RU2065445C1 - Peptides showing regenerative-reparative action - Google Patents

Abstract

FIELD: medicine, peptides. SUBSTANCE: product: new bioactive peptides of the general formula: R-Glu-Leu-Lys-Pro-Leu-X-Glu-Val-Leu-Asn-Leu-R1 where R - CH₃-CO-(Ac), Ac-Glu-, Ac-Leu-Glu-Glu-, HCO-(Form), Form-Glu-, Form-Glu-Glu-, Form-Leu-Glu-Glu-, CH-(CH)_n-CO-, CH-(CH)_n-CO-Glu-, CH-(CH)_n-CO-Glu-Glu-, CH-(CH)_n-CO-Leu-Glu-Glu-; n = 8-18; X - L-Glu, D-Glu; R1 - OMe or NH₂,. Proposed peptides are relatively short (11, 12, 13 and 14 amino acid residues) fragments of human IL-2 (interleukin-2). Peptides were synthesized by chemical synthesis according classic methods in solution using maximal protection of polyfunctional amino acids followed by protective groups removal and isolation as free form by HPLC method. Peptides have protective groups at molecule termini that enhances stability to proteolysis and provides favorable conditions for necessary conformation making in the process of their effect on target-cells due to neutralization of N- and C-terminal charges as it takes place in natural protein. Synthesized peptides are inductors and/or potentiating agents of growth-stimulating activity of macrophages in vitro, show regenerative-reparative action in vivo and can be used in medicine for treatment diseases accompanying generalized macrophage activation (hepatitis, toxic liver damages, liver cirrhosis, skin wounds, burns, diabetes and others). EFFECT: enhanced effectiveness of peptides. 12 tbl

Description

The invention relates to new biologically active compounds, specifically to peptides of the general formula:
R-Glu-Leu-Lys-Pro-Leu-X-Glu-Val-Leu-Asn-Leu-R ',
where R CH ₃ CO- (Ac), Ac-Glu-, Ac-Glu-Glu-, Ac-Leu-Glu-Glu-,
HCO- (Form), Form-Glu-, Form-Glu-Glu-, Form-Leu-Glu-Glu-,
CH ₃ - (CH ₂ -) _n CO-, CH ₃ (CH ₂ -) _n CO-Glu-Glu-,
CH ₃ - (CH ₂ -) _n CO-Leu-Glu-Glu-, n 8 18,
X L-Glu, D-Glu
R 'OMe or NH ₂
which are inducers and / or potentiators of growth-promoting activity of macrophages in vitro, have a regenerative and reparative effect in vivo and can be used in medicine for the treatment of a number of diseases accompanied by generalized activation of macrophages (hepatitis, toxic liver damage, liver cirrhosis, skin wounds, burns, polytrauma, diabetes, etc.).

 Currently, the study of the problem of the participation of the immune system in the regulation of regenerative and reparative processes allows us to conclude that macrophages are the final effector of the regulation of tissue homeostasis in general, and regenerative and reparative processes in particular. They regulate the proliferation and functions of the main components of connective tissue, and as effectors are directly involved in maintaining tissue homeostasis by eliminating spent cells, their debris, and other tissue degradation products (1,2,3), providing tissue elements with growth factors and other compounds necessary for normal growth and functioning of tissue (4).

Using the biologically active compounds synthesized by him, the macrophage transforms signals from the higher parts of the homeostasis maintenance system to the cellular elements of tissues. On the other hand, the macrophage is capable of incorporating the higher links of the homeostasis system, synthesizing key cytokines, thus closing the feedback system, including with the central nervous system [5]
Due to such a wide range of properties, macrophages are involved in the pathogenesis of almost all diseases, and accordingly, pharmacological modulation of their activity opens up new possibilities for intervention in pathological processes.

 Macrophages occupy a special place in the processes of tissue regeneration and repair, in particular during the wound process. To date, it has been shown that tissue destruction products are macrophage activators (6), and activation is accompanied by the sequential inclusion of the synthesis of a number of monokines mobilizing the main systems for maintaining homeostasis (from the central nervous to immune), as well as the direct inclusion of these cells in the regeneration process.

 Thus, in the set of functions of macrophages, two main ones can be distinguished: cytotoxic, caused directly by the effector properties of the cell, and growth-stimulating, in the implementation of which the macrophage manifests itself as a secretory cell.

In recent years, it has become apparent that, both in terms of the synthesis of growth factors and their role in various pathologies, the productive functions of macrophages are no less important than the destructive ones. In particular, the ability of activated macrophages to stimulate tumor growth is already as well proven as their ability to inhibit such growth [7]
The results of numerous studies of recent years indicate the fundamental importance of macrophage hyperactivation in the pathogenesis of many diseases or their associated syndromes, in connection with which a wide study of macrophage activity inhibitors has begun. In particular, tumor cell products, tumor growth factor (TGF-β)), interleukin-10 (IL-10), tumor necrosis factor receptor antagonists (TNF) and interleukin-1 (IL-1) are considered potential drugs.

 Currently, a significant number of drugs are known that activate and regulate the cytotoxic effect of macrophages [8], but agents that selectively activate productive, growth-stimulating activity are not actually described.

