RU2716819C1 - Protein-peptide complex and drug preparation agent, which is protein-peptide complex - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: invention refers to biotechnology and medicine, namely to a protein-peptide complex and a pharmacological composition for preventing and treating retinal diseases of the human eye and farm animals. Protein-peptide complex is obtained by reacting equimolecular amounts of a mixture of regulatory peptides with molecular weights of 1,000–6,000 Da or a regulatory peptide with a molecular weight of 4372 Da and serum albumin with molecular weights of 66,362–66,898 Da, recovered from pigmented epithelium of the eyes of vertebrates. Pharmacological composition contains a protein-peptide complex and a pharmaceutically acceptable carrier.

EFFECT: protein-peptide complex described above and a pharmacological composition containing a protein-peptide complex have a protective effect on the state of tissues of the posterior sector of the eye - pigment epithelium, neural retina, sclera, choroid, which is manifested in maintaining the adhesion, polypheraphy and differentiation status of pigment cells, as well as in maintaining the viability of bipolar cells and Muller glia in the retina.

2 cl, 6 dwg, 4 tbl, 16 ex

Description

The invention relates to biotechnology and medicine, and specifically to a biologically active protein-peptide complex (hereinafter - BOD), formed by the interaction of a protein and a peptide isolated from the tissue of the pigment epithelium (hereinafter - PE) of the vertebrate eye, which can be used as a drug substance a drug for the prevention and treatment of retinal eye diseases in humans and farm animals.

Despite the successes of modern ophthalmology in the treatment of common eye diseases, their frequency currently continues to increase. This is due to the widespread use of television and computer equipment, which leads to a significant increase in the load on the visual apparatus, especially on the retina and retinal PE - the main tissues of the eye involved in the formation of the visual signal. Currently, in the practice of ophthalmology, drugs that contribute to the restoration of PE in violation of its morphofunctional state are absent. Therefore, the aim of the present invention was a biologically active substance - BOD, which has a tissue-specific, but not species-specific protective effect on the retina and PE of the eyes of vertebrates.

The invention is known according to the patent of the Russian Federation No. 2073518 - a means of restoring the function of the retina of the eye MKI ⁶ A61K 35/44, A61F 9/00, priority from 06/17/93. The present invention describes an agent that is a complex of low molecular weight polypeptides isolated from mammalian retina by extraction with a 3% solution of acetic acid with the addition of zinc chloride and precipitation of the supernatant protein with acetone. The tool contributed to the restoration of the function of the retina of the eye. The disadvantages of this invention is the use of a mixture of peptides with different molecular weights, the lack of identification among them of biologically active peptides exhibiting regenerative activity.

The known method [RU patent of the Russian Federation No. 2373904 (2009)] for the treatment of various pathologies of the posterior segment of the eye with peptide bioregulators retinalamine and cortexin, which are polypeptide fractions of the retina of livestock. The disadvantage of the invention is the intramuscular use of solutions of bioregulators in high concentration: 0.1% retinalamine solution and 0.2% cortexin solution. The possibility of developing allergic reactions with the use of retinalamin and age restrictions up to 18 years. If cortexin is also used, individual hypersensitivity to the components of the drug is possible.

Known tetrapeptide that stimulates the function of the retina of the eye, a pharmacological agent based on it and the method of its use [RU patent of the Russian Federation No. 2161982 (2001)]. The disadvantage of this invention is the injection into the eye area, which can cause traumatic injuries, the drug is also used in the form of a high concentration solution, due to which adverse reactions from the body, including the development of allergic reactions, are possible.

There is also known a method for producing a biologically active complex and a biologically active protein-peptide complex (patent RU2428196), which has a neuroregenerative and neuroprotective effect and stimulates the physiological and reparative regeneration of nervous tissue, for use in the manufacture of drugs for diseases of the central and peripheral nervous system and the method for its preparation . This method allows to obtain a mixture of proteins and polypeptides from the brain of embryos of ungulates. The disadvantages of this invention are the incomplete purification of the final substance, which is a protein-peptide complex, as well as the use of embryonic material, which may contain other biologically active substances that can cause adverse reactions in the tissues of an adult body. The disadvantages of this invention include obtaining only from the brain of ungulates of ungulates of animals a protein-peptide complex that does not affect the state of the eye tissues, including pigment epithelium.

From RU 2311915, December 10, 2007, a method is known for producing a medicament for treating diseases of the retina and PE of the eye, as well as the choroid, as a result of which a fraction containing regulatory peptides with a molecular weight of less than 8 Da is isolated from pigment epithelium of the vertebral animals, which exhibits activity in doses corresponding to concentrations of 10 ^-10 - 10 ^-12 mg / ml (in physiological saline-water solution). The invention provides a preparation containing regulatory peptides with a small molecular weight, which at low doses have a protective effect on the state of the tissues of the posterior retina. However, the use of the obtained peptide-containing preparation is not sufficiently effective for the treatment of eye diseases associated with impaired retinal function and PE.

From the article by V.P. Yamskova et al. “Structural and functional features of a new bioregulator isolated from pigment epithelium tissue of the bull’s eye” (Biochemistry, 2009, vol. 74, issue 9, pp. 1195-1203), a bioregulator operating in ultra-low doses of 10 ^-17 mg protein. The functional basis of this bioregulator is a regulatory peptide with a molecular mass of 4372 Da and a mixture of proteins with a molecular mass of 14980-66283 Da), which modulates the activity of the regulatory peptide. It was noted that the regulatory peptide is responsible for the manifestation of membrane-active activity by the bioregulator, and the protein-containing fraction is responsible for its biological effect in ultra-low doses.

The technical problem solved by the present invention is to create BOD by interacting with highly purified preparations of the regulatory peptide and a protein isolated from intact, containing the largest number of physiologically active proteins and peptides of the regions of the retinal PE of the vertebrate eye, which can be used as a substance of a new drug for treatment of ocular retinal diseases.

The technical result of the claimed invention is to obtain a biologically active complex by the interaction of a peptide and a protein isolated from PE tissue of the eye of vertebrate animals, which has a protective effect on the state of the tissues of the posterior sector of the eye - PE, neural retina; sclera, a choroid, which is expressed in maintaining the status of adhesion, polyperation and differentiation of pigment cells, as well as in maintaining the viability of bipolar cells and Mueller glia in the retina.

The stated technical problem and the technical result are achieved by a group of inventions, which includes a protein-peptide complex obtained by the interaction of equimolecular amounts of vertebrate animals extracted from tissue PE (pigment epithelium) of a mixture of regulatory peptides with molecular weights of 1000-6000 Da or a regulatory peptide with a molecular weight of 4372 Yes, and protein - serum albumin with molecular weights of 66362-66898 Yes.

The stated technical problem and the technical result are also achieved by the pharmacological composition for the prevention and treatment of retinal diseases of the human eye and farm animals, including the protein-peptide complex described in the previous paragraph, and a pharmaceutically acceptable carrier and exhibiting biological activity in doses corresponding to 10 ^{-3-10 -19} mg protein / ml.

