RU2526825C1 - Pharmaceutical antiangiogenic composition for treating eye diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: pharmaceutical antiangiogenic composition for treating eye diseases contains an active substance that is a fragment of one of recombinant polypeptides: endostatin fragment with an amino acid sequence from 1 to 49 or tumstatin fragment with an amino acid sequence from 69 to 95 or pigment epithelium-derived factor (PEDF) fragment with an amino acid sequence from 44 to 77 having stabilising ProGlyPro cluster on C-terminal, and a pharmaceutically acceptable carrier. The active substance and pharmaceutically acceptable carrier are found in the following proportions, wt %: active substance - 3.9×10^-7 to 53×10^-7 and pharmaceutically acceptable carrier - up to 100 wt %.

EFFECT: using this composition provides the antiangiogenic effect mediated by various biological targets at different stages of angiogenesis.

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Description

The invention relates to medicines for the correction of disorders of angiogenesis in ophthalmology and relates to a pharmaceutical composition containing an active substance - a fragment of one of the recombinant polypeptides: a fragment of endostatin with an amino acid sequence from 1 to 49 or a fragment of tumstatin - with an amino acid sequence from 69 to 95 or a fragment of pigment epithelial factor (PEDF) - with an amino acid sequence of 44 to 77, having a stabilizing ProGlyPro cluster at the C-terminus, and a pharmaceutical administration removable media.

Angiogenesis is a process in which the migration, attachment and adhesion of endothelial cells, and then their proliferation and organization into vessels with the formation of the vasculature, necessary for tissue development.

Normally, the processes of angiogenesis in the body proceed with moderate intensity and are activated during the regeneration of damaged tissues, the dissolution of blood clots, the elimination of foci of inflammation, scarring and other recovery processes, as well as during the growth and development of the body.

Violations of angiogenesis occur in various diseases, including eye diseases.

The main endogenous angiogenesis stimulants include cytokines: vascular endothelial growth factor, placenta growth factor, fibroblast growth factor, platelet growth factor, transforming growth factor, hepatocyte growth factor, insulin-like growth factor, tumor necrosis factor, IL-8, erythropoietin, granulocyte colony and granulocyte-macrophage colony-stimulating factor (Griffioen AW, Molema G. Angiogenesis: Potentials for Pharmacologic Intervention in the Treatment of Cancer, Cardiovascular Diseases, and Chronic Inflammation // Pharmacological Reviews. - 2000. - Vol.52, Issue 2. - P. 237-268; Carm eliet P. Angiogenesis in health and disease // Nature Medicine. - 2003. - Vol. 9. - P.653-660).

Neovascularization accompanies a wide range of eye diseases, such as: diabetic retinopathy, retinopathy of premature infants, age-related macular degeneration, central retinal vein thrombosis, neovascular glaucoma, high grade myopia, histoplasmosis, tumors of the organ of vision, which occupy a leading position in the list of diseases leading to disability blindness (Neroev V.V., Sarygina O.I., Levkina O.A., Slepova O.S. Study of local secretion of pro- and anti-angiogenic factors before and after laser coagulation tissue in patients with proliferative diabetic retinopathy // Medical Immunology. - 2009. - Volume 11, No. 4-5. - P.369-370. Sarygina OI, Neroev VV, Levkina OA Role of the vascular endothelial growth factor in the pathogenesis of diabetic retinopathy // Ophthalmology. - 2009. - Volume 6, No. 2. - P.58-62. Agnes Noël, Maud Jost, Vincent Lambert, Julie Lecomte and Jean-Marie Rakic Anti-angiogenic therapy of exudative age-related macular degeneration: current progress and emerging concepts // Trends in Molecular Medicine. - 2007. - Vol.13. - Issue 8.- P.345-352)

