The means of stimulating the proliferation of fibroblasts

 

The invention relates to medicine and medical industries and is the use of a peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu as a means of stimulating the proliferation of fibroblasts. The peptide Lys-Glu-Glu-Leu-Asn-Glu capable of stimulating the proliferation of fibroblasts in 50 times smaller dosages, which indicates its higher specific activity and makes it possible to reduce the risk of side effects. The proposed tool can be used as a stimulator of proliferation in various pathologies requiring activation of the regeneration potential, and provides increased biological activity. 3 table.

The invention relates to medicine and medical industries and is the use of a peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu as a means of stimulating the proliferation of fibroblasts.

Known substance, obtained from whole blood derived glycolipids and stimulates the regeneration of cells and tissues (patent of the USSR 1396958, ÁL4A 61 K 35/14); tool peptide isolated from the placenta and has anti-inflammatory and reparative action (as the USSR 948001, ÁL4A 61 K 35/50). These drugs have R the th cattle and stimulating the proliferation of fibroblasts (Biochim. et Biophys. Acta (P), 626 (1980), 397-411, L. Kelly, D. Puett Preparation). The use of this drug as a drug is difficult due to the complexity of the production method, a small release of active substances, considerable variability in physico-chemical properties of peptides that are included in its composition, but also due to the presence of unwanted side effects, due to the presence of ballast macromolecular components.

Known Monokini - polypeptide molecules produced by cells of the macrophage-macrophage number and belonging to the class of cytokines that possess a wide spectrum of biological activity, including the capacity to stimulate the proliferation of fibroblasts. These include heparinase epidermal growth factor (HB-ECF), produced by macrophages and stimulate the proliferation of smooth muscle cells and fibroblasts; transforming growth factor produced by the brain cells, macrophages, keratinocytes and stimulates the proliferation of fibroblasts and induce the development of cancer and angiogenesis; platelet-derived growth factor that stimulates the proliferation of fibroblasts, glial cells, smooth muscle cells, epithelial cells and endothelial (Gibert E. B., Silver N. B. Ka is the cue S. A., Kalinina N. M. Cytokines in mononuclear phagocytes in the regulation of the reaction of inflammation and immunity // Immunology. - 1995. - 3. - S. 30-44). Known fibroblast growth factor (FGF), comprising a polypeptide with a molecular mass of 50 kDa and chain length of 267 amino acids; its analogues (RF patent 97105853, IPC6With a 61 K 38/18, 1999); peptides, which are 60% identical FGF from amino acid residue 28 (Glu) to amino acid residue 207 (Ala) (RF patent 99109987, IPC7A 61 K 38/16, 2001), and basic fibroblast growth factor (bFGF), which is part of the natural molecules of fibroblast growth factor 10 and 155 amino acid (RF patent 2093519, IPC6FROM 07 TO 1/12, 1997). These tools are capable of stimulating the proliferation of fibroblasts. However, their use as drugs is hampered by the complexity and high cost of industrial synthesis, the possibility of allergic reactions, due to the high molecular weight of the drug. It is preferable to use low molecular weight synthetic peptides. They are effective in very small doses and do not have harmful effects on the body.

Known periodontal peptides having the ability to stimulate the proliferation of fibroblasts [1]. the rise and is its prototype. According to the prototype of periodontal peptide dose-dependently and selectively stimulate the proliferation of fibroblasts. This effect is manifested in the increase in cell number and proliferation index. At the same time, under the influence of periodontal peptides increases the content of DNA and RNA in the culture of fibroblasts, which also indicates more intense cell proliferation. Periodontal peptides active in the concentration range from 0.5 to 50 μg/ml And observed dose-effect - by increasing the concentration of periodontal peptides in the culture medium increases their potency. However, this peptide has low biological activity. Thus, the maximum activity of the substances in fibroblast cultures is evident in the concentration of 50 µg/ml of This requires the use of large doses of the drug and possible causes allergic and other adverse side effects.

The present invention is directed to expanding Arsenal and increase biological activity means of stimulating the proliferation of fibroblasts.

The solution of this problem is achieved by the fact that as a means of having the ability to stimulate the proliferation of fibroblasts, preeten previously as a means of having the ability to stimulate the differentiation of T-lymphocytes [2, 3]. The peptide was synthesized by standard methods of solid-phase synthesis by increasing peptide chain from N-Terminus [4].

