RU2441906C2 - Composition comprising low-molecular fragments of peptidoglicane of gram-negative bacteria for preventing or treating of human diseases - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: invention relates to the area of biotechnology. The pharmaceutical composition comprising three components of bacterial nature are described: (1) beta-N-acetyl-D-glucosaminyl-(1-4)-N-acetyl-D-muramoyl-L-alanine-D-isoglutaminyl-meso-diaminopimyelinic acid (GMtri), (2) beta-N-acetyl-D-glucosaminyl-(1-4)-N-acetyl-D-muramoyl-L-alanine-D-isoglutaminyl-meso-diaminopimyeloid-D-alanine (GMtetra), (3) GMtetra dimer (di-GMtetra) in which tetrameric residues of GMtetra are held together by an amide bond between carboxyl group of terminal D-alanine of one residue and w-amino group of meso-diaminopimyelinic acid of the other. The composition is proposed for obtaining a medicinal product for preventing or treating of diseases and conditions caused by bacteria that require enhancing nonspecific resistance of the organism to bacteria, diseases caused by Human Immuno-deficiency Virus (HIV) and herpes simplex virus, or diseases and conditions that require the organism's properties of malignant tumor rejection or myelopoiesis stimulation. A set for preventing or treating of diseases and conditions that comprses the above composition in a dosage form is disclosed, too.

EFFECT: increased effectiveness of certain medicines.

16 cl, 8 tbl

Description

FIELD OF THE INVENTION

The present invention relates to the field of pharmaceuticals and to the field of medicine. In particular, it discloses the use of a biologically active composition (hereinafter referred to as polyuramyl, abbreviated PM), intended to stimulate the mammalian immune system and consisting of three components of a bacterial nature, namely: (1) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D- from glutaminyl- meso- diaminopimelic acid (GMtri), (2) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl -D-muramoyl-L-alanyl-D- from glutaminyl- meso- diaminopimeloyl-D-alanine (GM tetra) and (3) a GM tetra dimer (diHM tetra), in which the bond between the monomer A GM tetra is carried out due to the carboxyl group of the terminal D-alanine of one GM tetra and the ω-amino group of the meso- diaminopimelic acid of another GM tetra. The invention also relates to pharmaceutically acceptable salts of PM. PM and its salts according to this invention are suitable for the treatment and prevention of diseases of mammals, in which it is necessary to stimulate the immune system.

BACKGROUND OF THE INVENTION

Application RU 2008144644 discloses a composition and a method for producing PM. This text is incorporated into this application by reference.

BACKGROUND

The decrease in human resistance to infectious agents, which is currently observed in many countries of the world, leads to an increase in the prevalence of chronic infectious and inflammatory diseases that are difficult to treat with traditional methods of treatment. Examples of such diseases are chronic bronchitis, chronic prostatitis, chronic recurrent pyoderma, chronic recurrent herpes virus infection. In these diseases, stimulation of the immune system is required in order to enhance the effects of traditional antimicrobial therapy and either eradicate a persistent infectious agent or reduce the frequency, duration and severity of exacerbations.

Recently, the problem of bioterrorism has become increasingly important. As a biological weapon, pathogenic bacteria, such as Yersinia pestis, Bacillus anthracis, Salmonella typhi, etc. can be used. In case of a threat of biological attack, measures may be required to quickly increase the population's resistance to bacterial infection. The protection given by traditional vaccines develops slowly (within weeks after the start of the vaccination course), since it is based on the activation of a slowly responding adaptive immune system. Under conditions of a biological threat, it is preferable to use immunostimulants that activate the innate immune system and give quick, albeit transient, protection against a wide range of pathogenic microorganisms.

Another important problem of medicine and society as a whole is acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV). Although the currently used highly active anti-retroviral therapy (HAART) allows for a long time to control HIV infection in patients, it is expensive and highly toxic. The issue of preventing HIV infection in risk groups has not been fully resolved either. Therefore, the use of new classes of drugs, in particular immunostimulants, for the prevention and treatment of HIV infection is justified and relevant.

An important problem of modern immunology is the ineffectiveness of the immune response against malignant tumors. A malignant tumor, as a rule, produces a number of factors that suppress the activity of cells of the innate immune system, designed to fight the tumor. Recently, intensive work has been conducted on the search for immunostimulants capable of “reactivating” cells of the immune system that are suppressed by the tumor process [Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002; 20: 709-760].