In particular, the indicated protein-peptide complex was obtained by the interaction of equimolecular amounts of the regulatory peptide of the vertebrate eye pigment epithelium (PE) tissue, the regulatory peptide, which is an N-terminal fragment of phosphodiesterase of cyclic guanazine monophosphate with a molecular weight of 4372 Da, and bovine serum albumin g6060602 molecular weight 66367 Yes.

The resulting complex has a pronounced protective effect in doses corresponding to 10 ^-3 - 10 ^-19 mg of protein / ml on the state of the tissues of the posterior sector of the eye - PE, neural retina, sclera, choroid.

In general terms, the method for isolation and purification of BOD according to the invention consists in isolating the macular region of retinal PE from freshly enucleated eyes of a bull or other vertebrate animal, washing it in physiological saline, keeping the PE tissue in aqueous saline, centrifuging the tissue extract, and collecting the supernatant liquid, precipitated proteins in a 100% solution of ammonium sulfate, centrifuged to collect the fraction floating at 100,000 g - 105,000 g, dialyzed until complete removal of ammonium ions, separated by highly efficient by liquid chromatography in a gradient of acetonitrile in 0.1% trifluoroacetic acid at a pH of 2.2-3.0, a temperature of 22-25 ° C for 30-60 minutes, fractions of hydrophilic peptides and protein, a member of the serum albumin family, are collected, from which acetonitrile is removed and mixed in the presence of 10 ^-3 M CaCl ₂ in equimolecular amounts.

The invention provides a highly purified BOD having activity in doses corresponding to 10 ⁻³ to 10 ⁻¹⁹ mg of protein / ml.

Another object of the claimed invention is a pharmacological composition for the preparation of a medicament for the prevention and treatment of retinal diseases of the human eye and farm animals, comprising a protein-peptide complex and a pharmaceutically acceptable carrier.

This protein-peptide complex (hereinafter - BOD) has a protective effect on the state of the tissues of the posterior sector of the eye - PE, neural retina, sclera, choroid, which is expressed in maintaining the status of adhesion, proliferation and differentiation of PE cells, as well as in maintaining the viability of bipolar cells and Müller glia in the retina.

The invention is illustrated by the following examples, which describe a method for producing a protein-peptide complex (example 1) and determination of specific activity (examples 2-13), as well as FIG. 1-6.

FIG. 1. A histological section of the posterior part of the eye of the Pleurodeles waltl newt during organotypic cultivation of tissues of the neural retina, pigment epithelium, choroid, sclera in vitro on the 3rd day (control series).

Oh. 10 × approx. ten;

Bean. 10 × approx. 20.

With this type of cultivation, gradual degradation of all tissues of the eye occurs: there is a violation of adhesion between the retina and PE, a significant number of apoptotic cells are observed in the retina, as well as a change in the state of the pigment epithelium cells, their differentiation status, which is estimated by the pigment displacement on the apical side of the monolayer cells, violation of adhesion between them and the death of the cells themselves. In the sclera, cavities are observed that have arisen as a result of the degradation of collagen fibers.

FIG. 2. Detection of specific proteins and the state of cells on histological sections of the posterior part of the eye of the Pleurodeles waltl newt during organotypic cultivation of tissues of the neural retina, PE, choroid, sclera in vitro on the 3rd day (control series).

A - immunohistochemical staining for retina photoreceptor protein reverin vol. × 20 approx. × 10;

B - immunohistochemical staining for protein of Mueller glia cells in the retina GFAP vol. × 10 approx. × 10;

B - histochemical staining for the retinal enzyme acetylcholinesterase vol. × 20 approx. × 10;

G - normal and apoptotic nuclei of bipolar cells in the retina about. × 100, approx. × 10.

FIG. 3. Histological section of the posterior part of the eye of the Triton Pleurodeles waltl during organotypic cultivation of tissues of the neural retina, PE, choroid, sclera in vitro on the 3rd day in the presence of a polypeptide with a molecular weight of 4372 Da, representing N-, isolated from tissue extract of pigment epithelium of the bull’s eye terminal fragment of phosphodiesterase of cyclic guanosine monophosphate (concentration of 10 ^-7 mg protein / ml).

Oh. 10 × approx. ten;

Bean. 10 × approx. 20.

FIG. 4. Histological section of the posterior part of the eye of the Triton Pleurodeles waltl during organotypic cultivation of tissues of the neural retina, PE, choroid, sclera in vitro on the 3rd day in the presence of serum albumin (gi | 367460260) with a molecular weight of 66367 Yes isolated from PE tissue extract of the eye , (concentration of 10 ^-7 mg protein / ml). The complete lack of protective action of this fraction. The condition of all tissues of the eye of the posterior part of the eye does not differ from that in the control series.

Oh. 10 × approx. ten;

Bean. 10 × approx. 20.

FIG. 5. Histological section of the posterior part of the eye of the Triton Pleurodeles waltl during organotypic cultivation of tissues of the neural retina, PE, choroid, sclera in vitro on the 3rd day in the presence of a restored protein-peptide complex (concentration of 10 ^-17 mg protein / ml). In this case, there is the greatest safety of the tissues of the posterior part of the eye. Between the processes of photoreceptors, a monolayer of PE and choroid dense adhesive interactions are noted. Unlike other experimental series, a small amount of apoptotic cells was observed in the retina. Stabilization of the status of differentiation of pigment epithelial cells occurs, which is expressed in the uniform distribution of pigment in these cells and in maintaining a holistic monolayer structure. The condition of the sclera tissue is close to the state of intact tissue: the compact structure of collagen fibers is preserved.

Oh. 10 × approx. ten;

Bean. 10 × approx. 20.

FIG. 6. Histological section of the posterior part of the eye of the Triton Pleurodeles waltl during organotypic cultivation of tissues of the neural retina, PE, choroid, sclera in vitro on the 3rd day in the presence of a restored protein-peptide complex (concentration of ^10-7 mg protein / ml).

Oh. 10 × approx. ten;

Bean. 10 × approx. 20.

The protective effect of the reduced protein-peptide complex in the indicated concentration is expressed in maintaining adhesion between the retina, PE and choroid over an extended section of the posterior part of the eye with minor disturbances in some areas. Most of the retinal cells, as in the control, are in an apoptotic state. It should be noted that adhesive interactions were maintained in the monolayer of PE cells. Pigment granules were distributed evenly in the cytoplasm of cells, which indicates a stable state of differentiation of these cells under these conditions compared with the control. The condition of the scleral membrane did not differ from that in the control series.