Known use in ophthalmology drugs Makugen and Lutsentis; Avastin (Anti-VEGF. Editors F. Bandello, M. Battaglia Parodi. KARGER. Basel (Switzerland). 2010. P.144. ISSN 978-3-8-55-9530-8. Carl DR, David MB, Prema A. et al. Randomized double-masked, sham-controlled trial of ranibizumab for neovascular age-related macular degeneration: PIER Study year 1. // Am J Ophthalmol. - 2008 - Vol.145 - P.239-248. Costa RA, Jorge R. Calucci D. t al: Intravitreal bevacizumab for choroidal neovascularization caused by AMD (IBNA Study): results of a phase I dose-escalation study. // Invest Ophthalmol Vis Sci - 2006 - Vol.46 - P.4569-4578) . These drugs have a narrow molecular focus: they inhibit, block, or inhibit the realization of the biological effects of one pro-angiogenic molecule, vascular endothelial growth factor (VEGF), aimed at triggering angiogenesis. Their target is VEGF or its receptor, that is, they exert their action through a VEGF-dependent mechanism of action (Anti-VEGF. Editors F. Bandello, M. Battaglia Parodi. KARGER. Basel (Switzerland). 2010. P.144. ISSN 978-3-8-55-9530-8).

Multicenter clinical trials to study the efficacy of Lucentis and Avastin in AMD have proven the existence of natural tachyphylaxis in 2% of patients for whom these drugs were not initially effective, 10% of the population revealed the formation of pharmacokinetic tolerance with a decrease in the therapeutic effect of the drugs with repeated injections, and the therapeutic effect of Avastin halves after three injections (Ziemssen F, Neuhann IM, Voelker M. Tachyphylaxis and bevacizumab. Ophthalmology 2009; 116: 1591-2; author reply 1592-3. Avgikos KN, Horgan SE, Sivaraj RR, et al. Tachyphylaxis and bevacizumab. Ophthalmology 2009; 116: 1831-2 ; author reply 1832. Kopal Mithal, Raja Narayanan Switching anti-VEGFs in Tachyphylaxis Br J Ophthalmol published online January 17, 2012).

In this regard, there is a need for the search and development of pharmaceutical compositions, the use of which is based on the strategy of combining VEGF inhibitors with other natural inhibitors of angiogenesis. The targets for such anti-angiogenic therapy may be endothelial cells, regulatory peptides involved in angiogenesis, receptors to them, or genes responsible for the synthesis of pro- and / or anti-angiogenic peptides.

In this aspect, the development of more effective regulators of angiogenesis disorders for ophthalmology based on recombinant peptides of targeted antiangiogenic purpose is relevant.

The technical result of the invention is to obtain an anti-angiogenic effect at different stages of angiogenesis, while being mediated through various biological targets.

Purpose peptides fragments of three recombinant peptides: tumstatin with an amino acid sequence of 69 to 95, endostatin with an amino acid sequence of 1 to 49 and pigment epithelial differentiation factor (PEDF) with an amino acid sequence of 44 to 77 were obtained using biotechnology. Artificial genes of target peptides optimized for efficient expression in bacterial cells by codon composition were cloned into plasmid vectors containing the intein gene sequence under the control of a strong phage promoter. As the carrier strain, we used the collection strain E.coli ER2566, which provides superexpression of hybrid genetic constructs and high production of the hybrid protein (Beirakhova K.A. Recombinant polypeptides for the treatment of eye diseases accompanied by pathological angiogenesis. Author's abstract. Diss. Ph.D. M., 2012).

The biological activity of the tested peptides was studied in vitro and in vivo using experimental models of eye disease accompanied by corneal angiogenesis in laboratory animals with an assessment of the angiostatic potential of the studied peptides.

The biological effects of target peptides were studied on two types of endothelial cell (EC) cultures: SVEC4-10 obtained from regional lymph nodes of C3H / HeJ mice (H-2k) transformed with SV40 virus and human EC-EA line. Well, 926.

In vitro methods for studying the antiangiogenic activity of peptides included: cytotoxic, proliferation, and migration tests; as well as a test for the formation of capillary-like structures (CPS).

