RU2236257C1 - Synthetic immunogen for therapy and prophylaxis of addiction with narcotic and psychoactive substances - Google Patents

Abstract

FIELD: medicine, immunology, narcology.

SUBSTANCE: invention can be used for treatment of narcomania, intoxication with different poisons in industry and in homes, in technogenic catastrophe and so on. Synthetic immunogen represents conjugate as the natural or artificial protein (egg albumin or human gamma-globulin, or bacterial toxins) as a carrier and haptene - narcotic or psychotropic compound taken among the following groups of low-molecular compounds: aromatic propaneamines or isoquinolines, or diphenylmethanes, or indoleamines, or derivatives of barbituric acid, or derivatives of substituted piperidines. Conjugate is modified with an immunocompetent polyelectrolyte - polyoxidonium in the ratio, weight %: conjugate : polyoxidonium = 1:(1.5-5.0). The advantage of invention involves the high immunological activity, reduced doses and treatment periods.

EFFECT: valuable properties of immunogen.

1 tbl, 16 ex

Description

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

Currently, synthetic immunogens are widely used in domestic medical practice, consisting of a carrier - polyoxidonium (an N-oxidized derivative of high molecular weight polyethylene piperazine) and hapten - a high molecular weight compound, for example, the enzyme hyaluronidase, or a mixture of hemagglutin and neuramidase, or cholera toxin, or triton extract (glycopeptide) of the cell walls of BCG microbacteria, or others.

[see, for example, s. No. 97103034, IPC A 61 K 38/43, declared 02/28/1997, publ. 09/10/1998; p. No. 1580617, IPC A 61 K 39/145, declared 05/13/1988, publ. 02/10/1997; Sec. No. 2021816, IPC A 61 K 39/106, s. 06/05/1991, publ. 10/30/1994; Sec. No. 2153354, IPC A 61 K 39/04, head office 03/31/1999, publ. 07/27/2000; g. “Immunology” No. 6, 2002, p. 344-333; G. “The attending physician” No. 4, 1998].

Despite the fact that polyoxidonium, which is part of the known immunogens, has unique properties (reproducibility of the chemical structure, the absence of ballast impurities, pronounced immunostimulating activity, pronounced detoxifying, antioxidant and membrane stabilizing properties), the preparations described above can only be used to treat acute and chronic bacterial or viral infections due to the presence of specific haptens.

Known for the technical essence and the achieved result that is closest to the claimed invention is a synthetic immunogen 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.

[cm. Kovalev I.E., Field O.YU. “Biochemical basis of immunity to low molecular weight chemical compounds”, M .: Nauka, 1985].

The disadvantages of the prototype are as follows:

- the presence of ballast impurities;

- low therapeutic (immunostimulating) activity;

- the need for large doses (up to 20 mg / kg) and the duration of immunization (up to 6 months);

- the inability to achieve stable and / or prolonged immunity.

The objective of the present invention is to provide an easily reproducible, affordable and harmless synthetic immunogen that can protect a person from the effects of a wide range of toxic substances, which forms a stable, prolonged immune response in his body.

The problem is solved in that in the known synthetic immunogen for the treatment and prevention of abuse of narcotic and psychoactive substances, which is a conjugate in the form of a carrier - a natural or artificial protein and hapten - a narcotic or psychotropic compound, according to the invention, the conjugate is modified by immunocompetent polyelectrolyte-polyoxidonium in the ratio , wt.%: conjugate: polyoxidonium = 1: 1.5-5.0,

moreover, it contains, as a carrier, egg albumin, or human gamma globulin, or microbial toxins, and as a hapten, low molecular weight compounds from the following groups: aromatic propanamines, or isoquinolines, or diphenylmethanes, or indolamines, or derivatives of barbituric acid, or derivatives substituted piperidines.

Modification of the proposed conjugate with polyoxidonium ensures the absence of ballast impurities in the synthetic immunogen, while the claimed conjugate: polyoxidonium ratio is necessary and sufficient to achieve high immunostimulating activity with a stable prolonged action.

