RU2634576C1 - Method for tissue regeneration stimulation - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: tissue regeneration is stimulated by modeling a cutaneous wound in a mouse with external application of SDF-1 from the 1st to the 7th day of the wound process once a day at a dose of 5 ng and subcutaneous administration of G-CSF for 3 days 1 time per day in a dose of 125 mcg/kg (microgram/kilogram) starting from the 5th day after wound modeling.

EFFECT: stimulation of skin tissues regeneration.

2 tbl, 1 ex

Description

The invention relates to medicine, specifically to pharmacology and cell technology.

There are a large number of ways to stimulate tissue regeneration using various pharmacological agents [1, 2].

A known method of stimulating the regeneration of tissues (lung tissue) by introducing SDF-1 - factor (stromal cell derived factor-1) into the damage zone against the background of a preliminary course systemic introduction of G-CSF (granulocyte-colony stimulating factor) into the body [3]. This method is the closest to the claimed and selected as a prototype.

The disadvantage of the prototype method is its lack of effectiveness and limitation of indications for use in case of damage to lung tissue.

The problem solved by this invention is to increase the efficiency of the method, as well as expanding the indications for its use.

The task is achieved by a technical solution, which consists in stimulating tissue regeneration when modeling a skin wound using external application of SDF-1 from 1 to 7 days of the wound healing process 1 time per day at a dose of 5 ng (nanograms) and subcutaneous administration of G-CSF for 3 days 1 time per day at a dose of 125 mcg / kg (micrograms / kilogram) starting from 5 days after modeling the wound.

New in the present invention is the stimulation of tissue regeneration with the introduction of G-CSF 1 time per day at a dose of 125 μg / kg for 3 days in the body starting 5 days after modeling the wound on the background of external use of SDF-1 from 1 to 7 days of wound healing at a dose of 5 ng (nanograms).

The information obtained in recent years about the properties and patterns of life of poly (multi) potent progenitor cells has opened the possibility of developing a new direction in the treatment of many diseases - cell therapy. The most physiological approach to solving the problems of regenerative medicine is the pharmacological stimulation of endogenous stem cells (SC) [4].

There is a known possibility of stimulating the mobilization of stem cells of depot tissues into peripheral blood using granulocyte colony stimulating factor (G-CSF, G-CSF) [3, 4], as well as the ability of SDF-1 to bind progenitor cells of various classes and thereby affect their migration and homing [3]. Given these circumstances, a method was developed to stimulate the regeneration of damaged lung tissue by introducing SDF-1 into the zone of lung damage after a course of systemic administration of G-CSF [3]. The use of the described sequence of use of these substances, namely the preliminary introduction of G-CSF, seems reasonable and appropriate [3]. Since it is assumed that SDF-1 provides homing already mobilized into the blood using G-CSF stem cells (SC). Moreover, the possibility of increasing the efficiency of the method by changing the sequence and timing of the use of these pharmacological substances has not been studied. In addition, the known significant specificity of the functioning of various body tissues [4, 5] does not allow one to rely on the universality of the known method and the possibility of its effective application in relation to the skin.

The fact of stimulation of tissue regeneration with the introduction of G-CSF after using SDF-1 to achieve pronounced therapeutic effects for a specialist is not obvious. The experiment showed unpredictable results.

New properties do not follow explicitly from the prior art in this field. An identical set of features was not found in the study of the prior art in patent and medical literature.

The present invention can be used in medicine.

Based on the foregoing, the claimed technical solution should be considered relevant criteria: "Novelty", "Inventive step", "Industrial applicability".

The method is as follows.

From 1 to 7 days after modeling a skin wound, a SDF-1 is applied externally to a laboratory animal at a dose of 5 ng starting 5 days after modeling a skin wound, G-CSF is administered subcutaneously for 3 days once a day at a dose of 125 μg / kg

The proposed method was studied in experiments on outbred mice in the amount of 56 pieces, weighing 20-22 g. Animals were obtained from the Department of Experimental Biological Models NIIIFiRM them. E.D. Goldberg Tomsk Scientific and Technical Center. The studies were carried out in accordance with the rules of laboratory practice (GLP), Order of the Ministry of Health and Social Development of the Russian Federation No. 708n dated 08/23/2010 “On approval of the rules of laboratory practice”, Federal Law of April 12, 2010 No. 61-FZ “On the circulation of medicines”, “Guidelines on preclinical studies of new drugs ”(Moscow, 2013).

Example 1

The effectiveness of tissue regeneration of the proposed method was studied on a model of planar skin wounds [6, 7]. A skin flap 10 × 10 mm in size was cut out on the depilated back area in mice under mild ether anesthesia. To simulate longer healing, the scabs were removed from the experimental wound regularly (every other day).

The substances used were administered to experimental animals in three regimens.

1 circuit. After modeling the skin wound, laboratory microbes from day 1 to day 7 externally applied 50 microliters of a solution of SDF-1 (SDF-1 alfa, STEMCELL Technologies, Canada) at a dose of 5 ng. Starting from the 5th day of the experiment, G-CSF solution (Neupogen, Hoffman-la Roche, Switzerland) was injected subcutaneously for 3 days once a day at a dose of 125 μg / kg (0.2 ml). Solutions of pharmacological substances were prepared in accordance with the instructions for use by manufacturers.

2 circuit. According to the prototype method, after modeling a skin wound, laboratory microbes from day 1 to day 7 externally applied 50 microliters of SDF-1 solution (SDF-1 alfa, STEMCELL Technologies, Canada) at a dose of 5 ng. Before modeling the skin wound, the animals were injected subcutaneously with G-CSF solution (Neupogen, Hoffman-la Roche, Switzerland) once a day at a dose of 125 μg / kg (0.2 ml) for 5 days.