 However, it is known that in the process of activation of many cells of the immune system, including macrophages, the key role is played by interleukin-2 (IL-2) - one of the central mediators of the immune system. In addition, this lymphokine is involved in the development of the inflammatory response in the case of many of the diseases described above [9] In recent years, several attempts have been made to recreate the fully or partially unique biological properties of interleukin 2 using its peptide fragments.

 A recombinant IL-2 [10] polypeptide is known, consisting of 153 amino acid residues (including 20 amino acids of the signal peptide) and retains the full activity of the natural polypeptide despite the absence of a carbohydrate moiety.

Known peptides fragments of IL-2, consisting of 110-120 amino acid residues and having growth activity for lymphocytes, obtained by genetic engineering methods [11]
At the same time, short peptide fragments with growth-promoting activity of IL-2 have not been described to date.

 However, IL-2 itself and its fragments obtained by genetic engineering methods are difficult to isolate and purify to a homogeneous state, and it is difficult to control their purity and compliance with the specified formula. Impurities that cannot be separated often negatively affect cell cultures (not to mention living organisms) and reveal biological functions that are not characteristic of IL-2 itself and its polypeptide fragments.

 In addition, in some cases, it is advisable to isolate from the whole set of diverse biological properties of human IL-2, only one or several specific ones (for example, growth-stimulating, cytotoxic, etc.). Peptide fragments with such properties could be used in medicine. It is known that in many cases the use of relatively short peptide fragments is preferable to the use of long proteins that cause allergic reactions and other undesirable side effects, especially when doctors do not have a natural glycosylated protein, but its genetically engineered non-glycosylated analogue (as in the case of with IL-2 and many other proteins).

 The present invention relates to new biologically active compounds.

где R CH₃CO-(Ac), Ac-Glu-, Ac-Glu-Glu-, Ac-Leu-Glu-Glu-,
HCO-(Form), Form-Glu-, Form-Glu-Glu-, Form-Leu-Glu-Glu-,
CH₃-(CH₂-)_nCO-, CH₃-(CH₂-)_nCO-Glu-, CH₃-(CH₂-)_nCO-Glu-Glu-,
CH₃-(CH₂-)_nCO-Leu-Glu-Glu-, n 8 18,
X L-Glu, D-Glu
R'- OMe или NH₂,
состоящие из 11, 12, 13 и 14 аминокислотых остатков, которые можно получить с помощью химического синтеза и легко выделить в свободном виде и очистить до гомогенного состояния.The inventive compounds are relatively short peptide fragments of the sequence 62-72, 61-72, 60-72, 59-72 of human IL-2:

where R CH ₃ CO- (Ac), Ac-Glu-, Ac-Glu-Glu-, Ac-Leu-Glu-Glu-,
HCO- (Form), Form-Glu-, Form-Glu-Glu-, Form-Leu-Glu-Glu-,
CH ₃ - (CH ₂ -) _n CO-, CH ₃ - (CH ₂ -) _n CO-Glu-, CH ₃ - (CH ₂ -) _n CO-Glu-Glu-,
CH ₃ - (CH ₂ -) _n CO-Leu-Glu-Glu-, n 8 18,
X L-Glu, D-Glu
R'- OMe or NH ₂ ,
consisting of 11, 12, 13 and 14 amino acid residues that can be obtained using chemical synthesis and can be easily isolated in free form and purified to a homogeneous state.

 The inventive peptides are unique modulators of macrophage activity in vitro, inducing and / or potentiating the growth-promoting effect of preactivated macrophages, while almost completely suppressing their cytotoxic activity.

 The inventive substances have a regenerative and reparative effect in vivo, and in relation to unstimulated macrophages they are completely inert, which makes them especially attractive for medical use for the treatment of a number of diseases accompanied by generalized activation of macrophages.

 The inventive peptides are the shortest of all previously known fragments of human IL-2, capable of exhibiting one of the types of diverse activity of IL-2.

 Blocking by an acetyl or alkyl protective group at the N-terminus and methyl ether or amide at the C-terminus of the molecule increases the resistance of the peptides to proteolysis, as well as provides more favorable conditions for creating the necessary conformation during the action on target cells, neutralizing the charges at the ends of the molecules, as is the case in natural protein.

The proposed peptides are obtained by classical methods of peptide synthesis in solution according to Scheme 1-2, which includes the condensation of two protected peptide blocks selected in such a way that the block activated during condensation has a proline residue that is practically not racemic at the C-terminus under these conditions. To block the side chains of Glu and Lys residues, benzyl type protections were chosen; for N ^a -function tert. butyloxycarbonyl group (Boc); for the C ^a function methyl ester (OMe).

 The synthesis of each of the fragments is carried out by sequential chain extension from the C-terminus by the methods of penta-fluorophenyl and p-nitrophenyl ethers.

Boc-protecting groups are removed using a mixture of acetic and trifluoroacetic acids in a ratio of 3: 7; under these conditions, the side reaction of the cleavage of the Z-group with the N ^e Lys function did not occur.

