RU2495048C1 - Peptide possessing antiatherosclerotic action and composition for preventing and treating atherosclerotic vascular disease - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to immunology and medicine, namely to new peptides of general formula: X-CC-C(Y)-CC-Z, wherein X is a B-cell epitope of apolipoprotein B100 protein; C is an amino acid residue specified in K or R; Y is an immunoadjuvant specified in a group of Pam₁CSS-; Pam₂CSS-, Pam₃CSS-; Z - T-helper epitope specified in AKFVAAWTLKAAA, KLIPNASLIENCTKAEL, QYIKANSKFIGITE.

EFFECT: peptide preparations possess higher antisclerotic activity as compared to their analogues, and the compositions thereof are of practical interest as a vaccine for preventing and treating atherosclerotic vascular disease.

9 cl, 1 dwg, 6 tbl, 7 ex

Description

The invention relates to the field of immunology and medicine, namely to new peptides of the general formula: X-CZ-C (U) -TsZ-Z,

where X is the B-cell epitope of the apolipoprotein B 100 protein;

C is an amino acid residue selected from K or R;

Y - immunoadjuvant selected from the group Pam ₁ CSS-; Pam ₂ CSS, Pam ₃ CSS.

Z - T-helper epitope selected from AKFVAAWTLTCAAA, KLI-PNASLIENCTKAEL, QYIKANSKFIGITE;

and preparations based on these peptides for the prevention and treatment of atherosclerosis.

Atherosclerosis is a chronic disease that results in a variable combination of changes in the inner lining (intima) of the arteries, which causes a thickening of the inner layers (inner lining) of the large and medium arteries, as well as the accumulation of lipids, complex carbohydrates, fibrous tissue, blood components, calcification. Atherosclerosis reduces blood flow and can cause ischemia and tissue necrosis in organs supplied with vessels that have been affected. Atherosclerosis is a major cause of cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. It is the leading cause of death in the Western world and is predicted to become the leading cause of death worldwide in two decades (RU 2322260, 2006).

The disease begins with the accumulation of lipoproteins, primarily low-density lipoproteins (LDL), in the extracellular matrix of the vessel. LDL particles undergo modification as a result of oxidation. Products produced by the oxidation of LDL are toxic to vascular cells, cause inflammation and initiate plaque formation. In many ways, atherosclerosis is a response to damage, including inflammation and fibrosis. Epitopes in oxidized LDL are recognized by the immune system and cause stimulation of the formation of antibodies, which are usually directed against structures based on certain peptides, in particular, against Aiolipoprotein B (ApoB) and its fragments (RU 2322260, 2006).

ApoB is a protein that is constantly associated with LDL. ApoB contains 4563 amino acids. Oxidized LDL leads to degradation of ApoB into fragments, apolipoprotein B is fragmented, and aldehyde adducts combine with positively charged amino acids, in particular lysine. In this case, peptide sequences that are usually not exposed due to the three-dimensional structure of apolipoprotein B (immune reactions are almost exclusively directed against peptide sequences 5-6 amino acids in length) become accessible to immune cells and become immunogenic due to haptenylation with aldehydes. Based on this, it can be assumed that natural and artificially synthesized peptides based on ApoB can be used to create vaccines against atherosclerosis.

It has now been shown (George et al. Atherosclerosis, Volume 138, 1998: "Hyperimmunization of apo-E-deficient mice with homologous malondialdehyde low density lipoprotein suppresses early atherogenesis", p.147-152) that the introduction of some of these peptides against oxidized LDL reduces the development of atherosclerosis. However, studies have shown that this does not always happen. In particular, when studying the effect of peptides included in ApoB (RU 2296582, 2007) on the development of atherosclerosis, it was found that immunization using a mixture of peptides # 10, 45, 154, 199, and 240 gives impetus to increased development of atherosclerosis. Immunization using other peptide sequences, for example, peptide sequences # 1 and from 30 to 34, generally does not affect the development of atherosclerosis. Those. immune responses against oxidized LDL can protect against development, contribute to the development of atherosclerosis and have no effect at all, depending on which structures of oxidized LDL they are directed to.

In this regard, the search for peptide structures of ApoB that can prevent the development of atherosclerosis and serve as the basis for the creation of vaccines against atherosclerosis is quite relevant and practically important.

The closest in technical essence and the achieved solution to the claimed group of inventions are fragments of ApoB, as well as pharmaceutical compositions and vaccines based on them, proposed for the prevention and treatment of atherosclerosis (RU 2296582, 2007). In the framework of the invention, 38 peptide sequences were obtained that are promising for the diagnosis and treatment of atherosclerosis. The most promising for the treatment of atherosclerosis, according to the authors, are the peptides p1-EEEMLENVSLVCPKDATRFK, p2-ATRFKHLRKYTYNYEAESSS, p74-VISIPRLQAEARSEILAHWS and p301-HTFLIYITELLKKLQSTTVM. As shown by studies conducted by the authors of the present invention, among other peptides, the peptides p45-IEIGLEGKGFEPTLEALFGK and p210-KTTKQSFDLSVKAQYKKNKH proposed in the closest analogue are promising.

Along with the possibility of using individual peptides, the possibility of using mixtures of peptides with canonized bovine serum albumin and alumina hydrate as vaccines against atherosclerosis was noted.

