RU2557971C2 - Method of influencing functional activity of cannabinoid receptor - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, namely to therapy and pharmaceutics, and deals with an influence on the functional activity of a cannabinoid receptor. For this purpose an activated-potentiated form of antibodies to the human cannabinoid receptor is introduced into an organism with the application of an activated-potentiated form of antibodies to an intact molecule or a polypeptide fragment of the human cannabinoid receptor of type I.

EFFECT: method ensures the effective correction of endocannabinoid system impairments with the absence of side effects.

4 cl, 1 tbl, 1 ex

Description

The invention relates to medicine and can be used to increase the effectiveness of the functional activity of the cannabinoid receptor.

The human cannabinoid receptors are known in the art (Ken Mackie. Cannabinoid receptors as therapeutic targets. The Annual Review of Pharmacology and Toxicology, 2006. 46: 101-122), which are present both in the central nervous system and in the peripheral nervous system, and together with endocannabinoids are a single endocannabinoid system.

The drug “Rimonabant” (Acomplia) is known from the prior art, by its mechanism of action it is an antagonist of cannabinoid receptors (CB 1), which has a central effect on the brain, reducing appetite (Despres JP, Golay A, Sjostrom L; Rimonabant in Obesity-Lipids Study Group. Effects of rimonabant on metabolic risk factors in overweight patients with dyslipidemia. The New England Journal of Medicine. 2005 Nov.17; 353 (20): 2121-34). However, this drug, which affects the functional activity of the cannabinoid receptor, has many side effects.

The invention is aimed at creating an effective way of influencing the functional activity of the cannabinoid receptor, which provides correction of disorders of the endocannabinoid system.

The solution to this problem is provided by the fact that in the method of influencing the functional activity of the cannabinoid receptor according to the invention, an activated - potentiated form of antibodies to the human cannabinoid receptor is obtained, which is obtained by sequential multiple dilutions of the matrix solution of antibodies to the human cannabinoid receptor in aqueous or aqueous alcohol solvent and intermediate external mechanical stress.

In this case, an activated - potentiated form of antibodies to the whole molecule or polypeptide fragment of the human type I cannabinoid receptor is used.

Preferably, an aqueous or aqueous-alcoholic solution of an activated — potentiated form of human type I cannabinoid receptor antibody is used, obtained by repeated serial dilution — uniformly reducing the concentration of the matrix solution of affinity-purified human type I cannabinoid receptor with a concentration of 0.5 ÷ 5.0 mg / ml and intermediate external exposure.

In this case, a mixture of various, mainly hundred, homeopathic dilutions obtained by repeatedly sequentially uniformly reducing the concentration of the matrix solution of affinity-purified antibodies to the cannabinoid receptor type I person and an intermediate external effect is used.

It is preferable to use an activated - potentiated form of antibodies to the whole molecule of the human cannabinoid receptor type I or a polypeptide fragment with a sequence selected, for example, from the following group:

QRGTQKSIII;

EKLQSVCSDIFPHIDETYL;

IQRGTQKSIIIHTSEDGKVQVTRPDQARM;

KAHSHAVRMIQRGTQKSIIIHTSEDGKVQVTRPDQARMDIRLAKT;

MSVSTDTSAEAL;

TEFYNKSLSSFKENEENIQCGENFMDIECFMVLNPS;

AQPLDNSMGDSDCLHKHAN;

GTQKSIIIHTSEDG;

MTAGDNPQLVPADQVNITEFYNKSLSSFKENEENIQCGENFMDIEC FMVLN;

VVAFCLMWTIAIVI;

EFYNKSLSSFKENEENIQCGENFMDIECFMVLNPSQQLAIAVLSLTL;

NEENIQCGE;

GSPFQEKMTAGDNPQLVPADQVNITEFYNKSL;

AYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWN.

In this case, the activated - potentiated form of antibodies to the human type I cannabinoid receptor is used in an ultra-low dose obtained by super-dilution of the initial matrix solution in 100 ¹² , 100 ³⁰ and 100 ²⁰⁰ times.

