RU2123859C1 - Peptide showing immunosuppressive property - Google Patents

Abstract

FIELD: chemistry of peptides, medicine. SUBSTANCE: invention relates to new biologically active substances. Peptide of the formula H-A- -T p- where A is D-Glu or D-iGlu and Y is OH or substituted (C₁-C₃),-amide shows immunosuppressive property and can be used in medicine, veterinary science and experimental biochemistry. EFFECT: broadened pattern of immunoreagents of protein origin. 4 ex, 8 tbl

Description

 The invention relates to medicine, namely to new biologically active substances with immunoregulatory properties, and can find application in medicine, veterinary medicine, as well as in experimental biochemistry.

 The relevance of developing new harmless immunoregulating peptides that can stop the progressive growth of diseases such as malignant neoplasms, sepsis, chronic and sluggish infections developing against the background of immunodeficiency, is confirmed by a large number of studies conducted in this area.

 The most common way to identify new peptides is to isolate active peptide fractions from total tissue extracts, fractionation and purification up to the isolation of an individual substance and its identification (SU N 1600047, SU N1638849, SU N 1737798). Thymic extracts, in particular, thymosin fraction 5 / Goldstein A.L., Guna A., Latz M. M., Hardy H.A., are widely known as regulators of immune processes in practical medicine. White A. /, thymalin / CH, N 6595-86 /. These extracts consist of a complex of substances of a polypeptide nature and their preparation from natural sources is limited by the complexity of production, the low yield of active substances and the significant variability of their physicochemical characteristics and biological properties. In addition, due to the presence of ballast components in the natural preparations of the thymus, side effects sometimes occur in patients with them. The latter circumstance was an incentive for the creation of synthetic peptide preparations. Currently, a number of peptides having immunoregulatory properties have been synthesized: SU N 1582393, SU N 1541821, SU N 1518956, EP N 230052, EP N 406931, US N 5021551, US N 5013723. Each of the obtained synthetic peptides with a limited set of necessary properties It has high activity, low toxicity, and the absence of side effects that determine their possible use in medicine.

The present invention provides a new peptide as an agent having an immunosuppressive property of the formula
HAD-Trp-Y,
Where
AD-Glu or D-iGlu;
Y is OH or a substituted amide (C ₁ -C ₃ ).

 The method for producing the peptide is illustrated by the following example.

 Example 1

Obtaining H-iD-Glu-D-Trp-OH and HD-Glu-D-Trp-OH
1. Obtaining Boc-D-Glu-OH
14.7 (0.1 mol) of HD-Glu-OH was dissolved with 200 mm distilled water, the pH was adjusted to 10.2 with a 1 molar KOH solution and a solution of 33.0 g (0.3 mol) of BOC ₂ O in dioxane was added with vigorous stirring . We monitored the pH value on the pH-stat. After completion of the reaction, the mixture was placed in a separatory funnel, extracted from an alkaline medium with 3 × 150 mm ethyl acetate, the aqueous phase was slowly acidified with a 0.2% sulfuric acid solution to pH 3.0 and extracted with Boc-D-Glu-OH into the organic phase (3 200 mm). The organic layer was washed with 3 x 200 mm saturated Na ₂ SO ₄ solution until neutral, dried over Na ₂ SO ₄ . Evaporated in vacuo to an oily state. Yield: 16.7 g (68%).

2. Preparation of a mixture of Boc-D-Glu-D-Trp-OH and Boc-iD-Glu-D-Trp-OH
16.7 g (0.068 mol) of Boc-D-Glu-OH was dissolved in 200 ml of dimethylformamide, cooled to 0 ^° and a solution of 20.6 g (0.1 mol) of N, N '-dicyclohexylcarbodiimide in 100 ml of dimethylformamide was added with stirring . The mixture was stirred for 4 hours at + 4 ^o C and left for 8 hours at room temperature. The precipitated dicyclohexylurea was filtered off, the precipitate was washed with 2 x 50 mm with dimethylformamide, the filtrate was evaporated in vacuo to 1/2 volume and added with cooling to +4 ^o C and vigorous stirring, 24.3 (0.1 mole) HD-Trp-OK. The solution was allowed to gradually warm to room temperature. The completion of the reaction was monitored by TLC in the system (chloroform: ethyl acetate: methanol 6: 3: 1) until the spot of internal BOC-D-Glu acid anhydride disappeared. The residues of dicyclohexine urea were filtered off, dimethylformamide was evaporated in vacuo. To the oily residue were added 200 mm ethyl acetate and 200 ml of a 0.2% sulfuric acid solution. The organic layer was separated, washed to a neutral medium with saturated Na ₂ SO ₄ solution, dried over Na ₂ SO ₄ , and the ethyl acetate solution was evaporated in vacuo. The resulting oily precipitate was a mixture of Boc-D-Glu-D-Trp-OH and Boc-iD-Glu-D-Trp-OH. The total oil yield was 25.4 g (70%).

