RU2643329C1 - Synthetic immunogen for protection and treatment of psychoactive substances addiction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is an immunogen made in the form of a conjugate of a macromolecular carrier, a hapten and a synthetic polymer non-covalently bound to the conjugate, characterized by the synthetic polymer being a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of general formula I, where n is 25 to 50 mol %, and the viscosity average molecular weight Mη is 15-45 kDa. Or it is a copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of general formula II, where (m+k) is 20-90 mol %, wherein the proportion of the monomeric units of 4-vinylpyridine is 0.05 (m+k) to 0.30 (m+k), and the viscosity average molecular weight Mη is equal to 15-250 kDa.

EFFECT: formation of antibodies and a stable immune response, which leads to body detoxification with regard to psychoactive and narcotic substances by adding a synthetic polymer to the conjugate.

4 cl, 27 ex, 3 tbl

Description

The invention relates to medicine, in particular to immunology, and can be used for the treatment of drug addiction, poisoning with various poisons in industry and everyday life, in industrial accidents, etc.

The invention is a synthetic immunogen for protection against the toxic effects of narcotic and psychoactive substances. The immunogen is made in the form of a conjugate of a macromolecular carrier selected from the series: natural or artificial protein, oligo - and polypeptide, carbohydrate, lipid or nucleotide, and hapten - a narcotic or psychotropic compound associated with a synthetic polymer, characterized in that it is used as a synthetic polymer copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula I

where n is equal to 25-50 mol. %, and the average viscosity molecular weight Mη is 15-45 kDa, or a copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula II

where (m + k) is 20-90 mol. %, while the proportion of monomer units of 4-vinylpyridine is from 0.05 (m + k) to 0.30 (m + k), and the average viscosity molecular weight Mη is 15-250 kDa. A synthetic immunogen is known from the prior art for the treatment and prevention of drug abuse (in particular, opiate addiction), which is a conjugate in the form of a carrier - a natural or artificial protein and hapten - a narcotic or psychotropic compound. Moreover, it contains bovine serum albumin or synthetic polypeptides and glycoproteins as a carrier, and morphine, or codeine, or heroin, or methadone, or the like as a hapten. (see Kovalev I.E., Polevaya O.Yu. Biochemical basis of immunity to low molecular weight chemical compounds. M: Nauka, 1985).

However, this immunogen has ballast impurities that reduce its therapeutic (immunostimulating) activity and requires the use of large doses (up to 20 mg / kg). In addition, the duration of immunization reaches 6 months, and the achievement of stable and / or prolonged immunity is not always possible.

An immunogen is known, which is a conjugate of morphine derivatives (6-hemisuccinyl and 3-O-carboxymethyl) with a carrier protein (tetanus toxoid), capable of, according to the results of preclinical tests in rodents, trigger the synthesis of polyclonal antibodies to opiates - morphine and its structural analogue of heroin - as a result of repeated administration of these drugs during active immunization (EP 1767221 A2, 03/28/2007; US 20080241183 A1, 02/10/2008; US 008,008,457 B2, 08/30/2011). With the introduction of such an immunogen in the blood, antibodies to these opiates appear in high titers, which can bind drugs that enter the body from the outside in the bloodstream and thereby prevent their entry into the brain through the blood-brain barrier. As a result of a decrease in positive reinforcement, the attraction to opiates (morphine and its derivatives, in particular heroin) is dying away, which was shown on the model of intravenous self-administration of these drugs by rodents.

The disadvantage of this immunogen is the instability of such a compound, leading to spontaneous decomposition into the original components, even in small quantities.

A synthetic immunogen is known for the treatment and prevention of drug and psychoactive substance abuse (patent RU 2236257 C1, September 15, 2003), which is a conjugate in the form of a carrier - natural or artificial protein (egg albumin, human gammaglobulin, microbial toxins) and narcotic or psychotropic compounds of the following groups of low molecular weight compounds: aromatic propanamines, or isoquinolines, or diphenylmethanes, or indolamines, or derivatives of barbituric acid, or derivatives substituted x piperidines. The conjugate is modified by an immunocompetent polyelectrolyte - polyoxidonium.

The disadvantages of this immunogen can be attributed to the fact that obtaining the proposed conjugate requires the use of either highly toxic and carcinogenic benzidine, or succinyl dichloride, which has lacrimator properties, high toxicity, and corrosive activity.