Example 1. Isolation from the tissue of the pigment epithelium of the eye of a bull of Bos taurus protein-peptide complex, the determination of its composition and structure

From the freshly enucleated bull’s eyes, the macular zone of the PE retina is microsurgically isolated at 20 ° C, washed and extracted in aqueous saline saline: 1.5 × 10 ^-1 M NaCl, 1.0 × 10 ^-3 M CaCl ₂ , 5 × 10 ^{- 3} M KCI, 1 × 10 ^-3 M HEPES, 4-8 ° C, 3-4 hours. After centrifugation at 3000 g for 30 minutes, dry ammonium sulfate was added to the resulting tissue extract with constant stirring to obtain a saturated solution (780 g / l), the resulting protein suspension is left at 4-8 ° C for 120 hours, then centrifuged at 105,000 g for 60 minutes. A supernatant, a precipitate and a float fraction are obtained, which are further dialysed against water (with a volume ratio of the dialyzed fraction and the dialysis fluid 1: 100) at 4-8 ° C. until ammonium ions are completely removed. The presence of ammonium ions in the solution is determined by reaction with the Nessler reagent. The supernatant fraction was concentrated using a rotary vacuum evaporator at a temperature below 40 ° C, then it was separated using the isoelectric focusing method (IEF) in the sucrose density gradient on a LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10 0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution — 60% sucrose in 0.1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of the supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The column is layered with solutions in the following order: heavy electrode solution, heavy and light gradient solutions, which are supplied from a gradient mixer, and then a light electrode solution. IEF is carried out at + 4 ° C for 96 hours at a voltage of 500-1500 V. Collect fractions with a volume of 5-10 ml. Protein detection is carried out on any spectrophotometer at 280 nm, the pH value is determined for each fraction using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:10 with a five-fold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix; a number of peptides with molecular weights in the range of 1000-6000 Da are detected. Electrophoresis of the IEF fraction collected in the range of pH <3.0 in a 15% polyacrylamide gel under denaturing conditions is carried out. A Coomassie-stainable band corresponding to a molecular weight of approximately 66,000 Da was isolated from the gel. The performed tryptic hydrolysis of the protein in a gel followed by a mass spectrometric study of the hydrolysis products shows that the isolated protein with a molecular weight of 66367 Da is a protein of the mammalian albumin family (under the number gi | 367460260 in the SwissProt database). The partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE-), which has a 100% homology with the N-terminal sequence of a mature bovine serum albumin molecule, was established by Edman's method.

Thus, the IEF fraction collected in the pH range <3.0 is a BOD, consisting of protein gi | 367460260 - a representative of the bovine blood serum albumin family with a molecular weight of 66367 Da, pl in the pH range of 3.90-4, 00 and mixtures of small peptides with a molecular weight of 1000-6000 Da.

Example 2. The formation of the protein-peptide complex by the interaction of the peptide with a molecular weight of 4372 Da and albumin (qi | 367460260) with a molecular weight of 66367 Da, isolated from the tissue of the pigment epithelium of the eye of a bull B. taurus

From the freshly enucleated bull’s eyes, the macular zone of the retinal pigment epithelium is microsurgically isolated at 20 ° C, washed and extracted in physiological saline: 1.5 × 10 ^-1 M NaCl, 1.0 × 10 ^-3 M CaCl ₂ , 5 × 10 ^-3 M KCI, 1 × 10 ^-3 M HEPES, 4-8 ° C, 3-4 hours. After centrifugation at 3000 g for 30 minutes, dry ammonium sulfate is added to the resulting tissue extract with constant stirring until a saturated solution is obtained (780 g / l), the resulting protein suspension is left at 4-8 ° C for 120 hours, then centrifuged at 105,000 g for 60 minutes. A supernatant, a precipitate, and a float fraction are obtained, which are further dialysed against water at 4-8 ° C. until ammonium ions are completely removed.

The desalted floating fraction was further separated by reverse-phase HPLC on a hydrophobic column Biochemak C ₈ (Russia) (4.6 × 250 mm) using an Agilent 1200 high-pressure chromatograph (USA). Elution is carried out in a concentration gradient of acetonitrile (5-80%) in a 0.1% aqueous solution of trifluoroacetic acid (pH 2.2) at a rate of 0.5 ml / min for 30 min at 25 ° C. Detection is carried out at 214 nm and 280 nm. A number of fractions are obtained with a retention time of 3-5 minutes and 14-17 minutes. HPLC fractions with a retention time of 3-5 minutes were analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix. A fraction is isolated which is characterized by a mass spectrometric signal corresponding to a molecular weight of 4372 Da. A tryptic analysis of protein in solution is carried out, followed by a mass spectrometric study of the hydrolysis products and show that the polypeptide with a molecular mass of 4372 Da has the following amino acid sequence:

Ac-MNLEPPKAEIRSATRVMGGPVTPRKGPPKFKQRQTRQF.

Analysis of this sequence shows that this is the N-terminal fragment of cyclic GMF phosphodiesterase from the retina of cattle [Ovchinnikov Yu.A., Lipkin V.M., Kumarev V.P., Gubanov V.V., Khramtsov N.V., Akhmedov N.B., Zagranichny V.E., Muradov K.G. FEBS Letters, 1986, 204, 288-292.].

The HPLC fraction with a retention time of 14-17 minutes was examined in a 15% polyacrylamide gel under denaturing conditions. A fraction is obtained which is characterized by a Coomassie-dye band corresponding to a molecular weight of approximately 66,000 Da. The carried out tryptic analysis of the protein of this fraction followed by mass spectrometric analysis of the hydrolysis products is carried out mass spectrometrically using an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as the matrix. The results obtained show that this the fraction is bovine serum albumin (gi | 367460260 in the SwissProt database) with a molecular weight of 66367 Da. The partial N-terminal amino acid sequence of this protein (DTHKSEIAHRFKDLGE-), which has 100% homology with the N-terminal sequence of the mature bovine serum albumin molecule, is determined by the Edman method. Acetonitrile and trifluoroacetic acid are removed from the solution of the HPLC fractions of the peptide and albumin and dissolved in physiological saline.

Example 3. Obtaining a protein-peptide complex by the interaction of a peptide with a molecular weight of 4372 Da and serum albumin (qi | 367460260) with a molecular weight of 66898 Yes of its solutions in various concentrations isolated from pigment epithelium of a bull’s eye

An HPLC fraction (retention time 3-5 min) containing an N-terminal cyclic GMF phosphodiesterase fragment with a protein concentration of 90 μg / ml and an HPLC fraction (retention time 14-17 min) containing bovine serum albumin gi | 367460260 with a concentration protein 200 μg / ml, mixed in a ratio of 1:10 in the presence of 10 ^-3 M CaCl ₂ . Thus, BOD is obtained, which is used as a biologically active substance exhibiting morphofunctional activity in relation to the pigment epithelium and retina.

Example 4. The selection of the protein-peptide complex from the tissue of the pigment epithelium of the mouse eye, the determination of its composition and structure

The experiment was performed on C57BI / 6 mice of both sexes, weighing 18-22 g.