The use of specific carriers and haptens in the proposed synthetic immunogen protects the human body from the action of a wide range of toxic substances (including drugs, industrial poisons, various carcinogens).

The inventive synthetic immunogen stimulates the processes within the immune system: migration and interaction of T and B lymphocytes, production of antibodies in response to foreign antigens, the functioning of NK cells and macrophages. Under its influence, the synthesis of antibodies is enhanced, the number of which increases exponentially during the first two weeks, and by the end of the second month from the start of administration, antibodies accumulate in the body that can bind doses of narcotic or other toxic substances that are 5-10 times higher than lethal ones. In particular, along with the production of anti-heroin antibodies, the formation of mononuclear cells of the spleen that binds heroin is induced. Thus, immunization of the patient with the proposed drug leads to the development of prolonged tolerance to specific substances in the absence of any negative effects.

Analysis of the known technical solutions allows us to conclude that the proposed invention is not known from the level of the studied technology, which indicates its compliance with the criterion of “novelty”.

The essence of the present invention for specialists does not follow explicitly from the prior art, which allows us to conclude that it meets the criterion of "inventive step".

The possibility of preparing a synthetic immunogen from available commercially available components using traditional equipment using known techniques indicates that the invention meets the criterion of “industrial applicability”.

To prepare the inventive synthetic immunogen used industrially produced or self-synthesized components are given in table. 1.

The preparation of the synthetic immunogen was carried out according to well-known traditional methods (see the end of the description).

Method 1.

To a mixture of 1.2 g of benzidine, 2 ml of formic acid and 70 ml of water at minus 10 ° C was added 1 g of sodium nitrate in 2 ml of water. The reaction mixture was kept under cooling and stirring for one hour. Then the resulting solution was added at 0 ° C to a solution of 2 g of fentanyl (carfentanil, methadone, cocakine, nicotine, heroin, etc.) in 15 ml of water, maintaining a pH of 7.0-8.0 by adding a 10% caustic solution soda. The precipitate was filtered off, washed with water and repeatedly with chloroform.

To 200 mg of prepared para-aminodiphenylazofentanyl (β-carfentanil, methadone, cocaquine, nicotine, heroin, etc.) and 10 ml of concentrated hydrochloric acid at minus 10 ° C, 29 mg of sodium nitrite in 2 ml of water was added. The reaction mixture was kept under cooling and stirring for 30 minutes. The resulting solution was added in the cold to a solution of human gamma globulin or a synthetic polypeptide in water, maintaining a pH of 7.0-8.0 by the addition of sodium hydroxide. The mixture was left for 12 hours in the refrigerator. Purification of the resulting conjugate was carried out through semipermeable membranes against the flow of distilled water for 5 days or Sephadex. The conjugate was modified with polyoxidonium, previously activated in a known manner (see, for example, No. 1580617) with the conjugate: polyoxidonium ratio = 1: 1.5-5.0.

The structural formula of the obtained synthetic immunogen is presented at the end of the description.

Method 2.

They took stoichiometric amounts of naltrexone (morphine, codeine, etc.) 0.01 mol (3.8 g) in pyridine 0.01 mol (0.8 g) and succinyl dichloride 0.01 mol (1.6 g). Pyridine was used in the reaction as an eliminator of hydrogen chloride released during the addition of naltrexone to one of the chlorocarboxy groups of succinyl dichloride. The second similar group remained free for subsequent attachment to the protein - human gamma globulin or a synthetic polypeptide. The conjugate was purified and modified with polyoxidonium in the same manner as described in Method 1.

The structural formula of the obtained synthetic immunogen is presented at the end of the description.

The inventive synthetic immunogen was synthesized under the conditions of the Department of Pharmacology of the Ural Medical Academy, which has permission from the Russian Ministry of Health for scientific work with narcotic and psychotropic substances in accordance with the following documents: Order of the Ministry of Health of the USSR No. 1311 of 12/30/1982 and the Federal Law “On Narcotic Drugs and Psychotropic” substances ”No. 3 - Federal Law from 8.01.98. The synthesized immunogens were repeatedly tested on mice, as well as on groups of volunteers in accordance with Article 124 of the Criminal Code of the Russian Federation, Article 43 of the Fundamentals of Legislation on the Protection of Public Health and Article 21, Part 2 of the Constitution of the Russian Federation.