Thus, immunostimulants have several important fields of application in modern medicine. The search for new, effective immunostimulants with a minimum of side effects is an urgent task of immunopharmacology.

Most immunostimulants known today are either substances of bacterial origin, or their (semi) synthetic analogues. Among them, oligodeoxynucleotides (ODN) containing unmethylated tandems of cytosine guanine (CpG-ODN), non-pyrogenic lipopolysaccharides (LPS), and also peptidoglycan derivatives (PG) of the bacterial cell wall can be noted.

PG is a heteropolymer formed by linear polysaccharide chains in which the residues of N-acetyl-D-glucosamine (GlcNAc) alternate with the residues of N-acetyl-D-muramic acid (MurNAc) and are connected to each other β- (1 → 4) - glycosidic bonds. Short peptides are associated with the lactyl groups of MurNAc residues, which in the case of gram-negative bacteria, as a rule, are tripeptides of the type L-alanyl-D- iso- glutaminyl- meso- diaminopimelic acid or tetrapeptides of the type L-alanyl-D- iso- glutaminyl- meso- diaminopimeloyl- D-alanine. Tetrapeptides departing from adjacent polysaccharide chains can be linked together by covalent bonds (formed between the carboxyl group of the D-alanine residue of one tetrapeptide and the ω-amino group of the mesodiaminopimelic acid residue of the other tetrapeptide), which ensures the stability of the three-dimensional structure of PG.

The immunostimulating activity of GHGs is provided by both whole GHG macromolecules and its relatively small fragments (muropeptides), which arise either during the enzymatic hydrolysis of GHGs in human cells, or during the biosynthesis or degradation of GHGs in the bacteria themselves. The first of these fragments was discovered N-acetylmuramyl-L-alanyl-D-glutamic acid (muramyl dipeptide, MDP) [Ellouz AF, Ciorubaru R, Lederer E. Minimal structural requirements for adjuvant activity of bacterial peptidoglycan subunits. Biochem Biophys Res Commun 1974; 59: 1317-25]. MDP has a powerful immunostimulating and adjuvant effect, but was unsuitable for clinical use due to pronounced pyrogenicity. Other highly active natural muropeptides have been described later, in particular N-acetylmuramyl-L-alanyl-D- iso- glutaminyl- meso- diaminopimelic acid (muramyl tripeptide) [Girardin SE, Boneca IG, Carneiro LA, Antignac A, Jehanno M, Viala J, Tiala J, Viala J, Tiala J, K, Taha MK, Labigne A, Zahringer U, Coyle AJ, DiStefano PS, Bertin J, Sansonetti PJ, Philpott DJ. Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science 2003; 300: 1584-7]. A number of (semi) synthetic non-pyrogenic muropeptides have also been obtained that have obtained approval for medical use or are currently undergoing clinical trials [Werner GH, Jolles P. Immunostimulating agents. Eur J Biochem 1996; 242: 1-19]. Such muropeptides include 6-O-tetradecylhexadecanoyl-N-acetylmuramyl-L-alanyl-D-isoglutamine (B30-MDP); an MTP-PE preparation in which dipalmitoylphosphatidylethanolamine is covalently attached to the muramyl tripeptide, and the whole complex is incorporated into liposomes; N-acetylmuramyl-L-alanyl-D-isoglutamine-an-butyl ether (murabutide); N-acetylmuramyl-L-alanyl-D-isoglutamine-stearoyl-L-lysine (romurtide); β-N-acetylglucosaminyl- (1 → 4) -N-acetylmuramyl-L-alanyl-D-isoglutamine (GMDP or Lycopid).

In contrast to the compounds described above, which are fully or partially synthetic, we decided to create a muropeptide immunostimulant of a completely natural nature. We proceeded from the fact that the immune system of humans and mammals in the process of evolution "tuned" to the recognition of substances of natural origin, and therefore the effect of such substances on the immune system will be more effective and balanced in terms of the ratio of therapeutic and side effects.

