Synthetic immunogen for the treatment and prevention of the abuse of narcotic and psychoactive substances

 

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, when man-made disasters, etc., the invention consists in that the synthetic immunogen is conjugated to a carrier is a natural or synthetic protein (egg albumin, or human gamma globulin, or microbial toxins) and hapten - narcotic or psychotropic compounds of the following groups of low molecular weight compounds: aromatic propanamine, or isoquinolines, or diphenylmethane, or indolamine, or derivatives of barbituric acid, or derivatives substituted piperidino. Conjugate modified immune polyelectrolyte - polyoxidonium at the ratio, wt.%: conjugate polyoxidonium = 1:1,5-5,0. The advantage of the invention lies in the high immunological activity, the reduction of doses and duration of treatment. table 2.

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, when man-made disasters, etc.,

It is now widely COI is of (N-oxidized derivative polyethyleneimine with high molecular weight) and hapten - high-molecular compounds, such as enzyme hyaluronidase, or a mixture of hemagglutin and neuramidase, or cholera toxin, or titanoboa extract (glycopeptide) cell walls of microbes BCG, or other

[see, for example, Z. No. 97103034, IPC And 61 To 38/43, Appl. 28.02.1997, publ. 10.09.1998; p. No. 1580617, IPC And 61 To 39/145, Appl. 13.05.1988, publ. 10.02.1997; p. No. 2021816, IPC And 61 To 39/106, Z. 05.06.1991, publ. 30.10.1994; p. No. 2153354, IPC And 61 To 39/04, Saul. 31.03.1999, publ. 27.07.2000; W. “Immunology”, No. 6, 2002, S. 324-333; W.“The doctor” No. 4, 1998].

Although part of the famous immunogenic polyoxidonium has unique properties (reproducibility of the chemical structure, the lack of ballast impurities, pronounced immunostimulating activity, expressed detoxifying, antioxidant and membrane-stabilizing properties) described above drugs can only be applied for the treatment of acute and chronic bacterial or viral infections due to the presence of specific haptens.

Known closest to the technical essence and the achieved result to the claimed invention of synthetic immunogen for the treatment and prevention of the misuse of drugs (in particular, opiate dependent on the economic or psychotropic compounds. And as the media it contains bovine serum albumin or synthetic polypeptides and glycoproteins, and the hapten - morphine, or codeine, or heroin, or methadone, or etc.

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

The disadvantages of the prototype the following:

- the presence of ballast impurities;

low therapeutic (immunostimulirutuyu) activity;

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

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

The present invention is the creation of legkovozvodimih, accessible and friendly synthetic immunogen capable of protecting the person from the actions of a wide range of toxic substances that form in the body by a persistent, prolonged immune response.

The problem is solved in that in the known synthetic immunogen for the treatment and prevention of the abuse of narcotic and psychoactive substances, representing conjugate as a carrier is a natural or artificial protein and hapten - narcotic or psychotropic with the m 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 is a low-molecular-weight compounds from the following groups: aromatic propanamine, or isoquinolines, or diphenylmethane, or indolamine, or derivatives of barbituric acid, or derivatives substituted piperidino.

Modification of the proposed conjugate with polyoxidonium ensures the absence of synthetic immunogen ballast impurities, while the claimed ratio of conjugate polyoxidonium is necessary and sufficient for achieving a high immunostimulatory activity stable for prolonged action.

Using the proposed synthetic immunogen specific media and haptens protects the human body from a wide range of toxic substances (including drugs, industrial poisons, various carcinogens).

The inventive synthetic immunogen stimulates the processes within the immune system: the migration and interaction of the T - and b-lymphocytes, production of antibodies in response to foreign antigens, the functioning of NK-cells and macrophages. Under its influence increases the synthesis of antibodies, if the ACA from the beginning of the introduction in the body accumulate antibodies able to link a dose of narcotic or other toxic substances, 5-10 times lethal. In particular, along with the products antiheroine antibodies, induced the formation of mononuclear cells in the spleen, linking heroin. Thus, immunization of the patient offered by the drug leads to the development of long-lasting tolerance to specific substances in the absence of any negative effects.

