Application of novel oligopeptide fragments of protein s100b as stimulators and modulators of regenerative processes in eye cornea

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to ophthalmology and can be applied for treatment of traumatic and dystrophic injuries of eye cornea. For this purpose peptide fragments of protein S100b - SP2 and/or SP3 in concentrations 10-6 M are introduced in conjunctive cavity or in endonasal way. Introduction is realised daily two times per day during 10 days.

EFFECT: method ensures fast and high-quality restoration of injured zone of cornea due to stimulation of regenerative-reparative processes in cornea.

3 ex, 2 cl

 

Institution of Russian Academy of medical Sciences Scientific research Institute of normal physiology Anokhin

The invention relates to medicine, in particular to ophthalmology, and can be used for the treatment of various traumatic and degenerative lesions of the cornea.

Requirements to quality and rapid rehabilitation of patients after various traumatic injuries and degenerative diseases of the cornea stimulated the development of modern microsurgery of the eye (see, in particular, Belyaev V.S., "Operations on cornea and sclera" (M, Medicine. 1984), the Operations on cornea and the sclera (in kN.:

The guide eye surgery, M., 1988); Kasparov A.A. and others, "a New method of surgical treatment of bullous keratopathy)and pathogenetically justified pharmacotherapy of reparative processes of the cornea (Isaeva GT and other "New opportunities impact on reparative processes in penetrating wounds of the cornea" Ophthalmological journal., 1981, No. 4, p.75-81); Michuk Û.F. and other "New approaches to therapy of primary corneal dystrophy", tezy dopovidey dev ATAGO s SDU oftalmologia Ukrani, 1996).

In the structure of eye injuries corneal injuries and their consequences are 31-34%of the severe group pathology leading to significant snizeni the vision, represent lesions of the cornea, chemical, thermal and thermo-chemical agents, which range from 30 to 62.8% of the total number of eye injuries. According to various data, the percentage of disability due to injury of the cornea in the Russian Federation varies from 22,8%to 26%.

Despite the ever-expanding Arsenal of drugs and the introduction of microsurgical techniques, the frequency of corneal complications remains high. In this regard, along with the improvement of methods of surgical treatment, there still remains the search for new, more effective drugs.

The development and implementation of medicines, can have a positive impact on the reparative processes of the cornea, the subject of many works. Research was conducted to study the effect on the regenerative processes of various drugs - see Aznabaev M.L., and others, "Effectiveness of Caracol in the treatment of burns of eyes and skin. Eye injury in experimental situations. M 1995; Bezugly BSI others, "Effectiveness of thiotriazoline in treatment of traumatic keratitis", tezy dopovidey dev ATAGO s SDU oftalmologia Ukraine, 1996; Zubarev SF and others, "Influence of eye drops Aronia on the regeneration of damaged cornea" (ibid); Kozlova T. and others, "Effect for the e units proteoglycans on postoperative regeneration of the cornea", VI Congress of ophthalmologists, M., 1994, and some others. Based on a comprehensive morphological studies have found a stimulating effect on the regenerative processes of fibronectin and collagen (see, in particular, Polunin GSI other "Inhibitors plasminogen enzymes and autofilllocation in the treatment of epithelial defects of the cornea," journal of ophthalmol. 1993., No. 4, p.14-16). Of particular interest are biologically active products obtained from natural raw materials, which, in particular, are having an immunostimulating action apilak, solution chlorfilipt and ointments containing extracts of calendula flowers and rose hips. (Masurenkov VP and others, "Herbal medicine in ophthalmology". The honey. assistance, 1998, No. 2, p.27-29; Week A.F., and others, "Experimental study of eye ointments on the basis of herbal remedies in cases of penetrating injury of the cornea". New in the treatment of eye burns, Abstracts, M., 1989, p.48-49). However, despite the wide range of medications used to treat traumatic and degenerative lesions of the cornea, the effectiveness of such funds, in most cases, does not meet modern requirements.