The proposed introduction into the body of an activated - potentiated form of antibodies to the human cannabinoid receptor (i.e., forms of antibodies to the human cannabinoid receptor, prepared according to the homeopathic technology of potentiation by repeated serial dilution and intermediate external action - vertical shaking, which is active in pharmacological models and / or clinical methods of influencing the functional activity of the cannabinoid receptor) provides an effective core rection of disorders of the endocannabinoid system without any side effects, which is confirmed experimentally on adequate models.

In addition, the claimed invention expands the arsenal of technical means for correcting disorders of the endocannabinoid system without side effects, including as part of complex therapy.

The claimed method is implemented as follows.

To prepare a homeopathically activated - potentiated form of antibodies to the human cannabinoid receptor, monoclonal or, mainly, polyclonal antibodies are used, which can be obtained using known technologies - methods described, for example, in the book: Immunological methods, ed. G. Frimel, M., "Medicine", 1987, S. 9-33; or, for example, in an article by Laffly E., Sodoyer R. Hum. Antibodies. Monoclonal and recombinant antibodies, 30 years after. - 2005 - Vol.14. - N 1-2. P.33-55.

Monoclonal antibodies are obtained, for example, using hybridoma technology. Moreover, the initial stage of the process includes immunization, based on the principles already developed in the preparation of polyclonal antisera. Further stages of the work include obtaining hybrid cells producing clones of antibodies of the same specificity. Their isolation in an individual form is carried out by the same methods as in the case of polyclonal antisera.

Polyclonal antibodies can be obtained by active immunization of animals. To do this, according to a specially developed scheme, the animals are given a series of injections of the substance required by the invention, an antigen: a human type I cannabinoid receptor. As a result of such a procedure, a monospecific antiserum with a high content of antibodies is obtained, which is used to obtain an activated - potentiated form. If necessary, the antibodies present in the antiserum are purified, for example, by affinity chromatography, using fractionation by salt precipitation or ion exchange chromatography.

For immunization of rabbits, you can use as an immunogen (antigen) adjuvant and the whole molecule of the cannabinoid receptor type I person of the following sequence:

MKSILDGLAD TTFRTITTDL LYVGSNDIQY EDIKGDMASK

LGYFPQKFPL TSFRGSPFQE KMTAGDNPQL VPADQVNITE

FYNKSLSSFK ENEENIQCGE NFMDIECFMV LNPSQQLAIA

VLSLTLGTFT VLENLLVLCV ILHSRSLRCR PSYHFIGSLA

VADLLGSVIF VYSFIDFHVF HRKDSRNVFL FKLGGVTASF

TASVGSLFLT AIDRYISIHR PLAYKRIVTR PKAVVAFCLM

WTIAIVIAVL PLLGWNCEKL QSVCSDIFPH IDETYLMFWI

GVTSVLLLFI VYAYMYILWK AHSHAVRMIQ RGTQKSIIIH

TSEDGKVQVT RPDQARMDIR LAKTLVLILV VLIICWGPLL

AIMVYDVFGK MNKLIKTVFA FCSMLCLLNS TVNPIIYALR

SKDLRHAFRS MFPSCEGTAQ PLDNSMGDSD CLHKHANNAA

SVHRAAESCl KSTVKIAKVT MSVSTDTSAE AL

It is possible to obtain polyclonal antibodies to the cannabinoid receptor of human type I as the immunogen use a polypeptide fragment of the cannabinoid receptor of human type I with a sequence selected, for example, from the following group:

QRGTQKSIII;

EKLQSVCSDIFPHIDETYL;

IQRGTQKSIIIHTSEDGKVQVTRPDQARM;

KAHSHAVRMIQRGTQKSIIIHTSEDGKVQVTRPDQARMDIRLAKT;

MSVSTDTSAEAL;

TEFYNKSLSSFKENEENIQCGENFMDIECFMVLNPS;

AQPLDNSMGDSDCLHKHAN;

GTQKSIIIHTSEDG;

MTAGDNPQLVPADQVNITEFYNKSLSSFKENEENIQCGENFMDIEC

FMVLN;

VVAFCLMWTIAIVI;

EFYNKSLSSFKENEENIQCGENFMDIECFMVLNPSQQLAIAVLSLTL;

NEENIQCGE;

GSPFQEKMTAGDNPQLVPADQVNITEFYNKSL;