Analysis of the known technical solutions allows us to conclude that the invention is not known from the level of the investigated technique that demonstrates its compliance with the criterion of “novelty”.

The essence of the present invention for professionals not obvious from the prior art, which allows to make a conclusion about its compliance with the criterion of “inventive step”.

The possibility of preparation of synthetic immunogen of the available industrially produced components on conventional equipment using known techniques demonstrates compliance invention, the criterion of “industrial applicability”.

For preparation of the inventive synthetic immunogen used industrially manufactured or independently synthesized comnum methods (see at the end of the description).

Methodology 1.

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

To 200 mg prepared para-aminodiphenylamine (carfentanil, methadone, - kokkina, nicotine, heroin, etc) and 10 ml of concentrated hydrochloric acid at -10With added 29 mg of sodium nitrite in 2 ml of water. The reaction mixture was stirred under cooling and stirring for 30 minutes. The resulting solution was added in the cold to the solution of human gamma-globulin or synthetic polypeptide in the water, maintaining a pH of 7.0 to 8.0 by adding sodium hydroxide. The mixture was left for 12 hours in the refrigerator. Purification of the conjugate was performed through a semi-permeable membrane against the current of distilled water for 5 days or the emer, p. No. 1580617) at a ratio of conjugate polyoxidonium = 1:1,5-5,0.

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

Method 2.

Took stoichiometric amount of naltrexone (morphine, codeine, etc) 0.01 mol (3.8 g) in pyridine 0.01 mol (0.8 g) and succinyldicholine 0.01 mol (1.6 g). Pyridine was used in the reaction as eliminator of hydrogen chloride released in the accession process of naltrexone to one of chlorocarbonyl of succinyldicholine. The second similar group remained free for subsequent addition to protein - human gamma globulin or synthetic polypeptide. Purification of the conjugate and its modification by polyoxidonium was carried out as described in method 1.

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

The inventive synthetic immunogen synthesized under conditions of the Department of pharmacology of the Ural medical Academy, with the permission of Ministry of health of Russia on scientific work with narcotic and psychotropic substances in accordance with the following documents: the Order of the USSR Ministry of health No. 1311 from 30.12.1982, and the Federal law “On narcotic drugs and psychotropic substances” No. 3 - FZ of 8.01.98. with Art. 124 of the criminal code, Art. 43 of the fundamentals of legislation on health protection of citizens and century 21, including 2 of the Constitution of the Russian Federation.

Synthetic immunogen was administered to patients in the form of a solution subcutaneously as a 0.5 ml two weeks later the procedure was repeated and then continued introduction of the immunogen 1 time per week for six weeks. An active immune response was determined by standard methods: TPHA, RTPA, ELISA, RIA. The patients were regularly monitored for changes in their health status within 5 years.

Example 1. Synthetic immunogen for the treatment and prevention of the abuse of narcotic and psychoactive substances.

Synthetic immunogen is a conjugate as a carrier - natural protein - human gamma-globulin and hapten - low molecular weight compounds from the group of isoquinoline-naltrexone.

Conjugate modified immune polyelectrolyte-polyoxidonium at the ratio, wt.%: conjugate polyoxidonium = 1:1,5, 10.0 mg and 15.0 mg, respectively.

In examples 2-8 presents a synthetic immunogen to the variation of media, haptens and the ratio (conjugate polyoxidonium) in the claimed range.

Formulations of synthetic immunogen known and taken for the prototype [see Science, 1985], as well as their pharmacological actions are shown in the table. 2.

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

- reproducible without ballast impurities;

- accessibility and safety;

- high immunological activity;

- reduction of doses and duration of treatment;

- stable prolonged immunity;

- protection from a wide array of toxic substances (narcotic and psychoactive).