In this regard, of particular interest for the treatment of various traumatic and degenerative lesions of the cornea caused by so-called regulatory peptides, which principle is tliat launchers, regulatory and modulatory functions in metabolic processes, as well as participation in inflammatory processes (see, for example, Ashmarin I.P. and others, "Cascade unidirectional regulatory processes short-lived peptides (thyrotropin-releasing hormone)". Physiologist, journal of the USSR. Sechenov, 1989, T, No. 5, s-632). So, in the literature there are reports on the impact of regulatory peptides on regenerative processes in various organs and tissues. In the pathologically changed tissues such peptides exhibit the properties of endogenous protectors, contributing to the conservation cooperative intercellular interactions, increasing the viability of cells and stimulating the regenerative and reparative processes (Borisenko AV, stem cell Biology, fundamental aspects. Abstracts, 2005, p.17-18). However, in connection with the polyfunctional action of biologically active Oligopeptide connection, which is implemented by cascading principle, currently actively developing direction of synthetic analogues of short peptides that retain the biological activity and selectivity of their protein precursors.

During previous studies, the authors have obtained new evidence about the positive incentive effect of CA2+- binding protein SlOOb, with the code in the main neirotroficescoe activity on the healing of corneal wounds in rabbit, that resulted in restoration of the basal membrane, a layer of epithelial cells, corneal plates and damaged nerve endings. [Krasnov, M., V.V. Sherstnev, Zigangirov GG, Bocharov, V.E., Maleeva L.V., Alinevich V.B. have been // Vestnik of ophthalmology. 1994. - T. - N4. - P.29-32; Sherstnev VV, Krasnov MM, Ziangirova GG, Malaeva LV, Bocharov VE, Olinevich VB, Gruden' MA. The stimulating effect of S100beta protein on regeneration of the rabbit cornea]. Biull Eksp Biol Med. 1994 Jul; 118 (7): 98-101. Zigangirov GG, Alinevich V.B. have been // Izvestiya an. The USSR, Biological Series. 2000. No. 1. - P.27-32).

Objective behind the creation of the claimed solution is the ability to use in ophthalmology new synthetic analogues of endogenous protein fragments Sl00b as promoters regenerative reparative processes in the cornea, in particular, after partial keratectomy. The technical result achieved when the task is quick and high-quality restoration of the damaged zone of the cornea after treatment of acute and/or chronic injury of the cornea, in particular, after partial keratectomy, while maintaining simplicity and ease of introduction of the drug.

Put the result in the claimed method of treatment of acute and/or chronic injury of the cornea, including the stimulation of regenerative reparatory processes in the cornea of the eye is achieved by the fact that stimulation of the Vedas is t by introducing the synthesized peptide fragments of the protein S100b is called - SP2 and SP3 at a concentration of 10-6M twice a day for 10 days conjunctival cavity or endonasal.

Preferred but not required embodiment of the inventive method provides for its use after partial keratectomy.

The ability to achieve results in the claimed method of treatment of acute and/or chronic injury of the cornea and retina due to the fact that the above synthesized peptide fragments have strong selective properties in comparison with the matrix molecule protein S100b, which is involved in a wide spectrum of biological activity, such as growth, development and differentiation of cellular elements, the exchange of ions CA 2+, Zn2+, has expressed neurotrophic growth and activity, is actively involved in various pathological processes in the nervous system. Introduction into the nasal cavity or into the conjunctival cavity provides ease of use while maintaining the biological activity of the synthesized peptide fragments of the protein S100b.

To confirm the achievement of results when using the inventive method studies have been conducted, the object of which was of the cornea obtained from sexually Mature male rats "Wistar (n=90), weighing 180-200 grams. In this work, we used the experiment the optimum model partial keratectomy, which have been subjected to all the animals after which the rats were divided into 6 groups, two groups of rats were control, four groups of animals were used to study the effects of two methods of administration of the peptide fragments of the protein S100b-SP2 and SP3 obtained by peptide synthesis. Group No. 1 (n=15) was the rats of the control group, which was introduced in 25 µl of 0.9% aqueous sodium chloride solution (saline) twice a day in the conjunctival SAC of the damaged right eye for 10 days; Group No. 2 was the rats that were injected with saline into the nasal cavity twice a day for 25 ál for 10 days. Group No. 3 entered rats, which were administered twice daily 25 ál of peptide SP2, dissolved in saline at a concentration of 10-6M in the conjunctival SAC of the damaged right eye for 10 days. Group No. 4 entered rats, which were administered twice daily 25 ál of peptide SP2, dissolved in saline at a concentration of 10-6M in the nasal cavity twice a day for 25 ál for 10 days.. In group No. 5 entered rats, which were administered twice daily 25 ál of peptide SP3, dissolved in saline at a concentration of 10-6M in the conjunctival SAC of the damaged right eye for 10 days. Group No. 6 entered rats to the m was administered twice daily 25 ál of peptide SP3, dissolved in saline at a concentration of 10-6M in the nasal cavity twice a day for 25 ál for 10 days.

