EA025690B1 - Wound healing agent possessing immunostimulatory and antioxidant effects - Google Patents

Abstract

The invention is related to medicine (traumatology, surgery) and veterinary and can be used for stimulation of skin wound healing. The invention consists in use of a peptide of a formula HN-D-Ala-L-Glu-L-Trp-COOH differing from timogen HN-L-Glu-L-Trp-COOH in the presence of an additional amino acid (D-alanine) near the N-end of the peptide. Peptide was administered once a day parenterally (intraperitoneally or intracutaneously) in a single dose equimolar with timogen (HN-D-Ala-L-Glu-L-Trp-COOH - 1,2 μg/kg, timogen - 1 μg/kg of body weight). In case of parenteral administration of the peptide during 10 days from the moment of appearance of a skin wound, the experimental animals (Wistar rats) showed stimulation of reparative skin regeneration (the number of fibroblasts increased) and activation of blood neutrophil function. Further, in such conditions, the peptide HN-D-Ala-L-Glu-L-Trp-COOH had an antioxidant effect, which was confirmed by an increase in superoxide dismutase activity after intraperitoneal and intracutaneous administration, also the level of malon dialdehyde lowered after intracutaneous administration of the peptide. Thus, the synthetic peptide of general formula HN-D-Ala-L-Glu-L-Trp-COOH can be used as an agent stimulating healing of skin wounds, immune and oxidant protection of an organism.

Description

The invention relates to medicine (pharmacology, traumatology, surgery), veterinary medicine and can be used as a means of stimulating the healing of skin wounds and the immune function of the body.

Known reporters that stimulate the healing of skin wounds: methyluracil, pentoxyl, betamecil, dexpanthenol and others. However, with injuries, many of them affect only certain links of pathogenesis (Russian Drug Register (RLS). Encyclopedia of Medicines, 2004, 11th issue, pp. 1155-1156). In this regard, it is often necessary to use complex therapy. However, this approach to the treatment of wounds and injuries can have unpredictable consequences of drug interactions with polypharmacy. At the same time, many drugs in this group have side effects that limit their use (Belousov Yu.B., Moiseev V.S., Lepakhin V.K. Clinical Pharmacology and Pharmacotherapy. M .: Universum Publishint, 1997, 532 pp.) .

Regulatory peptides are also known that have a reparative and wound healing effect (Ryukai, L. Thy. And Tibetan SNK aib Ikky tetoboebd // 1. Vyutaet δει. Ролут. Еб. 2008. Уо1. 19, No. 8. P. 969-988) . The advantage of peptide agents is the fact that they are inactivated by proteolysis and break down to ordinary amino acids, which are not foreign (xenobiotics) for the body and, therefore, do not have side effects (Belousov Yu.B., Kukes V.G., Lepakhin V .K., Petrov V.I., Clinical Pharmacology, National Guide, M. Geotar-Media, 2009, 965 pp.).

The well-known peptide preparation - thymogen (dipeptide Н ₂ Н-Е-С1ц-Е-Тгр-СОО) is an immunomodulator, but it is often used as a repair agent for stimulating wound healing, as well as for treating injuries of various etiologies (Russian Drug Register (RLS) ). Encyclopedia of Medicines, 2004, 11th issue, pp. 849-850). An example of the use of thymogen for stimulating the healing of skin wounds and activating the function of blood neutrophils (the synergistic immunotropic and reparative effect of thymogen, dalargin and the regulatory peptide ^ Υ-ΗΙδ-6Υδ in experimental skin injury / V. Yu. Cherdakov can be given as an analogue of the same purpose). , M.Yu. Smakhtin, G.M. Dubrovin, I.I. Bobyntsev // Kursk Scientific and Practical Bulletin Man and His Health. 2013. No. 1. P. 38-42). But the disadvantages of this drug include fast inactivation under the action of proteolytic enzymes (proteases), the level of which is especially high in the inflammation zone (Pohkop, M.C. \ Waipb есηGesOop: and Gabige o! Teoibb Lea baed saikeb ba ap 1tba1apse o! Bacepa S.C. Rohkop // 8th ed. Cbn.Ob.At. 1997. Wo1. 77. For proteases, it is enough to destroy only one peptide bond between two amino acids, which explains the short half-life of this peptide (TA. Vegdeop, H. p. Chap, V.S. Chab1ec, I. So1b Opa1 akotrbop enbapsetep! OG b1rebbe b-Cb-b-b- Ttr-OH Lb Lb1b apb d1usoku sofidabop // Vyuro1utetk. 2008.V. 90, No. 5. P. 633-643). Therefore, this drug may not be effective enough, especially when applied topically (8a1bakbteboua, Ζ.Έ 1tipotobi1abpd asbubu oG 1todep sh asi! E rapsgeabbk / Ζ.Έ 8a1bakbteboua // Worth. Rbap. 2000. T. 69, No. 6. P. 35 -36).