There is also known a tool for the prevention and treatment of opium addiction (patent RU 2264228, 11/20/2005), which is an immunogen - primary monoclonal antibodies to opiates, in particular to the morphine-3BSA conjugate, when immunized with which anti-idiotypic (secondary ) antibodies. The mechanism of action of such a tool, according to the author, is associated with the modification of peripheral opioid receptors by the formed anti-idiotypic antibodies, which in a reflected manner can affect the brain's opioid system.

The disadvantage of this tool is the rather complicated and expensive technology for producing monoclonal antibodies (obtaining polyclonal antibodies is simpler and cheaper), as well as the fact that the effect of immunization will be observed only if a certain level of secondary (anti-idiotypic) antibodies to opiates (morphine) is present in the blood . The use of opiates, which may occur despite immunization, will not further stimulate the production of secondary (anti-idiotypic) antibodies to opiates (morphine).

As the closest analogue of the present invention, the authors consider the hapten-carrier conjugate, which is capable of producing anti-hapten antibodies for treatment of dependence on psychoactive substances, which is a conjugate of a macromolecular carrier, a hapten, which is made in the form of copolymer particles, non-covalently bound to the conjugate (European patent EP 1329226 A2, 07/23/2003).

The objective of the present invention is to provide an immunogen that does not require the use of poly (4-nitrophenyl) acrylate, hard-to-remove and flammable organic solvents, and at the same time it can cause a stable immune response in the body and reduces the toxic effect of drugs and psychoactive substances due to a steady increase in titer specific antibodies to them.

The problem is solved in that in the known synthetic immunogen for protection against the toxic effects of narcotic and psychoactive substances, which is a conjugate of a macromolecular carrier selected from a natural or artificial protein, oligo - and polypeptide, carbohydrate, lipid or nucleotide, and hapten - narcotic or psychotropic of the compound, it is new that a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone or a copolymer of 4-vinylpyridine, 2-methyl-5-vinyl is used as a synthetic polymer iridina and N-vinylpyrrolidone.

Thus, the invention provides a synthetic immunogen that induces the production of antibodies specific for psychoactive substances, which is a conjugate of a macromolecular carrier selected from the range: natural or artificial protein, oligo- or polypeptide, carbohydrate, lipid or nucleotide, and hapten - a narcotic or psychotropic compound , and associated with the conjugate synthetic polymer, characterized in that as a synthetic polymer using a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolide she is of general formula I

where n is equal to 25-50 mol. %, and the average viscosity molecular weight Mη is 15-45 kDa, or a copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula II

where (m + k) is 20-90 mol. %, while the proportion of monomer units of 4-vinylpyridine is from 0.05 (m + k) to 0.30 (m + k), and the average viscosity molecular weight Mη is 15-250 kDa.

Preferably, as the macromolecular carrier selected from the group of natural proteins, the immunogen contains human gamma globulin or albumin.

As a hapten, it may contain a narcotic or psychotropic compound selected from opiates, for example morphine, heroin, codeine; opioid receptor antagonists, for example, naltrexone naloxone; cannabinoids, for example, delta-9-tetrahydrocannabinol; amphetamines, for example amphetamine, methamphetamine, ecstasy; benzoylegonin and its derivatives, for example cocaine; barbiturates, for example barbamyl, phenobarbital; benzodiazepines, for example gidezepam; methadone, phencyclidine, tricyclic antidepressants, cotinine and their derivatives and metabolites.

Preferably, the immunogen contains naltrexone as a hapten.

The technical result of the invention is a more effective immune reaction than the closest analogue, which the claimed synthetic immunogen provides by adding to the conjugate a synthetic polymer, which is used as a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula I or copolymer 4- vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula II. Thus, under standard conditions without the use of difficult to remove and flammable substances, it is possible to reproduce the structure of the immunogen in a given range of component ratios, achieving an increase in the duration of persistent humoral immunity and an increased content of specific antibodies. In addition, as a result of using the invention, a decrease in the toxicity of the immunogen is observed, which is associated with the composition of the chemical compounds used in its preparation.

The range of ratios from 2 to 20 moles of conjugate per mole of synthetic polymer provides standardization and control of the pharmacological effect in predetermined proportions during the formation and enlargement of the structure of the immunogen. A ratio of 10: 1 mol / mol is preferred, which gives the highest possible reproducibility and stability of the synthetic immunogen.

The use of specific carriers and haptens in the proposed synthetic immunogen protects the human body from the effects of a wide range of psychoactive and narcotic substances.

