Method for connective tissue density correction

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, namely to cardiology and can be used for connective tissue density correction following myocardial infarction and pericardiotomy. The method involves triple administration of an inhibitor of a polypeptide sequence containing fragments -Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly- and -Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu- and having 100 to 240 amino-acid residues in 1.5 and 6 hours and in 3 days after infarction and pericardiotomy started.

EFFECT: use of the invention allows restructuring the forming connective tissue due to changing of the polypeptide sequence concentration in certain distances of a pathological process and thereby reducing fibrosis following myocardial infarction and pericardiotomy.

4 cl, 2 tbl, 5 dwg, 2 ex

 

The present invention relates to the field of medicine and biology.

It is known that the basis of a number of pathological processes, such as infarction, cirrhosis, adhesive disease and other conditions is the formation of excessive and unnecessary rigidity of the newly formed connective tissue.

There is a method of wound healing and promote healthy skin, including the use of peptides that originate from prefermented forms the matrix metalloproteases and their inhibitors. In the known technical solution is disclosed the use of a composition for the manufacture of medicines, promoting the healing of wounds and prevent scarring. (Anti-aging and wound healing compositions: U.S. Pat. 2318873 Grew. Federation: IPC512N 9/64, 9/99; AC 38/04, 38/43; 61L 15/32; A61P 43/00 / Quark S., Malik S., Villanueva ZH.M.; applicant and patentee, Kimberly-Clark worldwide, Inc. No. 2004103071/13; Appl. 15.08.02; publ. 10.03.08, bull. No. 7. - 76 S.).

The disadvantage of this method is only for external use, which limits the scope of their use in treatment of wound surfaces.

The closest in technical essence to the present invention is a method of obtaining growth factors protein nature, growth factor protein nature and inhibitor of proliferation of fibroblasts (the Way of gaining the growth factors protein nature, growth factor protein nature and inhibitor of proliferation of fibroblasts: Pat. 2283654 Grew. Federation: MCP AC 35/48, 35/54, 35/32, 38/02 / Afanas'ev, B.V., Wagemaker,, Zubrowska PS; applicant and patentee Afanas'ev, B.V., Wagemaker,, Zubrowska PS No. 2005109195; Appl. 30.03.05; publ. 20.09.06, bull. No. 26. - 13 S.).

The known method is as follows. Embryonic skin fibroblast line V cultured with conditioned medium obtained by culturing the embryonic fragments of human bone tissue within 8-10 weeks. The protein fraction with the fibroblast - and fibrosis-inhibiting activity, allocate chromatography, gel-filtration and centrifugation. Derived growth factor is a purified protein having the fibroblast-inhibitory activity, the severity of which ranges from 40% to 0%. The inhibition of proliferation of fibroblasts find reduction include radioactive labels.

The disadvantage of this method is the possibility of achieving suppression of proliferation of fibroblasts only in the culture medium, and low probability of achieving the target effect (from 0 to 40%), a narrow range of effectiveness - no effect at a concentration of conditioned medium to 0.5%, the stimulation of proliferation of fibroblasts at a concentration of from 1 to 5% and under the pressure of activity at a concentration of more than 20%, as well as the lack of influence on the synthetic activity of fibroblasts.

The task of the claimed invention to provide an method of controlling the morphogenesis of the connective tissue.

The technical result of the proposed method is the possibility of purposeful changes in the structure formed connective tissue, due to changes in the quantitative composition of connective tissue cells and the density of collagen fibers.

The technical result is achieved by a method for correcting density of the connective tissue is carried out by effects on fibroblasts.

Distinctive techniques of the proposed method lies in the fact that to reduce the density of the formed connective tissue in vivo injected a substance that blocks the biological activity of the polypeptide containing 3 amino acid sequence-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu- and having from 100 to 240 amino acid residue three times in 1.5, 6 hours and 3 days after the onset of myocardial infarction or pericardiotomy.

Comparative analysis of the proposed technical solutions to the prototype allows to make a conclusion on the conformity of the proposed technical solution the criteria of the invention of "novelty."

The authors of the proposed method is not known none technical the information and communications technology, the implementation of which would provide the specified density of collagen fibers in forming connective tissue.

Targeted changes in the concentration of the polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residue, at certain time intervals of the formation of the pathological process allowed the authors of the proposed method to achieve programmed density formed connective tissue.

The inventive method achieves perceived by the applicant of the technical result, namely the provision of opportunities for purposeful change patterns formed connective tissue, due to a change in the density of collagen fibers under the influence of artificially altered the concentration of the polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residue.

The difference of the proposed method lies in the fact that made an impact on the production of extracellular matrix connective tissue cells, migration and proliferation of cells.

Distinctive techniques of the proposed method consist in reducing the Broza the individual in the myocardium after myocardial infarction and pericardial after pericardiotomy.

Differences between the proposed method from famous authors find that for its implementation uses a pharmaceutical composition incorporating the inhibitor polypeptide sequence fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-, having from 100 to 240 amino acid residue, as well as pharmaceutically acceptable carrier. When this inhibitor polypeptide sequence is an antibody obtained from a sensitized animal, or artificially synthesized substance having a chemical affinity to the specified polypeptide.

Distinctive technique is that the introduction of pharmaceutical compositions spend the needy in this subject, condition or disease which is a disease or condition of the heart (myocardial infarction) or serous membranes (pericardiotomy).

The above can conclude that the technical solutions according to the criterion of "inventive step".

The method constituting the invention, intended for use in experimental and clinical medicine and biology. The possibility of its fulfillment is confirmed as described in the application techniques and equipment.

The foregoing leads to the conclusion that the claimed technical solution soo is in accordance with the criteria of the invention "industrial applicability".

Implementation of the proposed method is illustrated by examples of specific performance.