In a mouse EC culture, the antiangiogenic properties of peptides were studied in a concentration range from 0.1 to 20 nM. In experiments with the human EC line, concentrations from 2.5 to 10,240 nM were studied. EC migration for tumstatin was determined in the concentration range of 1280–10240 nM, for endostatin, 640–10240 nM, and for PEDF, 20–320 nM. The choice of concentrations was based on the results of a previous test to evaluate their antiproliferative activity. The formation of CPS was studied by adding those concentrations of peptides that suppressed EC migration. They constituted 1250, 2500, 5000 and 10000 nM for tumstatin and endostatin; for PEDF - 20, 40, 80 and 160 nM. Peptide activity in EC EA culture. Well, 926 was compared not only with each other, but also with the activity of the Avastin preparation, in a concentration from 10,240 nM to 2.5 nM.

In vivo experimental studies were performed on the eyes of chinchilla rabbits (mature males weighing 2.5-3 kg). The first model of angiogenesis was formed by flashing the cornea with vikril seams in two eyes of 30 rabbits (60 eyes). On the day of corneal flashing, peptides began to be administered subconjunctively in the estimated doses: 7.2 ng PEDF, 96 ng endostatin, 70.2 ng tumstatin. Injections were given daily for 10 days. The right eyes of animals served as control; the left eye was experienced. Animals were removed from the experiment on day 15.

As a second model, post-burn corneal angiogenesis was used, injecting 0.1 ml of a 10% alkali solution into the corneal apex intrastromally. A model was formed on the right eye of 33 rabbits (n = 33, 10 eyes for each peptide). Five experimental rabbits were given subconjunctival preparations at the calculated dose starting from the 1st day of the experiment (early angiogenesis). They were withdrawn from the experiment on the 12th day. The remaining five rabbits were injected with peptides in a ten-fold dose, starting from the 7th day, when the vasculature was already formed (late angiogenesis, avascular therapy). Injections were given for 10 days. The experiment was withdrawn on the 30th day.

Angiostatic activity of peptides in the eyes of experimental animals was evaluated clinically (biomicroscopically, colorimetrically), angiographically and morphologically. The state of the vascular wall was studied using electron microscopy.

The use of 2 models of corneal angiogenesis in the eyes of experimental animals made it possible to verify the presence of the ability of the tested peptides to interrupt or suppress the process of neoplasm of the vasculature, as well as to give a quantitative assessment of them.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained in the course of research.

To obtain a pharmaceutical composition according to the invention, a fragment of one of the recombinant polypeptides: endostatin 1-49 or tumstatin 69-95 or pigment endothelial factor (PEDF) 44-77, having at the C-terminus a stabilizing ProGlyPro cluster as an active substance is mixed with a pharmaceutically acceptable carrier, while the active substance and a pharmaceutically acceptable carrier are in the following ratio in wt.%:

active substance - 3.9 × 10 ^-7 - 53 × 10 ^-7

pharmaceutically acceptable carrier - up to 100 wt.%.

The set of in vitro experimental data clearly illustrated the biological activity of tumstatin, endostatin, PEDF. It manifested itself in their ability to suppress the different stages of angiogenesis: proliferation, migration, and the formation of EC CPS. It was found that in the early stages of angiogenesis, all 3 recombinant peptides within the analyzed doses have a dose-dependent effect. The most powerful anti-angiogenic potential was shown by tumstatin (at a dose of 70.2 ng) and PEDF (at a dose of 7.2 ng). Experimental data confirmed the angiostatic activity of tumstatin, endostatin, PEDF. In vitro, this activity was manifested in their ability to suppress proliferation (endostatin, tumstatin, PEDF), migration (endostatin) and the formation of EC capillary-like structures (endostatin, tumstatin, PEDF) at specific concentrations and culturing conditions.

In vivo, the anti-angiogenic potential of tumstatin, endostatin, and PEDF was expressed by a reduction in the number of newly formed vessels per unit area, a narrowing of their lumen diameter and length while administration of angiogenesis inducers. The avasculogenic potential of tumstatin was manifested by apoptosis of endothelial cells, followed by resorption of the vascular wall.

As a pharmaceutically acceptable carrier, various substances can be used, depending on the dosage form of the drug introduced into the body and the route of administration. In particular, for insertion into the conjunctival or nasal cavity, distilled water can be used intravitreal, it is also possible to use other carriers, in particular eye films. As a preservative, in particular, benzalkonium chloride (0.005-0.01%) can be used.