All of the above necessitates the search for new peptide reparative agents with higher biological activity.

The technical result of the invention is to create a new synthetic wound healing agent with higher biological activity.

The technical result of the invention is achieved in that a peptide of general formula H ₂ UN-A1a-E-C1i-E-Ttr-COOH is used as a wound healing agent.

Due to the introduction of the peptide from the Ν-terminus of the amino acid E-A1a (Ό-alanine), the wound healing activity is enhanced, probably due to the fact that the introduction of Ό-amino acids increases their resistance to proteases (Steuep N.M., Ee \ U1eb Ό. 1piepse og rerbbek k1gis1iga11u te1a! Eb 1o ASTN apb M8N op asbuye auo1bapsse ta1k // Rtopbegk oG Nogtop geekebs. 1977, ν. 4, p. 140-152).

The peptide proposed as a wound healing agent is obtained by chemical synthesis from individual amino acids. The peptide can be synthesized in any peptide synthesis laboratory. We used a preparation synthesized at the Research Institute of Chemistry of St. Petersburg State University. As a comparison drug, we used thymogen having the formula H ₂ Yu E-C1i-L-Tgr-COOH, which is a publicly available pharmaceutical product and is manufactured by MBNPK CITOMED ZAO (Russia).

Identification of the effects of the drug was carried out on Wistar rats weighing 180-220 g. The wound process was modeled under chloral hydrate anesthesia (300 mg / kg ip). In this case, full-layer wounds of standard size (1 cm ² ) were applied to the withers of animals (Amiraslanov Yu.A., Matasov V.M., Khotinyan V.F. Treatment of wounds in a controlled abacterial environment. M., 1981. 157 p.).

Peptides were administered once a day parenterally (intraperitoneally or intradermally): thymogen in the recommended therapeutic dose of 1 μg per 1 kg of body weight (Russian Drug Register (RLS). Encyclopedia of drugs, 2004, 11th issue, pp. 849-850), and thymogen analog (peptide with the general formula H ^ -O-AN-E-SSh-E-Ttr-COOH) was administered in an equimolar single dose of 1.2 μg / kg. The corresponding weighed peptide was dissolved in a sterile isotonic sodium chloride solution and was administered to animals in a volume of 0.1 ml ten times (once a day) with an interval of 24 hours between injections. The last injection was carried out 12 hours before the animals were withdrawn from the experiment. Rats control

- 1,025,690 groups were injected with an isotonic solution of sodium chloride in an equivalent volume over the same period of time.

To assess the healing activity of skin wounds, their histological examination was performed. The material taken for the study was fixed in a 10% solution of neutral formalin. After dehydration and paraffin embedding, sections 7–10 μm thick were made, which were then stained with hematoxylin-eosin. When morphometry was performed using a LeCa CME light microscope, the percentage of fibroblasts, which are known to be directly involved in the wound healing process, was determined in the papillary layer of the skin per 100 cells (G. Savostyanov, Fundamentals of structural histology. Spatial organization of epithelia. St. Petersburg: Nauka, 2005. 375 from.).

An important role in wound healing is also played by the immune function and especially the activity of neutrophils. As you know, they are the first of the phagocytes to come to the focus of inflammation, and the probability of suppuration of the wound and, consequently, the duration of the wound process (Freidlin I.S., Totolyan A.A. Cells of the immune system. St. Petersburg, Nauka, 2001. 390 p. .). The phagocytic activity of blood neutrophils was investigated after their incubation with latex (Medvedev, A.N. A method for studying the absorption phase of phagocytosis // Lab. Business. 1991. No. 2. P. 19-20), determining the phagocytic number (PS) (average number of absorbed particles of latex per phagocyte) in smears stained according to Romanovsky. In each smear, 100 neutrophils were counted. (Fremel G. Immunological methods. M .: Medicine, 1987. 472 p.). Oxygen-dependent activity of neutrophils was determined by the nitro-blue tetrazolium reduction reaction (NBT test) using MiShzsap MS 400 (Eab8u81et8, Finland) (Zinkin V.Yu., Godkov M.A. diagnostician. 2004. No. 8. P. 26-29).

