.

4. The composition according to p. 3, characterized in that it is suitable for oral administration.

 

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FIELD: medicine, cardiology.

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EFFECT: higher efficiency of therapy.

3 ex

FIELD: medicine, purulent surgery.

SUBSTANCE: one should perform generally accepted medicinal therapy, moreover, one should prescribe additional chemotrypsin during the first 1-2 d after operation to be locally injected twice-thrice during day-time period at concentration of 0.5-1.0 mg/ml of 10%-sodium chloride solution at exposure of 1.5-2 h at the quantity of one fourth up to one third against the purulent volume removed out of abscess cavity, then therapy course should be supplemented with bacteriophage which should be locally injected twice during day-time period at daily dosage being 200 ml, not more at exposure of 1.5-2 h at the quantity of one tenth up to one fifth against purulent volume removed out of abscess cavity. As for bacteriophage type, it should be matched in accordance to the results of bacteriological survey of abscess cavitary content. Therapy course lasts for 6-9 d.

EFFECT: higher efficiency of therapy.

2 ex

FIELD: medicine.

SUBSTANCE: invention proposes agent for suppression of snores noise designated for intramuscular administration. Agent comprises the highly purified clostridium toxin BONT/A or highly purified clostridium toxins BONT/B, BONT/C1, BONT/D, BONT/E, BONT/F and/or BONT/G. Also, agent can be used comprising a fused protein that involves light subunit of clostridium toxins among the group BONT/A, BONT/B, BONT/C1, BONT/D, BONT/E, BONT/F, BONT/G, TeNT, and heavy subunit of another clostridium toxin from the same group, or mixture of fused proteins, or complex comprising clostridium toxin or a fused protein, and one or more therapeutically good tolerable hemagglutinin, and/or one or more pharmaceutically good tolerable nontoxic protein. The proposed therapeutic agent expands assortment of medicinal agent used in the snore therapy. Invention can be used for suppression of snore noise.

EFFECT: enhanced effectiveness of agent.

10 cl, 3 ex

FIELD: medicine, hepatology.

SUBSTANCE: invention relates to a method for treatment of chronic hepatitis. Method involves applying granocyte that is administrated every day intravenously in the dose 1-10 mcg/kg of patient weight per 24 h for 7 days, not above, in combination with conventional complex used in treatment of this disease. Method provides normalization of activity of blood enzymes that indicates the liver function recovery and improvement of the liver structure indices.

EFFECT: improved method for treatment.

2 ex

FIELD: pharmaceutical chemistry.

SUBSTANCE: invention relates to (i) essentially crystalline melagatran in the form of hydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 21.1, 10.5, 7.6, 7,0, 6.7, 6.4, 6.2, 5.7, 5.4, 5.3, 5.22, 5,19, 5.07, 4.90, 4.75, 4,68, 4.35, 4.19, 4.00, 3.94, 3.85, 3.81, 3.73, 3.70, 3.63, 3.52, 3.39, 3.27, 3,23, 3.12, 3.09, 3.06, 2.75, 2.38, and 2.35 Å and/or water content 4.3%; and (ii) essentially crystalline melagatran in the form of anhydrate, which is characterized by x-ray diffraction pattern on powder having crystalline peaks with following d values: 17.8, 8.9, 8.1, 7.5, 6.9, 6.3, 5.9, 5.6, 5.5, 5.4, 5.3, 5.2, 5.0, 4.71, 4.43, 4.38, 4.33, 4.14, 4.12, 4.05, 3.91, 3.73, 3.61, 3.58, 3.56, 3.47, 3.40, 3.36, 3,28, 3.24, 3.17, 3.09, 3.01, 2.96, 2.83, 2.54, 2.49, 2.41, 2.38, and 2.35 Å. Invention also relates to a method for preparation of indicated form, a method for interconversion of anhydrite form, to use of indicated compounds as pharmaceutical agent, and to preparation of drugs. Pharmaceutical preparation is suitable for treatment of condition, in case of which inhibition of thrombin is needed or desirable. Invention provides a method for treatment of such condition.

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14 cl, 4 dwg, 3 tbl, 9 ex

FIELD: veterinary science.

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EFFECT: higher efficiency.

1 dwg, 1 ex, 1 tbl

FIELD: medicine, pediatrics.

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EFFECT: higher efficiency of therapy.

2 ex

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EFFECT: valuable properties of compositions.

35 cl, 11 tbl, 7 ex

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EFFECT: valuable biological properties of composition.

3 cl, 16 tbl, 9 ex

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