The disadvantage of the above peptides and vaccines is their lack of effectiveness in atherosclerosis.

The problem solved by the authors was to obtain more effective drugs for the prevention and treatment of vascular atherosclerosis based on peptides, as well as expanding the range of possible peptide drugs based on ApoB fragments.

The use of a fragment of an immunoadjuvant in the structure was to

to provide easier delivery of the vaccine construct to immunocompetent cells.

The use of a fragment of the T-helper epitope in the claimed complex seemed appropriate due to the possibility in its presence of reducing or eliminating genetically determined variations in the immune response to the B-epitope, based on the fact that peptides that encode a T-cell determinant that stimulates Th cells mammals, bind to the products of the main histocompatibility complex of the second type of most haplotypes and cause a strong CD4 + Th response in most members of the outbred population. This, in particular, was observed with the use of the QYIKANSKFIGITE fragment of tetanus toxin, the KLIPNASLIENCTKAEL peptide from the dog plague virus protein, the GALNNRFQIKGVELKS peptide from the influenza hemagglutinin light chain peptide, or the AKFVAAWTLKAAA peptide, which was named P. Pordigne et al. Pordign.aldon.ald.onordon.ald.onordon.ald. J. Immmunol. (1989) 19, 2237-2242).

The problem was solved by using complex preparations for these purposes, having in their structure both fragments of ApoB-100 and additionally fragments of the immunoadjuvant and T-helper epitope.

The technical result was achieved by creating a peptide of the General formula:

X-CC-C (Y) -CC-Z,

where X is the B-cell epitope of the apolipoprotein B 100 protein;

C is an amino acid residue selected from K or R;

Y - immunoadjuvant selected from the group Pam ₁ CSS-; Pam ₂ CSS, Pam ₃ CSS.

Z is a T-helper epitope selected from AKFVAAWTLKAAA, KLI-PNASLIENCTKAEL, QYIKANSKFIGITE;

and compositions based on them.

As B-cell epitopes of Apo-B 100 protein, it contains peptide fragments of Apo B 100, in particular, described in (RU 2296582, 2007). The best results were obtained using the following peptides for these purposes: a peptide identical to the sequence of 43-62 apolipoprotein B 100 person (p2); a peptide identical to the sequence 661-680 of human apolipoprotein B 100 (p45); a peptide identical to the sequence of 3136-3155 apolipoprotein B 100 person (p210).

As an immunoadjuvant, as a rule, known compounds are used that are capable of activating an adaptive immune response with the formation of antigen-specific clones of T and B lymphocytes, as well as memory T cells, with respect to a particular pathogen.

These include, in particular, peptides from the group Pam ₁ CSS-; Pam ₂ CSS, Pam ₃ CSS.

The best results with the use of peptides were achieved using the compound (Pam ₂ Cys) having the structural formula as an immunoadjuvant

Between the B-epitope and the immunoadjuvant is a sequence of two residues of positively charged amino acids (for example, lysine or arginine). A similar sequence is found between the immunoadjuvant and the T-epitope. These sequences serve as sites through which intracellular processing of the vaccine construct passes to free determinants.

Peptides used as T-helper epitope

AKFVAAWTLKAAA,

KLIPNASLIENCTKAEL, and

QYIKANSKFIGITE.

The peptides are prepared using standard solid phase peptide synthesis technology, in solution, or using coherent synthesis methods (e.g., as described by J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969). In solid-phase synthesis, the first amino acid is attached to an insoluble polymer, the polypeptide chain is built up sequentially or protected fragments are used at the condensation stage, excess reagents are removed by filtration, followed by washing. Synthesis can be carried out using Boc / Bzl or Fmoc / tBu technology, or any other system orthogonal protective groups. Upon completion of the assembly of the target sequence, the peptide is cleaved from the polymer, usually accompanied by removal of permanent protecting groups. Purification of the final product is carried out, as a rule, by chromatographic methods, for example, using reversed-phase or ion-exchange high-performance liquid chromatography.

Tests have shown that, along with individual complex peptides, mixtures of them can be used to prevent the development of atherosclerosis. In particular, the best results were obtained when using a drug containing a mixture of peptides as a vaccine against atherosclerosis:

- P2-IRP2-TE1:

Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam ₂ CSS) KK-AKFVAAWTLKAAA

- R45-IRP2-TE1:

Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam ₂ CSS) KK-AKFVAAWTLKAAA

- P210-IRP2-TE1:

Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam ₂ CSS) KK-AKFVAAWTLKAAA

in the ratio of 0.25-0.5: 0.25-0.5: 0.25-0.5. The greatest efficiency was achieved with a peptide ratio of 1: 1: 1. A decrease in the proportion of one of the peptides in the mixture to less than 0.25 led to a significant decrease in antiatherosclerotic activity.

Figure 1 shows micrographs of preparations of the descending aorta en face in Apo E (- / -) mice after staining with oil red O Original increase of 40 times.

A) group of mice No. 4 (immunization with a mixture of peptides), B) group of mice No. 1 (control, PBS administration), C) group of mice No. 2 (control, administration of AK-FVAAWTLKAAA-TE 1). Appendix 1 provides the formulas of the main compounds used.