 Protected peptides are purified by silasorb adsorption chromatography (Silasorb 600; 30 μm, Chemapol, Czechoslovakia) in a gradient of ethyl acetate: ethanol solvents, and also crystallization. Their individuality and purity are controlled by thin layer chromatography on plates with a fixed layer of silica gel (Merck, West Germany) and amino acid analysis (on a Durrum instrument, USA).

 Protected undeca, dodeca, trideca and tetradeapepeptides are obtained by condensation of blocks (4 + 7, 5 + 7, 6 + 7 and 7 + 7), respectively, with 4-toluenesulfonate N-cyclohexyl-N'-2- (N-methylmorpholino ) -ethylcarbodiimide in the presence of 1-hydroxybenzotriazole, and a 1.1-fold excess of activated tetra-, penta-, hexa-, heptapeptides is used.

The introduction of the acetyl group is carried out after removal of the N ^a- Boc-protection with acetic anhydride. The removal of the side protective groups is carried out by HBr / CH ₃ COOH or trifluoromethanesulfonic acid in the presence of anisole. The resulting products were purified by high performance liquid chromatography on a SynChropak RP-4 column (250x21.2 mm, SynChrom, Inc.).

The purity of the final products is controlled by thin layer chromatography (see table. 1), amino acid analysis (see table 2.) FAB-mass spectrometric and ^1H NMR studies confirm the compliance of the peptides with the above formulas.

 For a better understanding of the invention, the following are examples of its specific implementation.

Example 1. Obtaining the target product (XVII)
a. Obtaining Boc-Asn-Leu-OMe (I). 10 g (0.055 mol) of HCl-Leu-OMe are dissolved in 35 ml of dimethylformamide, 6.00 ml (0.055 mol) of N-methylmorpholine, 7.26 g (0.55 mol) of 1-hydroxybenzotriazole and 20.00 g (0.057) are added. mol) Boc-Asn-ONp. The reaction mixture was stirred for 12 hours at 24 ^{° C.} , evaporated to a volume of 10 ml and poured into 1 L of 2% H ₂ SO ₄ with stirring. The precipitate formed is filtered off, washed on a filter with 2% Н ₂ SO ₄ , water, us. a solution of KHCO ₃ , again with water, a solvent mixture of ether-methanol (5: 1, 50 ml), ethanol (100 ml). The yield of dipeptide (I) 18 u (91%).

 b. Obtaining Boc-Leu-Asn-Leu-OMe (II).

18.00 g (0.050 mol) of Boc-Asn-Leu-OMe are dissolved in 200 ml of a mixture of acetic and trifluoroacetic acids (3: 7), incubated for 1.5 hours at 20 ^o C, evaporated in vacuo, the residue is crystallized from hexane, washed ether (500 ml). The yield of trifluoroacetate (Ia) is 18.65 g (quantitative).

18.65 g (0.050 mol) of trifluoroacetate (Ia) are dissolved in 100 ml of dioxane, 5.49 ml (0.050 mol) of N-methylmorpholine, 6.80 g (0.050 mol) of 1-hydroxybenzotriazole, 19.38 g (0.055 mol) are added. ) Vos-Leu-ONp. The reaction mixture was stirred vigorously for 1 h at 20 ^{° C.} , 100 ml of dimethylformamide was added, stirred again for 12 hours at 20 ^° C. The dioxane was evaporated, the residue was diluted with 2% H ₂ SO ₄ to a volume of 1.5 L. The precipitate formed is filtered off, washed on the filter sequentially: with a 2% solution of H ₂ SO ₄ (200 ml); water (500 ml); us. NaHCO ₃ solution (250 ml); water (500 ml). The precipitate was dried under vacuum and washed with 50 ml of a mixture of methanol-ether (1: 5) and 200 ml of ethanol, and dried again under vacuum. The yield of tripeptide (II) was 18.34 g (63%).

 Compounds (III), (IV), (V), (VI) are obtained as in b, the reaction yields and physicochemical characteristics are shown in Tables 1 and 2.

in. Preparation of Boc-Lys (Z) -Pro-OH (VII)
6.9 g (60.0 mmol) of Pro are dissolved in 14 ml of pyridine, 6.6 ml (60.0 mmol) of N-methylmorpholine are added. 16.4 g (30.0 mmol) of Boc-Lys (Z) -OPfp and 4.0 g (30.0 mmol) of HOBt were dissolved in dioxane and added to the reaction mixture dropwise at 2 ^{° C.} at 10 ^{° C.} under vigorous stirring. After that, the reaction mixture was stirred for another 12 hours at 25 ^o C, poured 1.5 l of a 2% solution of H ₂ SO ₄ . The precipitated oil is extracted with ethyl acetate, the ethyl acetate layer is washed with a 2% solution of H ₂ SO ₄ , (3 • 300 ml), water, dried over Na ₂ SO ₄ , the desiccant is filtered off, and the filtrate is evaporated. The residue is dissolved in 20 ml of us. KHCO ₃ solution, washed with ether. The aqueous layer was acidified with citric acid to pH 5, the precipitated oil was extracted with ethyl acetate, the ethyl acetate layer was washed with water, dried over Na ₂ SO ₄ . The desiccant is filtered off, the filtrate is evaporated, the residue is chromatographed on a column (35 x 100 mm) with silasorb 600 using elution in a gradient of solvents ethyl acetate ethanol with 10% acetic acid. The yield of depeptide (VII) is 10.7 g (75%).

 d. Preparation of Boc-Leu-Lys (Z) -Pro-OH (VIII).