Before instillation, the solution should be diluted with buffer to a final concentration.

Drops for the eyes should be isotonic. The acidity of human tears is 7.14-7.82. An isotonic solution is one that corresponds to an osmotic pressure of 305 mOsm / l. For this, dextran 40 and 70, dextrose, propylene glycol are used.

The invention is illustrated by drawings and illustrated in tables.

Table 1 shows the formation of capillary-like structures of the EC line of EA.An 926 in the presence of fragments of tumstatin, endostatin and PEDF.

Figure 1 shows a morphological assessment of biological activity, reflecting that at a concentration of 160 nm PEDF inhibits the proliferation of EC EA. Well, 926 and 4 times stronger than Avastin; in high concentrations, its anti-migration activity exceeds the activity of therapeutic doses of Avastin.

Table 2 shows the results of experimental in vitro studies that made it possible to calculate the estimated therapeutic dose of the tested peptides with a subconjunctival route of administration. The dose of the peptide per injection was calculated by multiplying three components: the optimal concentration of the peptide obtained in in vitro experiments in SVEC 4-10 cell culture, the molar mass of the peptide, and the rabbit's eye volume (1.8 cm ³ ).

Table 3 presents a quantitative morphometric and colorimetric assessment of the biological activity of antiangiogenic peptides.

The invention is illustrated by examples for the development and creation of models of various stages of angiogenesis to assess the biological activity of tumstatin, endostatin, PEDF (example 1); to develop methods for quantifying the biological activity of tumstatin, endostatin, PEDF in vitro and in vivo (example 2); on an experimental assessment of the biological activity of tumstatin, endostatin, PEDF in vitro (example 3); according to an experimental assessment of the biological activity of tumstatin, endostatin, PEDF in vivo (example 4), to prepare solutions for insertion into the conjunctival cavity, subconjunctival and intravitreal (examples 5-11).

Example 1. A two-level biological testing system was used, in which the first level was represented by in vitro studies, and the second level by in vivo experiments. Each of the levels was formed by several models of angiogenesis, representing the successive stages of the assembly of the vascular network. The evidence of the in vitro experimental base was enhanced using two types of cultures (mouse EC SVEC4-10 and human EC - EA. Well 926) and a series of controls (basic, positive and additional control).

The in vitro antiangiogenic potential of recombinant peptides revealed was studied in vivo in two experimental models of eye diseases accompanied by angiogenesis in the cornea. In the first model (30 rabbits, 60 eyes), potential angiostatics were administered simultaneously with angiogenesis inducers. Vicrylic filaments acted as an inductor. It is known that the reaction of surrounding tissues to vicryl is moderately pronounced; vikrilovy threads are absorbed during hydrolysis with the formation of glycolic and lactic acids, which are a natural component of tissue activity. This eliminates a systemic (general) reaction to vicryl. The pro-inflammatory and angiostimulating effects of Vicryl when flashing a rabbit’s cornea gradually increase, and then, they also gradually level due to resorption. The resorption process begins from day 12, which limits the time period for observing the angiogenesis model, and, as a result, affects the interpretation of long-term results. Therefore, this model was used to test the activity of peptides in the early stages of angiogenesis. In this case, the peptides began to be administered from the day the cornea was flashed. The day of induction of angiogenesis and the day of its blocking by angiostatic peptides coincided.

In the second model (36 rabbits, 10 eyes per peptide, 6 eyes control), the possibility of interruption of angiogenesis at the early and late stages (avascular therapy) was studied. The model of anterior angiogenesis was formed by injection of 0.1 ml of ³ 10% sodium hydroxide solution intrastromically into the corneal apex. Unlike the previous model, alkali is recognized as a more powerful inducer of angiogenesis. Toxic paresis of capillaries and a decrease in blood circulation during an alkaline burn of the cornea lead to the development of tissue and cell hypoxia. Stimulation of angiogenesis proceeds in 2 stages: at the stage of applying a burn (early angiogenesis) and at the stage of reparative processes in conditions of developing and increasing hypoxia. In conditions of hypoxia, which triggers the growth of new vessels, the severity of angiogenesis reaches a maximum. The formation of new blood vessels can be observed over a long period of time, which allows the use of this model as a model of both early and late angiogenesis.