The synthetic immunogen was administered to patients as a solution subcutaneously as 0.5 ml. After two weeks, the procedure was repeated and then continued the introduction of the immunogen once a week for another six weeks. The time of an active immune response was determined by standard methods: RPHA, RTPHA, ELISA, RIA. Patients were regularly monitored for changes in their health status for 5 years.

Example 1. Synthetic immunogen for the treatment and prevention of drug and psychoactive substance abuse.

A synthetic immunogen is a conjugate in the form of a carrier - a natural protein - human gamma globulin and hapten - a low molecular weight compound from the isoquinoline-naltrexone group.

The conjugate is modified by immunocompetent polyelectrolyte-polyoxidonium at a ratio, wt.%: Conjugate: polyoxidonium = 1: 1.5, 10.0 mg and 15.0 mg, respectively.

In examples 2-8, a synthetic immunogen is presented with a variation in carriers, haptens, and ratios (conjugate: polyoxidonium) within the claimed limits.

The compositions of the proposed synthetic immunogen and known, taken as a prototype [see Kovalev I.E., Field Yu.I. “Biochemical basis of immunity to low molecular weight chemical compounds”, M .: Nauka, 1985], as well as their pharmacological actions are given in table. 2.

As can be seen from the examples and data in the table, the use of the claimed synthetic immunogen in comparison with the known, taken as a prototype [see Kovalev I.E., Field O.YU. “Biochemical fundamentals of immunity to low molecular weight chemical compounds”, M .: Nauka, 1985], provides the following technical and socially useful benefits:

- easy reproducibility without ballast impurities;

- accessibility and safety;

- high immunological activity;

- reduction of doses and treatment time;

- stable prolonged immunity;

- protection against the action of a wide range of toxic substances (narcotic and psychoactive).

Preclinical Studies

In a special series of experiments by the method of biological tests, the possibility of neutralizing the toxic effects of heroin in animals was clarified. The control group of animals — four non-immunized rabbits, and the experimental group — four immunized rabbits, were injected with an absolutely lethal dose of heroin 100 mg / kg (LD100). It was found that all four rabbits in the control group died from the indicated doses. Among the immunized rabbits, death was not recorded in any of the four cases, i.e. a clear immunological defense was observed.

Serum studies of immunized animals were also performed.

Eight rabbits weighing 2 kg each were immunized with 3-hemisuccinylnaltrexone - human gamma globulin - polyoxidonium containing 45 moles of 3-hemisuccinylnaltrexone and 3 moles of polyoxidonium per protein molecule, which was administered intramuscularly at 30 mg / kg twice a week for 8 weeks . In the blood serum of animals before and after immunization, the presence of antibodies to the drugs was determined and their specificity was studied in the passive inhibition reaction

Immunization of rabbits with the drug according to the given scheme led to the appearance in the blood serum of high titers of antibodies to the immunocomplex of 3-hemisuccinylnaltrexone - human gamma globulin - polyoxidonium (1: 2560). In the reaction of inhibition of passive hemagglutination, it was found that the resulting antibodies bind heroin and 3-hemisuccinylnaltrexone: the titer of antibodies to the immunocomplex was reduced to 1: 320 when the concentration of heroin and 3-hemisuccinylnaltrexone was 0.001 mol, which indicates the presence of antibodies to these compounds.

Thus, the synthesized immunocomplex specifically binds heroin not only in vitro, but also in the whole body.

