Method for correction of endothelial dysfunction

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology and pharmacology, and concerns correction of endothelial dysfunction. That is ensured by administering an activated potentiated form of vascular endothelial growth factor (VEGF) antibodies in an effective amount.

EFFECT: correction of endothelial dysfunction.

5 cl, 1 tbl, 1 ex

 

The invention relates to medicine, in particular cardiovascolari, and can be used for the correction of endothelial dysfunction.

The prior art method of correction of endothelial dysfunction with a mixture of solutions of homeopathic dilutions of polyclonal antibodies to endothelial synthase nitric oxide person (EN 2306953 C1, A61K 39/395, 2007). However, the disadvantage of this invention is that the claimed method is effective in endothelial dysfunction associated with low levels of endothelial NO-synthase, and may not provide therapeutic efficacy in endothelial dysfunction with normal production of NO-synthase.

The invention is aimed at creating an effective and safe method of correction of endothelial dysfunction in preventing inflammation of the vessel wall and the development of endothelial dysfunction.

The solution of the problem provided by the fact that the method of correction of endothelial dysfunction in the body enter the activated-potentiated form of antibodies to growth factor vascular endothelium.

Moreover, the activated-potentiated form of antibodies to factor a vascular endothelial growth used in the form of activated-potentiated aqueous or aqueous-alcoholic solution, the activity of which is due to the process many who Kratovo serial dilutions in water or water-alcohol solvent in combination with an external mechanical impact - vertical shaking of every dilution.

Water or water-alcohol solution of the activated-potentiated form of antibodies to factor a vascular endothelial growth can be obtained by repeated consecutive dilution in combination with external mechanical impact - vertical shaking of every dilution of the matrix solution of antibodies to factor a vascular endothelial growth with a concentration of 0.55.0 mg/ml

Moreover, the activated-potentiated form of antibodies to factor a vascular endothelial growth used in the form of a mixture of various, mainly centesimal, in homeopathic dilutions technology.

The claimed method can be used for the correction of L-Name-induced endothelial dysfunction in rats male Wistar caused by daily intraperitoneal injection of 2 times per day L-Name at a dose of 12.5 mg/kg 4.5 mg/kg (9 ml/kg/day)by intragastric introduction of the activated-potentiated form of antibodies to the growth factor of vascular endothelium in the form of a mixture of solutions of homeopathic dilutions C12, C30, C200.

In addition, the activated-potentiated form of antibodies to factor a vascular endothelial growth used in the form of a mixture of different dilutions, prepared according to homeopathic technology, mainly in the form of a mixture of the three solutions, the preparation is different from the matrix solution, diluted, respectively, in the 10012, 10030and 100200once that is equivalent to boast of dilutions C12, C30, C200, prepared according to homeopathic technology.

Proposed an activated-potentiated form of antibodies to factor a vascular endothelial growth (i.e. the form of antibodies, prepared according to homeopathic technology exponentiation by repeated consecutive dilution in combination with external mechanical impact - vertical shaking of every dilution (see, for example, Usabe "Homeopathic medicinal product", M, 1967, p.14-29), which has activity in pharmacological models and/or clinical correction of endothelial dysfunction)provides the activation of eNOS, increased neovascularization of ischemic tissues, resulting normalized vascular tone and blood circulation, increases tolerance to physical activity, improves the General health.

The activated-potentiated form of antibodies to growth factor vascular endothelial prepared as follows. For making an activated-potentiated form of antibodies using monoclonal or polyclonal antibodies, which can be obtained by known technologies - techniques are described, for example, in the book: Immunological methods, edited by Primes, M, "Medicine", 1987, p.9-33; or, for example, article Laffly E., R. Sodoyer Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

To obtain antibodies to factor a vascular endothelial growth as an immunogen (antigen) for immunization of rabbits can be used adjuvant and whole molecule growth factor vascular endothelial following sequence:

It is possible to obtain antibodies to factor a vascular endothelial growth using as immunogen (antigen) polypeptide fragment of a growth factor, a vascular endothelial selected, for example, of the following sequences:

41-60:

71-91:

111-120:

191-210:

221-232:

Preferred for the preparation of activated-potentiated form of antibodies to growth factor vascular endothelium is the use of antibodies to the growth factor of vascular endothelium, which as a matrix (primary) solution with a concentration of 0.55.0 mg/ml is used for the subsequent preparation of the activated-potentiated form.