As a source of PG, we took the classic gram-negative microorganism Salmonella typhi (S. typhi). Since natural PG is insoluble, its solubilization by hydrolysis in the presence of lysozyme, which cleaves the polysaccharide chains of PG, was required. At the initial stage of work, by partial lysozyme hydrolysis, a substance was obtained, which is a polymer fragments of PG, where the number of repeating units of GlcNAc-MurNAc ranged from 5 to 20. The method of preparation and some properties of this substance are described in application RU 2006124330. Despite the pronounced immunostimulating effect, this substance had significant drawbacks, namely: 1) the difficulty in selecting the time of incubation of GHG with lysozyme, and therefore the reaction products could contain on average both> 20 and <5 repeating units, 2 ) the uncertainty of the chemical structure, in particular the uncertainty of the content of peptide chains in the substance, which made it difficult to prepare a preparation with standard properties.

In the future, we began to strive to ensure that the hydrolysis of GHG went to the end, which was achieved by prolonged treatment with lysozyme. This led to a biologically active immunostimulating composition consisting of only three components (muropeptides) with a specific chemical structure:

compound 1 : β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D- from glutaminyl- meso- diaminopimelic acid (GMtri),

compound 2 : β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D- from glutaminyl- meso- diaminopimeloyl-D-alanine (GM tetra),

Compound 3: a GMetter dimer (diHMtetra), in which the bond between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetetra residue and the ω-amino group of the meso- diaminopimelic acid of the other GMtetra.

The specified composition is referred to in this application as "Polymuramil" (abbreviated PM). The method of preparation and some properties of PM were disclosed in the application RU 2008144644, where they are mentioned as low molecular weight products of the hydrolysis of PCB, for example, with a molecular weight of 1-2 kDa. With the targeted isolation of PM as the target product, it is necessary to carry out the process of enzymatic hydrolysis at a temperature of at least 35-37 ° C, the weight ratio of PCB: lysozyme 10: 0.3-1.0, for at least 3 days with the periodic addition of a fresh portion of the enzyme and, as described in the above application, with the constant removal of low molecular weight hydrolysis products, and their further purification is carried out using preparative gel chromatography. However, a detailed study of the effect of PM on various parts of the immune system has not been conducted. As a result, the specific pharmacological activity of PM was not determined. This was the reason that in the application RU 2008144644 was presented for protection only the way to obtain PM.

At the time of application RU 2008144644, the immunostimulatory activity of PM was studied fragmentarily. It was known that PM induces the production of tumor necrosis factor-alpha (TNF-α) by human mononuclear cells and also enhances the humoral response of experimental mice to a T-dependent antigen (sheep erythrocytes). However, this information disclosed in the application RU 2008144644 was not enough to justify the method of using PM in specific nosological forms. Based on the available data and on the properties of the known analogues of PM, one could assume a wide scope of the drug, in particular, for the treatment and / or prevention of infectious diseases of various etiologies. However, it turned out that PM does not induce the production of interferon alpha (IFN-α), a cytokine that plays a central role in protecting against most known viruses pathogenic for humans and mammals, which made it difficult to use PM for non-specific therapy or prevention of viral infections. These negative data served as the reason for a detailed study of the immunotropic properties of PM, which made it possible to clearly determine the spectrum of diseases in which the drug is able to exert a therapeutic effect based on experimentally confirmed types of its activity. The data of this study, which determine the scope and methods of application of PM, are the basis of this application.

SUMMARY OF THE INVENTION

The present invention relates to the use of PM and pharmaceutical compositions based on it for the treatment and prevention of diseases in which it is necessary to stimulate the immune system. The list of these diseases is determined based on the types of PM activity we have established. According to studies conducted by us after the filing of application RU 2008144644, PM has the following types of activity: 1) induces the production of activation cytokines (interleukin [IL] -1β, IL-6, IL-17, IFN-γ, TNF-α) and inflammatory chemokines (IP-10, IL-8, MIP-1α, MIP-1β, RANTES) by the cells of the innate immune system, which leads to a rapid increase in anti-infection resistance of the body; 2) enhances the ability of white blood cells to kill bacteria; 3) enhances the production of natural antibodies to common antigenic determinants of bacteria; 4) protects experimental animals from lethal infections caused by gram-positive and gram-negative bacteria; 5) induces the production of myelopoietins and enhances myelopoiesis; 6) induces the production of chemokines, which are natural antagonists of the human immunodeficiency virus; 7) increases the functional activity of NK cells playing one of the key roles in protecting the body from viral infections and malignant neoplasms; 8) slows the growth of transplantable malignant tumors in experimental animals. Moreover, PM does not have pyrogenic and other serious side effects with parenteral administration to humans at a dose of up to 10 μg / kg / day. The examples will illustrate the most important properties of PM, justifying the methods and scope of the drug and pharmaceutical compositions based on it. These properties of the active substance have not been described in the documentation of the prior art, including and in RU 2008144644.