10.7 g (22.0 mmol) of dipeptide (VII) are dissolved in 50 ml of a mixture of acetic and trifluoroacetic acids (3: 7), cooled to 10 ^o C, incubated for 30 min at 20 ^o C, evaporated in vacuo at 30 ^o C , the residue was washed with hexane, dried under vacuum. The yield of trifluoroacetate (VII) is 10.8 (quantitative).

10.8 g (22.0 mmol) of trifluoroacetate (VIIa) are dissolved in 20 ml of dioxane, 4.8 ml (44.0 mmol) of N-methylmorpholine are added, 11.9 g (30.0 mmol) of Boc-Leu-OPfp . The reaction mixture was stirred for 20 hours at 25 ^° C., poured into 1.5 L of a 2% solution of H ₂ SO ₄ , dioxane was evaporated, the residue was dissolved in ethyl acetate and washed successively with a 2% solution of H ₂ SO ₄ (100 ml); water (300 ml); us. NaCl solution with 10% NaHCO ₃ (150 ml); water (300 ml), dried over Na ₂ SO _{4, the} desiccant is filtered off, the filtrate is evaporated. The residue was dried in vacuo. The yield of tripeptide (VIII) was 12.3 g (95%).

 Compounds (IX), (X), (XI), (XII) are obtained as in step d, the reaction yields and physicochemical characteristics are shown in Tables 1 and 2.

 d. Obtaining Boc-Leu-Glu (OBzl) -Glu (OBzl) -Glu (OBzl) -Leu-Lys (Z) -Pro-Leu-Glu (OBzl) - Val-Leu-Asn-Leu-OMe (XIII) .

114 mg (0.10 mmol) of trifluoroacetate (VIa); 204 mg (0.15 mmol) of heptapeptide (XII); 22 mg (0.16 mmol) of HObt are dissolved in 1 ml of dimethylformamide, 11 μl (0.10 mmol) of N-methylmorpholine are added, cooled to -10 ^° C with stirring, and a solution of 42.4 mg (0%) cooled to 0 ^° C is added. , 10 mmol) of N-cyclohexyl-N'-2- (N-methylmorpholino) ethylcarodiimide 4-toluenesulfonate in 200 μl of dimethylformamide. The reaction mixture was stirred 1 hour at 0 -5 ^o C x 12 hours at 20 ^o C, then recooled to -10 ^o C was added and cooled to 0 ^o C solution of 20 mg (0.047 mmol) of 4-toluenesulfonate N-cyclohexyl-N '-2- (N-methylmorpholino) ethyl carbodiimide in 100 μl of dimethylformamide. The reaction mixture was stirred for 1 h at -5 ^o C and 48 h at 20 ^o C, diluted with 2% H ₂ SO ₄ 10 times. The precipitate formed is filtered off, washed on a filter with 2% Н ₂ SO ₄ (3x6 ml), water, us. NaHCO ₃ , water, dried in vacuo, washed with ethyl acetate (10 ml), ethanol (10 ml), dried again in vacuo. The yield of tetradecapapeptide (XIII) is 150 mg (64%).

 Undecapeptide (XIV) is obtained by condensation of two protected fragments of tetrapeptide (IX) and heptapeptide (VIa) similarly to tetradeapeptide (XIII), see paragraph d.

 Dodecapeptide (XV) is obtained by condensation of two protected fragments of pentapeptide (X) and heptapeptide (VIa) similarly to tetradecapapeptide (XIII), see paragraph e.

 Tridecapeptide (XVI) is obtained by condensation of two protected fragments of hexapeptide (XI) and heptapeptide (VIa) similarly to the preparation of tetradeapeptide (XIII), see paragraph e.

 e. Obtaining Ac-Leu-Glu (OBzl) -Glu (OBzl) -Glu (OBzl) -Leu-Lys (Z) -Pro-Leu-Glu- (OBzl) -Glu (OBzl) -Val-Leu-Asn-Leu- OMe (XIIIa).

88 mg (0.037 mmol) of tetradecapapeptide (XIII) are dissolved in 3 ml of a mixture of acetic and trifluoroacetic acids (3: 7), incubated for 1.5 hours at 20 ^{° C.} , evaporated. The residue is crystallized from ether, washed with ether (50 ml), dried under vacuum. The yield of tetradecapapeptide (XIII) trifluoroacetate is 88 mg (quantitative).

88 mg (0.037 mmol) of tetradecapapeptide (XIII) trifluoroacetate are dissolved in 2 ml of trifluoroethanol, 8.0 μm (0.074 mmol) of N-methylmorpholine 352 μl (0.37 mmol) of acetic anhydride are added. The reaction mixture was kept for 30 minutes at 20 ^{° C.} , the solvent was evaporated, the residue was washed with water (10 ml), and dried under vacuum. The yield of tetradecapapeptide (XIIIa) is 86 mg (99%).