Polyclonal antibodies can be obtained by active immunization of animals. To do this, specially the bit is botanas scheme animals make a series of injections required in accordance with the invention substances-antigens: growth factor vascular endothelium. As a result of this procedure is to obtain monospecific antisera with high content of antibodies that is used to produce the activated-potentiated forms. If necessary, conduct the purification of antibody present in anticigarette, for example, by the method of affinity chromatography by the application of salt fractionation by precipitation or ion exchange chromatography.

Before taking the blood sample for 7-9 days spend 1-3 intravenous injection for raising antibodies. In the process of immunization in rabbits take small blood samples to estimate the number of antibodies. The maximum level of immune response to the introduction of most soluble antigens is achieved in 40-60 days after the first injection. After completion of the first cycle immunization of rabbits for 30 days to give to restore health and are reimmunization including 1-3 intravenous injection. To obtain antisera from immunized rabbits collect the blood in a centrifuge tube with a volume of 50 ml using a wooden spatula to remove from the walls of the tube formed clots and put the wand in the clot formed in the center of the tube. The blood is placed in a refrigerator (4C) overnight. The next day remove the clot adhered to the spatula, and centrifuged remaining liquid at 13000g at those who tell 10 minutes The supernatant (supernatant) is anticorodal. The resulting anticavity should be yellow. Add to anticigarette 20% (weight concentration) NaN3to a final concentration of 0.02% and stored until use in a frozen state at -20C (or without addition of NaN3- at -70C). For selection of antisera antibody to factor a vascular endothelial growth produce absorption in the solid phase in the following sequence:

1) 10 ml of rabbit antisera diluted 2 times with 0.15 M NaCl, type of 6.26 g of Na2SO4, mixed and incubated for 12-16 h at 4C;

2) the precipitation is removed by centrifugation, dissolved in 10 ml of phosphate buffer and then cialiswhat against the same buffer overnight at room temperature;

3) after removing the precipitate by centrifugation, the solution was applied to the column with DEAE-cellulose, equilibrated with phosphate buffer;

4) the fraction of antibodies determined by measuring the optical density of the eluate at 280 nm.

Then make a clearance antibodies, for example, by the method of affinity chromatography obtained by attaching antibodies to the growth factor of vascular endothelium, which is insoluble matrix followed by elution with concentrated salt solutions.

Thus obtained buffer solution p is likeonline antibodies to growth factor a vascular endothelial with a concentration of 0.55.0 mg/ml, preferably 2,03,0 mg/ml is used as the matrix (primary) solution for the subsequent preparation of the activated-potentiated form of the drug.

Monoclonal antibodies can be obtained, for example, using hybridoma technology, which involves several steps: sequential immunization of animals, preferably mice, the allocation of the immunized animal an antibody-producing b-lymphocytes from lymphoid organs of the animal (spleen, lymph nodes); the creation of a hybrid (antibody-producing b-lymphocytes hybridizing with myeloma cells); the cultivation of hybrid and selection of productive hybrid cells; screening secreted by hybridomas antibodies; cloning hybridoma cells; isolation of antibodies from the culture fluid and purification of monoclonal antibodies.

Mice make the immunization antigen - factor vascular endothelial growth mixed with adjuvant. After a few days in immunized mice produce lymphoid organs and prepare a suspension of cells. Choose the myeloma cells for hybridization with cells of immunized animals.

Cells of the immunized animal and myeloma cells are mixed and centrifuged. After centrifugation the supernatant was removed and to the precipitate add Aut polyethylene glycol (0.5 ml 50%PEG solution) and incubated. Then washed the cells from the PEG. Next, cells with myeloma cells were placed in GAT environment and cultivated.

After cultivation was allocated surviving hybridoma cells with myeloma cells of the immunized animal, which retained the ability to synthesize and secrete antibodies.

Then spent the hybrid cloning and selection of the desired clones. Survivors GAT cells subcultured in a special plastic tablets, usually containing 96 wells with a capacity of about 0.2 cm3. To each well was put in an average of 10 hybridoma cells and cultured. After several days of cultivation, the content of each well was examined for the presence of antibodies of the desired specificity. Cells from the wells containing the desired antibodies are cloned, i.e. repeatedly subcultured on the same hole, but the rate of 1 cell per well, again, were cultured and tested for the presence of the desired antibodies. The procedure was repeated 1-2 times. Thus, selected clones producing antibodies only one of the desired specificity, i.e. monoclonal antibodies.

Then spent the selection of monoclonal antibodies from the culture fluid of hybridoma cells and carried out the purification of monoclonal antibodies using methods of chromatography, preferably affinity chromatography.

Thus obtained monoclonal anti-Christ. ate to factor a vascular endothelial growth with a concentration of 0.55.0 mg/ml, preferably 2,03,0 mg/ml is used as the matrix (primary) solution for the subsequent preparation of the activated-potentiated form of the drug.