The present application also relates to the use of PM for the preparation of pharmaceutical compositions for the treatment and prevention of diseases in which the stimulation of the immune system is necessary. PM, as a mixture of amphoteric compounds, can be used in neutral, anionic and cationic forms. In the anionic form, all or part of the protons in the terminal carboxyl groups are replaced by other cations, for example, sodium, potassium, ammonium, triethylammonium cations can be used. In the cationic form, all or part of the free amino groups are converted to the ammonium form, and, for example, chloride, sulfate or acetate anions can be used as a counterion.

The preferred pharmaceutical composition for the use of PM is a ready-made sterile solution for parenteral administration. The concentration of PM in the specified solution can vary from 0.01 to 20 mg / ml. The solution may optionally contain pharmacologically acceptable additives, including substances that increase the stability of the drug, pH regulators, tonicity regulators. The solution is packaged in sterile ampoules or ready-to-use injectors containing PM in an amount of 0.01 to 20 mg per ampoule / injector. Injectors for parenteral administration are described in the prior art and are marketed by various companies. These injectors are equipped with an injection solution, sterilized if necessary, and packaged in boxes, containers, etc.

The advantage of the injector is that it is a ready-to-use device that even an unprepared person can use.

In the course of studies it was found that muropeptide preparations, which include PM, can penetrate through the epithelial barriers into the bloodstream [Andronova TM, Pinegin BV Likopid (GMDP) is a modern domestic highly effective immunomodulator. Russia, 2005]. This makes it possible to release PM in the form of pharmaceutical compositions for transmucosal and transdermal use. So, PM can be produced in the form of pharmaceutical compositions for oral and oral administration, in particular in the form of tablets, capsules, oral solutions, troches and buccal forms. Capsules may contain mixtures of PM with inert excipients and / or diluents, such as pharmaceutically acceptable starches, sugars, synthetic sweeteners, powdered celluloses, various types of flour, gelatins, gums. Suitable tablet preparations may be prepared by conventional methods of compression, wet granulation or dry granulation using pharmaceutically acceptable excipients, diluents, binders, lubricants, disintegrants, surface modifiers (including surfactants), suspending or stabilizing agents. An oral preparation may also consist of an active ingredient that is administered in water or fruit juice, containing, if necessary, suitable solvents or emulsifiers. Packing of the drug can vary from 0.01 to 20 mg per tablet or capsule or from 0.01 to 20 mg per 1 ml of solution.

PM can be produced in the form of a solution for intranasal administration with a concentration of the active substance from 0.01 to 20 mg / ml in combination with pharmacologically acceptable additives.

PM pharmaceutical compositions for external use include ointments and creams containing PM in a concentration of from 0.01 to 20 mg / ml, optionally in combination with traditional substances that improve the penetration of the drug through the skin barrier (penetrators).

Finally, another form of PM release can be suppositories for rectal or vaginal administration containing PM in a dose of 0.01 to 20 mg per suppository, in combination with pharmacologically acceptable additives. The composition of the carrier compositions for suppositories is known in the art.

Finished forms of drugs and compositions based on PM are packaged in boxes, bags, tubes, bottles, etc. depending on the traditions and needs of the market.

For example, tablets may be packaged in a blister for one or more course doses. The blister may contain printed instructions for use and be packaged in a container or box in which the instructions for use are separately enclosed.

Various packaging options are known in the art and do not require further description.

The appointment of PM or pharmaceutical compositions based on it, as well as monitoring the patient's condition during treatment, is carried out by the attending physician. Based on the mechanism of action of the drug discovered by the authors, PM or pharmaceutical compositions based on it are shown in the groups of diseases listed below.

I. Treatment and prevention of diseases caused by bacteria:

1) treatment of acute and chronic diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract, female genital organs and prostate gland caused by bacteria;

2) prevention and treatment of purulent complications of surgical operations;

3) the prevention of infectious infectious diseases caused by pathogenic bacteria and manifesting themselves in the form of epidemics.