 Undeca (XIVa), dodeca- (XVa), tridecapeptides (XVIa) are obtained as described in Unblocking followed by acetylation of undeca- (XIV) dodeca- (XV) and tridecapeptide (XVI), respectively.

 g. Preparation of Pal-Leu-Glu (OBzl) -Glu (OBzl) -Glu- (OBzl) -Leu-Lys (Z) -Pro-Leu-Glu (OBzl) - Val-Leu-Asn-Leu-OMe (XIIIb).

50 mg (0.021 mmol) of tetradecapapeptide (XIII) trifluoroacetate are dissolved in 1.5 ml of freshly distilled, anhydrous dimethylformamide, 2.31 ml (0.021 mmol) of N-methylmorpholine and 31.89 ml (0.105 mmol) of palmitoyl chloride (Pal-Cl) are added with stirring ) 4.62 ml (0.042 mmol) of N-methylmorpholine are added portionwise to the reaction mixture in vigorous stirring. The reaction mixture was kept under stirring for 1 h at 25 ^{° C.} , the demethylformamide was evaporated, the residue was washed with hexane, water, and dried in vacuo. The yield of tetradecapetide (XIIIb) is 49.64 mg (94%).

 h. Preparation of Form-Leu-Glu (OBzl) -Glu (OBzl) -Glu (OBzl) -Leu-Lys (Z) -Pro-Leu-Glu (OBzl) -Val-Leu-Asn-Leu-Ome (XIIIc).

100 mg (0.42 mmol) of tetradecapapeptide (XIII) trifluoroacetate are dissolved in trifluoroethanol, 46.2 μl (0.42 mmol) of N-methylmorpholine are added, they are kept for 15 minutes, and trifluoroethanol is evaporated. The residue was dried to dryness in vacuo, dissolved in 882 μl of 98% formic acid, cooled to 0 5 ^° C and 294 μl (3.11 mmol) of acetic anhydride were added in 3 μl portions for 1 h. The reaction mixture was kept under stirring for 1 h at 0 ^o C and 48 h at 25 ^o C. The formic acid is evaporated, the residue is washed with ether (2 • 10 ml), water (3 • 10 ml), dried in vacuum. The yield of tetradecapapeptide (XIIIb) is 82.21 mg (85%).

 and. Preparation of Ac-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe (XVIIa).

86 mg (0.037 mmol) of tetradecapapeptide (XIIIa) are dissolved in HBr / AcOH (37), incubated for 1 h at 25 ^o C, evaporated at t no higher than 35 ^o C, washed repeatedly with ether, dried in vacuum, subjected to purification by HPLC on SynChropak RP-4 column (250x21.2 mm, SynChrom, Inc.). The yield of tetradecapapeptide (XVII) is 27 mg (43%).

 K. Preparation of Ac-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe (XVIIa).

92 mg (0.040 mmol) of tetradecapapeptide (XIIIa) are dissolved in 400 μl of TFA, 12 μl (0.114 mmol) of anisole are added and 17 μl (0.190 mmol) of trifluoromethanesulfonic acid, incubated for 1 h at 25 ^° C. TFA is removed in vacuo at no higher than 35 ^o С, the residue was washed repeatedly with ether, dried in vacuo, and purified by HPLC on a SynChropak RP-4 column (250x21.2 mm, SynChrom, Inc.). The yield of tetradecapapeptide (XVII) is 37 mg (53%).

 Undeka- (XVIII), dodeca- (XIX), tridecapeptides (XX) are obtained by the release of protected peptides (XIVa), (XVa), (XVIa), respectively, as described in par. or to.

l Preparation of Ac-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-NH ₂ (XXI).

27 mg (0.016 mmol) of tetradecapetide (XVIIa) are dissolved in methanol saturated with ammonia (anhydrous), kept in vacuum (10 mmHg) at 25 ^o C for 48 h, methanol is removed in vacuum (t no more than 30 ^o C), the residue is dissolved in water and lyophilized. The yield of tetradecapapeptide (XXI) is 27 mg (quantitative).

 m. Preparation of Form-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe (XVIIc).

80.0 mg (0.035 mmol) of tetradecapapeptide (XIIIb) are dissolved in 2 ml of trifluoroethanol, 50 μl of formic acid, 197 μl (2.1 mmol, 60 equiv) of 1,4-cyclohexadiene and freshly prepared palladium black are added with stirring. The reaction mixture was stirred for 12 hours at 25 ^{° C.} , the catalyst was filtered off, and the filtrate was evaporated. The residue was washed with ether (3 • 15 ml), ethyl acetate (3 • 15 ml), dissolved in water, and purified by HPLC on a SynChropak RP-4 column (250x21.2 mm, SynChrom, Inc.). The yield of tetradecapapeptide (XVIIc) is 41.30 mg (69%).

 n Preparation of Pal-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-Glu-Glu-Val-Leu-Asn-Leu-OMe (XVIIb).

 Tetradecapeptide (XVIIb) is obtained by the release of the protected peptide (XIII) as described in p. And or to. The yield and constants of the obtained tetradecapapeptide (XVIIb) are given in Tables 1 and 2.

 Preparation of Ac-Leu-Glu-Glu-Glu-Leu-Lys-Pro-Leu-D-Glu-Glu-Val-Leu-Asn-Leu-OMe (XXII).