The following examples serve to illustrate but not limit the invention.

Example 1.

Laboratory animals - Wistar rats was carried out by simulation of myocardial infarction by simultaneous damage bipolar electrocoagulation kolokowski the arteries of the heart in the region of the middle third. Control of myocardial damage after modeling was performed using electrocardiography.

The natural concentration of the polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residue was changed by the introduction of inhibitor obtained from sensitized animal (rabbit). 40 Wistar rats intracardiac spent the injection of the indicated inhibitor in a dose of 2 µg (volume injection 0.1 ml) three times in 1.5, 6 hours and 3 days after myocardial infarction (main group).

In the control group used animals (45 rats), which pre-the same method was simulated myocardial infarction, and during the pathological process happened naturally.

The decrease in the concentration of the active polypeptide in the main group of animals. is in the control group was confirmed by the dynamics of its concentration in the blood. The measurements were carried out after 2, 6, 12 hours, 1, 3, 7, 14, 30 the day after surgery, enzyme-linked immunosorbent assay.

Dynamics of the level of active polypeptide in the blood of experimental animals are shown in figure 1 (Appendix to the description on the application, figure 1). Animals of the control group showed a significant increase in the level of active polypeptide with reaching a maximum at 6 hours after surgery (494.18±58.78 PG/ml), and then decrease. In animals the main group noted natural significant reduction in the level of active polypeptide in comparison with the control group at time 2 hour (is at 24.33±2.59 PG/ml in the study group compared with 159.75±14.45 PG/ml in the control group, p<0.01) and 6 hours (at 28.58±2.05 and 494.18±58.78 PG/ml, respectively, p<0.01) after surgery.

Dynamics of formation of connective tissue in the area of ischemic damage. So, in the main group within 30 days after myocardial infarction in the emerging area of postinfarction cardiosclerosis density of collagen fibers in 2 times below the control values: 19.15±1.22% compared to 41.71±1.30% (M±SE) in the control group (p<0.05). This is confirmed by the data given in table 1 and sections histological zones of infarction control (figure 2) and the core (figure 3) groups of animals (painting by van Gison, an increase of 600).

Table 1.
The relative amount of collagen fibers in the formation of post-infarction scar 30 days after myocardial infarction
No.GroupThe relative amount of collagen, % (median, 25%-75% quartiles)
1ControlAt 40.51 [40.33-44.30]
2Main18.50 [17.42-21.52]*;
Note: * - p<0.05 compared with the control group

From the presented data it follows that the inhibition activity of the polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residue, resulted in the decrease in the density of the emerging connective tissue in the field of post-infarction scar. The decrease in the density of collagen fibers in animals the main group compared to the control group allowed us to achieve a lower stiffness of the left ventricle (table 2), which reduces the likelihood of developing dia-stoliczkai heart failure.

Table 2.
The rigidity index of infarction in the groups of experimental animals at 30 days after surgery
GroupRigidity, PA/ml (median, 25% - 75% quartiles)Intergroup differences in test Mann-Whitney
ZP
Control7535.04 [7342.33-7584.95]-
Main5976.83 [5673.62-6437.64]Z=-2.19338p=0.02828

Example 2.

Laboratory animals (Wistar rats) underwent thoracotomy, the dissection of the pericardium. The natural concentration of the active polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-, modified by the introduction of the inhibitor. The inhibitor was administered by intracardiac dose of 2 mg (the amount of injection of 0.1 ml), three times in 1.5 and 6 hours and 3 days after surgery (10 animals of the main group). As a control group used animals with the natural flow after pericardiotomy (12 animals).

On the 14th and 30th day after pericardiotomy analysed and exp is the size of adhesions in the cavity of the pericardium. It is revealed that in animals of the control group adhesions in the cavity of the pericardium registered in 66.7% of cases in the study group - 10% (p<0.05) (application to the description of the application, figure 4 and 5), while its expression was significantly lower. Thus, figure 4 shows a histological slice of the heart of the animal of the control group at 30 days after surgery simulation. Paint hematoxylin-eosin, magnification 150. Adhesions in the pericardium (position A). Figure 5 - the main group, the animal on the 30th day after pericardiotomy, the absence of adhesions in the cavity of the pericardium (the position). Paint hematoxylin-eosin, magnification 150.

From the presented data it follows that the inhibition activity of the polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Lys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residue leads to inhibition of the formation of connective tissue in serous cavity after damage to the serous membranes.

Thus, the proposed method allows the correction process of the formation of connective tissue due to deliberate changes in the characteristics of the emerging connective tissue. This method can be used in medicine and biology.

1. The method for correcting density of the connective tissue in the Le of myocardial infarction or pericardiotomy, including the effects on fibroblasts, characterized in that for reducing the density of the connective tissue three times in 1.5 and 6 h and 3 days after the onset of heart attack or pericardiotomy enter the inhibitor polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu - and having from 100 to 240 amino acid residues.

2. The method according to claim 1, characterized in that the use of a pharmaceutical composition comprising the inhibitor polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-with from 100 to 240 amino acid residues, as well as pharmaceutically acceptable media.

3. The method according to claim 2, characterized in that the inhibitor polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-with from 100 to 240 amino acid residues, is an antibody obtained from a sensitized animal.

4. The method according to claim 1, characterized in that the inhibitor polypeptide sequence containing fragments-Asp-Pro-Lys-Arg-Leu-Tyr-Cys-Asn-Gly-Gly-, -Glu-Arg-Gly-Val-Val-Ser-Ile-Lys-Gly - Ala-Asn-Arg-Tyr-Leu-Ala-Met-Lys-Glu-Asp-Gly-Arg-Leu-with from 100 to 240 amino acid residues, is synthesized in vitro substance having a chemical affinity is to the specified polypeptide sequence.



 

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