AYKRIVTRPKAVVAFCLMWTIAIVIAVLPLLGWN

Before blood sampling for 1-3 days, 1-3 intravenous injections are performed to increase the level of antibodies. During immunization, small blood samples are taken from rabbits to evaluate the amount of antibody. The maximum level of the immune response to the administration of most soluble antigens is achieved 40-60 days after the first injection. After the end of the first cycle of immunization of rabbits for 30 days, they restore health and re-immunization, including 1-3 intravenous injections. To obtain antisera from immunized rabbits, blood is collected in a 50 ml centrifuge tube. Using a wooden spatula, the formed clots are removed from the walls of the tube and the wand is placed in the clot formed in the center of the tube. Blood is placed in a refrigerator (temperature 4 ° C) at night. The next day, the clot attached to the spatula is removed and the remaining liquid is centrifuged at 13000 g for 10 minutes. The supernatant (supernatant) is an antiserum. The resulting antiserum should be yellow. Add to the antiserum 20% (weight concentration) NaN ₃ to a final concentration of 0.02% and store until use in a frozen state at a temperature of -20 ° C. To isolate antibodies to the cannabinoid receptor type I from an antiserum of a person, type 1 absorption is performed on the solid phase in the following sequence:

1) 10 ml of rabbit antiserum is diluted 2 times with 0.15 M NaCl, 6.26 g of Na ₂ SO ₄ are added, stirred and incubated for 12-16 hours at 4 ° C;

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

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

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

The antibodies are then purified by affinity chromatography by attaching the obtained antibodies to the human type I cannabinoid receptor, which is located on an insoluble matrix, followed by elution with concentrated salt solutions.

Thus obtained buffer solution of polyclonal rabbit antibodies to cannabinoid receptor type I human, purified on antigen, with a concentration of 0.5 ÷ 5.0 mg / ml, preferably 2.0 ÷ 3.0 mg / ml, is used as a matrix (primary) solution for the subsequent preparation of the activated - potentiated form.

An activated - potentiated form of the active substance - antibodies to the human type I cannabinoid receptor in an ultra-low dose is prepared by uniformly reducing the concentration as a result of successive dilution of 1 part of the matrix solution in 9 parts (for decimal dilution), or in 99 parts (for hundredth dilution C) , or in 999 parts (for a thousandth dilution of M) of a neutral aqueous or hydroalcoholic solvent with multiple vertical shaking-potentiation (or "dynamization") of each gender chennogo dilution and using separate containers for each subsequent dilution until the required potency - the multiplicity of homeopathic dilution method (see, e.g., W. Schwabe "Homeopathic medicines", M., 1967, at s.14-29.).

For example, to prepare the 12th hundredth dilution of C 12, one part of the aforementioned matrix solution of antibodies to the cannabinoid receptor type I person with a concentration of 3.0 mg / ml is diluted in 99 parts of a neutral aqueous or aqueous-alcoholic solvent and many times (10 or more times) shake vertically-potentiate the obtained 1st hundredth C1 dilution. From the 1st hundredth C1 dilution prepare the 2nd hundredth dilution C2. This operation is repeated 11 times, obtaining the 12th hundredth dilution of C12. Thus, the 12th hundredth dilution of C12 is a solution obtained by diluting successively in different containers 12 times of the 1st part of the initial matrix solution of antibodies to human cannabinoid receptor type I with a concentration of 3.0 mg / ml in 99 parts of a neutral solvent , i.e. the solution obtained by super-dilution of the initial matrix solution in 100 to ¹² times. Similar operations with the appropriate dilution ratio are carried out to obtain dilutions of C30 and C200.

When using a mixture of various homeopathic, mainly hundred, dilutions of the activated - potentiated form of antibodies to the human type I cannabinoid receptor in an ultra-low dose of the active substance, each component of the composition (for example, C12, C30, C200) is prepared separately according to the technology described above to their penultimate dilution (respectively until C11, C29, C199) and then, in accordance with the composition of the mixture, one part of each component is introduced into one container and mixed with the required amount of solvent (with Responsibly, with 97 parts for one hundred breeding). In this case, an activated - potentiated form of antibodies to the human type I cannabinoid receptor is obtained in an ultra-low dose obtained by super-dilution of the initial matrix solution 100 ¹² , 100, 100 ²⁰⁰ times, which is equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200.