The use of PM for the treatment and prevention of diseases of bacterial origin is justified by the following types of its activity: 1) increased ability of phagocytes to kill bacteria; 2) the induction of the production of cytokines (IL-1β, IL-6, IL-17, IFN-γ, TNF-α) and chemokines (IP-10, IL-8, MIP-1α, MIP-1β, RANTES), necessary for development of an optimal innate immune response against bacteria; 3) the induction of the production of antimicrobial peptides - defensins; 4) increased production of natural antibodies to common antigenic determinants of bacteria; 5) induction of production of myelopoietins (G-CSF, GM-CSF) and increased output of myeloid cells (monocytes, granulocytes) from the bone marrow; 6) protection of experimental animals from infection with lethal doses of pathogenic gram-positive and gram-negative bacteria. As will be demonstrated in the Examples, the effect of PM develops rapidly, appearing already on the 1st - 3rd day of daily administration, which is important when using the drug for prophylactic purposes in risk groups.

II. Treatment of diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract and female genital organs caused by pathogenic and conditionally pathogenic fungi.

The use of PM for the treatment and prevention of diseases of fungal genesis is justified by the following types of its activity: 1) induction of the production of cytokines (IL-1β, IL-6, IL-17) and chemokines (IL-8), necessary for the development of an optimal innate immune response against fungi ; 2) the induction of the production of antimicrobial peptides - defensins; 3) induction of the production of myelopoietins (G-CSF, GM-CSF) and increased output of myeloid cells (monocytes, granulocytes) from the bone marrow.

III. Prevention and treatment of diseases caused by viruses:

HIV prevention and treatment;

treatment of chronic recurrent herpes virus infection (HVHVI) caused by the herpes simplex virus type 1 and 2 (HSV-1 and -2).

The use of PM in infections with these viruses is due to the ability of the drug to increase the activity of NK cells, which are one of the key effectors of antiviral immunity, to induce the production of chemokine IP-10, which is a chemoattractant for natural interferon-producing cells - the most important effectors of antiviral immunity, and also to induce the production of defensins - antimicrobial peptides capable of inhibiting the penetration of HSV and HIV into target cells and / or intracellular replication of these viruses [Mackew icz C et al. alpha-Defensins can have anti-HIV activity but are not CD8 cell anti-HIV factors. AIDS 2003; 17: F23-32. Hazrati E et al. Human α- and β-defensins block multiple steps in herpes simplex virus infection. J Immunol 2006; 177: 8658-66]. In addition, PM induces the secretion of chemokines RANTES, MIP-1α and MIP-1β, which block the cellular coreceptors for HIV and prevent the entry of HIV into cells.

IV. Adjuvant immunotherapy of solid malignant neoplasms of various localization. The use of PM for the treatment of malignant tumors is due to the following types of activity: 1) suppression of the growth of solid malignant tumors in experimental animals; 2) the induction of the production of cytokines involved in tumor rejection, in particular TNF-α; 3) increased activity of NK cells.

V. Treatment of conditions accompanied by suppression of myelopoiesis (acute and chronic radiation sickness, poisoning with myelotoxic substances, conditions during or after radiation therapy or chemotherapy of malignant neoplasms). The use of PM in this group of diseases is due to the ability of the drug to cause the production of colony-stimulating factors (G-CSF, GM-CSF), which are necessary for the differentiation of myeloid cells in the bone marrow, which leads to an increase in the levels of neutrophils and monocytes in the bloodstream.

The examples presented below, based on the use of adequate and evidence-based models for this area, confirm the proposed scope of the use of drugs and PM compositions.

However, the above list of diseases is not restrictive. The choice of specific conditions in which the appointment of PM or pharmaceutical compositions based on it is required is within the competence of a specialist, based on the identified and described biological activity of PM.

The preferred way to use PM in most nosological forms is parenteral. As a rule, PM is administered intramuscularly, however, depending on the nosological form, severity of the patient's condition and other factors, intradermal, subcutaneous and intravenous administration of the drug is possible. It was experimentally established that with intramuscular administration, the drug is safe at a dose of up to about 10 μg / kg / day. This dosage can vary from about 0.1 to 100 mcg / kg / day, depending on the patient's condition and individual PM tolerance.