Preclinical studies

In a special series of experiments method, a biological sample has been clarified the possibility of neutralizing the toxic effects of heroin on animals. Group of control animals four unimmunized rabbit and experienced group of four immunized rabbit was injected intravenously absolutely lethal heroin dose of 100 mg/kg (LD100). It was found that all four of the rabbit of the control group died from the indicated doses. Among the immunized rabbits death is not registered with the I blood serum of immunized animals.

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

Immunization of rabbits drug on the scheme resulted in the serum of high titers of antibodies to immunocomplex 3 hemicylindrical - human gamma-globulin - polyoxidonium (1:2560). In response inhibition passive haemagglutination assays, it was found that the resulting antibodies bind heroin and 3 hemicylindrical: the titer of antibodies to immunocomplex decreased to 1:320 when using a concentration of heroin and 3-hemisuccinate 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.

Obtained from the blood serum of rabbit antibodies studied the binding specificity of oonam and other ligands of opiate prescriptions, conducted inhibitory enzyme linked immunosorbent assay. As inhibitors used morphine, heroin, codeine, N-allelomorphic and methadone. The results indicate that active immunization of animals with immunocomplexes 4 hemicylindrical - human gamma-globulin - polyoxidonium induced antibodies that specifically bind morphine, heroin, codeine, N-allelomorphic and practically do not react with methadone.

The specificity of antibodies against heroin in rabbit immune serum.

The data obtained show that the binding of opiates is dependent on the presence in their molecules of certain functional groups and radicals. So, in particular, the high specificity of antibodies to heroin due to the presence immunocomplexes complex essential communication naltrexone with succinic acid, and N-allylprodine - attached to the nitrogen atom cyclopropylmethyl radical in the chemical structure of naltrexone. Thus, the resulting antibodies have a high specificity only to opiates phenantrene series (heroin and its structural analogues).

Given the experimental results, the authors concluded that 3-hemisuccinate the prigoda the th response and synthesis of high-affinity antibodies to opiates phenantrene series.

Clinical studies.

Similar studies specificity were carried out with antibodies isolated from the serum of volunteers - addicts immunized with a synthetic 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 injected subcutaneously in the upper third of the right shoulder and left hand alternately, once a week, 8-10 injections per course.

From the source 22 sera addicts antipathie antibodies selected in all samples. According to ELISA in the sera contained antibodies of IgG - and IgM-class against morphine, heroin and codeine, reacting to the same extent with each of the metabolites.

The specificity of antibodies against heroin in human immune serum

As in the case of immune rabbit antiobiotic antibodies, antibodies isolated from the serum of the blood of drug addicts, practically do not interact with drugs refinancinga series. Antibodies isolated from the serum of patients with addiction compared to antibodies from rabbit immune sera have a wider range of specificity.

To confirm d is s for more information.

Specific examples of the application of synthetic immunogens presented in table. 2.

Example 9 (corresponds to part 1 of the table)

The patient is 25 years old, 7 years old opium addiction.

Initial laboratory data: 1 ml plasma - 2570 pmol germinomatous antibodies defined by radioimmune assay (RIA).

Patients were immunized with a synthetic immunogen - human gamma-globulin-naltrexone (hapten from the group isoquinolines) - polyoxidonium.

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

Control and laboratory data (12 months from the start of treatment with immunogen): RIA 1 ml - 50120 pmol.

Example 10. (2)

The patient was 32 years old, 9 years old opium addiction.

Initial laboratory data: 1 ml - 1520 pmol; sorption capacity of erythrocytes (SSE) - detoxification function of the body 64,1±5,7% (the definition of rapid absorption by red blood cells vital dyes methylene blue, see W.“Laboratory work”, 1988, No. 9, item A. A. Tojibaeva, what immunogen - human gamma-globulin-naltrexone - polyoxidonium.