The essence and advantages of the claimed invention are illustrated by the following examples.

Example 1. Synthesis of starting peptides.

The peptides were synthesized by the solid state method on an Applied Biosystems 430A synthesizer according to the in situ method using noc-boc protected amino acid derivatives (Schnolzer M. et. Al. Int. J. Pept. Prot. Res. - 1992. - v.40. - N2 - p. 180-193). The following derivatives of trifunctional amino acids Boc- (Mts) -Arg-OH, Boc- (Bzl) -Ser-OH, Boc- (Bzl) -Thr-OH, Boc- (Dnp) -His-OH, Boc- were used in the work (OBzl) -Glu-OH, Boc- (OBzl) -Asp, Boc- (ClZ) -Lys, Boc- (Cl2Bzl) -Tyr-OH. The peptides were synthesized using 0.1 mM Boc- (Bzl) -Ser-PAM-polymer, Boc- (Dnp) -His-PAM-polymer, Boc- (ClZ) -Lys-PAM-polymer and Boc-βAla-PAM- polymer. As an insoluble matrix used 1% copolymer of styrene and divinylbenzene.

The release was carried out with undiluted trifluoroacetic acid (TFA) twice in 1 minute. The neutralization during the first condensation was carried out by adding a 3 ^x excess of diisopropylethylamine directly to the reaction mixture, at the stage of addition of the amino acid residue; re-condensation was carried out after additional washing of the peptidyl polymer with 10% solution of diisopropylethylamine (DIPEA) in dimethylformamide (DMF). The addition of amino acid residues was carried out by the method of 1-hydroxybenzotriazole esters using 2-5-fold excess of reagents. A mixture of amino acid derivatives was prepared immediately before being added to the reaction vessel.

The completeness of the condensation reaction was monitored using ninhydrin and bromophenol tests.

After completion of the peptide chain extension, the peptidyl polymers containing histidine were treated with a mixture of DMF: ethanedithiol (EDT): DIPEA = 7: 2: 1 for 30 minutes and washed with DMF and methylene chloride (DCM). All peptidyl polymers were treated with TFA twice in 1 minute, washed with DCM and diethyl ether, removed from the reactor and dried.

The peptides were cleaved from the polymer and the side protective groups were removed using liquid hydrogen fluoride via Sn1 / Sn2 mechanisms in the presence of scavengers. For this, 1 g of the peptidyl polymer was placed in a hydrogen fluoride (HF) reactor, 0.8 ml of m-cresol, 0.2 ml of EDT, 6.5 ml of dimethyl sulfide (DMS) and 2.5 ml of HF were added, and stirred for 2 hours at 0 ° C. Then, DMS and HF were evaporated, and 10 ml of HF was distilled into the reactor, and the mixture was stirred at 0 ° C for 1 hour. After evaporation of HF, the residue was transferred with ether to a filter, washed with acetonitrile and ether. The product was isolated by washing off TFA in 200 ml of ether.

The isolated coarse products were purified by semi-preparative reverse-phase HPLC on a Waters Prep Nova-Pak HR C-18, 6µ, 60Å, 19 × 300 mm column in an acetonitrile gradient of 5-75% in 30 minutes. Detection at 220 nm. The fraction corresponding to the peak of the main product, after freeze drying, was subjected to amino acid, mass spectral and HPLC analyzes:

- HPLC conditions: Gilson analytical chromatograph, France, DeltaPak C-18 column, 5µm, 100A °, 3.6 × 150 mm; gradient (10-50)% acetonitrile in 0.1% TFA in 20 minutes;

- Amino-slot analysis conditions: LKB Alpha Plus 4151 amino acid analyzer, Sweden; hydrolysis:

- 4M methanesulfonic acid containing 0.2% tryptamine, 110 ° C, 22 hours;

- 12N hydrochloric acid, propionic acid (1: 1), 110 ° C, 22 hours.

- conditions for mass spectral analysis: Voyager-DE Bio-Spectrometry Workstation mass spectrometer, Per Sepetive Biosystems, USA. All synthesized peptides had the amino acid composition and molecular weight corresponding to theoretical values. The purity of the compounds according to analytical RP-HPLC was at least 95%.

As a result of applying this methodology, peptides similar to those obtained in the closest analogue were obtained, namely p2-ATRFKHLRKYTYNYEAESSS; p45-IEIGLEGKGFEPTLEALFGK and p210-KTTKQSFDLSVKAQYKKNKH, which were then further modified to produce peptides

p2MAP- (ATRFKHLRKYTYNYEAESSS) ₄ K ₂ KβA;

p45MAP- (IEIGLEGKGFEPTLEALFGK) ₄ K ₂ KβA and

p210MAP- (KTTKQSFDLSVKAQYKKNKH) ₄ K ₂ KβA

Example 2. Obtaining target (complex) peptides

The peptides were synthesized by the solid phase method using the Boc / Bzl strategy in manual mode /J.M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969 /. For temporary protection of the α-amino function, a tert-butyloxycarbonyl group was used. To block the side radicals of arginine, lysine and histidine, mesitylenesulfonyl, 2-chlorobenzyloxycarbonyl and dinitrophenyl groups were used, respectively. The series, threonine, aspartic and glutamic acids were introduced into the polypeptide chain as the corresponding benzyl esters, and tryptophan as a formyl derivative along the indole ring. The side chain of the lysine residue, to which dipalmitoyloxyprolylcysteine was subsequently attached via a spacer, was blocked by the fluorenylmethyloxycarbonyl group.