In some cases, for example, for the treatment of inflammatory infectious diseases and malignant neoplasms of the gastrointestinal tract, PM can be used in the form of tablets, capsules or oral solutions. The dosage of the drug when taken orally is, as a rule, from 0.1 to 300 mcg / kg / day, depending on the patient's condition and individual PM tolerance.

For the treatment of diseases of the rectum, prostate and female genital organs, as well as for the prevention of HIV infection, PM can be used in the form of rectal and vaginal suppositories.

For the treatment of skin diseases, PM can be applied externally in the form of creams and ointments.

For the treatment of diseases of the upper respiratory tract, PM can be used in the form of drops for intranasal use.

When prescribing PM adhere to the traditional tactics of dose selection. The factors influencing the choice of treatment parameters are the type and severity of the underlying disease, individual susceptibility to the action of PM, the presence of concomitant diseases. The duration of the use of the drug for medicinal purposes is usually 5-7 days, however, depending on the nosological form, condition and reactivity of the patient, it can vary from 1 to 30 days. The duration of the use of the drug for prophylactic purposes is, as a rule, 1-5 days. The entire daily dose of the drug, as a rule, is given once, however, it is possible to divide the daily dose into 2-3 doses. PM can be used in combination with antimicrobial drugs for the treatment or prevention of infectious diseases, as well as in combination with standard antitumor chemotherapeutic and immunotherapeutic agents. The drug is compatible with antimicrobial, anti-inflammatory and antitumor drugs that were available to the authors, among those traditionally used in the treatment of the above diseases and approved for use in the Russian Federation as of March 2009 (see the Drug Register).

Examples

The following examples, given in order to detail and illustrate the invention, are not intended to limit the claims of the present invention.

Example 1 Obtaining the active substance

The active substance (PM) was obtained on the basis of the method disclosed in the application RU 2008144644. The control of the received batch was carried out by mass spectrometry (MALDI-TOF). From the data of MALDI-TOF it followed that the batch of the preparation contains three components - GMtri, GMtetra and diHMtetra with mol. masses (calculated values in brackets) are 868.4 (868.8), 939.4 (939.8) and 1860.8 (1861.7), respectively.

The resulting active principle is characterized by low reactivity due to the absence of highly reactive groups in it. This gives reason to expect that the resulting substance will have good storage properties and allow various manipulations with it. This assumption was verified and confirmed upon receipt and evaluation of the characteristics of the resulting preparations, compositions and dosage forms.

Various variants of medicinal compositions and forms were prepared from the obtained PM preparation.

In particular, under aseptic conditions, a PM solution in physiological saline was prepared.

The solution was packaged in ampoules and vials. The activity check after 6 months of storage, as well as after accelerated aging for an equivalent period showed the absence of sediment and PM degradation products.

In addition, on the basis of the obtained PM, dry preparations were prepared, including a lyophilisate for reconstitution before injection (with and without lactose supplementation), a dry powder composition with excipients, from which capsules, tablets and sachets intended for oral administration were then prepared.

The dry PM preparation was mixed with the composition for the preparation of suppositories based on cocoa butter, obtaining forms for rectal and vaginal administration.

PM-based creams and emulsions were conventionally prepared using conventional fat carriers and emulsions in the art. Glycerin and dimethyl sulfoxide were used as penetration activators (penetrants).

Lozenges for resorption were prepared using tragacanth and gums.

All prepared forms after 6 months of storage under normal climatic conditions showed the preservation of their functional properties both in terms of biological activity, and in terms of maintaining the characteristics of the dosage form.

Example 2. PM induces the production of a wide range of activation cytokines and chemokines by dendritic cells (DCs) and human macrophages (MFs) in vitro.

In the application RU 2008144644, the ability of PM to induce the production of TNF-α cytokine, one of the main activation cytokines, by mononuclear blood cells (MNCs), was disclosed. However, an effective immunostimulant should not induce a single cytokine, but a specific set of cytokines and chemokines that transmit an activation signal to different parts of the immune system. In this example, the ability of PM to induce the synthesis of cytokines and chemokines by dendritic cells and macrophages, which are one of the main targets of immunostimulants in vivo, was investigated. This model is adequate for assessing the effect of immunostimulants on the cytokine status in vivo. DK and MF were obtained from MNCC of healthy donors in vitro using the generally accepted technique [Sallusto F, Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte / macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 1994; 179: 1109-18]. DC and MF were cultured without PM or with PM concentration of 10 μg / ml for 24 h, after which supernatants were collected and cytokine levels were analyzed in them by multiplex analysis (table 1).