PE is extracted microsurgically from freshly enucleated eyes of mice, tissue is washed in physiological saline (1.5 × 10 ^-1 M NaCl; 1.0 × 10 ^-3 M CaCl ₂ ; 5 × 10 ^-3 M KCI; 1 × 10 ^{- 3} M HEPES) and then extracted in a fresh solution at 4-5 ° C for 5 hours. The resulting tissue extract was centrifuged (3000 g, 30 min), dry ammonium sulfate was gradually added with stirring until a saturated solution was formed (780 g / l) , add sodium azide (10 μg / l) and leave the suspension of proteins for 72 hours at 4-5 ° C. By centrifugation (10000 g, 4-5 ° C, 30 min), the supernatant fraction is collected, dialyzed against water (with a volume ratio of dialyzed fraction and dialysis fluid of 1: 100). The presence of ammonium ions in the solution is determined by reaction with the Nessler reagent. The supernatant fraction was concentrated using a rotary vacuum evaporator at a temperature below 40 ° C, then it was separated using the isoelectric focusing method (IEF) in the sucrose density gradient on a LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10 0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution — 60% sucrose in 0.1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of the supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The column is layered with solutions in the following order: heavy electrode solution, heavy and light gradient solutions, which are supplied from a gradient mixer, and then a light electrode solution. IEF is carried out at + 4 ° C for 96 hours at a voltage of 500-1500 V. Collect fractions with a volume of 5 ml. Protein detection is carried out on any spectrophotometer at 280 nm, the pH value is determined for each fraction using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:10 with a five-fold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range of 1000-6000 Da, as well as protein are detected with a molecular weight of 66472 Yes. Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the range of pH <3.0. A strip corresponding to a molecular weight of approximately 66,000 Da is cut out from the gel. The performed tryptic hydrolysis of the protein in a gel followed by a mass spectrometric study of the hydrolysis products shows that the isolated protein with a molecular weight of 66472 Da is a protein of the mouse albumin family (under qi | 163310765 in the SwissProt database). Thus, the IEF fraction collected in the pH range <3.0 is a BOD consisting of mouse serum albumin with a pi value in the range of pH 4.0-4.1 and a mixture of small peptides with a molecular weight of 1000-6000 Yes .

Example 5. Isolation of the protein-peptide complex from the tissue of pigment epithelium of the chicken eye

The experiment is carried out on chickens of the Leggorn breed of both sexes, weighing 400-500 g.

From freshly enucleated eyes of chickens, pigment epithelial tissue is microsurgically isolated, washed in saline (1.5 × 10 ^-1 M NaCl; 1.0 × 10 ^-3 M CaCl ₂ ; 5 × 10 ^-3 M KCI; 1 × 10 ^-3 M HEPES) and then extracted in a fresh solution at 4-5 ° C for 5 hours. The resulting tissue extract is centrifuged (3000 g, 30 min), dry ammonium sulfate is gradually added with stirring until a saturated solution is formed (780 g / l ), add sodium azide (10 μg / L) and leave the suspension of proteins for 72 hours at 4-5 ° C. By centrifugation (10000 g, 4-5 ° C, 30 min), the supernatant fraction is collected, dialyzed against water (with a volume ratio of dialyzed fraction and dialysis fluid of 1: 100). The presence of ammonium ions in the solution is determined by reaction with the Nessler reagent. The supernatant fraction was concentrated using a rotary vacuum evaporator at a temperature below 40 ° C, then it was separated using the isoelectric focusing method (IEF) in the sucrose density gradient on a LKB-440 column (LKB, Sweden) using ampholines in the pH range 3.5–10 0 (Serva, Germany) (Osterman, 1983). The following solutions are prepared for separation: heavy electrode solution — 60% sucrose in 0.1% solution of H ₃ PO ₄ ; light electrode solution - 0.1% NaOH; heavy gradient solution - 75 g of sucrose, 100 ml of the supernatant, adjusted to 200 ml with distilled water; light gradient solution - 15 g of sucrose, up to 200 ml of distilled water. The column is layered with solutions in the following order: heavy electrode solution, heavy and light gradient solutions, which are supplied from a gradient mixer, and then a light electrode solution. IEF is carried out at + 4 ° C for 96 hours at a voltage of 500-1500 V. Collect fractions with a volume of 5 ml. Protein detection is carried out on any spectrophotometer at 280 nm, the pH value is determined for each fraction using a pH meter. According to the obtained separation pattern, a fraction is collected in the range of pH <3.0, which is then dialyzed against distilled water until sucrose is removed in a volume ratio of 1:10 with a five-fold change of water. This fraction is analyzed by mass spectrometry on an UltraFlex 2 time-of-flight mass analyzer (Bruker Daltonic, Germany) using α-cyano-4-hydroxycinnamic acid as a matrix, a number of peptides with molecular weights in the range 800-5000 Da, as well as protein are detected with a molecular weight of 67155 Yes. Electrophoresis is carried out in a 15% polyacrylamide gel under denaturing conditions of the IEF fraction collected in the pH range <3.0. A strip corresponding to a molecular weight of approximately 68,000 Da is cut out from the gel. The performed tryptic hydrolysis of the protein in a gel followed by mass spectrometric analysis of the hydrolysis products shows that the isolated protein is a protein of the chicken albumin family (under the number gi | 766944282 in the SwissProt database). Thus, the IEF fraction collected in the pH range <3.0 is a BOD consisting of chicken serum albumin with a pi in the pH range of 3.9-4.0 and a mixture of small peptides with a molecular weight of 800-5000 Yes .

Example 6. Obtaining solutions of BOD in various concentrations of its solutions in various concentrations

Two preparations are used as the initial solution of BOD found in the tissue of the pigment epithelium of the bull’s eye: BOD1 — IEF fraction collected in the region of pH <3.0 (Example 1) with a protein concentration of 100 ± 10 μg / ml and BOD2 formed the interaction of equimolecular amounts of a regulatory peptide with a molecular weight of 4372 Da and protein (gi | 367460260) with mol. mass 66367 Yes, in the presence of ions 10^-3 M CaCl₂ (Example 2), in the form of a solution with a protein concentration of 100 ± 10 μg / ml.

In a vial containing 0.1 ml of BOD solution, 0.9 ml of distilled water is added and vigorously shaken 15 times. 0.1 ml of the resulting solution was taken into a vial, 0.9 ml of distilled water was added, and shaken 15 times. This procedure is repeated, thus obtaining solutions of BOD.

Similarly receive solutions of BOD with 10-fold sequential dilution from 1 to 20 degrees in physiological saline with a composition of 0.15 M NaCl; 10 ^-3 M CaCl ₂ . In experiments on the study of specific activity on the model of culturing eye tissues in the posterior sector of the eye, solutions with concentrations corresponding to 10 ^{-3 were} studied; 10 ^-7 - 10 ^-17 ; 10 ^-19 mg protein / ml.

Example 7. The effect of solutions of the protein-peptide complex found in pigment epithelium of the bull’s eye, in various concentrations, as well as its individual components — the regulatory peptide and the modulator protein, on the state of the eye tissue of the posterior part of the Pleurdeles waltl newt eye. with organotypic in vitro cultivation

The effect of BOD, found in the pigment epithelium tissue of the bull’s eye in various doses, as well as its individual components, on the state of the retina, PE, and sclera, was studied on the model of organotypic cultivation of the posterior sector of the newt eye in vitro, since the activity of BOD is characterized by the presence of tissue, but not specific specificity.