Damage to tissues and organs causes the development of oxidative stress with increased formation of reactive oxygen species (V.K. Kazimirko, V.I. Maltsev, V.Yu. Butylin, NI Gorobets. Free radical oxidation and antioxidant therapy. Morion, 2004. 160. 160 from.). Therefore, in injuries, it is also important to identify the effects of new drug substances in relation to the activity of free radical reactions and the antioxidant system of the body.

To identify antioxidant effects, the activity of superoxide dismutase (SOD) was determined (Kostyuk V.A., Potapov A.N., Kovaleva Zh.V. (1990). Issued by Chemistry No. 2) and the level of malondialdehyde (MDA) in the blood ( IFAT, M. TYoagIshts Asl ua1ie op AEGIS otodepa1e og ta! az ratashe1et og Ιίρίά retoh16a1yup ίη adschd, CC1 ₄ sh1ohyua1yup APB uyatsh beytepez E / M. IFAT, M. isYata // Vyusyet. MEB. 1980. Uo1. 23, number 3 P. 302-311) using an Are1 330 спект spectrophotometer (Japan).

An example of a specific implementation.

Example 1. The effects of the peptide H ₂ X-O-A1a-B-C1i-B-Tze-COOH with intraperitoneal administration.

To Wistar rats (10 animals) under chloral hydrate anesthesia (300 mg / kg intraperitoneally), standard-size full-layer wounds (1 cm ² ) were modeled at the withers (Amiraslanov Yu.A., Matasov V.M., Khotinyan V.F. Treatment of wounds in a controlled abacterial medium. M., 1981. 157 p.). A peptide with the general formula H ₂ X-O-A1a-B-S1i-B-Tgr-COOH was dissolved in a sterile isotonic sodium chloride solution and was administered to animals in a volume of 0.1 ml intraperitoneally in a single dose of 1.2 μg / kg of weight ten times ( once a day) with an interval of 24 hours between injections. The last injection was carried out 12 hours before the animals were withdrawn from the experiment. The day after the last administration, rats were sacrificed by bleeding under chloral hydrate anesthesia. The objects of study were blood and skin tissue.

The revealed effects are presented in table. 1 and 2.

Table 1

The effect of the peptide H ₂ X-E-A1a-B-C1i-B-Tg ^ e-COOH on the number of fibroblasts (M ± t, n = 10) in the papillary layer of the skin with intraperitoneal administration under conditions of skin wounds

No. p / p Experience Conditions Fibroblasts,% one. Control (No. C1) 29.3 ± 1.2 2. Thymogen 29.6 ± 1.7 3. H ₂ P-O-A1a-E-O1i-b-Tgr-COOH 34.5 ± 2.4 * ¹ · ²

Note: * shows significant differences, p <0.05, and the adjacent figure of the number of comparison groups; p - the number of animals in the group.

Under conditions of skin wounds with intraperitoneal administration, the peptide H ₂ X-E-A1a-B-C1i-B-Tg ^ e-COOH increases the number of fibroblasts in the skin, which indicates its wound healing activity (Table 1).

- 2 025690

table 2

Immunotropic and antioxidant effects (M ± m, n = 10) of the peptide H ₂ X-O-A1a-T-O1i-L-Tgr-COOH in conditions of skin wounds with intraperitoneal administration

No. Experience Conditions Immune function Antioxidant function PS, abs. HCT, op Superoxide MDA P/ P dismutase conv. units μmol / l one. The control (NaC1) 4.53 ± 0.43 0.22 ± 0.015 2.32 ± 0.12 2.26 ± 0.21 2. Thymogen 6.54 ± 0.89 * ' 0.242 ± 0.023 2.71 ± 0.11 * ‘ 2.04 ± 0.11 3. Η ₂ Ν-ϋ-Αΐ3-b-C1i-b-Tgr- COOH 5.7 ± 0.44 * ' 0.314 ± 0.009 * '' ² 3.21 ± 0.16 * ^1.2 1.94 ± 0.15

Note: * shows significant differences, p <0.05, and the adjacent figure of the number of comparison groups; p - the number of animals in the group.