The angiostatic potential of recombinant fragments of tumstatin, endostatin and PEDF was tested at the stage of early and late angiogenesis. In the first case, the test peptides were administered simultaneously with angiogenesis inducers, which were vicryl sutures (1 model) or 10% sodium hydroxide solution (alkaline burn, 2 model). In the second case, tumstatin, endostatin and PEDF were administered starting from the 7th day, when the vasculature was already formed (late angiogenesis, avascular therapy, model 2). The vessels that grew in the cornea already formed a “corolla”, in which nearly full “ mature "vessels with blood flow.

The use of 2 models of corneal angiogenesis in the eyes of experimental animals made it possible to verify the presence of the ability of the tested peptides to interrupt or suppress the process of neoplasm of the vasculature, as well as to give a quantitative assessment of them.

Example 2. When conducting experimental studies in vitro, the following expert criteria were developed and tested to allow a comprehensive quantitative assessment of the biological activity of peptides:

- EC survival index (% of control, cytotoxic test);

- proliferation index (% of control, proliferation test);

- change in the line width of the monolayer (pixels,% of the original width, migration test);

- change in the number of migrated cells (pcs. migration test);

- the length and number of cords (test for the formation of the KPS);

- the number of units (test for the formation of the KPS).

When conducting in vivo experimental studies, several of the following expert criteria were used to provide a comprehensive quantitative assessment of the biological activity of peptides:

- density of corneal vascularization (in points, pixels; clinically, angiographically and colorimetrically);

- area of corneal opacity (in pixels, colorimetric);

- the number of newly formed vessels per unit area of the slice in the viewing area (morphometrically);

- an indicator of the total lumen of blood vessels on the slice (in pixels, colorimetric and morphometric);

- vascularization index of the limb-corneal zone (morphologically);

- semi-quantitative assessment of the IHC reaction with the marker EC-CD31.

The developed testing system, which together includes 4 models of in vitro angiogenesis and 2 in vivo models, made it possible to obtain an evidence base (13 expert quantitative indicators) to substantiate approaches to an alternative strategy of antiangiogenic therapy using recombinant endogenous antiangiogenic peptides intended for the treatment of eye diseases associated with angiogenesis, excluding or supplementing the treatment with drugs of VEGF-dependent mechanism of action.

Example 3. In in vitro experimental studies on a culture of EC line SVEC 4-10, all 3 tested peptides showed antiangiogenic activity in the range of studied concentrations (0.1 nm, 1 nm, 10 nm and 20 nm):

Endostatin in concentrations of 0.1 nM, 1 nM, 10 nM and 20 nM did not have cytotoxic activity and the ability to suppress EC proliferation, but blocked the formation of CPS at a concentration of 10 nM, recommended for further studies. Tumstatin had a weak cytotoxic effect, but blocked the proliferation of EC and the formation of CPS at a concentration of 10 nM, this concentration was used for subsequent studies. PEDF exerted the maximum cytotoxic effect, blocked the proliferation of EC and the formation of CPS at a concentration of 1 nM, it was chosen for further studies.

On the culture of EC human line EA. Well, 926 all 3 tested peptides in the range of studied concentrations showed biological activity. The nature of the biological effect depended on the concentration of the peptide and the percentage of ETS in the culture medium; as well as from the stage of angiogenesis (proliferation / migration / formation of CPS).

Endostatin inhibited the proliferation of EC EA. Well, 926 at concentrations of 2.5, 5, 5120 and 10,240 nM (5% ETS, p <0.05), inhibited migration at concentrations of 1280 and 10,240 nM (5% ETS, p <0,01), 2560 and 5120 nM (10% ETS, p <0.05); inhibited the formation of CPS at concentrations of 1250 and 2500 nM (5% and 10% ETS p <0.05 and p <0.05, respectively).