In experimental study of the specific activity of the claimed funds held in comparison with the prototype, revealed a new, previously unknown to the peptide Lys-Glu-Glu-Leu-Asn-Glu ability to stimulate the proliferation of fibroblasts. This is confirmed by the following examples.

Example 1. The influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu on the proliferation of fibroblasts in cell culture Biological activity of the proposed tools studied for its effect on the culture of fibroblasts. Fibroblasts were isolated by the standard method of 8-12 week human embryos, taken in artificial termination of pregnancy [5], and was made in medium 199 containing lactalbumin hydrolysate and 20% fetal calf serum in a 1:1 ratio. The cell concentration was brought to 3105/ml In culture fibroblasts contributed peptide Lys-Glu-Glu-Leu-Asn-Glu in concentrations of 0.005 to 0.5 μg/ml as a control in the fibroblast cultures were added medium 199 in the same volume, as well as periodontal peptides in the concentration range from to has a stimulating effect on the proliferation of fibroblasts in culture. Thus, microscopic evaluation, under the influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu increases the number of fibroblasts, and marked dose-effect (table 1). The peptide Lys-Glu-Glu-Leu-Asn-Glu stimulates the proliferation of fibroblasts in a concentration range from 0.01 to 0.5 μg/ml Periodontal peptides (prototype) have a similar effect, however, they are effective in concentrations of from 0.5 to 50 μg/ml Thus, the minimum concentration at which the inventive tool enhances proliferation of fibroblasts in cell culture, 50 times less than that of the periodontal peptides, indicating a higher specific activity of the peptide Lys-Glu-Glu-Leu-Asn-Glu.

Example 2. The influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu on the content of DNA and RNA in the culture of fibroblasts, the Level of DNA and RNA in the culture of fibroblasts indicates the intensity of cell proliferation and is an additional confirmation of the specific activity of the proposed drug. The content of DNA and RNA in the culture of fibroblasts was determined by the standard method M, Hardworking [6].

Under the influence of the peptide Lys-Glu-Glu-Leu-Asn-Glu in the culture of fibroblasts increased the content of DNA and RNA due to an increase in cell mass. The proposed tool will be active in the to the Nations from 0.5 to 50 μg/ml (table 2, 3). Thus, the peptide Lys-Glu-Glu-Leu-Asn-Glu increases the level of nucleic acids at doses 50 times smaller than periodontal peptides, which is a reflection of its high specific activity.

The results of experimental study of the proposed drug attest to its high biological activity exceeding that of a known drug - periodontal peptides. Compared with the prototype, the claimed peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu capable of stimulating the proliferation of fibroblasts in 50 times smaller doses. The reduced dosage of the proposed drug to achieve the same effect indicates a higher activity of the proposed tools and gives the possibility to reduce the risk of side effects.

Thus, in experimental study of the specific activity of the claimed funds established that the peptide Lys-Glu-Glu-Leu-Asn-Glu has the property to stimulate the proliferation of fibroblasts.

The proposed tool can be used in medicine as a stimulator of proliferation and regeneration in various pathologies requiring stimulation of regenerative potential.

Sources of information 1. Turner D. L., Stepanov A. C., Kuznik B. I., Tsybikov The 1637077, IPC6A 61 K 35/37, application 4683547/14 from 25.04.1989.

2. Tsepelev, S. L. bioactive peptides Bursa fabritsiusa and their influence on the immune system: Dis... Kida. the honey. Sciences. - Chita, 2001. - S.

3. Tsepelev, C. L., Tsepelev, S. L., Tsybikov N. N., Korepanov S. I. a Means of stimulating the differentiation of T-lymphocytes // application for the invention 2001103670 from 07.02.2001.

4. Steward J. M. , Young, J. D. Solid phase peptide synthesis, San Francisco, 1969.

5. New methods of culture of animal tissues./ Edited by j. Wasli - M., Mir, 1976.

6. Modern methods in biochemistry./ Ed. by C. N. Orehovica, M, Medicine,1977, S. 313-316.

Claims

The use of a peptide of the formula Lys-Glu-Glu-Leu-Asn-Glu as a means of stimulating the proliferation of fibroblasts.

 

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