The corresponding PAM polymers with an initial capacity of ~ 1 mMol / g were used as an insoluble matrix. A 1% copolymer of styrene and divinylbenzene was used as the polymer. The extension of the polypeptide chain was carried out by in situ method. The release was carried out undiluted TFA twice in 1 minute.

The neutralization during the first condensation was carried out by adding a 3-fold excess of DIPEA directly to the reaction mixture at the stage of addition of the amino acid residue; re-condensation was carried out after additional washing of the peptidyl polymer with a 10% solution of DIPEA in DMF. The addition of amino acid residues was carried out by the method of 1-hydroxybenzotriazole esters using 3-fold excess of reagents. After the addition of amino acid residues corresponding to the sequence of synthesized peptides, the N-terminal amino group was acetylated using 40 equivalents of acetic anhydride in DMF. Formyl groups from tryptophan residues and histidine dinitrophenyl protection were removed by treatment with 20% piperidine in DMF for 30 minutes.

The fluorenylmethyloxycarbonyl group from the side chain of the lysine residue at position 23 was removed by treating the peptidyl polymer with a 50% solution of piperidine in DMF. Serine (introduced as the corresponding benzyl ester, and cysteine as Fmoc (Trt) Cys-OH. After removal of trityl from the sulfhydryl cysteine group, the residue of 1-bromo-2,3-hydroxy propane was added, which was then acylated with a 1-hydroxybenzotriazole derivative of palmitic acid in methylene chloride. Fmoc grouping with the alpha amino group of cysteine was removed either after the introduction of palmitic acid residues (upon receipt of diacyl derivatives), or before their introduction (upon preparation of triacyl derivatives).

Permanent protecting groups were removed and peptides were cleaved from the insoluble matrix by anhydrous liquid hydrogen fluoride in the presence of scavengers. 9.5 ml of hydrogen fluoride and 0.3 ml of m-cresol and 0.2 ml of ethanedithiol were transferred into a vessel with a peptidyl polymer under stirring on a magnetic stirrer at 0 ° C for 1 hour. Then, hydrogen fluoride was evaporated, and the peptide was precipitated with ether from trifluoroacetic acid.

The isolated products were purified by semi-preparative reverse-phase high-performance liquid chromatography (HPLC) on a Waters Prep Nova-Pak HR C-18, 6µ, 60Å, 19 × 300 mm column in an acetonitrile gradient of 5-75% in 30 minutes. Detection at 220 nm . The fraction corresponding to the peak of the main product, after freeze drying, was subjected to oxidation. The obtained target compounds were characterized by mass spectrometry, amino acid and HPLC analyzes.

Conditions for HPLC analysis: Gilson analytical chromatograph, France, DeltaPak C-18 column, 5µm, 100A °, 3.6 × 150 mm; gradient (15-65)% acetonitrile in 0.1% TFA in 25 minutes;

Amino -lot analysis conditions: LKB Alpha Plus 4151 amino acid analyzer, Sweden; hydrolysis by sequential administration:

- 4M methanesulfonic acid containing 0.2% tryptamine, at 110 ° C, for 22 hours;

- a mixture of 12N hydrochloric and propionic acids in a ratio of 1: 1, at 110 ° C, for 22 hours.

Mass spectral analysis conditions: Voyager-DE Bio-Spectrometry Workstation mass spectrometer, Per Sepetive Biosystems, USA.

All synthesized peptides had the amino acid composition and molecular weight corresponding to theoretical values. The purity of the compounds according to analytical RP-HPLC was at least 90%. The structures of the obtained peptides, molecular weights, and exit times under the conditions of analytical RP HPLC are shown in Table 1.