The study was conducted on adult sexually mature individuals of the Pleurodeles waltl newt contained in the aquarium Institute of Development Biology. N.K. Koltsova RAS.

Microsurgically excrete eyes from pre-anesthetized in 0.1% solution of MS-222, and cultivate in medium containing 350 ml of 199 medium, 150 ml of bidistilled water, 0.15 ml of 1M Hepes and 1 ml of 4% sulfate solution gentamicin. Before cultivation, cold sterilization of the medium is carried out through GV membrane filters with a pore diameter of 0.22 μm (Millipore, USA). Triton eye tissue is isolated with a binocular. First, the eye is exempted from skin and fat, and then cut along the circumference proximal to the limb. The growth region of the retina is discarded along with the iris, cornea and lens. For subsequent cultivation, the posterior sectors of the eyes are used, each of which includes the retina, PE, choroid and sclera. The obtained posterior parts of the eyes are placed in penicillin vials filled with serum-free medium on A / E type cellulose filters (Gelman, USA), washed in 70% ethanol and rinsed in culture medium. Before the cultivation, the protein-peptide complex purified after reverse-phase HPLC in various concentrations is added to the bottles of the experimental series. In the control series, the appropriate amount of saline is added to the culture medium. All vials are closed with sterile caps, Parafilm M film, aluminum foil and cultured without changing the culture medium in an incubator in the dark at 20-22 ° C for 72 hours.

Cultures of the posterior sector of the eye are immersed in a 4% formalin solution prepared on phosphate buffer (0.1 M PBS, pH 7.4) and fixed for 6 hours. In addition to formalin fixation, 96% ethanol supercooled in liquid nitrogen is used. After fixation, the material is washed in 0.1 M PBS 2 times for 24 hours at 4 ° C, dehydrated and embedded in paraffin. For research by the method of indirect immunostaining (Engelhardt, 1968), sections with a thickness of 6-7 microns are prepared. Before incubation with antibodies, the sections were freed from paraffin, dehydrated and washed in cold 0.1 M PBS buffer for 30 minutes. To study photoreceptors, recombinant polyclonal monospecific anti-Reverin photoreceptor protein antibodies are diluted 1:20 (final concentration 0.011 mg / ml) in PBS containing X-100 Triton (0.3%) and bovine serum albumin (0.5%). To study retinal glial cells, commercial polyclonal antibodies against acidic glial fibrillar protein (GFAP, Sigma, USA) at a 1:50 dilution are used.

After incubation with the first antibodies, the sections are washed three times for 5 min in 0.1 M PBS buffer, and then treated with the second, conjugated with peroxidase (IgG, complete molecule, Sigma, USA), diluted anti-rabbit (for reverin and GFAP) antibodies 1:50. Sections of cultured fragments, as well as sections of the normal eyes of the newt, are used in antibody staining reactions. In addition, sections stained with only second antibodies are used for control. Immunoreaction studies and imaging are performed using an Olympus microscope (VANOX, ANVT3, Japan), an Olympus digital camera (U-PMTVC, Japan) and a computer console equipped with programs (ViewfinderLite, StudioLite).

Analysis of control preparations in all cases shows the absence of specific staining. Explants are described on a series of conventional and semi-thin sections. To prepare a series of conventional sections, explants of the posterior wall of the eye containing the retina, immediately after cultivation, are placed in the Buena fixative. Paraffin sections 7 μm thick are stained with hematoxylin-eosin. To prepare semi-thin sections of explants, the method is used (Grigoryan E.N., Ivanov I.P., Poplinskaya V.A. Izv. RAS. Ser. Biol., 1996, 3, 319). Series of semi-thin sections are made as strictly as possible along the long axes of the outer segments of the photoreceptors, glued to glass, stained with toluidine blue and analyzed with a 1000x magnification of the Jenaval microscope (Carl Zeiss, JENA, Germany). For morphological studies using fragments of the eyes of the newt, cultivated only in serum-free medium (control) and in the same medium, but with the addition of fractions isolated from PE of the bull’s eye.

To determine the viability of the cells of the cultured retina, processed and untreated by the studied fractions, direct counting of viable cells and the total number of cells in semi-thin sections is used. Clear morphological criteria are used: the shape and density of the staining of the bodies of bipolar cells. On a series of semi-thin sections using an eyepiece ruler, select equal sections of the layer. And they determine the ratio of unchanged, morphologically normal cells to the total number of cells and conduct statistical processing of the data obtained using the Origin 6.0 Professional program. Muller glial cell counts were also determined after staining with anti-GFAP antibodies on 7 μm sections of explants. In the calculations, the approach described above and the ocular mesh are used. Since the bodies of Müller cells are difficult to distinguish on sections of 7 μm, we consider the number of their stained long fibers, clearly visible even when the main orientation of the section is shifted. The number of these processes is additionally estimated on planar sections of the retina. In quantitative studies, posterior fragments of the eyes of newts, cultured only in serum-free medium and in the same medium, but with the addition of fractions isolated from PE of the bull’s eye, are used. For histochemical staining, cryosections 14 μm thick are used. Cultured retinal explants are fixed in supercooled alcohol. The cultivation of newt eye tissue in serum-free medium leads to a gradual degradation of all eye tissues (Fig. 1). A significant number of apoptotic cells are present in the retina, there is a violation of adhesion between the retina and PE, and a change in the state of PE cells is observed: the status of differentiation of PE cells, which is estimated by the shift of the pigment to the apical side of the monolayer cells, the violation of adhesion between them and the death of the cells themselves. In the sclera, cavities that arise as a result of the degradation of collagen fibers are observed. However, with this type of cultivation as part of the posterior wall of the eye, the retinal cells demonstrate both viability and expression of specific proteins of glial cells and neurons (glial acidic fibrillar protein), photoreceptor protein - reverin and the enzyme of the cholinergic transmission modulator - acetylcholinesterase (Fig. 2A-D )

The cultivation of the posterior part of the eye of the newt in a medium containing a fraction of the peptide mol. mass of 4372 Yes in doses corresponding to 10 ^-7 ; 10 ^-9 ; ^10-11 ; 10 ^-13 ; ^10-15 ; ^10-17 mg of protein does not lead to a change in the state of the tissues of the eye of the newt compared with the culture of the posterior sector of the eye, incubated in serum-free medium (Fig. 3).

The cultivation of the posterior part of the eye of the newt in a medium containing a protein fraction with a molecular weight of 66367 Da in doses corresponding to 10 ^-7 ; 10 ^-9 ; ^10-11 ; 10 ^-13 ; ^10-15 ; ^10-17 mg of protein does not lead to a change in the state of the tissues of the eye of the newt compared with the culture of the posterior sector, incubated in serum-free medium (Fig. 4).