Tumstatin suppressed proliferation of EC EA. Well, 926 at concentrations of 2560 and 10240 nM (5% ETS, p <0.05), did not affect EC migration under any conditions, inhibited the formation of CPS at concentrations of 1250, 2500, 5000, and 10000 nM, significantly reducing the length (2, 5% ETS, p <0.05), the number of strands (p <0.01) and cell aggregates (p <0.01), which indicated inhibition of both direct / and branched angiogenesis.

PEDF inhibited EC proliferation at concentrations of 20, 40, 80, and 160 nM (5% ETS, p <0.01), at a concentration of 160 nM blocked proliferation (10% ETS, p <0.05), suppressed the formation of CPS at concentrations of 20 and 80 nM (2.5% ETS), decreasing the number of strands (p <0.01) and EC aggregates (p <0.01) and inhibiting branched angiogenesis.

Avastin inhibited the proliferation of EC EA.Nu926 at concentrations of 2.5, 5, 10 and 20 nM (2.5%, 5% and 10% ETS, p <0.01), 640, 1280, 2560, 5120 and 10240 nM ( 5% and 10% ETS, p <0.01), inhibited migration at a concentration of 5 nM (10% ETS, p <0.01) and inhibited KPS at a concentration of 5 nM, reducing the length of strands (2.5% ETS, p <0.01, table 1).

Example 4. It was found that in the early stages of angiogenesis, all 3 recombinant peptides within the analyzed doses have a dose-dependent effect. The most powerful anti-angiogenic potential was shown by tumstatin (at a dose of 70.2 ng) and PEDF (at a dose of 7.2 ng) (Table 2). The effect was manifested by a significant decrease in the vascularization index of the limb and the cornea (p <0.001), a decrease in the number (p <0.001) and the length of the vessels extending from the limb into the optical zone of the cornea of the experimental rabbit eyes compared with the control ones. Clinically (ad oculus) vessels in the experimental eyes were not detected. They could be identified biomicroscopically at a magnification of × 16. In terms of the severity of the biological effects, endostatin was inferior to them. Morphological studies confirmed clinically detectable effects. In addition, at the light level, a different degree of severity of edema (density and depth of distribution) and cellular infiltration of the cornea around the experimental sutures attracted attention. The infiltrate mainly consisted of lymphocytes, plasmocytes and eosinophils (Table 3). Peptides significantly reduced the number of cellular elements in the infiltrate that developed around the vicryl joints. Comparative aspects of the quantitative assessment of the decongestant and anti-inflammatory effect of antiangiogenic peptides demonstrated the advantages of tumstatin and PEDF over endostatin. The apparent biological effect of tumstatin and PEDF can apparently be explained by inhibition of VEGF secretion, which is responsible for permeability of the vascular wall.

The ability to suppress late stages of angiogenesis or avascular potential was evaluated in the second model of angiogenesis. It was detected in one tumstatin in a 10-fold therapeutic dose. A clinically significant effect developed in the eyes of rabbits after 5 injections. It was manifested by a decrease in the density of newly formed vessels in the paralymbal zone of the cornea, regression of the end of the most branched section of the newly formed vessels, a reduction in the diameter of “mature” vessels with circulating blood flow, and neglect of small vessels. Endostatin exerted an effect exclusively in the early stages of post-burn angiogenesis (1-5 days) and also in a 10-fold dose. It was manifested by a decrease in the density of the vascular limbal network. As the vasculature completed its formation (from day 7), the activity of the peptide was leveled.

Example 5. The preparation of a solution for injection into the conjunctival cavity using a fragment of endostatin.

As an active principle, a fragment of endostatin 1-49 is used in concentrations from 53.4 μg of the peptide to 534 μg per 1 liter of solution; which corresponds to 5.3 × 10 ^-7 - 53 × 10 ^-7 wt.% the active substance. As a carrier, you can use distilled water, which is added up to 100%. Before instillation, the solution should be diluted with buffer to a final concentration.

Drops for the eyes should be isotonic. The acidity of human tears is 7.14-7.82. An isotonic solution is one that corresponds to an osmotic pressure of 305 mOsm / l. For this, dextran 40 and 70, dextrose, propylene glycol are used.