Preferred for the preparation of activated-potentiated form of antibodies to growth factor vascular endothelium is the use of a mixture of three water-alcohol dilution of initial matrix solution of antibodies, diluted, respectively, in the 10012, 10030and the 100200time, which corresponds to boast of dilutions 12, C30 and C200, prepared according to homeopathic technology.

The activated-potentiated form of antibodies to factor a vascular endothelial growth prepared by uniform reduction of the concentration in the serial dilutions 1 of the above matrix solution of 9 parts (for decimal dilution (D), or in 99 parts (for centesimal dilution), or in 999 parts (for the thousandth breeding M) neutral solvent with multiple vertical shaking ("dynamics") of each received cultivation and use separate containers for each subsequent breeding until you get the desired potency - ratio cultivation in homeopathic method (see, for example, Usabe "Homeopathic medicinal product", M, 1967, the.14-29).

External processing in the process of reducing the concentration can be realized by ultrasound, electromagnetic or other physical effects.

For example, for the preparation of the 12th centesimal dilution With 12 one part of the mentioned matrix solution of antibodies to factor a vascular endothelial growth with a concentration of 3.0 mg/ml diluted in 99 parts of a neutral aqueous or aqueous-alcoholic solvent and repeatedly (10 times or more) vertically shaken - potentiate received 1st somenoe C1 breeding. From the 1st centesimal 1 breeding prepare 2nd somenoe breeding C2. This operation is repeated 11 times, getting 12th somenoe breeding 12. Thus, 12th somenoe breeding With 12 represents the solution obtained by diluting consistently in different tanks 12 times the 1st part of the initial matrix solution of antibodies to factor a vascular endothelial growth with a concentration of 3.0 mg/ml in 99 parts of a neutral solvent, a solution prepared by dilution of the matrix solution in the 10012time. Similar operations with a corresponding multiplicity of cultivation is performed to obtain a dilution C30 and C200.

When used as a biologically active liquid component of the mixture of various, mainly centesimal, dilution of the active substance prepared according to homeopathic those is technologies each component of the composition (e.g., 12, C30, C200) prepare separately for the above-described technology to their penultimate cultivation (respectively, to obtain C11, s, 199) and then applied in accordance with the composition of the mixture in one container, one part of each component and mixed with the required amount of solvent (respectively, with 97 parts for centesimal dilution). You get the activated-potentiated form of antibodies to the growth factor of vascular endothelium in midget doses received by Sverrisdottir matrix solution, respectively, in the 10012, 10030and 100200time equivalent mixture of centesimal dilutions C12, C30 and C200, prepared according to homeopathic technology.

You can use the active substance in the form of a mixture of other various dilutions, such as decimal or centesimal, (D20, C30 C100 or C12, C30, with50 etc.), prepared according to homeopathic technology, the effectiveness of which is determined experimentally.

When potentiation instead of shaking in the process of reducing the concentration can also be external effects of ultrasound, electromagnetic or other physical effects.

For experimental studies have used monoclonal antibodies, cooked to order specialized questions than answers is logicheskoi firm.

Example 1. For experimental study of correction of endothelial dysfunction experiments were carried out on white rats male Wistar weighing 250-300 g were divided into 3 groups: group 1,control, received distilled water 9 ml/kg/day (daily intragastric administration.), group 2 received L-NAME (12.5 mg/kg), group 3 received L-NAME (12.5 mg/kg) + mixture of solutions of homeopathic dilutions of monoclonal antibody to VEGF - C12, C30, C200 (9 ml/kg/day)

Endothelial dysfunction in rats caused a (simulated) by the intraperitoneal administration of N-nitro-L-arginine methyl ester (L-NAME) at a dose of 12.5 mg/kg/day for 28 days. On the 29th day from the beginning of the experiment under General anesthesia (sodium thiopental (50 mg/kg) catheter in the left carotid artery for recording blood pressure (BP), a bolus of pharmacological agents in the right femoral vein. Hemodynamic parameters: systolic blood pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR) was measured continuously by a sensor TSD104A and hardware-software complex MR, production Biopac System, Inc., USA. Functional tests: endothelium-dependent vasodilatation (ESV) - intravenous administration of acetylcholine (ach) at a dose of 40 mcg/kg, endothelium-independent vasodilatation (ANSW) - nutrivene the e introduction of sodium nitroprusside (NP) at a dose of 30 g/kg

The degree of development of endothelial dysfunction was assessed by the coefficient of endothelial dysfunction, which is the ratio of the area of the triangle above the trend of the reduction reaction of blood pressure (BP) in response to the introduction of sodium nitroprusside (NP) to the area of the triangle above the trend of the reduction reaction of blood pressure in response to the introduction acetylcholine (ach).