It is possible to use an activated - potentiated form of antibodies to the human type I cannabinoid receptor in an ultra-low dose of the active substance in the form of a mixture of other various homeopathic dilutions, for example decimal and / or hundred (D 20, C30, C100 or C12, C30, C50, etc. ), the effectiveness of which is determined experimentally.

When potentiating instead of shaking in the process of decreasing the concentration, it is also possible to exert external influence by ultrasound, electromagnetic or other physical effect.

The claimed method can be implemented using an activated - potentiated form of antibodies to the human type I cannabinoid receptor in an ultra-low dose in solid dosage form, which is prepared by tabletting from a tablet mass, which, for example, contains 8-30 mass. parts of lactose granules with a particle size of 150–300 μm, irrigated in a fluidized bed with an aqueous or aqueous-alcoholic solution of an activated — potentiated form of antibodies to the human type I cannabinoid receptor, and pharmaceutically acceptable additives, including 75–95 wt. parts of pure lactose, 8 ÷ 15 mass. parts of microcrystalline cellulose and 0.8 ÷ 1.2 mass. parts of magnesium stearate. The weight of the tablet may be 150 ÷ 500 mg. It is preferable to use tablets weighing 250–300 mg, which include 3.0–6.0 mg / tablet of an activated — potentiated form of water-alcohol dilutions of a substance — the active substance of polyclonal rabbit antibodies to human cannabinoid receptors of type I, purified on antigen, in an ultra-low dose obtained by super-dilution according to the above homeopathic technology of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, equivalent to a mixture of hundred-percent homeopathic dilutions C12, C30, C200 (ultra-low doses - SMD anti-KR).

To conduct experimental studies, polyclonal antibodies were used, prepared by order of a specialized pharmaceutical company.

Example 1

The activated, potentiated form of antibodies to the human type I cannabinoid receptor claimed in accordance with the invention in an ultra-low dose (the direction of action is the type I cannabinoid receptor is central) in connection with the use of special potentization technology has a sparing effect on the receptor, leading to its sensitization and, as a result this is an increase in sensitivity to endogenous cannabinoids.

This effect is confirmed by the following experiment: a study was made of the effect of the drug on the basis of an activated - potentiated form of polyclonal rabbit antibodies to the human type I cannabinoid receptor, purified on antigen, in an ultra-low dose obtained by super-dilution of the initial matrix solution 100 ¹² , 100 ³⁰ , 100 ²⁰⁰ times, the equivalent mixture of hundreds of homeopathic dilutions C12, C30, C200 (ultra-low doses - SMD anti-KR), to the functional state of type I cannabinoid receptor (in vitro) in 2 stages: in agonist mode and in mode IU antagonist.

Agonist mode

Prior to insertion into the wells of a microplate (96-well plate, well volume 250 μl), the cells were suspended in HBSS buffer (Invitrogen) containing 20 mM HEPES (pH 7.4). Cells were preincubated for 10 minutes at room temperature with the addition of 20 μl of anti-Raman SMD. After pre-incubation, NKH477 adenylate cyclase activator was added to the wells. Cells were incubated for 10 minutes at 37 ° C and lysed. A fluorescent acceptor (cAMP labeled with D2) and a fluorescent donor (antibodies to cAMP labeled with europium cryptate) were added to the wells.

As a basal control, instead of the SMD anti-CR preparation, the cell suspension was preincubated in the presence of HBSS buffer (20 μl). As a stimulated control, instead of SMD anti-CR, the cell suspension was preincubated in the presence of the reference agonist CP 55940 (20 μl).

Functional activity (by cAMP concentration) was evaluated by time-resolved homogeneous fluorescence (HTRF). The fluorescence intensity in the basal control is taken as the background, and its value is subtracted from the fluorescence intensities in the experiment (SMD anti-KR) and control (CP 55940):

The measured specific response of the cell to the introduction of SMD anti-Raman is calculated by the formula: the fluorescence intensity in the experiment (SMD anti-Raman) is the fluorescence intensity in the basal control.

The measured specific response of the cell to the introduction of a reference agonist (CP 55940) is calculated by the formula: fluorescence intensity in the control (CP 55940) - fluorescence intensity in the basal control.

The results are expressed as a percentage of the specific response of the cell to the introduction of a reference agonist in stimulated control:

% response of the reference agonist = ((measured specific response / specific response in the control on the introduction of the reference agonist) × 100%).