Analysis of the known technical solutions allows us to conclude that the invention is not known from the prior art, which indicates its compliance with the condition of patentability "novelty".

According to the authors, the specialist is not able to come to a technical solution proposed in accordance with the present invention solely on the basis of general knowledge and analysis of the prior art, which allows us to conclude that it meets the patentability condition of "inventive step".

The possibility of preparing a synthetic immunogen from available commercially available components on traditional equipment using well-known techniques with the achievement of technical results indicates the compliance of the invention with the criterion of "industrial applicability".

The following are examples of the invention, contributing to a more accurate and complete understanding of its essence. Possible modifications and replacements are obvious to those skilled in the art, for example, those related to research protocols, which do not fall outside the scope of the invention as defined by its claims.

These examples confirm, but do not limit the scope of the claimed claims of the present invention.

1. Brief description of tables and terms

Table 1 presents data on the specificity and accessibility of epitopes of the immunogen binding to antibodies in ELISA.

Table 2 summarizes the data on the specificity of antibodies obtained by immunizing rabbits with synthetic immunogens.

Table 3 presents the compositions of the proposed synthetic immunogen and known, taken as a prototype, as well as data on the determination of immunological activity and their pharmacological action.

In the present invention, the term "inhibitor" refers to a substance that is used in a competitive enzyme-linked immunosorbent assay (ELISA) to assess the specificity and affinity of antibodies. As inhibitors, compounds are used that are structurally related to the hapten that is part of the claimed immunogen, as well as narcotic substances and a number of psychotropic drugs of a different chemical nature: methadone, pseudoephedrine, norephedrine, barbamil, canabinol, morphine, heroin, codeine, etc.

2. Synthesis of conjugates of macromolecular carriers with haptens

The introduction of carboxyl-containing derivatives that contribute to the hapten binding reaction with a macromolecular carrier for opiates, cocaine, barbiturates, benzodiazepines is carried out by their interaction with succinic acid anhydride in an absolute solvent.

The synthesis of carboxyl-containing derivatives of cannabinoids, amphetamine, methadone, phencyclidine, tricyclic antidepressants, cotinine is carried out by the azo coupling reaction with diazotized 4-aminobenzoic acid.

Conjugates are prepared on the basis of modified narcotic and psychotropic substances in which these substances are non-covalently bound to macromolecular carriers.

Condensation reactions of carboxyl-containing hapten derivatives with a macromolecular carrier are carried out using water-soluble carbodiimide, glutaraldehyde or isobutyl chloroformate. Macromolecular carriers contain the haptens listed above.

The resulting conjugate was isolated by gel chromatography on a column (2 × 50 cm) with Sephadex G-25, equilibrated in 0.05 M phosphate buffer (pH 7.2). The elution rate was 50 ml / h, and the protein fraction of the conjugate was withdrawn in the free volume of the column (250 ml). The chromatographic process was monitored using a Uvicord II flowing quartz cell from Pharmacia (Sweden) at a wavelength of 280 nm using a recording device and a fraction collector of the same company. The concentration of the protein present in the eluted fraction was determined at a wavelength of 280 nm in a Genesys 10UV spectrophotometer (Thermo Fisher Scientific, USA).

The number of moles of hapten bound to a macromolecular protein carrier is determined by comparative spectral analysis in the ultraviolet and visible regions.

Example 1. The synthesis of the conjugate of morphine with human serum albumin (M-HSA)

A solution of 65 mg (0.001 mmol) of human serum albumin (HSA) in 5.0 ml of distilled water is mixed with 3.0 ml of 0.1 M phosphate buffer containing 15.0 mg (0.039 mmol) of 6- (3- (aminohexylaminocarbonyl) propionate ) of morphine and upon cooling, a solution of 15 mg (0.055 mmol) of water-soluble carbodiimide in 3 ml of distilled water is added dropwise. The reaction mixture was incubated for 5 hours at 4 ° C. The resulting conjugate was isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

Get 25 mg of the conjugate of morphine with HSA (M-HSA) containing 5 moles of morphine per mole of protein.

Example 2. The synthesis of conjugates of amphetamine with human serum albumin (A-HSA).

D, L-1-phenyl-2-aminopropane (5 mg; 0.0369 mmol) and (14 mg; 0.00022 mmol) HSA was dissolved in 3.0 ml of 0.1 M phosphate buffer. 50 μl (0.4 mmol) of glutaraldehyde solution are added dropwise at room temperature with stirring. Gradually, the reaction mixture acquires a characteristic yellow color, indicating the end of the reaction. To stop the reaction, 40 μl of a 1M lysine solution was added to the mixture and incubated for 1 hour.