Table Characteristics of the resulting peptides Cipher Peptide structure Molecular weight Exit time, min Theor Exp. IRP2 TE1 Ac-KKK (SCPam ₂ ) KKAKFVAAWTLKAAA 2858.74 2859.48 7.75 P2-IRP1-TE1 Ac-ATRFKHLRKYTYNYEAESSS-KKK (SSCPam ₁ ) KKAKFVAAWTLKAAA 5050.02 5050.94 10.56 r45-IRP1-TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (SSCPam ₁ ) KKAKFVAAWTLKAAA 4750.82 4752.07 9.72 r210-IRP1-TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (SSCPam ₁ ) KKAKFVAAWTLKAAA 4982.08 4983.15 9.65 P2-IRP2-TE1 Ac-ATRFKFILRKYTYNYEAESSS-KKK (SSCPam ₂ ) KKAKFVAAWTLKAAA 5292,43 5,292.40 16.89 r45-IRP2-TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (SSCPam ₂ ) KKAKFVAAWTLKAAA 4989,23 4991.07 12/14 r210-IRP2-TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (SSCPam ₂ ) KKAKFVAAWTLKAAA 5220,49 5221.29 11.28 P2-IRP3-TE1 Ac-ATRFKHLRKYTYNYEAESSS-KKK (SSCPam ₃ ) KKAKFVAAWTLKAAA 5530.84 5532.12 23.18 p45-IRP3-PADRE Ac-IEIGLEGKGFEPTLEALFGK-KKK (SSCPam ₃ ) KKAKFVAAWTLKAAA 5227.64 5527.98 20.54 r210-IRP3-TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (SSCPam ₃ ) KKAKFVAAWTLKAAA 5458.90 5460.26 17.86 IRP2-TE2 Ac-KKK (SSCPam ₂ ) KKKLIP-NASLIENCTKAEL 3368.31 3369.78 6.78 P2-IRP2-TE2 Ac-ATRFKHLRKYTYNYEAESSS-KKK (SSCPam ₂ ) KKKLIP-NASLIENCTKAEL 5802.00 5802.61 15.13 r45-IRP2-TE2 Ac-IEIGLEGKGFEPTLEALFGK-KKK (SSCPam2) KKKLIP-NASLIENCTKAEL 5499.81 5502.22 13.36 P210-IRP2-TE2 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (SSCPam ₂ ) KKKLIP-NASLIENCTKAEL 5730.06 5731.36 11.97 IRP2 * -TE3 Ac-RRK (SSCPam ₂ ) RRQYI-KANSKFIGITE 3235.03 3235.88 7.20 P2-IRP2 * -TE3 Ac-TRFKHLRKYTYNYEAESSS-RRK (SSCPam ₂ ) RRQYIKANSKFIGITE 5668.72 5668.70 14.28 r45-IRP2-TE3 Ac-IEIGLEGKGFEPTLEALFGK-RRK (SSCPam ₂ ) RRQYIKANSKFIGITE 5365.52 5365.67 12.26 P210-IRP2 * -TE3 Ac-KTTKQSFDLSVKAQYKKNKH-RRK (SSCPam ₂ ) RRQYIKANSKFIGITE 5596.78 5597.85 10.84

The following notation is used in the table:

TE1 is a universal T-cell epitope of the sequence AK-FVAAWTLKAAA (PADRE); TE2 is the universal T-cell epitope of the KLIPNASLIENCTKAEL sequence from the fusion protein of the dog plague virus; TE3-universal T-cell epitope of the QYIKANSKFIGITE sequence from tetanus toxin;

IRP (1-3) - immunoregulatory peptides of the sequence Pam _n CysSerSer-, characterized by the number of palmitic acid residues, where n = 1-3, flanked by lysine residues; IRP2 * - - immunoregulatory peptide of the sequence Pam ₂ CysSerSer-, flanked by arginine residues;

Ac is acetyl, the residue is acetic acid; K is the lysine residue; I - isoleucine residue; E is the remainder of glutamic acid; D is the remainder of aspartic acid; C is the cysteine residue; T is the threonine residue; Q is the glutamine residue; S is the residue of serine; V is the remainder of valine; Y is the tyrosine residue; P is the remainder of proline; R is the remainder of arginine; L is the leucine residue; F is the residue of phenylalanine; W is the remainder of tryptophan; A is the residue of alanine; G is the residue of glycine; N - asparagine residue: H - histidine residue.

Example 4. Evaluation of the effect of immunization with peptides on atherosclerotic aortic plaques in mice

Obtaining aortic preparations and staining of plaques was performed according to the method (Branen L. et. Al. // The histichemical Journal, 2001, v.33, p.227-229). The aorta, the aortic arch, and the adjacent third of the heart were isolated after careful perfusion with phosphate-buffered saline and then with a 4% buffered solution of para-formaldehyde. Fat from the preparation was carefully removed. Then the aorta was cut lengthwise, opened and placed on a new glass previously coated with ovalbumin, dried in air for 30 minutes and fixed for one day in formalin. A solution of ovalbumin for coating the glasses was prepared by diluting 1:20 a stock solution of 15% filtered chicken egg albumin, 50% glycerol and 0.2% sodium azide. Plaque staining was performed using a solution of the dye of fatty red O in methanol and enclosed under a coverslip.

Plaque areas were measured using computerized histomorphometry. The preparations were photographed using an imaging system including a DMLB light microscope and a DC 300 digital camera (Leica, Germany). Pictures of each field of view were taken at a magnification of 40 times, while about 20 pictures were taken from each preparation to obtain an entire aorta, then they were combined to obtain a photograph of the whole aorta using the Canon Fotostish program on a computer. For morphometric studies, the photographs were calibrated, measured using the Image J. program. Each aorta was examined under a microscope at a magnification of 160-320 times; if in doubt, the borders of the plaques were enlarged at a higher magnification. Plaques encircled each individually. Then, using the Image J program, we measured: the number of plaques located at the sites of vascular discharge, 2) the number of aortic plaques not connected with the vessels, and 3) the area of each plaque in micrometers.