3. Preparation of Boc-D-Glu-D-Trp-OH and Boc-iD-Glu-D-Trp-OH
25.4 g (0.048 mol) of the mixture were dissolved in 200 ml of formic acid, stirred at 40 ^° C. for 1 hour and evaporated in vacuo to an oily state.

 Peptides were separated and purified by ion exchange chromatography on a Sephadex SP-PEA column in a gradient of 0.01-0.2 M pyridine acetate buffer. Yield: 5.7 g (35%) of H-iD-Glu-D-Trp-OH and 5.7 g (35%) of H-D-Glu-D-Trp-OH.

As a result of studying the physicochemical properties of the peptide, the following characteristics were obtained:
Primary Structure - H-iD-Glu-D-Trp-OH
Gross formula - C ₁₆ H ₂₀ N ₃ O ₅
Molecular Weight - 334.35
Appearance - white with a yellowish tint or gray powder.

 Solubility - readily soluble in water, moderately in alcohol, practically insoluble in chloroform.

 The UV spectrum in the region of 250-300 nm has a maximum of 280 ± 2 mm, the shoulder 287 ± 2 nm.

The values of R _f1 = 0.30 in the system (chloroform-methanol-32% acetic acid = 60:45:20) and R _f2 = 0.52 (in the system butanol-pyridine-water-acetic acid = 5: 5: 4: one).

The biological activity of the new peptide HAD-Trp-Y, where AD-Glu or D-iGlu, Y-OH or substituted amide (C ₁ -C ₃ ).

 Example 2

 Effect on the population of hematopoietic progenitor cells. As a test, stem-forming hemopoietic cells formed colonies on the spleens of irradiated mice.

 a) In vitro treatment of the H-D-Glu-D-Trp-Y peptide with intact bone marrow cells.

Intact bone marrow cells were incubated with the peptide at various concentrations (0.0025-10 μg / ml) for 1 hour at 37 ^° C. Then, the suspension was introduced without washing with irradiated lethal dose to mice-recipients. After 8 days, the animals were killed and counted colonies on the spleen. Table 1 shows the results.

 As can be seen from the data, incubation of bone marrow cells with a peptide in a concentration range of 0.0025–0.01 μg / ml leads to a decrease in the yield of colonies by about 30–40%; a preparation at concentrations of 0.02–10 μg / ml inhibits colony growth by more than than 60%. A group of mice along with bone marrow cells treated with a peptide (0.02 μg / ml) were injected with thymocides. In this case, the abolition of the suppression of colony growth by a peptide was registered, which indicates the effect of the peptide not directly on hematopoietic progenitor cells, but on regulatory cells participating along with them in splenic colony formation.

 b) In vitro treatment of the H-D-iGlu-D-Trp-Y peptide with intact bone marrow cells.

 Processing was carried out analogously to example 2 (a). Table 2 shows the results.

 As can be seen, the peptide contributes to a more than twofold decrease in the yield of splenic colonies.

 c) In vivo treatment of the H-D-Glu-D-Trp-Y peptide with intact bone marrow cells.

 The peptide at various concentrations was administered intraperitoneally (ip) to intact mice. After 2 hours, 1 or 2 days, they were sacrificed, suspensions of bone marrow cells were prepared, and they were injected into lethally irradiated (8.5 Gy) mice. A group of recipients was injected with thymocytes along with the bone marrow. After 9 days, the number of colonies on the spleen was counted. The optimal dose of the peptide (0.02 μg / ml) was selected, with which all further experiments are carried out. Table 3 shows the results.

As can be seen from the data, the peptide introduced to bone marrow donor mice helps to reduce the number of exocolonies in the spleen and to reduce the size of the KOE-C pool. The greatest inhibitory effect is manifested 2 days after the introduction of the peptide; additional administration of thymocytes to recipients cancels it. The cellularity of bone marrow, thymus and spleen in mice after administration of the peptide was also analyzed. It was found that the number of cells in the bone marrow of mice is slightly reduced compared to the control level, the relative number of cells per mg of organ weight in the thymus remains unchanged, and in the spleen decreases (control - 28.0 • 10 ⁷ ± 3.0, peptide - 18 , 2 • 10 ⁷ ).

 d) In vivo treatment of the H-D-iGlu-D-Trp-Y peptide with intact bone marrow cells.

 Processing was carried out analogously to example 2 (c). Table 4 shows the results.

 As can be seen from the data, the inhibitory effect of the peptide on the bone marrow KOE-C population and their colony-forming activity appears already 2 hours after the peptide injection to mice and lasts 2 days. Thymocytes completely cancel the effect of the peptide.