The resulting conjugate was dialyzed against phosphate buffer overnight at 4 ° C. The conjugate is isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

Get 5 mg of conjugate (A-HSA) containing 12 moles of amphetamine per mole of protein.

Example 3. Synthesis of a conjugate of cocaine with human serum albumin (Kok-HSA)

The conjugate of cocaine with human serum albumin (Kok-HSA) is obtained according to the method of example 1 from 65 mg (0.001 mmol) of HSA and 4.55 mg (0.015 mmol) of benzoylegonin.

Allocate 29 mg of the Kok-HSA conjugate containing 14 moles of cocaine per mole of protein.

Example 4. Synthesis of a conjugate of tetrahydrocannabinol with human serum albumin (Δ ⁹ -THC-HSA).

To a solution of 21 mg (3.3 × 10 ^-3 mmol) of HSA in 3 ml of distilled water, a solution of 16.6 mg (0.05 mmol) of delta-9-tetrahydrocannabinol, modified by the reaction of azo coupling with diazotized 4-aminobenzoic acid in 2, is added. 0 ml of 0.1 M phosphate buffer, the reaction mixture is cooled to 0 ° C and 5 mg of water-soluble carbodiimide are added with stirring. The reaction mixture was kept in the refrigerator for 12 hours, the precipitated urea precipitate was separated, and the resulting conjugate was isolated by column gel chromatography (Sephadex G-25).

10 mg of tetrahydrocannabinol conjugate with HSA (Δ ⁹ -THC-HSA) containing 15 moles of hapten per mole of protein are obtained.

Example 5. Synthesis of conjugate of naltrexone with human serum albumin (N-HSA).

A solution of 65 mg (0.001 mmol) of HSA in 17.0 ml of distilled water is mixed with 4.0 ml of 0.1 M phosphate buffer containing 12 mg (0.03 mmol) of succinylnaltrexone, and a solution of 13 mg (0) is added dropwise. , 04 mmol) of water-soluble carbodiimide in 3 ml of distilled water. The reaction mixture was incubated for 5 hours at 4 ° C. The resulting conjugate was isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

Obtain 27 mg of naltrexone conjugate with human serum albumin (H-HSA) containing 17 moles of naltrexone per mole of protein.

Example 6. Obtaining a conjugate of morphine with lysozyme (M-Lys).

To a solution of 50 mg (3.33 × 10 ^-3 mmol) of lysozyme in 2.5 ml of a 0.3 M solution of NaHCO _3, 0.3 ml of a solution of mixed 6-succinylmorphine anhydride obtained from 19.2 mg (0, 05 mmol) of 6-succinylmorphine and 6.0 μl of isobutyl chloroformate. The reaction mixture was incubated for 18 hours. At 4 ° C, the resulting morphine-lysozyme conjugate was isolated by gel filtration on a Sephadex G-25 column and freeze-dried.

30 mg of morphine conjugate with lysozyme (M-Lys) are obtained. The amount of attached morphine is calculated based on the UV spectra of the starting protein and the resulting conjugate from the change in absorbance at 280 nm. The resulting conjugate contains 12 moles of morphine per mole of protein.

Example 7. Obtaining conjugates of morphine with polylysine (M-PolyLys)

A solution of 50 mg (0.001 mmol) of polylysine (peptide polystructure) in 5.0 ml of distilled water is mixed with 2.0 ml of 0.1 M phosphate buffer containing 15.0 mg (0.039 mmol) of 6-succinylmorphine, and dropwise upon cooling a solution of 15 mg (0.055 mmol) of water-soluble in 3 ml of distilled water is added. The reaction mixture was incubated for 5 hours at 4 ° C. The resulting conjugate was isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

Get 25 mg of the conjugate of morphine with polylysine (M-PolyLys) containing 10 moles of morphine per mole of protein.

Example 8. Obtaining conjugates of morphine with polylysine (M-PolyLys)

A solution of 27 mg (0.0005 mmol) of polylysine in 30 ml of a 0.25 M NaHCO ₃ solution is added to a mixed anhydride solution containing 1.0 mg (0.003 mmol) of 3-O-carboxymethylmorphine. The mixture was kept at 0 ° C. overnight. The conjugate was isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

Get 20 mg of a conjugate of morphine with polylysine (M-PolyLys) containing 2 mol of morphine per mole of protein.