 The tetradeapeptide (XXII) is prepared analogously to the tetradeapeptide (XVIIa), except that at the stage of the synthesis of hexapeptide (V), instead of Boc-Glu (OBzl) -ONp, Boc-D-Glu- (OBzl) -ONp is attached to the trifluoroacetate of the pentapeptide (IVa). The yield and constants of the obtained tetradecapapeptide (XXII) are given in Tables 1 and 2.

 Example 2. Determination of the growth-stimulating activity of peritoneal macrophages.

The growth-stimulating activity of peritoneal macrophages is determined by planting target cells at a suboptimal concentration for spontaneous proliferation, which is 10 ³ cells per ml.

 All peptides preparations: C-1-3, C-1-4, C-1-5 and C-1-6 (for chemical formulas see table 12) are dissolved in physiological saline at a concentration of 1 mg / ml, directly before the study, sterilized by filtration and diluted in a complete culture medium at concentrations of 200 μg / ml, 20 μg / ml, 2 μg / ml, 0.2 μg / ml, 0.02 μg / ml, 0.02 μg / ml, 0.002 mcg / ml.

Peritoneal macrophages are obtained from BALB / c and SZN mice of the Rappolovo nursery by washing the abdominal cavity with Hanks medium at t 4 ^o С (12). Human macrophages and monocytes are isolated from peripheral blood by sampling it from the cubital vein in 14 healthy donors.

The resulting suspension of cells is pooled from 3-4 donors of the corresponding group, after which it is washed twice with Hanks medium (10 min each), resuspended, stained according to Romanov-Giemsa and the number of adherent (spread) stained cells after incubation for 15 is counted min at t 37 ^o С in an atmosphere of 98% humidity containing 5% CO ₂ (gas-flow incubator GPI-01, experimental plant of analytical engineering, Lomonosov). The resulting suspension of cells was adjusted to a concentration of 5-10 ⁵ / ml (by spread, stained cells) in Hanks medium, seeded in the wells of a 96-well plate (NUNC, Denmark) in the amount of 5x10 ⁴ cells / well. The plates are incubated in a gas-flow incubator for 2 hours, then a monolayer of monocytes or macrophages is washed three times with Hanks medium at t 37 ^° C from non-adherent cells, followed by the addition of nutrient medium Needle with 10% cattle serum, 3% L-glutamine solution (1 ml ) and antibiotics (penicillin 100 units / ml, streptomycin - 100 μg / ml).

To conduct experiments to assess the effect of drugs on the ability of macrophages to produce growth factors for target cells, macrophages at a concentration of 5x10 ⁵ cells / ml are incubated in the presence of drugs (in the above dose range for 24 hours, in a complete culture medium. Then the macrophages are washed three times, serum-free Eagle medium (200 μl) was added to each well containing macrophages, and after 18 hours of incubation under standard culture conditions, the culture medium was collected and stored at -70 ^° C until use.

Target cells (mouse myeloma cells PXX63-Ag / 8, B-16 or the erythromyeloid cell line K-562) are placed in the wells of a 96-well plate at a concentration of 10 ⁴ cells / ml, 100 μl per well. 100 μl of Eagle medium are added to control cultures, and 100 μl of supernatants of intact macrophages or macrophages preincubated with the studied peptides are tested in experimental cultures. Cattle serum is used as a positive control (final concentration 5 $\genfrac{}{}{0ex}{}{\text{1}}{\text{2}}$ ) The target cells are incubated for 48 hours, after which 10 μl of ³ N-thymidine with a specific activity of 1.0 μCi / ml are added to them. After two hours, the cells are harvested by the harvester and the content of the included isotope is determined on a liquid scintillator counter (Delta, USA). The effect of supernatants is determined by the formula: (OK) / K • 100, where O is the average level of isotope incorporation into three parallel cell cultures in the presence of an appropriate dose of the drug, and K is the same in cells without the addition of the drug and compared with the effect of serum and the effect of unstimulated macrophages.

 The experimental results presented in tables 3, 4 and 5 indicate that the studied peptides C-1-3, C-1-4, C-1-5, C-1-6 show the ability to inhibit cytotoxicity and induce strong growth-stimulating the activity of preactivated macrophages, and the peptide, designated as C-1-6, most clearly shows these properties.

 Example 3. The study of the effect of synthetic peptides C-1-3, C-1-4, C-1-5, C-1-6 on regenerative and reparative processes in the liver.

 The most studied experimental models of liver repair regeneration are restoration after partial hepatectomy or toxic damage. It is important to note that these models allow us to analyze not only the regenerative process (cell proliferation aimed at compensating for the defect as a result of surgery or toxic damage), but also the reparative process (restoration of the physiological functions of the organ).

 We used white male mice of the HP line weighing 22–25 g and white outbred male rats weighing 180–200 g. The studied peptides were injected intraperitoneally in 0.2 ml of physiological saline for 3 days, starting 36–40 h after toxic damage or three days before hepatectomy.

 a. The study of the regenerative and reparative properties of synthetic peptides C-1-3, C-1-4, C-1-5, C-1-6 on a model of toxic liver damage.