Preparation and staining of sections was performed as described in the article (Fredrikson GN et al. Ingibition of atherosclerosis in ApoE-Null mict by immunization with Apo-100 peptide secuences // Atherosclerosis, Thrombosisand Molecular Biology, 2003, v. 23, p. 879-884). A section of the aortic arch and the adjacent third of the heart were enclosed in OCT (Japan) and frozen in liquid nitrogen. Serial cryostatic sections were prepared with a thickness of 10 μm. Sections began to be collected when aortic valve cusps appeared on the preparations. Slices in the order they were laid out on glass slides. Three to four glasses were prepared from one sample. To visualize plaques, sections were stained with a solution of the dye of fatty red O in isopropanol, stained with Carazzi hematoxylin and placed under a coverslip. Using a visualization system, micrographs of sections were obtained. Plaques encircled each individually. Then, using the Image J program, the area of each plaque was measured in micrometers, and the aortic lumen area was also measured. The area of plaques was taken into account on sections on which three valves were clearly visualized. Thus, in most cases, three sections from the animal were evaluated. On preparations using the Image J program, plaque areas were measured and expressed as a percentage of the aortic lumen area. Sections were stained using the Van Gieson method to evaluate collagen content. The percentage of collagen was evaluated using computerized histomorphometry.

In the statistical processing of data, differences between groups were considered significant at p <0.05.

Studies were conducted on 20 groups of mice, 12 pieces in each group:

Group 1 - control (PBS) Groups 2-19 - received the peptides obtained in example 2 at a dose of 50 mg.

The drug was administered in three stages: at the age of mice 7-8 weeks, at the age of 9-10 weeks and at the age of 11-12 weeks intraperitoneally. For 10-11 weeks, the mice were fed a high cholesterol diet (Western diet). At 24-25 weeks they were killed and aortic examinations were performed.

The results obtained are shown in table 2. The data obtained indicate that the best results were obtained using the peptide P210-IRP ₂ -TE1.

To confirm the effectiveness of the peptide in comparison with its constituent fragments, the antisclerotic activity of the peptide Р210-ИРП ₂ -ТЭ1 was compared with the peptide Р210 and a mixture of peptides Р210 and ИРП ₂ -ТЭ1. The results obtained (table 3) showed that the obtained peptide has increased efficiency compared to its counterparts.

Example 4. Obtaining a composition for the prevention and treatment of atherosclerosis of blood vessels based on a mixture of peptides. 10 mg peptides Р2-ИРП ₂ -ТЭ1, Р45-ИРП ₂ -ТЭ1 and Р210-ИРП ₂ -ТЭ1 were dissolved in 10 ml of 0.02M sodium phosphate buffer, pH 7.4. The resulting solution was poured into 1 ml ampoules, frozen and lyophilized. Using a different weight ratio of peptides in the preparation of the solution for drying, a mixture was obtained (mixture 1) with a ratio of components 1: 1: 1.

Example 5. In the conditions of example 4, mixtures were obtained containing peptides P2-IRP _2- TE1, P45-IRP _2- TE1 and P210-IRP _2- TE1 in a ratio of components

Mixture 2 - 0.25: 0.5: 0.25:

Mixture 3 - 0.5: 0.25-0.25;

Mixture 4 - 0.25: 0.25: 0.5.

Example 6. In the conditions of example 3 was a study of the preparations obtained in examples 4 and 5. The results are shown in tables 4 and 5.

As follows from the data presented, the number and area of “vascular plaques” in group 4 immunized with a mixture of peptides were statistically significantly lower compared to control group 2, which was administered P210 + RPI _2- TE1. The differences between group 4 and groups 1-3 were statistically significant.

Figure 1 shows micrographs of en face descending aorta preparations in Apo E (- / -) mice after staining with oil red O Original magnification of 40 times. A) a group of mice No. 4 (immunization with a mixture of peptides), B) a group of mice No. 1 (control, administration of PBS), C) a group of mice No. 2 (control, introduction of TE1). Micrographs of the preparations show that under the microscope, atherosclerotic plaques were clearly visualized as dark red objects on the inner surface of the aorta. Plaques were characterized by the location in the lumens of blood vessels extending from the aorta ("vascular plaques"). Some plaques turned out to be unconnected by arrangement with vessels (“plaques of the aortic wall”). There is a lower number of plaques (red staining) in group 4 (Fig. 1A) compared with control groups 1 and 2 (Fig. 1B and C, respectively).

Example 7. The study of collagen content in plaques of the aortic sinus

In addition to measuring the area of aortic sinus plaques, it was checked how much immunization with a mixture of peptides will affect the phenotype of plaques, namely, the amount of collagen. The strength of the plaque itself depends on the amount of collagen, and this, in turn, affects the likelihood of its rupture. Plaque destruction, especially in the aorta, is a dangerous complication of atherosclerosis in humans.

The mice were injected with peptide P210 (group 1), P210 + IRP _2- TE1 (group 2), P210-IRP _2- TE1 (group 3) and a mixture of peptides No. 1, As shown by the results of studies (table 6), immunization in groups 2- 4 led to a pronounced increase in the average relative amount of collagen in the plaque. The percentage of collagen in plaques increased on average 1.9 (group 2); 1.63 (group 3) and 1.82 (group 4) times compared with group 1.

The differences between groups 1 and 2, 1 and 3 are significant. In the fourth group, we observed a pronounced tendency to increase the amount of collagen in the plaques; the lack of statistical reliability can be explained by the fact that in this group there were fewer animals.

Table 6. Relative collagen content in aortic sinus plaques in ApoE (- / -) mice (%) Indicator Group one 2 3 four Average 12 22.8 19.6 21.8 St. off 1.17 3,1 2.7 9.7 Number of mice 10 10 eleven 5 T test 1 and 2 1 and 3 1 and 4 R 0.00829 0.01999 0.10315

Analysis of the results showed that the claimed peptides have a higher antisclerotic activity compared to analogues, and compositions based on them are of practical interest as a vaccine for the prevention and treatment of vascular atherosclerosis.