The data in table 1 show that already in the first 24 hours of contact with cells of the immune system, PM induces the production of a set of cytokines and chemokines by dendritic cells and / or macrophages. These cytokines are necessary for the successful response of the immune system to bacterial, fungal and some viral infections. So, interleukin-1β activates various cells of the innate and adaptive immune system, as well as endothelium; interleukin-6 induces the production of acute phase antimicrobial proteins in the liver and is necessary for the induction of an optimal adaptive immune response; interleukin-17 induces the production of chemokines necessary for attracting antibacterial and antifungal immunity effect cells (neutrophils, macrophages), and also causes the production of granulocyte colony-stimulating factor (G-CSF), which enhances the formation of neutrophils in the bone marrow; interferon-γ provides the activation of macrophages and cytotoxic T cells; TNF-α activates endothelium, various cells of the innate immune system, as well as - indirectly - cells of the adaptive immune system, enhances the production of chemokines. Chemokines induced by PM are necessary to attract effector populations of leukocytes to the injection site, which is important for local use of PM. So, IL-8 attracts neutrophils, macrophages, T cells, IP-10 - T cells and natural interferon-producing cells, also known as plasmacytoid dendritic cells. Data on the induction of chemokines RANTES, MIP-1α and MIP-1β will be shown in Example 6.

From table 1 it can also be seen that PM does not induce the production of the antiviral cytokine IFN-alpha, and therefore the drug is not expected to be highly effective in most viral infections. The exception is HIV and HSV infections, the use of PM for the treatment and / or prevention of which is justified in this application.

Example 3 PM enhances the bactericidal activity of blood leukocytes in healthy volunteers in vivo.

One of the main mechanisms by which the immune system fights bacterial and fungal infections is phagocytosis - the absorption of microbes by leukocytes, followed by the destruction (killing) of microbes inside the leukocyte. Intracellular killing of microorganisms plays a crucial role in protecting against bacterial and fungal infections. A direct illustration of this is chronic granulomatous disease (CGB), in which leukocytes cannot kill microbes due to a genetic defect, which leads to the development of chronic bacterial and fungal infections and ultimately to the death of the patient. Stimulation of the bactericidal activity of leukocytes is one of the main tasks of using immunostimulants in the treatment and prevention of infectious diseases. To study the effect of PM on this aspect of immunity, the drug was administered to 6 healthy volunteers intramuscularly at a dose of 400 μg in the form of an aqueous solution. Another 4 healthy volunteers received placebo (water for injection) instead of PM. All subjects were bled prior to injection (0 h), as well as 4, 24, and 120 h after injection. The bactericidal activity of the obtained blood leukocytes was evaluated according to a previously published method [Dambaeva SV, Mazurov DV, Golubeva NM, D'yakonova VA, Pinegin BV, Khaitov RM. Effect of Polyoxidonium on the Phagocytic Activity of Human Peripheral Blood Leukocytes. Russ J. Immunol 2003; 8: 53-60], measuring the percentage of Staphylococcus aureus cells killed inside leukocytes during 1 hour of in vitro incubation. This technique is a classic method for assessing the phagocytic immunity. PM significantly increased the bactericidal activity of leukocytes, which was manifested in an increase in the ability of leukocytes to kill staphylococcus 4 and 24 hours after injection (table 2). It is important to note that the introduction of PM at the indicated dose was not accompanied by any pyrogenic effect (an increase in body temperature above 37.5 ° C was not observed in any of the 6 subjects, up to 37.5 ° C in 1 of the tested) and others side effects.

Example 4 PM stimulates myelopoiesis

The ability to absorb and intracellular killing of microbes (see example 3) is possessed mainly by the so-called myeloid cells - neutrophils, monocytes and macrophages. In particular, a fall in neutrophil levels in the blood below 1000 / mm ³ (neutropenia) dramatically increases the risk of local and systemic bacterial and fungal infections, which is the higher, the more pronounced and prolonged neutropenia. In the absence of treatment, this condition leads to the death of the patient. The causes of neutropenia are radiation sickness, poisoning with myelotoxic substances, as well as radiation or chemotherapy for malignant neoplasms. For the treatment of neutropenia, the growth factors necessary for the differentiation of myelocytes in the bone marrow are relatively successfully used: granulocyte-macrophage colony-stimulating factor (GM-CSF, milgrastim) and granulocyte colony-stimulating factor (G-CSF, filgrastim). Both drugs are extremely expensive because their preparation is based on recombinant technology. Induction of the same factors in vivo seems to be an adequate and less expensive replacement for recombinant proteins.