Toxic liver damage is carried out by a single subcutaneous (in the cervical fold) administration of carbon tetrachloride in the form of a 50% solution in olive oil, at doses of 5 and 7.5 ml / kg of body weight. It is known that CCl _{4 itself is} non-toxic, but in microsomal liver systems that perform a detoxification function, it is metabolized within 24-36 hours with the release of free radicals that damage the organ parenchyma at the sites of their formation (13,14). Therefore, the test peptide begins to be used 40 hours after hepatotoxin injection and is administered for 3 days at the doses indicated above.

 The effectiveness of the studied drugs is evaluated by their effect on the duration of thiopental anesthesia. Thiopental short-acting barbiturate, which is metabolized exclusively in microsomal hepatocyte systems. It is believed that the duration of thiopental anesthesia, along with the functional bromosulfolein test, is an adequate integral criterion that allows us to assess the functional state of hepatocytes. Thiopental in an empirically selected dose of 31-34 mg / kg when administered to mice intravenously, in the lateral vein of the tail, induces a narcotic sleep for 3-4 minutes in intact animals. The introduction of thiopental is carried out one day after the last injection of the studied peptides. The duration of narcotic sleep is judged by the duration of the lateral position of the animals.

 For mathematical processing of the obtained experimental data, generally accepted methods of parametric and nonparametric statistics are used.

All studies of peptides, and especially effective C-1-6, demonstrated the ability to quickly restore liver function under conditions of its damage to CCl ₄ almost to an intact level (see table 6).

 To clarify the dose-dependent nature of the action of the most effective of the studied drugs (C-1-6), it is administered according to the above scheme in doses: 0.5; 2.5; 12.5; 62.5 mg / kg. The hepatoprotective effect is maximum for the peptide at a dose of 0.5 mg / kg of weight and a further increase in the dose of 5, 25 and 125 times does not change it (see Table 7).

 To analyze the mechanisms of hepatoprotective action of C-1-6, we study its effect on the activity of hepatospecific enzymes alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, alkaline phosphatase, as well as on protein synthesizing liver function. Enzyme activity and total protein in the blood are determined colorimetrically on an Expren-550 biochemical analyzer using standard commercial Ciba Corning kits (15).

 The degree of damaged hepatocytes is determined by changing the level of enzymes of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase in the above experiment (see table 8). In case of liver damage, the level of these enzymes in the blood increases significantly due to their release into the blood through the damaged hepatocyte membrane. In this experiment, in animals of the control group, the content of alanine and aspartate aminotransferase was increased by 1.8 and 4.5 times, respectively. Against the backdrop of the three-day therapeutic use of C-1-6, the level of enzymes decreases by 36 and 41%, respectively. The content of lactate dehydrogenase decreases to the level of intact animals, with an increase in the control group by 73%. These results indicate the reparative effect of the drug C-1-6 on damaged hepatocyte membranes.

During the experiment, the total protein content in the blood is also measured - an indicator of the protein synthesizing function of hepatocytes. Under the influence of CCl _4, the total protein content significantly decreases to 4.1 g / l,
while when administered to animals, C-1-6 it normalizes and amounts to 68.4 ± 2.9 g / l.

 Thus, it is obvious that the drug C-1-6 has a pronounced stimulating effect on reparative processes in the liver.

 b. Study of the effect of the synthetic peptide C-1-6 on regeneration processes.

To study the effect of C-1-6 on regeneration processes, the classical model of partial hepatectomy is used according to the method (16). The drug at a dose of 2.0 mg / kg is administered within 3 days before surgery. The liver for histological examination is taken 24 or 48 hours after surgery, fixed in 20% formalin and stained with hematoxylin-eosin. The metotic index is calculated for 3000 hepaticides and expressed in ppm. In some experiments, the effect of C-1-6 on the index of labeled hepatocyte nuclei is determined. For this, ³ H-thymidine is administered to rats 1 hour before slaughter. Isotope incorporation is evaluated autoradiographically.

 The mitotic index and the index of labeled nuclei in the control group of animals in the control group of animals 24 hours after hepatectomy increase compared with intact animals by 20 and 24%, respectively, and after 48 hours they decrease to 14 and 10% of the intact level, respectively. With the introduction of C-1-6, a decrease in both parameters is observed 24 hours after hepatectomy (i.e., inhibition of regeneration) and their subsequent increase after 48 hours by 2.5 and 2 times compared with the control, respectively. Moreover, in the first 24 hours after hepatectomy, a significant accumulation in the glycogen cells of the main energy substrate of hepatocytes is observed in comparison with the control (see Table 9).

 The data obtained suggest that on the first day after hepatectomy, C-1-6 promotes the mobilization of carbohydrate resources in hepatocytes, which accelerates proliferation at later stages of liver repair, and a later biologically driven adaptive peak observed against C-1-6 a process resulting from stimulation of the functional activity of hepatocytes in conditions of severe liver failure.

 in. Study of the effect of the drug C-1-6 on the healing of full-layer skin wounds in mice.