Example 9. Obtaining a conjugate of Δ ⁹ -tetrahydrocannabinol with gamma globulin (Δ ⁹ -THC-gamma-globulin)

To a solution of 15 mg (0.1 × 10 ^-3 mmol) of gamma globulin in 3 ml of distilled water, a solution of 2.0 mg (0.006 mmol) of 4-carbosiphenyl-azo Δ ⁹ -tetrahydrocannabinol derivative (cannabinoid) in 1.0 ml is added. 0.1 M phosphate buffer, cool the reaction mixture to 0 ° C and add 5 mg of water-soluble carbodiimide with stirring. The reaction mixture was kept in a refrigerator for 12 hours, the precipitated urea precipitate was removed, the conjugate was isolated by column gel chromatography (Sephadex G-25), and lyophilized.

12 mg of Δ ⁹ -tetrahydrocannabinol conjugate with gamma-globulin (Δ ⁹ -THC-gamma-globulin) containing 5 moles of Δ ⁹ -tetrahydrocannabinol per mole of protein are obtained.

Example 10. Synthesis of conjugated amphetamine antigens with diphtheria toxin (A-Dt)

Analogously to example 2, D, L-1-phenyl-2-aminopropane (2 mg; 0.014 mmol) and 10 mg (0.00016 mmol) of diphtheria toxin protein are used, which are dissolved in 2.0 ml of 0.1 M phosphate buffer.

Get 7 mg of conjugate containing 6 moles of amphetamine per mole of protein.

Example 11. Obtaining a conjugate of barbamil with human gamma globulin (B-hCG)

Analogously to example 9, from a solution of 15 mg (0.1 × 10 ^-3 mmol) of gamma globulin in 3 ml of distilled water, to which a solution of 1.0 ml of 0.1 M phosphate buffer containing 5.2 mg is added with cooling and stirring (0.02 mmol) of barbamyl (a derivative of barbituric acid) and 5 mg of water-soluble carbodiimide, 10 mg of a conjugate of barbamil with gamma globulin (B-hCG) containing 6 moles of barbamil per mole of protein are obtained.

Example 12. Obtaining a conjugate of naltrexone with human gamma globulin (N-hCG)

A solution of 50 mg (0.0003 mmol) of gamma globulin in 40 ml of distilled water is mixed with 15.0 ml of 0.1 M phosphate buffer containing 30.0 mg (0.07 mmol) of succinylnaltrexone, and the solution is added dropwise with cooling 45 mg (0.16 mmol) of water-soluble carbodiimide in 10 ml of distilled water. The reaction mixture was incubated for 5 hours at 4 ° C. The conjugate was isolated by column gel chromatography (Sephadex G-25) and freeze-dried.

35 mg of gamma globulin-naltrexone conjugate is obtained containing 43 moles of naltrexone per mole of protein (N-hCG).

3. Obtaining a synthetic immunogen

To obtain a synthetic immunogen, a complex consisting of a conjugate of a macromolecular carrier in the form of a natural or artificial protein and a hapten, a narcotic or psychotropic compound non-covalently linked to a polymer matrix, a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone or copolymer 4 is used -vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, it is necessary to observe the ratio of hapten and macromolecular carrier in accordance with the invention. The conjugate contains the above macromolecular carriers and haptens in various ratios (Examples 1-12).

Example 13. Obtaining an immunogen consisting of a naltrexone conjugate with HSA (N-HSA) bound to a synthetic polymer (copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 50 mol% and Mη = 15 kDa).

To a solution of 180 mg of the conjugate (H-HSA) obtained in Example No. 5 in 30 ml of 0.1 M phosphate buffer (pH 7.2), 1600 mg of a synthetic polymer representing a copolymer of 2-methyl-5-vinylpyridine and N- vinylpyrrolidone, with n = 50 mol. % and Mη = 15 kDa. The mixture is incubated for 24 hours at 20 ° C. The resulting immunogen is freeze-dried, as a result, an immunogen is obtained containing the hapten / H-HSA conjugate (12: 1) and a synthetic polymer in a ratio of 1:10, the structure of which is characterized by ELISA, determining the availability of hapten naltrexone epitopes for binding to opiate antibodies ( Table 1, No. 2).