A complex formed by the interaction of equimolecular amounts of the peptide with a mol. weight of 4372 Yes and protein with a molecular weight of 66367 Yes in the presence of ions of 10 ^-3 M CaCl ₂ (Example 2). The addition of BOD to the nutrient medium at a dose corresponding to ^10-17 mg of protein during organotypic cultivation of the tissues of the posterior part of the eye of the newt in vitro helps to maintain adhesion, the status of proliferation and differentiation of cells, and ensures the highest preservation of eye tissue (Fig. 5). Between the processes of photoreceptors, a monolayer of PE and the choroid, dense adhesive interactions are noted, a small number of apoptotic cells are observed. The differentiation status of PE cells is stabilized, which is expressed in the uniform distribution of pigment in these cells and in maintaining the integral structure of the monolayer. The condition of the scleral tissue is close to the state of intact tissue: the conservation of the compact structure of collagen fibers, as well as viable fibroblasts are observed.

The BOD solution is added to the nutrient medium at a dose corresponding to 10 ^-7 mg of protein during organotypic cultivation of the tissues of the posterior sector of the eye in vitro as described in Example 5. Cultures are fixed and histological sections are prepared. A solution of BOD in this dose affects the condition of the tissues of the posterior part of the eye of the newt, which is expressed in maintaining adhesive interactions between the retina, PE and choroid over an extended section of the posterior sector of the eye with minor disturbances in some areas (Fig. 6). Most of the retinal cells are in a state of apoptosis. In the monolayer of PE cells, adhesive intercellular interactions are supported, pigment granules are distributed evenly in the cell cytoplasm, which indicates a stable state of differentiation of these cells under these conditions compared to the control. The condition of the scleral membrane does not differ from that in the control series.

Thus, BOD at a dose corresponding to ^10-17 mg of protein / ml has a pronounced protective effect on the retinal tissue and PE of the eye: BOD not only supports the state of the PE cells itself, but also affects the state of the bipolar and Muller cells of the neural retina (cellular sources of regeneration ), as well as the state of the scleral membrane and choroid.

The obtained BOD can be used as a pharmacological composition for the treatment of eye diseases. The following examples 10-18 present the results of a safety study of a BOD solution isolated from a bull’s eye.

Example 8. Assessment of the irritating effect of the BOD solution on the rabbit eye in vivo

We studied the biological effect of BOD, formed by the interaction of equimolecular amounts of the regulatory peptide isolated from PE of the bull’s eye with a mol. weighing 4372 Yes, and protein with mol. weight 66367 Yes, in the presence of Ca ^{2+ ions} (10 ^-3 M), in the form of a solution in a concentration corresponding to 10 ^-12 mg of protein / ml prepared for physical. the solution of the composition is 0.9% NaCl and 0.01% CaCl ₂ .

The study was carried out on sexually mature Chinchilla rabbits weighing 2500 g. The instillation of BOD was performed daily 3 times a day for 4 months. Animals were divided into 2 groups of 6 rabbits. The second group is control. For feeding, granular combined feed and fresh vegetables were used. Access to water and feed is free. Vivarium artificial lighting, air temperature 18-22 ° С. Prior to testing, all animals were quarantined for two weeks.

Conducted clinical observations of the eyes of rabbits by biomicroscopy. The condition of the eye was evaluated by the severity of the iris reaction, the state of the epithelium, the transparency of the anterior chamber moisture, conjunctival redness, the pupillary reflex, and the choroid.

Animals were removed from the experiment by hexenal anesthesia. The eyes were enucleated, fixed in a 10% solution of neutral formalin, poured into celloidin, serial sections were made with a thickness of 15 μm and stained with hematoxylin-eosin. In addition, the cornea of each eye was tested under a scanning electron microscope. For this, the corneas were fixed with a 2.5% solution of glutaraldehyde, sprayed with gold and examined under a scanning electron microscope.

The data of each observation of animals that instilled the BOD solution were compared with the data of control animals.

Intravital biomicroscopic examination of the eye

Throughout the observation period, the eyes of the test animals were subjected to biomicroscopic examination. It consisted in examining the eyes with an ophthalmoscope and a slit lamp.

Control group. The conjunctiva is pale pink in color, without swelling and discharge. The cornea is transparent. When crevice observation with fluorescein flaws in the epithelium are not detected. Liquid media of the eye are transparent. Reflexes corresponded to physiological norm. The fundus vessels of moderate blood supply, without nodes. The retina lies over the entire surface.

The experimental group (BOD) eyes remained calm. The conjunctiva is slightly pink, without secretions. The cornea is transparent, no edema was observed. The medium of the eye is transparent. The pupillary reflex is normal. The vessels of the retinal membrane are not changed. The condition of the cornea of the eyes was examined using fluorescein in crevice lighting. In no case have keratopathic changes been detected. When observing through the lens, a vascular pattern of the retina was noted more clearly compared to the control.

Assessment of the sensitizing effect of BOD

We studied the biological effect of BOD, formed by the interaction of equimolecular amounts of a regulatory peptide with a molecular weight of 4372 Da and a protein of mol. weighing 66367 Yes, in the presence of Ca ^{2+ ions} (10 ^-3 M), in the form of a solution in a concentration corresponding to 10 ^-12 mg of protein / ml prepared in physiological saline with a composition of 0.9% NaCl and 0.01% CaCl ₂ .

Studies were performed on guinea pigs, white rats, and white mice. The animals were obtained from the Kryukovo nursery, according to the passport they are young and mature. They were kept in standard metal cages of 5 individuals. For feeding, granular combined feed and fresh vegetables were used. Access to water and feed is free. Vivarium artificial lighting, air temperature 18-22 ° С. Prior to testing, all animals were quarantined for two weeks.

In order to standardize, before the start of the experiment, the animals were not fed for a day.

The results of the experiment were processed by methods of variation statistics according to student criterion t.

Example 9. Evaluation of the ability of BOD. cause active cutaneous anaphylaxis in guinea pigs in vivo

The study was conducted on guinea pigs in an amount of 10 animals per group, a total of 2 groups. The animals were sensitized according to the scheme: the first injection was subcutaneous, the next two intramuscularly every other day into the thigh area at concentrations of 10 ^-7 , 10 ^-9 , 10 ^-10 , 10 ^-17 mg / ml. On day 14, intracutaneously, the drug was administered to guinea pigs on clipped back areas. To control the reactivity of the skin, saline was injected with the same animal on another clipped skin area. The control animals were given a permissible injection of the drug (equal to a sensitizing dose). Then animals were injected intravenously with 0.5 ml of a 1% solution of blue Evans. After 30 minutes, the animals were removed from the experiment by ether anesthesia and the size of the blue spot on the inner side of the skin at the injection site was determined. A reaction is considered positive if the spot size is above 6 mm, not more than 3 mm in the control.

The results of measuring the size of the stained skin spot are shown in table 1.

The results are not statistically reliable.

Example 10. The effect of BOD on the delayed-type hypersensitivity reaction in mice

The experiments were performed on nonlinear mice weighing 18-20 g, 10 animals per group. The total number of animals in the experiment was 30. Animals were sensitized once intradermally at the base of the tail with the emulsion of the drug at a concentration of 10 ^-7 , 10 ^-9 , 10 ^-10 , 10 ^-17 mg / ml in NAF (1: 1). The dose of administration was 60 μl. Control animals were sensitized with NAF emulsion with Hanks solution (1: 1). To detect sensitization after 5 days, mice were injected with 40 μl of the test drug solution in the Hanks solution in the paw pad of the hind paw. 24 hours after testing, the edema was measured using an MK-0-25 engineering micrometer.