Antagonist mode

Prior to insertion into the wells of a microplate (96-well plate, well volume 250 μl), the cells were suspended in HBSS buffer (Invitrogen) containing 20 mM HEPES (pH 7.4). Cells were incubated for 5 minutes at room temperature with the addition of 20 μl of anti-Raman SMD. After adding the reference agonist of CP 55940 to the wells, the cells were incubated for 10 minutes at room temperature. Next, NKH477 adenylate cyclase activator was added to the wells. Cells were incubated for 10 minutes at 37 ° C and lysed. A fluorescent acceptor (cAMP labeled with D2) and a fluorescent donor (antibodies to cAMP labeled with europium cryptate) were added to the wells.

As a basal control, instead of SMD anti-Raman cell suspension, the cell suspension was incubated in the presence of the reference antagonist AM 281 (20 μl). The reference agonist CP 55940 was not added to the wells with the basal control. As a stimulated control, the cell suspension was preincubated in the presence of HBSS buffer containing 20 mM HEPES (pH 7.4) and the reference agonist CP 55940 (20 μl).

Functional activity (by cAMP concentration) was evaluated by time-resolved homogeneous fluorescence (HTRF). The fluorescence intensity in the basal control is taken as the background and its value is subtracted from the fluorescence intensities in the experiment (SMD anti-CR) and control (CP 55940).

The measured specific response of the cell to the introduction of SMD anti-Raman is calculated by the formula: the fluorescence intensity in the experiment (SMD anti-Raman) is the fluorescence intensity in the basal control.

The measured specific response of the cell to the introduction of a reference agonist (CP 55940) is calculated by the formula:

fluorescence intensity in the control (CP 55940) - fluorescence intensity in the basal control.

The results are expressed as percent inhibition of the specific response of the cell to the introduction of a reference agonist (CP 55940) in the control:

% inhibition of the response of the reference agonist = 100% - ((measured specific response / specific response in the control on the introduction of the reference agonist) × 100%)

As a result of studies, it was shown (see Table 1) that SMD anti-Raman changes the functional activity of type I cannabinoid receptor, estimated by the concentration of intracellular cAMP.

The sample (ultra-low doses of antibodies to the cannabinoid receptor type I (a mixture of homeopathic dilutions C12 + C30 + C200)) exhibits the properties of an agonist to the cannabinoid receptor type I, the severity of the modifying effect of which is 21% of the effect of the standard agonist CP55940 (the effect of the standard agonist is taken as 100% )

Table 1 Receptor A drug The content of SMD anti-Raman in the well (% by volume) (total volume of all compounds in the well is 200 μl) Agonist mode Antagonist mode Explanation of the results % agonist response Standard agonist % inhibition of agonist response Standard antagonist CB1 Type I Cannabinoid Receptor SMD antibodies to cannabinoid receptor type I (C12 + C30 + C200) 10% 21 CP 55940 0 AM 281 SMD antibodies have an agonist effect, the severity of which is 21% of the effect of a standard agonist (taken as 100%)

Claims (4)

1. A method of influencing the functional activity of the cannabinoid receptor, characterized in that an activated - potentiated form of antibodies to the human cannabinoid receptor is introduced into the body, obtained in the process of sequential multiple dilution of the matrix solution of antibodies to the human cannabinoid receptor in an aqueous or aqueous-alcoholic solvent and an intermediate external mechanical impact. 2. The method according to claim 1, characterized in that they use an activated - potentiated form of antibodies to the whole molecule or polypeptide fragment of the human type I cannabinoid receptor. 3. The method according to claim 1 or 2, characterized in that an aqueous or aqueous-alcoholic solution of an activated — potentiated form of an antibody to a human type I cannabinoid receptor is used, obtained by repeated serial dilution — uniformly reducing the concentration of a matrix solution of affinity-purified cannabinoid receptor antibodies Type I human with a concentration of 0.5 ÷ 5.0 mg / ml and intermediate external exposure. 4. The method according to claim 3, characterized in that they use a mixture of various, mainly hundred, homeopathic dilutions obtained by repeatedly sequentially uniformly reducing the concentration of the matrix solution of affinity purified antibodies to the human type I cannabinoid receptor and intermediate external exposure.