The solution can be supplemented with viscoelastic substances that extend the duration of action and retention on the surface of the eyeball, for example, gels or methyl cellulose. Antioxidants, such as bisulfite or thiosulfate, etc. can be used to prevent the breakdown of the active substance. The volume of 1 drop for the eyes and nose is 10 μl.

Example 6. Preparation of a solution for subconjunctival administration using a fragment of endostatin.

As an active principle, a fragment of endostatin 1-49 is used in concentrations from 53.4 μg of the peptide to 534 μg per 1 liter of solution; physiological saline can be used as a solvent, which is added up to 100%. The calculation of the volume of the solution for a single injection is carried out taking into account the recommended dose: not more than 0.3-0.5 ml, the same volume is used for retrobulbar injections.

Example 7. Preparation of a solution for injection into the conjunctival cavity using a fragment of tumstatin.

A fragment of tumstatin 69-95 is used as an active principle in concentrations from 38.7 μg of the peptide to 387 μg per 1 liter of solution; which corresponds to 3.9 × 10 ^-7 - 39 × 10 ^-7 wt.% the active substance. As a solution, distilled water can be used, which is added up to 100%. The isotonicity of the droplet solution is achieved by adding dextran / dextrose / propylene glycol in an amount sufficient to achieve an osmotic pressure of 305 mOsm / L. The solution can be supplemented with viscoelastic substances that extend the duration of action and retention on the surface of the eyeball, for example, gels or methyl cellulose. Antioxidants such as bisulfite or thiosulfate can be used to prevent the breakdown of the active substance.

Example 8. Preparation of a solution for subconjunctival administration using a fragment of tumstatin.

As an active principle, a fragment of tumstatin is used in concentrations from 38.7 μg of the peptide to 387 μg per 1 liter of solution; which corresponds to 3.9 × 10 ^-7 - 39.0 × 10 ^-7 wt.% of the active substance. As a solvent, physiological saline can be added, which is added up to 100%. The calculation of the volume of the solution for a single injection is carried out taking into account the recommended dose: 0.1-0.3 ml.

Example 9. Preparation of a solution for injection into the conjunctival cavity using a PEDF fragment.

As an active principle, a PEDF 44-77 fragment is used in concentrations from 4.0 μg of peptide to 40 μg per 1 liter of solution; which corresponds to 4.0 × 10 ^-7 - 40.0 × 10 ^-7 wt.% of the active substance. As a solution, distilled water can be used, which is added up to 100%. The isotonicity of the droplet solution is achieved by adding dextran / dextrose / propylene glycol in an amount sufficient to achieve an osmotic pressure of 305 mOsm / L. The solution can be supplemented with viscoelastic substances that extend the duration of action and retention on the surface of the eyeball, for example, gels or methyl cellulose. Antioxidants such as bisulfite or thiosulfate can be used to prevent the breakdown of the active substance. The recommended dose for a single instillation is 10 μl.

Example 10. Preparation of a solution for subconjunctival administration using a PEDF fragment.

As an active principle, a tumstatin fragment is used in concentrations from 4.0 μg of peptide to 400 μg per 1 liter of solution; as a solvent, physiological saline can be added, which is added up to 100%. The calculation of the volume of the solution for a single injection is carried out taking into account the recommended dose: 0.1-0.3 ml.

Example 11. Preparation of a solution for intravitreal administration.

The dose determined for in vitro administration for PEDF is 1 nM (Mr 4043 Da), i.e. 4 μg of peptide per 1 L of the final volume (4 × 10 ^-7 wt.%). The dose determined for in vitro administration for Tumstatin is a molecular weight of 3872 Da. The calculated dose: 10 nm. 10 nanomoles of tumstatin weigh 39 mcg. The amount of tumstatin per injection is 39 μg / 1000 ml * 1.8 ml = 70.2 ng. As a solvent, physiological saline can be used, which is added up to 100%. Calculation of the solution volume for a single intravitreal administration is based on the recommended dose: 0.05 ml.

Thus, the proposed peptides are included in the universal mechanism of suppression of angiogenesis in ocular pathology at key positions: activation of endothelial cells and can be used as part of a pharmaceutical composition for administration into the conjunctival or nasal cavity and for subconjunctival or intravitreal administration.