Annex 1 STRUCTURES OF BASIC PEPTIDES 1. (Pam ₂ CysSerSer-):

2. TE1: AKFVAAWTLKAAA 3. P2-IRP2-TE1: Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam2CSS) KX-AKFVAAWTLKAAA 4. R45-IRP2-TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam2CSS) KK-AKFVAAWTLKAAA 5. P210-IRP2-TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam2CSS) KK-AKFVAAWTLKAAA

table 2 The effect of the nature of the peptide on the severity of atherosclerotic lesions of the descending aorta in ApoE - / - mice Cipher Peptide structure Indicator The number of plaque vessels, pcs The area of plaque vessels, μm ² The total number of plaques, pcs Pbs The control 11.1 ± 2.4 2155 ± 408 14.0 ± 2.5 IRP2TE1 Ac-KKK (SSCPam ₂ ) KKAKFVAAWTLKAAA 9.8 ± 1.3 1950 ± 400 12.8 ± 30 P2-IRP1-TE1 Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam ₁ CSS) KKAKFVAAWTLKAAA 9.41 ± 1.8 1950 ± 355 12.9 ± 3.7 R45-IRP1-TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam ₁ CSS) KK AKFVAAWTLKAAA 9.9 ± 1.75 1900 ± 310 12.2 ± 3.1 P210-IRP ₂ -TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam ₁ CSS) KK AKFVAAWTLKAAA 9.95 ± 1.6 1910 ± 305 12.1 ± 3.5 P2-IRP ₂ -TE1 Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam ₂ CSS) KKAKFVAAWTLKAAA 9.87 ± 1.8 1890 ± 299 12.05 ± 3.4 R45-IRP ₂ -TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam ₂ CSS) KKAKFVAAWTLKAAA 9.5 ± 1.5 1800 ± 310 11.8 ± 3.1 P210-IRP ₂ -TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam ₂ CSS) KKAKFVAAWTLKAAA 9.2 ± 1.5 1650 ± 354 11.2 ± 3.2 P2-IRP ₃ -TE1 Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam ₃ CSS) KKAKFVAAWTLKAAA 9.4 ± 1.8 1700 ± 330 11.8 ± 3.1 R45-IRP ₃ -TE1 Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam ₃ CSS) KK AKFVAAWTLKAAA 9.56 ± 1.55 1780 ± 315 12.0 ± 3.5 P210-IRP3-TE1 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam ₃ CSS) KKAKFVAAWTLKAAA 9.6 ± 1.7 1870 ± 330 12.05 ± 3.15 IRP2-TE2 Ac-KKK (Pam ₂ CSS) KKKLIPNASLIENCTKAEL 9.44 ± 1.54 1850 ± 310 12.5 ± 3.1 P2-IRP2-TE2 Ac-ATRFKHLRKYTYNYEAESSS-KKK (Pam ₂ CSS) KKKLIPNASLIENCTKAEL 9.6 ± 1.3 1810 ± 306 12.8 ± 3.0 R45-IRP2-TE2 Ac-IEIGLEGKGFEPTLEALFGK-KKK (Pam ₂ CSS) KKKLIPNASLIENCTKAEL 9.5 ± 1.8 1870 ± 340 12.9 ± 3.5 P210-IRP2-TE2 Ac-KTTKQSFDLSVKAQYKKNKH-KKK (Pam ₂ CSS) KKKLIPNASLIENCTKAEL 9.9 ± 1.4 1855 ± 299 13.2 ± 2.9 IRP2 * -TE3 Ac-RRK (Pam ₂ CSS) RRQYIKANSKFIGITE 9.7 ± 1, 1880 ± 290 13.0 ± 3.5 P2-IRP2 * - Ac-ATRFKHLRKYTYNYEAESSS-RRK (Pam ₂ CSS) RR 9.9 ± 1.8 1830 ± 300 13.5 ± 3.3 r45-IRP2-TE3 Ac-IEIGLEGKGFEPTEEAEFGK-RRK (SSCPam2) RRQYIKANSKFIGITE 9.86 ± 1.7 1850 ± 330 13.8 ± 3.4 P210-IRP2 * -TE3 Ac-KTTKQSFDLSVKAQYKKNKH-RRK (SSCPam ₂ ) RRQYIKANSKFIGITE 10.2 ± 1.6 1900 ± 350 14.2 ± 3.6 The following notation is used in the table: TE1 - universal T-cell epitope of the sequence AKFVAAWTLKAAA (PADRE); TE2 is the universal T-cell epitope of the KLIPNASLIENCTKAEL sequence from the fusion protein of the dog plague virus; TE3 is the universal T-cell epitope of the QYIKANSKFIGITE sequence from tetanus toxin; IRP (1-3) - immunoregulatory peptides of the sequence Pam _n CysSerSer-, characterized by the number of palmitic acid residues, where n = 1-3, flanked by lysine residues; IRP2 * - immunoregulatory peptide of the sequence Pam ₂ CysSerSer- flanked by arginine residues; Ac is acetyl, the residue is acetic acid; K is the lysine residue; I - isoleucine residue; E is the remainder of glutamic acid; D is the remainder of aspartic acid; C is the cysteine residue; T is the remainder of threonine; Q is a glutamine residue; S is the residue of serine; V is the remainder of valine; Y is the tyrosine residue; P is the remainder of proline; R is the remainder of arginine; L is the leucine residue; F is the remainder of fenshalanin; W is the tryptophan residue; A is the residue of alanine; G is the residue of glycine; N is the remainder of asparagine; H is the histidine residue.