Antibodies obtained from the blood serum of rabbits were studied for the specificity of the binding of opiates of the phenanthrene series. The study of the specificity of antibodies, i.e. the ability to bind to morphine, heroin and other ligands of opiate recipes was performed by enzyme-linked immunosorbent assay. Morphine, heroin, codeine, N-allyl morphine and methadone were used as inhibitors. The results obtained indicate that, when animals are actively immunized with the 4-hemisuccinylnaltrexone immunocomplex — human gamma globulin — polyoxidonium, antibodies are specifically induced that specifically bind morphine, heroin, codeine, N-allyl morphine and practically do not react with methadone.

The specificity of antibodies against heroin in rabbit immune serum.

It can be seen from the obtained data that the binding of opiates depends on the presence of certain functional groups and radicals in their molecules. So, in particular, the high specificity of antibodies to heroin is due to the presence in the immunocomplex of a complex ester bond of naltrexone with succinic acid, and to N-allylmorphine - with the cyclopropyl ethyl radical attached to the nitrogen atom in the chemical structure of naltrexone. Thus, the resulting antibodies are highly specific only to the opiates of the phenanthrene series (heroin and its structural analogues).

Given the results of the experiments, the authors concluded that 3-hemisuccinylnaltrexone is suitable for the synthesis of an immunogen. Its chemical structure contains branched radicals for the formation of an immune response and the synthesis of high-affinity antibodies to opiates of the phenanthrene series.

Clinical researches.

Similar specificity studies were conducted with antibodies isolated from the blood serum of volunteers - drug addicts immunized with a synthesized immunogen.

The immunogen was administered to 22 volunteers suffering from opium addiction, age: 21-36 years. Duration of drug use: 2-13 years. The immunogen was administered subcutaneously in the upper third of the shoulder of the right and left arm alternately, once a week, 8-10 injections per course.

Anti-opiate antibodies were isolated from the initial 22 serum of drug addicts in all samples. According to ELISA, serum contains IgG and IgM class antibodies against morphine, heroin and codeine, which react equally with each of the metabolites.

The specificity of antibodies against heroin in human immune serum

As in the case of rabbit immune anti-opiate antibodies, antibodies isolated from the blood serum of drug addicts practically did not interact with drugs of the nephenanthrene series. Antibodies isolated from the blood serum of patients with drug addiction, in comparison with antibodies from rabbit immune sera, have a wider spectrum of specificity.

To confirm the data given in the table “Compositions of synthetic immunogens and their pharmacological action”, additional information is provided.

Specific examples of the use of synthetic immunogens are presented in table. 2.

Example 9 (corresponds to the composition of 1 table)

Patient - 25 years old, 7 years old opium addiction.

Initial laboratory data: 1 ml of blood plasma - 2570 pmol of heroin-binding antibodies determined by radioimmunoassay (RIA).

The patient was immunized with a synthetic immunogen - human gamma-globulin-naltrexone (a hapten from the group of isoquinolines) - polyoxidonium.

Laboratory data (after 9 weeks from the start of treatment with the immunogen): RIA 1 ml - 77550 pmol - synthesis of heroin-binding antibodies. After 8 weeks, T-lymphocytes mature and the formation of antibodies that cause the maturation of B-lymphocytes is stimulated, a lifelong cellular immunity occurs; there are no allergic reactions.

Control laboratory data (12 months after the start of treatment with the immunogen): RIA 1 ml - 50 120 pmol.

Example 10. (2)

Patient - 32 years old, 9 years old opium addiction.

Initial laboratory data: 1 ml - 1520 pmol; erythrocyte sorption capacity (SSE) - detoxification function of the body 64.1 ± 5.7% (determination by the erythrocyte method of absorption of vital dyes - methylene blue by the erythrocyte method, see. Laboratory work, 1988, No. 9, Art. A .A. Togaybaeva, A.V. Kurguzkina and others. “Method for the diagnosis of endogenous intoxication”).

The patient was immunized with a synthetic immunogen - human gamma globulin-naltrexone - polyoxidonium.

Laboratory data: RIA 1 ml - 78610 pmol, SSE - 44.1 ± 5.8% - increased detoxification function of the body. Other characteristics are similar to those shown in example 1.