Synthesis of fragments of the protein S100b was carried out by solid-phase method using Boc/Bzl strategy in a flow reactor with a continuous recording of changes in the volume of peptidylarginine (svetlogradsky monitoring). Were obtained peptide drugs adequate amount of molecular mass and purity purification 92%.

In this work, we used an experimental model of partial keratectomy eyes of rats. The influence of the studied substances on regenerative healing processes in the cornea was evaluated by nature epithelization, the restoration of the basal membrane and the corneal plates, proliferative activity of fibroblasts and epithelial cells. Using mesh ocularocular with increasing 125 and 1250 was carried out by counting the density and row of cells of the epithelial layer over the entire wound surface. Evaluated the composition and activity of the cells of inflammation in the periphery, in the middle and the Central zone of ecomii. Morphological features of apoptosis include: weakening of intercellular contacts with the loss of desmosomes and separation from adjacent cells; fragmentation and condensation of nuclear chromatin at the periphery of the cells; the formation of large and small, spherical and oval f is RMI structures containing eosinophilic cytoplasmic fragments with pinnatisectum, basophilic fragments of nuclei. The clinical course of inflammatory process was evaluated biomicroscopic..

Example 1. Rats in the control group after the partial keratectomy right eye regardless of the method of introduction of saline for 10 days was observed, the formation of epithelial tube of non-uniform thickness covering the entire wound surface and comprising 5-8 rows, which can be explained by a pronounced polymorphism of epithelial cells. Closer to the center of the thickness of the epithelial layer was decreased. Cells were acquired elongated shape in the horizontal plane. Cell nuclei were differentiated only in the basal layer of the epithelium. Revealed the weakening of the epithelial-stromal and intercellular contacts all over. The density of epithelial cells averaged 10±2 in the field of view (p/C). In some areas it was higher and had 12-14 cells in s/C. Cells in these areas were diverse in shape and size. In the basal epithelium met vacuolation with sparse cytoplasm and fragmented nuclei of cells. In many cells, it was noted the absence of nuclei, while maintaining the cytoskeleton. In the epithelial layer of mitotic and apoptotic activity was manifested unevenly. Mitosis met on average from 2-6 at the periphery and 1-2 in C is ntre. Cells with signs of apoptosis were detected at the periphery of the wound surface 1-4 cells in the Central zone 0-2 in n/z.

The basal membrane is greater for differentiated poorly and had uneven thickness with areas polosnogo structure.

The proliferation of fibroblasts was observed on the periphery of the wound surface in the middle. The entire area keratectomy lysis corneal plate was accompanied by a chaotic arrangement of collagen fibers.

The newly formed blood vessels were detected mainly at the periphery of the wound surface. The infiltrate in the middle and deep layers of the stroma consisted of polymorphonuclear leukocytes, monocytes and macrophages and numbered from 8 to 10 cells per field.

If biophilosophy the peak neovasc laritate accounted for 3-5 days and took the periphery and Central portion of the wound surface. In the Central zone on the background of significant edema stromal vessels were not found.

In 30% of cases, it was noted ulceration stroma with formation of descemetocele and perforation of the cornea.

Example 2. Rats in the experimental group after the partial keratectomy right eye when posleduyushim endonasal administration (introduction into the nasal cavity) peptide SP2 at a concentration of 10-6M 10 day epithelium covers the entire wound surface. On separate sites noted the ü weakening of intercellular contacts. Cells were of a different shape and prickly appearance. Similar changes were noted in the plots, where the detected fate polosnogo structure of the basal membrane. The rows of epithelial layer on the periphery of the wound surface was 8-9, closer to the center of 5-7 layers of cells. The weakening of intercellular contacts in the basal epithelium in individual cells was accompanied by fragmentation of nuclei, vacuolization of the cytoplasm preserving the contours of the cytoskeleton. Most of the fibroblasts in the subepithelial layer was in the apoptotic lysis. In the deeper layers of the restoration of the damaged zone of the cornea occurred due to the formation of oriented corneal plates. In the superficial layers of the corneal plates were disoriented in consequence of lesions lysis. The process of recovery zone ectomy was accompanied by desolation and thrombosis of newly formed vessels throughout the area ecomii. Polymorphonuclear cells were detected in the surface and intermediate layers 4-8 cells in field of view. Clinically, with the introduction of peptide SP2 at a concentration of 10-6M was observed forming in the surface layers of turbidity of medium intensity. When compared with the effect of the peptide SP3 in the same concentration and methods of injection of peptide density of the corneal epithelium is damaged eye was lower (8±1 rows of cells).