Table 3 The effect of prophylactic immunization on the severity of atherosclerotic lesions of the descending aorta in ApoE - / - mice. Indicator Units rev. Group one 2 3 four P210 P210 + IRP2-TE1 P210-IRP2-TE1 W-IRP2-TE1 The number of plaque vessels PC. 11.1 ± 2.4 9.9 ± 2.4 9.0 ± 2.5 9.2 ± 1.5 Area of vascular plaques Μm ² 2155 ± 408 2159 ± 456 1738 ± 642 1650 ± 354 Total plaque count PC. 14.0 ± 2.5 12.6 ± 3.0 12.4 ± 4.4 11.2 ± 3.2

Table 4 The effect of the composition of the drug on the severity of atherosclerotic lesions of the descending aorta in AroE - / - mice. Indicator Units rev. Group R45-IRP2-TE1 P2-IRP2-TE1 P210-IRP2-TE1 Р2-Р2-ИРП2-ТЭ1 Р45-ИРП2-ТЭ1 Р210-ИРП2-ТЭ1 The number of plaque vessels PC. 8.1 ± 2.3 9.7 ± 2.6 9.0 ± 2.5 6.2 ± 1.3 p _m = 0.055 p ₂₄ = 0.0025 Area of vascular plaques Μm ² 1494 ± 483 2120 ± 486 1738 ± 642 P2-3 = 0.116 1190 ± 336 p _{2 · 4} = 0.00 () 9 Total plaque count PC. 10.0 ± 2.6 12.1 ± 3.1 12.4 ± 4.4 7.2 ± 2.6 p _m = 0.081 p ₂₄ = 0.009

Table 5 The effect of the ratio of the ingredients of the drug P2, P2-IRP _2- TE1, P45-IRP _2- TE1, P210-IRP _2- TE1 on the severity of atherosclerotic lesions of the descending aorta in ApoE - / - mice. Indicator Units rev. Mixture mixture one 2 3 four Ratio of ingredients 0.33: 0.33: 0.33 0.25: 0.5: 0.25 0.5: 0.25: 0.25; 0.25: 0.25: 0.5 The number of plaque vessels PC. 6.2 ± 1.3 p _m = 0.055 7.6 ± 1.1 8.0 ± 1.2 7.4 ± 1.1 Area of vascular plaques Μm ² 1190 ± 336 p _{2 · 4} = 0.009 1470 ± 352 1510 ± 315 1333 ± 176 Total plaque count PC. 7.2 ± 2.6 p _m = 0.081 8.4 ± 2.1 7.0 ± 2.4 8.8 ± 2.2

Claims (9)

1. The peptide of the General formula:
X-CC-C (Y) -CC-Z,
where X is the B-cell epitope of the apolipoprotein B 100 protein;
C is an amino acid residue selected from K or R;
Y - immunoadjuvant selected from the group Pam ₁ CSS-; Pam ₂ CSS; Pam ₃ CSS;
Z is a T-helper epitope selected from AKFVAAWTLKAAA, KLIPNASLIENCTKAEL, QYIKANSKFIGITE;
for the prevention and treatment of vascular atherosclerosis, which has an antisclerotic effect. 2. The peptide according to claim 1, characterized in that as the B-cell epitope of the protein Apolipoprotein B 100 it contains the peptide p2 of Apolipoprotein B 100. 3. The peptide according to claim 1, characterized in that as a b-cell epitope of the protein apolipoprotein B 100 it contains the peptide p45 of apolipoprotein B 100. 4. The peptide according to claim 1, characterized in that as a B-cell epitope of the protein Apolipoprotein B 100 it contains the peptide p210 Apolipoprotein B 100. 5. The peptide according to claim 1 p2-IRP1-PADRE having the general formula Ac-ATRFKHLRKYTYNYEAESSS-KKK (SSCPam ₁ ) KK-AKFVAAWTLKAAA. 6. The peptide according to claim 1 p45-IRP1-PADRE having the general formula Ac-IEIGLEGKGFEPTLEALFGK-KKK (SSCPam ₁ ) KK-AKFVAAWTLKAAA. 7. The peptide according to claim 1 p210-IRP1-PADRE having the general formula Ac-KTTKQSFDLSVKAQYKKNKH-KKK (SSCPam ₁ ) KK-AKFVAAWTLKAAA. 8. Composition for the prevention and treatment of vascular atherosclerosis based on the B-cell epitope of apolipoprotein B 100 protein containing a mixture of peptides according to claims 5, 6 and 7 in the ratio of 0.25-0.5: 0.25-0.5: 0 , 25-0.5. 9. The composition according to claim 8, characterized in that the ratio of peptides according to claims 5, 6 and 7 is 1: 1: 1.

Images