Control laboratory data: RIA 1 ml - 51100 pmol.

Example 11. (3)

Patient 21 years old, 4 years old opium addiction.

Initial laboratory data: RIA 1 ml - 1680 pmol; SSE - 63.8 ± 5.6%; determination of opiates (urine immunochromatographic tests) - positive.

The patient was immunized with a synthetic immunogen - human gamma globulin-naltrexone - polyoxidonium.

Laboratory data: RIA 1 ml-78900 pmol, SSE - 42.4 ± 5.8%; the definition of opiates is negative, i.e. increased sorption of metabolites and xenobiotics. Other characteristics are similar to those in examples 1 and 2.

Control laboratory data: RIA 1 ml - 52600 pmol.

Note to examples 1-3: the data are documented in patient histories.

Example 12. (4)

Six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen - egg - albumin-amphetamine (a hapten from the group of aromatic propanamines) - polyoxidonium at the rate of 50 mg / kg.

A lethal dose of the psychoactive substance Pervitin was administered.

Laboratory data: RIA 1 ml - 76500 pmol. Other characteristics are similar to those in examples 1-3.

Control laboratory data: RIA 1 ml - 51200 pmol.

Example 13. (5)

Six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen - egg albumin-methadone (hapten from the diphenylmethane group) - polyoxidonium at a rate of 50 mg / kg.

A lethal dose of a narcotic substance, heroin, was administered.

Laboratory data: RIA 1 ml - 75300 pmol. Other characteristics are similar to those in examples 1-3.

Control laboratory data: RIA 1 ml - 52600 pmol.

Example 14 (6)

Six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen - diphtheria toxin-harmin (a hapten from the indolamine group) - polyoxidonium at a rate of 50 mg / kg.

A lethal dose of a psychoactive substance, pervitin, was administered.

Laboratory data: RIA 1 ml - 77900 pmol. Other characteristics are similar to those in examples 1-3.

Control laboratory data: RIA 1 ml - 54700 pmol.

Example 15 (7)

Six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen — human gamma globulin — phenobarbital (hapten from the group of derivatives of barbituric acid) —polyoxidonium at a rate of 50 mg / kg.

A lethal dose of sleeping pills, barbiturate, was administered.

Laboratory data: RIA 1 ml - 72300 pmol. Other characteristics are similar to those in examples 1-3.

Control laboratory data: RIA 1 ml - 51100 pmol.

Example 16 (8)

Six sexually mature outbred rats of both sexes were immunized with a synthetic immunogen - human diphtheria gamma globulin-carfentanil (a hapten from the group of substituted piperidine derivatives) - polyoxidonium at the rate of 50 mg / kg.

A lethal dose of a narcotic substance, morphine, was administered.

Laboratory data: RIA 1 ml - 75400 pmol. Other characteristics are similar to those in examples 1-3.

Control laboratory data: RIA 1 ml - 57200 pmol.

Prototype.

Six sexually mature outbred rats of both sexes were immunized with a diphtheria bovine serum albumin heroin at a rate of 50 mg / kg.

Only a lethal dose of a narcotic substance, morphine, was administered.

Laboratory data: RIA 1 ml - 70 100 pmol. High allergenicity due to the formation of immunoglobulins of the reactive type IgE class. The death of animals after 3 months as a result of anaphylactic shock during re-immunization.

Claims (1)

A synthetic immunogen for the treatment and prevention of abuse of narcotic and psychoactive substances, which is a conjugate consisting of a carrier - a natural or artificial protein and a hapten - a narcotic or psychotropic compound, characterized in that the conjugate is modified by immunocompetent polyelectrolyte - polyoxidonium in the ratio, wt.%: Conjugate : polyoxidonium = 1: 1.5-5.0, moreover, it contains egg albumin, or human gamma globulin, or microbial toxins as a carrier, and as g ptena - low molecular weight compounds from the following groups: aromatic propanamine or isoquinolines or diphenylmethanes, or indoleamines, or derivatives of barbituric acid, or derivatives of substituted piperidines.

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