Example 14. Obtaining an immunogen consisting of a naltrexone conjugate with hCG (N-hCG) bound to a synthetic polymer (copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone)

To a solution of 300 mg of the conjugate (N-hCG) obtained in Example 12 in 80 ml of 0.1 M phosphate buffer (pH 7.2) was added 2500 mg of a synthetic polymer, a copolymer of 2-methyl-5-vinyl pyridine and N -vinylpyrrolidone, with n = 25 mol. % and Mη = 45 kDa. The mixture is incubated for 24 hours at 20 ° C. Purification of the immunogen and its characterization is carried out as described in example 13.

An immunogen is obtained containing the naltrexone / N-hCG conjugate (43: 1) and a synthetic polymer in a ratio of 1:10. The structure of the immunogen and the availability of hapten epitopes are characterized by ELISA (Table 1, No. 1).

Example 15. Obtaining an immunogen consisting of a conjugate of HSA with morphine (M-HSA) and a synthetic polymer bound thereto (copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, (m + k) = 20 mol %, the proportion of monomer units of 4-vinylpyridine is 0.05 (m + k), Mη = 250 kDa).

To a solution of 13.5 mg of a synthetic polymer in 2 ml of pyrogen-free water, 38 mg of M-HSA obtained in Example 1 containing 6- (3- (aminohexylaminocarbonyl) propionate) morphine as a hapten is added, in a ratio of 5: 1 the mixture is kept in during the day at 20 ° C. The resulting immunogen is characterized as described in example 13.

An immunogen is obtained containing the naltrexone / M-HSA conjugate (5: 1) and a synthetic polymer in a ratio of 1:10. The structure of the immunogen and the availability of hapten epitopes are characterized by ELISA (Table 1, No. 3).

Example 16. Obtaining an immunogen consisting of a conjugate of HSA (M-HSA) with morphine, and the associated synthetic polymer (copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, (m + k) = 90 mol %, the proportion of monomer units of 4-vinylpyridine is 0.3 (m + k), Mη = 15 kDa), with a ratio of 10 moles of synthetic polymer per mole of conjugate.

The preparation is carried out analogously to example 14, only 6 mg of a synthetic polymer (copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, (m + k) = 90 mol.%, The proportion of monomer units 4- vinylpyridine is 0.3 (m + k), Mη = 15 kDa) in 2 ml of pyrogen-free water and 2 mg of M-HSA obtained in Example 1 containing (6- (3- (aminohexylaminocarbonyl) propionate) morphine as a hapten .

An immunogen is obtained containing the morphine / M-HSA conjugate (5: 1) and a synthetic polymer in a ratio of 1:10. The structure of the immunogen and the availability of hapten epitopes are characterized by ELISA (Table 1, No. 4).

Example 17. Obtaining an immunogen consisting of a conjugate of polylysine (M-PolyLys) with morphine bound to a synthetic polymer (copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, (m + k) = 90 mol% , the proportion of monomer units of 4-vinylpyridine is 0.3 (m + k), Mη = 15 kDa), with a ratio of 1 mol of conjugate per 3 mol of polymer.

The preparation is carried out analogously to example 14, only 5 mg of a synthetic polymer (copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, (m + k) = 90 mol.%, The proportion of monomer units 4- vinylpyridine is 0.3 (m + k), Mη = 15 kDa) in 2 ml of phosphate buffer and 1.4 mg of the M-PolyLys conjugate obtained in example 7.

The resulting complex has a 3: 1 replacement of the polymer matrix with a conjugate of polylysine with a spacer (3-O-carboxymethylmorphine) at the 3rd position of the morphine molecule.

Example 18. The preparation of an immunogen consisting of a conjugate of gamma globulin with Δ ⁹ -tetrahydrocannabinol (Δ ⁹ -THC-gamma-globulin) bound to a synthetic polymer (copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 25 mol.% and Mη = 25 kDa) with a ratio of 1 mol of conjugate to 2 mol of polymer.

The preparation is carried out analogously to example 14, only 3.5 mg of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 25 mol. % and Mη = 25 kDa in 1 ml of phosphate buffer and 1.2 mg of the Δ ⁹ -THC-gamma-globulin conjugate obtained in Example 9. The resulting complex has a 2: 1 substitution of the polymer matrix with the Δ ⁹ -THC-gamma-globulin conjugate .

Example 19. Obtaining an immunogen consisting of a diphtheria toxin conjugate with amphetamine bound to a synthetic polymer (copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 23 mol% and Mη = 45 kDa) at a ratio of 1 mol of conjugate for 4 moles of polymer.