The results of measuring the thickness of the paws in the test animals are summarized in table 2.

When statistical processing of the obtained results was not found significant difference in the thickness of the paws of the experimental groups and the control (P> 0.05).

The introduction of a solution of BOD does not cause a delayed-type hypersensitivity reaction.

Example 11. The effect of BOD on the mast cell degranulation reaction in rats in vitro

The experiment was carried out on Wistar rats weighing 180-200 g, females. The total number of animals in the experiment was 30. The BOD solution was administered intraperitoneally at concentrations of 10 ^-7 , 10 ^-9 , 10 ^-10 , 10 ^-17 mg / ml, the intact group served as a control.

To obtain a suspension of mast cells, animals were decapitated, a buffer was introduced into the abdominal cavity; after massage of the anterior abdominal wall, the mast cell suspension was collected by centrifugation (3000 g, 30 min) in test tubes.

The preparations were prepared on slides stained with a 0.3% alcohol solution of neutral red. To 0.03 ml of mast cell suspension was added 0.03 ml of the serum of the experimental animal and 0.03 ml of the test solution of BOD in a dilution of 1: 100. In the control, degranulation did not exceed 5%. The preparations were covered with a coverslip, the edges of which were sealed with paraffin. Then incubated for 15 minutes in a thermostat at 37 ° C. The preparations were examined microscopically at a magnification of × 40, counting mast cells that underwent degranulation and normal, in the amount of 100. The reaction was considered positive if the degree of degranulation exceeded 20%.

The percentage of mast cell degranulation in the experimental group (BOD) is 4%, and in the control group - 3%. The results were not statistically significant differences (p> 0.05).

Thus, the introduction of the BOD solution does not cause mast cell degranulation: the degranulation parameter values did not differ from the control and did not exceed the threshold value of 5%.

Example 12. The influence of BOD. Phagocytic activity of rat macrophages in vitro

The experiments were carried out on white rats weighing 180-200 g. The total number of animals in the experiment was 20, 10 in each group.

The method is based on determining the optical density of a lysing solution after the destruction of phagocytes that have absorbed neutral red particles. Rats exposed to the study drug were decapitated. 5 ml of 199 medium containing 20% fetal calf serum was administered aseptically intraperitoneally. After massage of the abdominal cavity, the medium was taken with a syringe. Portions of cell suspension obtained from the entire group of animals were combined into a common pool. The concentration of living cells was adjusted to 1.5 × 10 ³ cells / ml. The cell suspension was sterilely poured into 2 ml into plastic Petri dishes with a diameter of 40 mm. The plates were incubated for 2 hours at 37 ° C. The supernatant fraction containing cells that did not adhere to the plastic was discarded, and the monolayer fixed on the plastic was washed twice with medium 199. A solution containing neutral red was poured into slightly dried plates and kept at 37 ° C for 1 hour, then the solution was drained and the cells were washed medium 199. In each dish 3 ml of lysing solution were added, which was carefully treated with cells, making a rotational movement of the dish. The solution was poured into test tubes and the optical density was measured on a spectrophotometer at a wavelength of 540 nm. The results of the study of light transmission of the lysing solution are summarized in table 3.

When administered at concentrations of BOD 10- ^7, 10 ^-9, 10 ^-10, 10 ^-17 mg / ml there were no changes in macrophage phagocytic activity compared to the control series. There are no statistically significant differences between the indicators in the experimental and control groups (p> 0.05).

conclusions . The results of the studies allow us to conclude that there are no allergenic, sensitizing and immunotoxic properties of the BOD solution.

The BOD solution can be recommended for clinical trials.

Example 13. Studies of the influence of BOD. instilled into the eye of a rabbit, on the state of the eye tissue

A solution of BOD in concentrations of 10 ^-7 , 10 ^-9 , 10 ^-10 , 10 ^-17 mg / ml was instilled 6 times a day into the eyes of rabbits (Chinchilla breed, both sexes, 2300-2500 g) for 4 months. In the control group (10 rabbits), saline was similarly instilled into the eyes. Rabbits were taken out of the experiment by embolism, eyes were microsurgically excreted. Histological sections were made along the equatorial part. All eye structures were analyzed.

Control group. The cornea has five layers: flat non-keratinizing epithelium (outer layer); Bowman's boundary membrane; its own substance, which is represented by properly located collagen plates and flattened fibroblasts; posterior border membrane (Descemet's membrane), consisting of collagen fibers and endothelium, having one layer of regular hexagonal cells, between which there are flattened fibroblasts.

The iris is the anterior outgrowth of the choroid. It separates the front and rear cameras of the eye. Histologically, it is presented from the anterior chamber by a single-layered squamous epithelium; front boundary layer; vascular layer; posterior border and pigment layers. In the thickness of the iris pigment cells are traced.

The lens has the appearance of a biconvex body. Its front wall consists of a single-layer cubic epithelium, which becomes higher towards the equator. Epithelial cells are connected by gap junctions. The main part of the lens is occupied by lens fibers. The lens capsule is a thick basement membrane.

The sclera is built of dense cords of collagen fibers, between the sclera and the cornea there is a network of communicating cavities for the outflow of intraocular fluid (Schlemm's canal). From the side of the posterior wall of the eye, three layers can be roughly divided: the retina, choroid, and sclera. Under the sclera and the inner nuclear layer lies a layer of pigment epithelium, then the area of the rods and cones, covered by the outer boundary membrane. Further, the outer nuclear, outer reticular, inner nuclear, inner reticular, ganglionic, nerve fiber layers are located and terminated by the inner border membrane.

The entry point of the optic nerve is a blind spot.

The experimental group is BOD. Histological examination: cornea, stroma and endothelium unchanged. The angle of the front camera is open. The vascular pattern of the iris and its processes is not changed, there is no edema of the stroma. Ciliary body without features. The structure of the lens is normal. Clearly pronounced nuclear layers of the retina, no dystrophic and or inflammatory phenomena were found. Choroid without features.

Electron microscopic examination.

In control groups of animals in corneal samples, the entire inner surface is covered with endothelium. The cells have a regular hexagonal shape and a rounded core, tightly adjacent to each other.

In the groups of animals treated with BOD, no dystrophic processes were noted. The state of the endothelium did not differ from the control.

Thus, clinical and pathomorphological, including ultrastructural studies of the membranes of the eyes, after administration of BOD to sexually mature animals for 4 months, indicate the absence of a damaging effect on eye tissue.

Example 14. The study of the mutagenic and carcinogenic effects of the solution of BOD on somatic mammalian cells in vivo

The study of the potential mutagenic activity of BOD was carried out on mammals in vivo using methods included in the standard test system in accordance with the Methodological Recommendations for the Verification of Mutagenic Properties of New Medicines approved by the Pharmacological Committee of the USSR Ministry of Health, 1981.

The experiment was carried out on C57BI / 6J mice (males weighing 20-22 g), in each group there were 5 animals.