 Example 3

The effect of HAD-Trp-Y, where A is D-Glu or D-iClu, Y is OH or a substituted amide (C ₁ -C ₃ ), on the restoration of the hematopoietic progenitor cell population after cytopenia of various origins.

 a) Ionizing radiation was taken as an agent causing depletion of bone marrow. Donor mice were irradiated at a dose of 4 Gy. The peptide was administered intraperitoneally, 10 μg / kg 1 hour, 2 days and 1 day before irradiation. The bone marrow was removed from the femur in animals on the 8th day after exposure to ionizing radiation, since it was at this time that the exponential recovery of both the total cell mass and the population of hematopoietic progenitor cells in the bone marrow devastated as a result of radiation began. Table 5 shows the quantitative values of nucleated cells in the bone marrow of mice irradiated with a dose of 4 Gy and treated with a peptide.

 It was found that the number of nucleated cells in the bone marrow after injection of H-D-iGlu-D-Trp-OH exceeds the control values for all three periods of use of the peptide, and the maximum, statistically significant effect is observed for 2 days. The introduction of the peptide H-D-Glu-D-Trp-OH did not cause a change in the value of this parameter in the experimental groups compared with the control.

 In addition, on day 8, a sharp statistically significant increase in the number of hematopoietic progenitor cells in the bone marrow of 4 Gy irradiated mice that received the peptide 2 days before exposure to ionizing radiation was also recorded. The content of KOE-C in the bone marrow of mice irradiated with a dose of 4 Gy and treated with peptides is presented in table 6.

 Studies were carried out to restore the number of karyocytes and hematopoietic progenitor cells in the bone marrow of the femur after irradiation at a dose of 4 Gy and peptide injection for 14 days in groups of mice, 6 in each. The timing of the introduction of the peptide is as follows: 1 day and 2 days before exposure to ionizing radiation, intraperitoneally, 10 μg / kg The results are presented in table 7.

 As shown by experiments, the preliminary introduction of peptides 2 days before irradiation leads to a pronounced intensification of bone marrow cell restoration, the number of karyocytes in this case reaches the values of intact control by 7 days after irradiation, and moreover, in the presence of the HD-iGlu-D-Trp peptide -OH continues to increase up to 14 days.

 b) As another agent causing the emptying of the hematopoietic system, we chose a cytostatic cytosar, which was administered once a day three times strictly after 24 hours. The mouse peptide of 12 in each group was obtained three times every 2 hours after the second injection of the cytosar intraperitoneally, 10 μg / kg. The animals were sacrificed 3 hours after the last injection of the cytosar and the karyocyte content and the amount of KOE-C were determined in the bone marrow of the femur. The results are presented in table 8.

 It was found that in the presence of a peptide that does not affect the total karyocyte content in the bone marrow, the viability of hematopoietic progenitor cells is almost 70% higher than in the control.

 As a result, studies on the effect of the peptide on the restoration of hematopoiesis after exposure to damaging factors (irradiation, cytostatics) suggest that the peptide, as an immunosuppressant, promotes intensive restoration of the pool of hematopoietic progenitor cells (KOE-C) when injected before exposure to damaging factors, i.e. . peptide protects against damage.

 Example 4

 The possibility of using a proliferation-inhibiting peptide to prevent the development of secondary disease in lethally irradiated recipients with whom allogeneic bone marrow was transplanted was studied.

 With two different peptide injection regimens, an almost twofold increase in the 45-day survival rate of the experimental mice was found in comparison with the control. In addition, mice treated with the peptide showed a slow development of secondary disease by the weight loss test by about 2 weeks, and also on the 45th day in surviving mice of the experimental groups, the spleen weight exceeded the value of this parameter in the control.

 The results indicate the possibility of using the peptide as an immunosuppressant.

In order to study the safety of the peptide, acute toxicity was studied. The study of acute toxicity was carried out in accordance with the Methodological Recommendations of the Pharmacological Committee of the Russian Federation "Requirements for the preclinical study of the general toxic effect of new pharmacological substances", M., 1985. The results of the studies showed that when the intraperitoneal administration of 1000 multiple doses of the peptide did not have acute toxic effects doses it was not possible to reach its LD ₅₀ . Thus, the new biologically active peptide is not toxic.

According to the present invention, a new peptide of the formula
HAD-Trp-Y,
Where
AD-Glu or D-iGlu;
Y is OH or a substituted amide (C ₁ -C ₃ ),
has the biological activity described in examples 2-4 and may find application in medicine.

Claims (1)

An immunosuppressive peptide of the formula
Had-trp-j
where AD-Glu or D-iGlu; J-OH or substituted amide (C ₁ -C ₃ ).

Images