The preparation is carried out analogously to example 14, only 3.5 mg of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 23 mol. % and Mη = 45 kDa in 1 ml of phosphate buffer and 0.9 mg of the amphetamine-diphtheria toxin conjugate obtained in Example 10.

The resulting complex has a 4: 1 substitution of the polymer matrix with the amphetamine-diphtheria toxin conjugate.

Example 20. Obtaining an immunogen consisting of a conjugate of gamma globulin with barbamil bound to a synthetic polymer (copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 20 mol% and Mη = 35 kDa) at a ratio of 1 mol conjugate per 6 moles of polymer matrix

The preparation is carried out analogously to example 14, only 3.5 mg of a copolymer of 2-methyl-5-vinylpyridine and N-vinylpyrrolidone, where n = 20 mol. % and Mη = 35 kDa in 3 ml of phosphate buffer and 0.5 mg of the conjugate of barbamyl-gamma-globulin obtained in example 11.

The resulting complex has a 6: 1 substitution of the polymer matrix with the conjugate barbamyl-gamma-globulin.

4. In vivo experiments

In animal experiments, it was shown that the obtained synthetic immunogens are able to induce immune responses with the appearance of antibodies that specifically interact with haptens and neutralize their activity in the body. In a series of experiments, the possibility of neutralizing the toxic effects of morphine on animals was clarified.

Example 21. Rabbit model of intoxication with morphine

Rabbits are divided into two groups based on age and body weight so that the average values are comparable. The control group of animals consists of eight non-immunized rabbits, and the experimental group includes eight rabbits previously immunized with the synthetic immunogen morphine-HSA-synthetic polymer obtained in example 13. To do this, for a month with an interval of 7 days, injections of 0.2 ml per 4 points near the spine - in the area of the scapula and in the sacral region at the rate of 200 μg / kg per rabbit.

A lethal dose of morphine (LD100) is administered intravenously to all animals. All eight control rabbits die from the indicated lethal dose. Among the immunized rabbits, death was not recorded in any of eight cases, i.e. high immunological protection is observed for specific antibodies formed during the immunization process.

Example 22. Rat model of pervitin intoxication

Analogously to example 21, studies are carried out in two groups of six sexually mature outbred rats of both sexes. Animals from the experimental group are immunized with a synthetic immunogen amphetamine-diphtheria toxin-synthetic polymer at a rate of 400 μg / kg All rats are given a lethal dose of the psychoactive substance Pervitin.

All animals of the control group that did not receive the immunogen died. Among the immunized rats, deaths were not recorded in any of the six cases, i.e. high immunological protection is observed for specific antibodies formed during the immunization process.

Example 23. Rat model of barbiturate intoxication

Analogously to example 21, six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen barbamyl-gamma-globulin-synthetic polymer (hapten from the group of derivatives of barbituric acid) at a rate of 200 μg / kg. A lethal dose of sleeping pills is introduced - barbiturate.

In the control group, all animals die. Among the immunized rats, deaths were not recorded in any of the six cases, i.e. complete immunological protection is observed for specific antibodies formed during the immunization process.

Studies according to the above scheme, performed for haptens included in the claimed composition of the immunogen, showed similar results.

Example 24. Determination of the specificity of antibodies generated by immunization with a synthetic immunogen

Specificity is determined in the ELISA competition test. For this, in solid-phase ELISA, as inhibitors, compounds are used that are structurally related to the hapten that is part of the claimed immunogen, as well as narcotic substances and a number of psychotropic drugs of a different chemical nature.

For each of these compounds, the concentration is determined at which 50% inhibition of the interaction of antigapten antibodies with the antigen in the solid phase occurs. Specificity is calculated as the ratio of molar concentrations of the test substance to the starting hapten and expressed as a percentage. Data on the specificity of antibodies (in%) are presented in table 2.

According to table 2, it is seen that only immune rabbit antibodies to haptens that are part of the inventive immunogen did not cross-react with compounds of other classes and largely reacted with structurally related substances. So, for an immunogen consisting of an albumin conjugate with naltrexone (N-HSA) bound to a synthetic polymer (Example 13), antibodies are formed that specifically react with morphine, heroin and, to a slightly lesser extent, with codeine. Cross-reactions are absent with methadone, amphetamine, cannabinoids, cocaine and other substances that are not classified as opiates.

Similar results were obtained for an immunogen consisting of a gamma globulin conjugate (N-hCG) with naltrexone bound to a synthetic polymer (Example 14).