Mice of the experimental group No. 1 for five days daily were injected intraperitoneally with 0.1 ml of the drug solution BOD in concentrations of 10 ^-7 , 10 ^-9 , 10 ^-10 , 10 ^-17 mg / ml;

animals of the control group No. 2 were injected intraperitoneally for 5 days with 0.1 ml of physical. solution;

group No. 3 - intact animals that received neither BOD nor physical. rr

After 7 days, animals of all groups were removed from the experiment by ether anesthesia, the brain was removed from the thigh bones and cytogenetic preparations were prepared according to the generally accepted technique, including preliminary administration of colchicine, hypotonic treatment with 0.6% KCI, fixation in a mixture of methanol: acetic acid (1: 3). The results were processed using Student's criterion.

The mutagenic effect was studied by micronucleus test and test the frequency of chromosomal aberrations in bone marrow cells (table 4).

conclusions . As a result of the studies, it was found that when exposed to BOD, the proportion of damaged cells and the number of aberrations per cell in the bone marrow of mice were the same as in intact and control animals treated with saline.

The experimental data obtained in the course of the assessment of the BOD genetic activity indicate the absence of the mutagenic effect of the studied solution in somatic and generative cells of mammals and the absence of potential carcinogenic activity of the studied drug.

Example 15. The study of the immunotoxic and allergenic properties of a solution of BOD

The study of the immunotoxic and allergenic properties of the BOD solution was carried out on guinea pigs. The BOD solution was applied to the skin of experimental animals at a dose of 0.1 ml at a concentration of 10 ^-12 mg protein / ml for 5 days. The effect of the drug was determined by evaluating the following parameters:

• condition of the skin in the field of application of drugs,

• reaction of specific lysis of leukocytes,

• the reaction of specific agglomeration of leukocytes during cutaneous use,

• thymus cell blast transformation reaction,

• reaction of blast transformation of spleen cells.

Analyzing the results of experiments to study the immunotoxic and allergenic properties of the BOD solution, the following conclusion can be made:

BOD solution:

• Does not cause any changes in the skin of guinea pigs.

• For cutaneous use, it does not affect the reaction of specific leukocyte lysis.

• Does not affect the reaction of specific agglomeration of leukocytes during cutaneous use (experiment 3.3 ± 1.3%, control 3.4 ± 1.7%).

• Does not affect the blasttransformation of thymus cells (stimulation index - experience 138.9 ± 6.7; control 141.0 ± 5.7).

• Does not affect the blasttransformation of spleen cells (stimulation index - experiment 267.1 ± 3.7, control 275.3 ± 4.3).

Considering that the BOD solution does not have immunotoxic and allergenic effects, one can note as close analogues such ophthalmic preparations also based on biologically active substances that are isolated from animal tissues (blood serum, bull-calf eyes) as Solcoseryl (registration certificate P. P. No. 013887 / 01-2002) and Oftalamine (Cytamines - dietary supplements, St. Petersburg, 2004). It was found that these drugs, Solcoseryl and Oftalamin, which are used in significantly higher doses, respectively, 8.3 mg / g and 10 mg three times a day than the BOD solution, do not have immunotoxic and allergenic activities.

Output . The BOD solution does not exhibit immunotoxic and allergenic properties.

Example 16. The study of embryotoxicity and teratogenicity of BOD

An experimental assessment of the toxic effect of BOD on embryonic development of embryos, as well as the identification of disorders of embryonic development, manifested only in the postnatal period of life, was carried out on Wistar rats. The study of the drug was carried out during the entire period of pregnancy - 20 days. The animals were injected with a BOD solution (0.1 ml) intramuscularly daily. The study of the embryotoxic and teratogenic properties of the BOD solution was carried out in two stages: the first - the effect of the drug on embryonic development in the prenatal period was examined, in the second - the offspring in the postnatal period were examined. To assess the effect of BOD on embryotoxicity and teratogenicity, the number of miscarriages in rats, the number of rat pups in the litter, the number of surviving rat pups were determined, the physical and emotional state of the offspring, the motor function, and the maturation rate of sensory-motor reflexes were evaluated. In addition, we studied the changes in the genital organs in rats after administration of the BOD solution.

It was shown that under the influence of the BOD solution, miscarriages were not observed in the experimental animals, offspring were born on time, the number of rat pups in litters did not differ from the number of rat pups in the control group. The pups of the experimental group had the same weight as the offspring of the control group, the survival rate of the pups who experienced the influence of the BOD solution was complete. Their motor and emotional activity did not differ from the rat pups in the control group. In experimental animals treated with a solution of BOD, no changes were found in the genitals. Thus, the conducted studies did not establish the absence of any pathological disorders during embryonic and postembryonic development of the offspring of experimental animals after taking the BOD solution.

Output . BOD, studied in the form of a solution does not have embryotoxic and teratogenic effects, does not affect the general physical development of the offspring, its motor activity and emotional state, as well as the maturation rate of sensory-motor reflexes.

BOD Safety Conclusion

Summing up the results of a toxicological study of BOD, intended for ocular practice as a bioregulator of metabolic processes in the tissues of the posterior sector of the eye, which has a stabilizing effect on cell differentiation in pigment epithelium, on the state of bipolar and Muller cells of the neural retina (cellular sources of regeneration), as well as on the state of scleral shell and choroid of the eye, it should be noted that BOD is a low-toxic substance when instilled into the conjunctival sac for 4 months s.

In connection with the alleged clinical use of the BOD solution, a study was conducted in a chronic experiment. The studies were performed on chinchilla rabbits, which were administered BOD solution 6 times a day for 4 months. As studies have shown, this solution is well tolerated by animals and does not affect their general condition, hematological parameters and body systems (according to the biochemical tests used). According to the research results, the BOD solution can be recommended for research in the clinic.

Thus, BOD, formed by the interaction of equimolecular amounts of bovine eye isolated from PE, for example, a regulatory peptide with a molecular weight of 4372 Da and serum albumin protein with a mol. weight 66367 Yes, in the presence of Ca ^{2+ ions} (10 ^-3 M), in the form of a solution in a concentration corresponding to, for example, ^10-12 mg of protein / ml prepared for physical. a solution of composition - 0.9% NaCl and 0.01% CaCl ₂ can be used as a bioregulator of metabolic processes in PE, in the retina, sclera and choroid of the eye, which has a stabilizing effect on cell proliferation and differentiation in PE.

Claims (2)

1. Protein-peptide complex for the prevention and treatment of retinal diseases of the human eye and farm animals, which is a biologically active substance, and obtained by the interaction of equimolecular amounts of a mixture of regulatory peptides with molecular weights of 1000-6000 Da or a regulatory peptide with a molecular weight of 4372 Da and serum albumin with molecular weights 66362-66898 Yes, isolated from pigment epithelium of the eye of vertebrate animals. 2. A pharmacological composition for the prevention and treatment of retinal diseases of the human eye and farm animals, comprising a protein-peptide complex according to claim 1 and a pharmaceutically acceptable carrier.

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