Under skin wounds with intraperitoneal administration, the peptide H ₂ L-O-L1a-T-O1i-T-Tgr-COOH stimulates the function of blood neutrophils (PS, HCT) and increases the activity of SOD, which indicates its immunostimulating and antioxidant activity. Moreover, it has more pronounced effects compared with thymogen (table. 2).

Example 2. The effects of the peptide H ₂ X-O-A1a-T-O1i-L-Tgr-COOH with intradermal (local) administration.

To Wistar rats (10 animals) under chloral hydrate anesthesia (300 mg / kg intraperitoneally), standard-size full-layer wounds (1 cm ² ) were modeled at the withers (Amiraslanov Yu.A., Matasov V.M., Khotinyan V.F. Treatment of wounds in a controlled abacterial medium. M., 1981. 157 p.). A peptide with the general formula H ₂ X-O-A1a-T-O1i-L-Tgr-COOH was dissolved in a sterile isotonic sodium chloride solution and was administered to animals in a volume of 0.1 ml intradermally at a single dose of 1.2 μg / kg ten times ( once a day) with an interval of 24 hours between injections. The last injection was carried out 12 hours before the animals were withdrawn from the experiment. The day after the last administration, rats were sacrificed by bleeding under chloral hydrate anesthesia. The objects of study were blood and skin tissue.

The revealed effects are presented in table. 3 and 4.

Table 3

The effect of the peptide H ₂ X-P-A1a-T-O1i-T-Tgr-COOH on the number of fibroblasts (M ± t, n = 10) in the papillary layer of the skin when administered intradermally under conditions of skin wounds

No. p / p Experience Conditions Fibroblasts,% one. Control (PaCl) 36.5 ± 4.4 2. Thymogen 45.9 ± 1.9 * ' 3. H ₂ H-O-A1a-b-C1i-b-Tgr-COOH 52.3 ± 1.6 * '' ²

Note: * shows significant differences, p <0.05, and the adjacent figure of the number of comparison groups; p - the number of animals in the group.

Under conditions of skin wounds with intradermal administration, the peptide H ₂ X-O-A1a-T-O1i-L-Tgr-COOH increases the number of fibroblasts in the skin, which indicates its wound healing activity. Moreover, it has a more pronounced effect compared to thymogen (table. 3).

- 3 025690

Table 4

Immunotropic and antioxidant effects (M ± t, n = 10) of the peptide Н ₂ Ы-О-А1а-Е-О1ц-Е-Тгр-СОО in conditions of skin wounds with intradermal administration

No. P/ P Experience Conditions Immune function Antioxidant function PS, abs. HCT, op Superoxide dismutase conv. units MDA μmol / l I. The control (Chas1) 4.53 ± 0.43 0.22 ± 0.015 2.32 ± 0.12 2.26 ± 0.21 2. Thymogen 6.89 ± 0.87 * ' 0.393 ± 0.017 * ' 3.0 ± 0.20 * ' 1.68 ± 0.17 * ‘ 3. H ₂ Y-O-A1-L-C1i-1.-Tgr-COOH 6.27 ± 0.84 * ' 0.347 ± 0.012 * ¹ 3.12 ± 0.16 * ‘ 1.0 ± 0.15 * '' ²

Note: * shows significant differences, p <0.05, and the adjacent figure of the number of comparison groups; p - the number of animals in the group.

When administered intradermally, the peptide Н ₂ Н-О-А1а-Е-О1и-Е-Тгр-СОО stimulates the function of blood neutrophils (PS, HCT), increases the activity of SOD and decreases the level of MDA, which indicates its immunostimulating and antioxidant activity. Moreover, in relation to the level of MDA, it has a more pronounced effect compared to the thymogen (table. 4).

Thus, the peptide having the formula H ₂ N-O-A1a-E-O1i-E-Tgr-COOH stimulates wound healing processes, and at the same time it has immunostimulating and antioxidant effects both with intraperitoneal and intradermal (local) introductions.

Claims (1)

CLAIM The use of a synthetic peptide of the general formula H ₂ M-O-A1a-E-O1c-E-Tgr-COOH as a wound healing agent with immunostimulating and antioxidant effects.