 When studying the effect of the drug on the healing of full-layer skin wounds in mice, the technique proposed by O.D. Barnaulov is used (17). Under hexenal anesthesia (80 mg), a layer-by-layer lesion is applied intraperitoneally with a 9-mm diameter round stamp and straightened, giving it a rounded shape. The scab formed on the surface of the wound is removed on day 4. Then, every other day until 13 days, and then daily until the end of the experiment. This procedure, on the one hand, due to repeated injuries, increases the healing time of wounds, and, on the other hand, when applied topically, it improves the contact of the drug with the surface of the wound. The drug C-1-6 is administered intraperitoneally or locally from 1 to 10 days after surgery at a dose of 2.5 mg / kg of body weight. When assessing the effectiveness of the drug, the area of the wounds is taken into account (conditionally taking it equal to the product of the longitudinal and transverse diameters), as well as the number of mice in the group whose wounds completely heal by this time.

 A study of the effect of C-1-6 on the regeneration of full-layer skin wounds is carried out with peripheral and intraperitoneal administration in vivo, and the applied concentrations are 20 times lower than in the case of standard preparations of this effect. Although the process of wound healing is much slower than restoration of the liver, with the introduction of the drug C-1-6 there is a significant acceleration of the regeneration process, especially with peripheral administration (see table. 10). One of the reasons for the relative delay in the stimulating effect of C-1-6 is the fact that when applying full-layer skin wounds, the number of macrophages in the defect zone is determined only by the degree of blood supply to the damaged skin area. The capillary network in the liver is more developed than in the skin and the specific blood flow is much higher, which creates conditions for more intensive migration of macrophages in the damage zone.

 Thus, the drugs induce the growth-promoting activity of macrophages, both in vitro and in vivo. Their biological effect is mediated by the stimulation of macrophages that secrete a number of growth factors, which, in turn, stimulate the processes of regeneration and repair. From the presented data, we can conclude that the studied peptides C-1-3 C-1-6 have a pronounced immunomodulating activity and can be widely used in medicine in the treatment of various liver lesions, full-layer skin wounds.

 The study of acute toxicity.

 In the study of acute toxicity, the effect of the drug on the general condition of animals in behavioral tests, hematological and biochemical blood parameters, and the drug's convulsive activity are determined.

The study of acute toxicity is carried out for 10 days in behavioral tests: an open field, audiogenic seizures and a psychotic reaction (climbing test) on sexually mature males and female F1-CBA / Black mice weighing 20 ± 0.8 g. Animals are kept in groups of 5 each free access to food and water under artificial lighting with a duration of 12 hours of light and dark phases. 4 groups of 10 animals each (5 females and 5 males) are formed:
1. The control group, the animals of which 1 time 20 minutes before the start of the first behavioral test received an intraperitoneal injection of 0.5 ml Dulbecco saline for mice weighing 20 g

 The 2,3 and 4 groups receive a single intraperitoneal injection of the drug C-1-6 at a dose of 250 mg / kg, 25 mg / kg, 2.5 mg / kg, respectively, in a volume of 0.5 ml 20 minutes before the start of the first behavioral test. Observation of the behavior continues for 4 minutes, with the last turning off the lighting. Horizontal (by the number of crossed squares) and vertical (by the number of rises on the hind legs) activities are estimated, which are an indicator of motor and orientational activity, respectively. The number of intersections by the animals of the center of the field is determined, which makes it possible to assess the level of reaction of anxiety and fear. Take into account the grooming time and the number of contaminants in the mink, indicators reflecting the excitement and the level of stress of the animal.

The experimental testing of the behavioral effects of different doses of the drug C-1-6 shows that:
the most pronounced activity on behavior has a dose of 2.5 mg / kg, proposed as a therapeutic,
the drug has some stimulating effect with the induction of elements of a psychotic reaction against the background of a decrease in the research-orientational reaction and motor activity,
males in most tests are more sensitive to the action of the drug than females,
the drug does not have convulsive activity,
for most behavioral indicators, the effects of the drug C-1-6 are short-lived and disappear by the 3rd day after its administration.

 In the process of studying acute toxicity, the effect of the drug C-1-6 on the biochemical and hematological parameters of blood of laboratory rats is also determined with daily intraperitoneal administration for 1 week at therapeutic doses of 1 and 10 (see table 11). A pathoanatomical study and a pathomorphological study of liver samples are also performed.

 Thus, the toxic effect of the drug was not found for all investigated parameters even at a dose 250 times higher than the therapeutic, which indicates a large therapeutic breadth of the drug C-1-6 (Table 12). TTT1 TTT2 TTT3 TTT4 TTT5 TTT6 TTT7 TTT8 NY2

Claims (1)

Peptides of the general formula:
R-Glu-Leu-Lys-Pro-Leu-X-Glu-Val-Leu-Asn-Leu-R ',
Where
R CH ₃ CO- (Ac), Ac-Glu, Ac-Glu-Glu, Ac-Leu-Glu-Glu-, HCO- (Form), Form-Glu-, Form-Glu-Glu, Form-Leu-Glu -Glu-, CH ₃ (CH ₂ ) _n CO-, CH ₃ (CH ₂ ) _n CO-Glu-, CH ₃ (CH> ₂ ) _n CO-Glu-Glu-, CH ₃ (CH ₂ ) _n CO- Leu-Glu-Glu-, n 8-18;
X L-Glu, D-Glu; R 'OMe or NH ₂ having a regenerative and reparative action.

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