For an immunogen consisting of a conjugate of gamma globulin with Δ ⁹ -tetrahydrocannabinol (Δ ⁹ -THC-hCG) bound to a synthetic polymer, a specific antibody reaction is observed only for cannabinoids (Example 18). For an immunogen consisting of an albumin conjugate with amphetamine (Example 19) bound to a synthetic polymer, the antibodies react with amphetamine and to a lesser extent with methamphetamine in the absence of reaction with ephedrine structures, as well as with compounds of other classes.

For an immunogen consisting of a conjugate of gamma globulin with barbamil B-hCG) bound to a synthetic polymer, the resulting antibodies interact with barbamil and to a lesser extent phenobarbital, without giving cross-reactions with compounds of other classes (Example 20).

Thus, antibodies obtained by immunizing animals with artificial synthetic immunogens can be used to neutralize the harmful effects of a psychoactive substance, which in turn can be used to protect against toxic effects in the abuse of narcotic and psychoactive substances.

To confirm the data obtained, table 3 shows additional information about the immunogens.

SP - synthetic polymer

KMNG is a conjugate of a macromolecular carrier with a hapten.

6. The immune response in the rat model

Example 25. Intoxication with Pervitinum

Six sexually mature rats of both sexes are immunized with a synthetic immunogen complex with a conjugate: diphtheria toxin: amphetamine and the synthetic polymer obtained in Example 19, at a rate of 400 μg / kg.

Laboratory data: ELISA 1 ml - 75000 pmol (5 weeks after the start of immunization). A lethal dose of the psychoactive substance Pervitin was administered.

Control laboratory data: ELISA 1 ml - 54000 pmol. Antibody synthesis, lack of allergic reactions.

Example 26. Intoxication with barbiturate

Six sexually mature rats of both sexes are immunized with a synthetic immunogen complex with a gamma-globulin: barbamyl conjugate: the synthetic polymer obtained in Example 20, at a rate of 500 μg / kg.

Laboratory data: ELISA 1 ml - 76000 pmol (5 weeks after the start of immunization). A lethal dose of sleeping pills, barbiturate, was administered.

Control laboratory data: ELISA 1 ml - 57000 pmol. Synthesis of antibodies; lack of allergic reactions.

Example 27. Heroin Intoxication

Six sexually mature rats of both sexes were immunized with a synthetic immunogen complex with an ovalbumin: morphine conjugate: the synthetic polymer obtained in Example 15, at a rate of 300 μg / kg.

Laboratory data: ELISA 1 ml - 85000 pmol of specific antibodies to heroin (4 weeks after the start of immunization). A lethal dose of a heroin narcotic of 100 mg / kg was administered.

Control laboratory data: ELISA 1 ml - 60,000 pmol. Synthesis of antibodies; lack of allergic reactions.

As can be seen from the above examples and the data of table 3, the introduction of the inventive synthetic immunogen can cause the formation of antibodies in the body and a stable immune response, which leads to detoxification of the body in relation to the corresponding psychoactive and narcotic substances.

Claims (8)

1. A synthetic immunogen for the protection and treatment of dependence on psychoactive substances, which is a conjugate of a macromolecular carrier, hapten and a synthetic polymer non-covalently linked to a conjugate, characterized in that a copolymer of 2-methyl-5-vinylpyridine and N- is used as a synthetic polymer vinylpyrrolidone of the general formula I

where n is equal to 25-50 mol. %, and the average viscosity molecular weight Mη is 15-45 kDa, or a copolymer of 4-vinylpyridine, 2-methyl-5-vinylpyridine and N-vinylpyrrolidone of the general formula II

where (m + k) is 20-90 mol. %, while the proportion of monomer units of 4-vinylpyridine is from 0.05 (m + k) to 0.30 (m + k), and the average viscosity molecular weight Mη is 15-250 kDa. 2. A synthetic immunogen for the protection and treatment of dependence on psychoactive substances according to claim 1, characterized in that as a conjugate of a macromolecular carrier, you can use a natural protein, or an artificial protein, or an oligopeptide, or a polypeptide, or a carbohydrate, or lipid, or nucleotide. 3. A synthetic immunogen for the protection and treatment of dependence on psychoactive substances according to claim 1, characterized in that human gamma globulin or albumin can be used as a macromolecular carrier. 4. A synthetic immunogen for the protection and treatment of dependence on psychoactive substances according to claim 1, characterized in that naltrexone can be used as a hapten.