Laboratory data: RIA 1 ml - 78610 pmol, SSE is 44.1±5,8% - increased detoxification function of the body. Other features are similar to the shown in example 1.

Control laboratory data: RIA 1 ml - 51100 pmol.

Example 11.(3)

The patient is 21 years, 4 years opium addiction.

Initial laboratory data: RIA 1 ml - 1680 pmol; SSE - 63,8±5,6%; determination of opiates (immunochromatographic tests urine) - positive.

Patients were immunized with a synthetic immunogen - human gamma-globulin-naltrexone - polyoxidonium.

Laboratory data: RIA 1 ml-78900 pmol, SSE - 42,4±5,8%; determination of opiate - negative, i.e., enhanced sorption of metabolites and xenobiotics. Other characteristics similar to those shown in examples 1 and 2.

Control laboratory data: RIA 1 ml - 52600 pmol.

Note examples 1-3: the data documented in the patient medical records.

Example 12.(4)

Six adult mongrel rats of both sexes were immunized with a synthetic immunogen - egg - albumin-amphetamine (hapten from the group of aromatic propanamine) - polyoxidonium at the rate of 50 mg/kg

Was administered the lethal dose of psychoactive weisheimer 1-3.

Control laboratory data: RIA 1 ml - 51200 pmol.

Example 13. (5)

Six adult mongrel rats of both sexes were immunized with a synthetic immunogen - egg albumin-methadone (hapten from the group difenilmetana) - polyoxidonium at the rate of 50 mg/kg

Was administered a lethal dose of drugs - heroin.

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

Control laboratory data: RIA 1 ml - 52600 pmol.

Example 14 (6)

Six adult mongrel rats of both sexes were immunized with a synthetic immunogen - diphtheria toxin-Garmin (hapten group indolamines) - polyoxidonium at the rate of 50 mg/kg

Was administered the lethal dose of psychoactive substance - meth.

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

Control laboratory data: RIA 1 ml - 54700 pmol.

Example 15 (7)

Six adult mongrel 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 the rate of 50 mg/kg

Was administered a lethal dose of sleeping pills - barbiturate.

Laboratory data: the data: RIA 1 ml - 51100 pmol.

Example 16 (8)

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

Was administered a lethal dose of drugs such as morphine.

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

Control laboratory data: RIA 1 ml - 57200 pmol.

The prototype.

Six adult mongrel rats of both sexes were immunized with immunogen diphtheria bovine serum albumin-heroin at the rate of 50 mg/kg

Was administered only intravenously with a lethal dose of drugs such as morphine.

Laboratory data: RIA 1 ml - 70100 pmol. High allergenicity caused by formation of immunoglobulins reagentnogo type of class IgE. The death of animals after 3 months as a result of anaphylactic shock during re-immunization.

Claims

Synthetic immunogen for the treatment and prevention of the abuse of narcotic and psychoactive substances representing a conjugate consisting of a carrier is a natural or artificial balconette the polyelectrolyte - the polyoxidonium at the ratio, wt.%: conjugate polyoxidonium = 1:1,5-5,0, moreover, it contains as a carrier of egg albumin, or human gamma globulin, or microbial toxins, and as a hapten is a low-molecular-weight compounds from the following groups: aromatic propanamine, or isoquinolines, or diphenylmethane, or indolamine, or derivatives of barbituric acid, or derivatives substituted piperidino.

 

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FIELD: immunology.

SUBSTANCE: the innovation deals with new immunogenic conjugates of beta-propionamide-bound polysaccharide and N-propionamide-bound oligosaccharide with protein, and the method to obtain these conjugates has been suggested, as well. Conjugates should be applied to obtain vaccines against infectious diseases and cancer that enables to broaden the number of preparations applied in treating the above-mentioned diseases.

EFFECT: higher efficiency.

1 dwg, 2 ex, 8 tbl

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