When the statistical processing of the data was calculated the mean value, the standard deviation value. The differences were considered significant at p<0,05.

Processing of experimental data when carrying out functional tests on endothelium-dependent (acetylcholine 40 g/kg) and endothelium-independent (nitroprusside 30 mg/kg/in) relaxation of blood vessels in experimental animals has allowed to establish that intraperitoneal injection of L-Name at a dose of 12.5 mg/kg for 28 days caused an increase of the coefficient of endothelial dysfunction to 3.50,5 used (table 1), whereas in the control group of animals (intragastric administration of distilled water at a dose of 9 ml/kg/day) QED was 1.20,1 usled

In the group of animals that within 28 days were administered L-Name at a dose of 12.5 mg/kg, intragastrically mixture of solutions of homeopathic dilutions of monoclonal antibody to VEGF - 12, C30, C200 QED was 1.70,1 used, which was significantly lower than in the group of animals who enter the whether L-Name.

Thus, the obtained results confirm the possibility of correction L-Name-induced endothelial dysfunction in rats by L-Name at a dose of 12.5 mg/kg by intragastric introduction of a mixture of solutions of homeopathic dilutions of antibodies to VEGF - C12, C30, C200 in the dose of 9 ml/kg, resulting in the reduction of QED in this group of animals to the level of 1.70,1 usled, close to the level of QED in the control group of animals - 1,20,1 usled

Table 1
Dynamics of parameters of blood pressure and coefficient of endothelial dysfunction in the simulation of L-Name-induced endothelial dysfunction and correct it with a mixture of solutions of homeopathic dilutions of monoclonal antibody to VEGF - 12, C30, C200 in the dose of 9 ml/kg/day
Groups of animalsFunctional testGARDEN, mm HgDBP, mm Hg's vascular reactions when conducting EDVD with AH and AND with NP, usledQED, the river. units
Control (daily intragastric administration of distilled water and 9 ml/kg/day)Source 159,25,4to 124.24,71,20,1
OHto 96.96,752,03,03071,2501,1
NP113,86,1552,43617,2560,1
Treated with L-NAME (12.5 mg/kg)Source204,810*164,25,9*3,50,5*
OH111,37,464,74,3*3700,2536,9
NP118,29,961,44,411922,81838,9*
L-NAME (12.5 mg/kg) + mixture of solutions of homeopathic dilutions of monoclonal antibody to VEGF - C12, C30, C200 (9 ml/kg/day)Source212,69,4,*167,06,9*1,70,1**
OH122,47,5*to 72.34,2* 3643,3309,7
NP106,88,553,82,26119,4486,6**
Note: * - p<0,05 in comparison with the control group; ** - p<0,05 in comparison with the group of L-NAME. GARDEN - systolic blood pressure, DBP - diastolic blood pressure, S is the area above the curve recovery of blood pressure when conducting pharmacological tests, CED - factor for endothelial dysfunction.

The data obtained allow us to conclude about the possibility of effective correction of endothelial dysfunction in humans.

1. Method of correction of endothelial dysfunction, characterized by the fact that in the body enter the activated-potentiated form of antibodies to factor a vascular endothelial growth (VEGF).

2. The method according to claim 1, characterized in that the activated-potentiated form of antibodies to factor a vascular endothelial growth used in the form of activated-potentiated aqueous or aqueous-alcoholic solution, the activity of which is due to the process of multiple serial dilutions in water or water-alcohol solvent in combination with an external mechanical impact - vertical shaking of every dilution.

3. The method according to claim 2, Hara is turizmisa fact, water or water-alcohol solution of the activated-potentiated form of antibodies to factor a vascular endothelial growth obtained by repeated consecutive dilution in combination with external mechanical impact - vertical shaking of every dilution of the matrix solution of antibodies to factor a vascular endothelial growth with a concentration of 0.55.0 mg/ml

4. The method according to claim 1 or 2, characterized in that the activated-potentiated form of antibodies to factor a vascular endothelial growth used in the form of a mixture of various, mainly centesimal, in homeopathic dilutions technology.

5. The method according to claim 1 or 2, characterized in that for the correction of L-Name-induced endothelial dysfunction in rats induced by injection of L-Name at a dose of 12.5 mg/kg, using the activated-potentiated form of antibodies to the growth factor of vascular endothelium in the form of a mixture of solutions of homeopathic dilutions With 12, 30, 200.



 

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