3 circuit. In addition, after modeling a skin wound, 50 microliters of SDF-1 solution (SDF-1 alfa, STEMCELL Technologies, Canada) were applied externally to laboratory animals from 1 to 7 days in a dose of 5 ng against the background of subcutaneous administration (starting simultaneously with the start of SDF application -1) for 3 days, a solution of G-CSF (Neupogen, Hoffman-la Roche, Switzerland) once a day at a dose of 125 μg / kg (0.2 ml).

Used doses of pharmacological agents were selected in preliminary experiments as the most effective.

Control animals in similar modes and similar methods received equivalent volumes of saline.

The criteria for stimulating regeneration of action were the average diameter of the wound throughout the experiment, as well as the healing rate in% and mm / day (millimeters per day) from 1 to 6 days and from 6 to 13 days of the wound healing process. The results were processed by the method of variation statistics using the Student t test and the nonparametric Mann-Whitney U test.

During the experiment, wound healing in control animals was noted by the 14th day of the experiment. The use of pharmacological substances according to the prototype method had practically no effect on the rate of skin regeneration, the only exception was the decrease in the average diameter of the wound on the 6th day of the experiment (table. 1, 2). At the same time, the simultaneous start of the use of pharmacological substances led to an accelerated course of reparative processes and the restoration of damage by 13 days (Table 1). However, there were no statistically significant differences with respect to the calculated rates of wound healing in the first (1-6 days) and in the second (6-13 days) between this experimental and control groups (Table 2).

At the same time, the use of pharmacological substances according to the proposed method caused a significant stimulation of skin regeneration. In this case, the healing of the defect was observed by the 11th day of the experiment. Moreover, throughout the experiment, a marked decrease in the size of wounds and an increase in the speed of their healing were recorded (Tables 1, 2).

In general, the results indicate the most effective use of SDF-1 and G-CSF to stimulate the healing process of the skin with the introduction of G-CSF into the body in the second period of the wound process - in the regeneration phase [2, 5] against the background of continued therapy with SDF- 1 in the affected area, starting immediately after modeling a skin defect. The basis for increasing the effectiveness of the proposed method, obviously, is to ensure maximum synchronization of the processes of epithelization and maturation of granulation tissue in the wound. Apparently, SDF-1 in this case promotes the migration of predominantly stromal progenitor cells into the affected area [4, 8] from nearby tissues, which ensures the rapid formation of granulations necessary for engraftment and growth of epithelial cells [9], which are formed in their own first of all, from the stem cells of tissues depot migrating under the influence of G-CSF [4, 8]. A change in the sequence of application of these funds is probably accompanied by the separation of the processes of epithelization and maturation of granulation tissue [9]. In the case of preliminary administration of G-CSF (or simultaneous with the initiation of therapy with SDF-1), mobilized G-CSF progenitor cells are likely to fall from the blood into conditions in which the realization of their regenerative potential is significantly difficult due to the lack of an adequate microenvironment - granulation tissue . Moreover, the low level of “survival” of SCs that migrated to the lesion zone ultimately leads to a rapid depletion of the pool of mobilized SCs circulating in the blood [4, 8] and a deficiency of the initial elements of the epithelium during the period of sufficient development of granulations.

Cited literature

1. Mashkovsky M.D. Medicines: 15th ed. - M.: Public Association "Publishing House New Wave", 2008. - 1206 p.

2. Kotelnikov V.P. Wounds and their treatment. - M .: Knowledge. 1991 .-- 123 p.

3. Hannoush E.J., Sifri Z.C., Elhassan I.O., e.a. Impact of enhanced mobilization of bone marrow derived cells to site of injury // J Trauma. 2011, V.71, N2. - P. 283-289.

4. Digay A.M., Zyuzkov G.N. Cell therapy: new approaches // Science in Russia. - Moscow: Publishing House "Science", 2009. - Volume. 169. - No. 1. S. 4-8.

5. Babaeva A.G. Regeneration: facts and perspectives. - M. Publishing House of the Russian Academy of Medical Sciences, 2009 .-- 336 p.

6. Guidelines for the study of dermatotropic drugs // Guidelines for preclinical studies of new drugs / Ed. A.N. Mironova. - M.: Grif and K, 2013 .-- S. 738-745 (944 p.).

7. Zyuzkov G.N., Povetieva T.N., Semenov A.A. and others. Patent (RU) for the invention No. 2475260 "Means with regenerative activity" (publ. 02.20.2013, Bull. No. 5).

8. Digay A.M., Zyuzkov G.N., Zhdanov V.V. and others. Immobilized granulocyte colony stimulating factor. Pharmacological properties and prospects of use. - Tomsk: Publishing House: LLC Printing Manufactory, 2011. - 149 p.

9. Anichkov N.N., Volkova K.G., Garshin V.G. Morphology of wound healing. - M .: Medgiz, 1951. - 123 p.

Claims (1)

A method of stimulating tissue regeneration, which consists in introducing SDF-1 into the damage zone and systemically introducing G-CSF into the mouse organism, in which tissue regeneration is stimulated when modeling a skin wound in a mouse using external application of SDF-1 from 1 to 7- e day of the wound healing process 1 time per day at a dose of 5 ng (nanograms) and subcutaneous administration of G-CSF for 3 days 1 time per day at a dose of 125 μg / kg (micrograms / kilogram) starting from 5 days after modeling the wound.