Table 1 Cultivation of EC line EA. Well 926 in the presence of: The average length of the cords formed by EC line EA. Well 926 (in pixels) The number of cords formed by EC line EA. Well 926 The number of aggregates formed by EC line EA. Well 926 DMEM / F12 medium with the addition of 2.5% ETS (spontaneous level) 182.7 ± 2.4 63.8 ± 2.1 50.1 ± 2.3 bFGF, 20 g / ml 258.6 ± 4.8 *** 81 ± 2.0 * 67.1 ± 2.1 * Avastin, 5 nM 165.2 ± 2.3 *** 73.3 ± 3.1 * 59.1 ± 2.6 ** Tumstatin 1250 nm 169.1 ± 5.6 * 40.8 ± 2.6 *** 30.9 ± 2.4 *** Tumstatin 2500 nM 160.6 ± 7.4 ** 34.6 ± 2.1 *** 24.3 ± 1.4 *** Tumstatin 5000 nM 154.1 ± 10.2 *** 28.0 ± 2.2 *** 20.9 ± 1.3 *** Tumstatin 10000 nM 213.7 ± 7.5 *** 41.4 ± 3.3 *** 32.1 ± 2.4 *** Endostatin 1250 nM 154.1 ± 2.7 *** 76.2 ± 5.6 * 65.1 ± 5.7 *** Endostatin 2500 nM 153.4 ± 3.4 *** 71.1 ± 7.0 59.8 ± 6.4 *** Endostatin 5000 nM 182.5 ± 4.0 66.5 ± 6.0 56.4 ± 5.4 *** Endostatin 10,000 nM 183.1 ± 4.0 55.9 ± 5.9 47.1 ± 5.3 PEDF, 20 nM 187.3 ± 4.4 *** 48.8 ± 3.9 ** 37.7 ± 4.2 ** PEDF, 40 nM 101.4 ± 11.1 *** 10.9 ± 5.6 *** 5.5 ± 3.4 *** PEDF, 80 nM 72.4 ± 14.0 *** 5.2 ± 3.9 *** 3.6 ± 2.9 *** PEDF, 160 nM 183.4 ± 8.1 28.8 ± 10.4 *** 22.2 ± 8.0 *** Reliability of differences from the spontaneous level: * - p <0.05; ** - p <0.01; *** - p <0.001.

Table 2 Peptide name Concentration, nm Molar mass, g / mol The number of peptide per 1 injection, ng Ten-fold dose, ng Tumstatin 10 3870 70,2 702 Endostatin 10 5338 96 960 Pedf one 4044 7.2 72

Table 3 Test peptide Test eye Colorimetric rating Morphometric evaluation Number of Pixels (Mcp. ± m) Vascularization Index (Mcp. ± m) The number of vessels on the cut, pcs. (Mcp. ± m) Tumstatin (n = 10) The control 128830 ± 1115 0,03 90 ± 17 Experience 73788 ± 1491 * 0.01 * 33 ± 5 * PEDF (n = 10) The control 243734 ± 1125 0.016 76 ± 8 Experience 64974 ± 1234 * 0.008 * 38 ± 5 * Endostatin (n = 10) The control 102882 ± 1112 0.02 83 ± 6 Experience 58886 ± 1265 * 0.01 24 ± 4 ** Note: * - the significance of differences in indicators in the experimental and control eyes p <0.001; ** p <0.01.

Claims (1)

A pharmaceutical anti-angiogenic composition for treating eye diseases, characterized in that it contains the active substance — a fragment of one of the recombinant polypeptides: an endostatin fragment with an amino acid sequence of 1 to 49 or a tumstatin fragment with an amino acid sequence of 69 to 95 or a pigment epithelial factor (PEDF) fragment - with an amino acid sequence from 44 to 77, having a stabilizing ProGlyPro cluster at the C-terminus, and a pharmaceutically acceptable carrier, wherein the active substance and a pharmaceutically acceptable carrier are in the following ratio in wt.%:
active substance - 3.9 × 10 ^-7 - 53 × 10 ^-7
pharmaceutically acceptable carrier - up to 100 wt.%.

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