Example 3 rats in the experimental group after the partial keratectomy right eye m the subsequent instillation into the nose of a solution of the peptide SP3 at a concentration of 10 -6M epithelial layer covered the entire wound surface cells mainly oval-shaped. It was noted the restoration of intercellular connections, the basal membrane throughout. On the periphery of the wound surface epithelial layer consisted of 8-10 rows. Closer to the center of the rows decreased and reached 6-8 rows. The loss of the top layer of the epithelium occurred through normal shedding of cells. Cell density was approximately the same throughout the epithelial layer and consisted of 12±1. Of these apoptotic cells on average there were 1-3 in the field of view, in mitosis 2-5. Against the background of recovery of intercellular contacts in the Central zone was observed polymorphism of the basal epithelium, the change in nuclear-cytoplasmic ratio in the direction of increasing the proportion of the cytoplasm, reducing cytochromes cells. Nuclear apparatus of cells was prokruchivala unevenly, less intense in the center than at the periphery and in the paracentral zone. In the cytoplasm of epithelial cells located closer to the Central zone, was observed fragmentation of nuclei in various shape and size glybki.

Restoring row and orientation of corneal plates in the zone keratectomy in the deep and middle layers was accompanied by the obliteration of newly formed vessels. In the surface layers were found on the AGI lysis and disorientation corneal plates. Marked the formation of fibroblast membranes consisting of oriented fusiform shape keratocytes, located mainly in the middle layer. Subepithelial in clone fibroblasts was observed a predominance of proliferative apoptotic processes over 3 to 1. Shaped corneal plates were observed throughout acetaminophenol zone. The structure of the corneal plates in deep and partially the surface layers in the area of ectomy was close to normal. In the surface layers on separate sites were detected foci disoriented collagen fibers.

The infiltrate was dominated by polymorphonuclear leukocytes located unevenly distributed predominantly in the surface layers 2 to 4 in n/z.

Fibroblast membrane with the introduction of SP3 at a concentration of 10-6M was detected in almost the entire surface area of ecomii and consisted of fusiform keratocytes. The intercellular space was filled with a thin, intensely dyed corneal plates. Polymorphonuclear leukocytes were detected in subepithelial from 2 to 4 in n/z.

Biophilosophy with the introduction of SP3 at a concentration of 10-6M in the cornea was observed the formation of a pronounced turbidity. The activity of the formation of neovasculature had for 3-4 days and remained stable in 2 days. the 10 days the vessels were detected in some areas on the periphery of ecomii and was in a state of desolation.

A distinctive feature of SP3 was its pronounced effect on the proliferative activity of fibroblasts.

It must be emphasized that significant differences influence SP2 and SP3 on for regenerative reparative process and method of administration were noted.

Thus, in experimental models of partial keratectomy shows a pronounced stimulating effect on the flowing regenerative repair processes in partially damaged the cornea adult rats synthesized new Oligopeptide fragments of the protein S100b SP2 and SP3. Proven expressed the proliferative activity of fibroblasts in the application of SP3, which ensured the restoration of the volume of the stroma of the cornea in the area of ecomii. Effects SP2 was characterized by a stabilizing effect on the cellular structure and the partial recovery of the corneal stroma. Introduction Oligopeptide fragments in ophthalmic practice will significantly reduce the effect of the damaging agent and to improve the quality of rehabilitation of patients with corneal damage.

1. A method of treating acute and/or chronic injury of the cornea, including the stimulation of regenerative reparatory processes in the cornea of the eye, while the stimulation lead through the introduction of synthesized peptide fragments of the protein S100b SP2 and/or SP3 at a concentration of 10-6

2. The method according to claim 1 applied after partial keratectomy.



 

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