We found that PM causes significant production of G-CSF and GM-CSF by dendritic cells and macrophages in vitro (Table 3). The experiments were performed as in Example 2.

To ensure that PM not only induces the production of CSF in vitro, but actually affects myelopoiesis in vivo, the drug was administered to healthy volunteers, as in example 3. Prior to injection, as well as 24 and 120 hours after injection, the levels of neutrophils, monocytes and lymphocytes in the blood. The data in table 4 show that 24 hours after intramuscular injection of PM at a dose of 400 μg, a selective and statistically significant increase in the levels of monocytes and young (stab) neutrophils occurs with a constant content of lymphocytes and “old” (segmented) neutrophils. These data confirm the ability of PM to stimulate myelopoiesis by inducing appropriate CSF, which justifies the use of the drug for hematopoiesis depression and for the treatment of infectious diseases of a bacterial and fungal nature.

Table 4
The content of various leukocyte populations in healthy individuals who received PM and placebo (× 10 ⁹ / l, M ± m) PM (n = 6) Placebo (n = 5) Neutrophils Lymphocytes Monocytes Neutrophils Lymphocytes Monocytes Nuclear wand Segmented Nuclear Nuclear wand Segmented Nuclear 0 h 0.05 ± 0.03 3.0 ± 0.04 1.8 ± 0.03 0.22 ± 0.04 0.12 ± 0.04 3.4 ± 0.17 2.1 ± 0.11 0.36 ± 0.03 24 h 0.29 ± 0.06 3.7 ± 0.16 1.9 ± 0.14 0.50 ± 0.10 0.17 ± 0.04 3.3 ± 0.16 2.1 ± 0.16 0.43 ± 0.06 120 h 0.12 ± 0.02 3.2 ± 0.08 1.8 ± 0.03 0.43 ± 0.06 0.01 ± 0.01 3.8 ± 0.18 1.9 ± 0.18 0.52 ± 0.06 * p <0.05 compared with the 0 h point (paired t-test)

Example 5 PM increases the resistance of mice to lethal infections with gram-positive and gram-negative bacteria.

An integral indicator of the immunostimulating activity of a substance is its ability to increase the resistance of a macroorganism to lethal infections caused by various types of pathogenic microorganisms. An increase in infection resistance may be due to several mechanisms, including those described in Examples 1-4. Bacterial immunostimulants, such as in this invention, act mainly on the innate component of the immune system and therefore cause an immediate, relatively non-selective, but transient resistance to pathogenic microorganisms. In this example, the effect of PM on nonspecific resistance was studied on outbred mice weighing 12-14 g. Groups of 10 mice were given PM once, subcutaneously, at doses of 100, 10 and 1 μg / mouse. The control group was injected with sterile saline. 24 hours after the injection, the mice were intraperitoneally infected with 1 DCL (absolutely lethal dose) of Salmonella typhimurium (strain 415) or Staphylococcus aureus (strain Wood 46). These microorganisms are typical representatives of gram-negative and gram-positive pathogenic bacteria, respectively, and are traditionally used in such experiments. In the experiment described, the most pathogenic strains of S. typhimurium and St. aureus. When using less pathogenic strains, the effect came faster (data not shown).

From table 5 it is seen that PM at a dose of 100 μg / mouse increases the resistance of mice to infections caused by S. typhimurium and St. aureus, protecting from death by 60% and 50%, respectively. Smaller doses have a weaker protective effect. Thus, PM has a pronounced ability to protect experimental animals from fatal infections caused by gram-negative and gram-positive bacteria.

Table 5
The effect of PM on the survival of mice after infection with lethal doses of pathogenic bacteria Dose PM (mcg / mouse) Bacteria The death of mice (day) Survived% 1st 2nd 3rd 4th one hundred S. typhimurium 0 0 3 one 60 10 0 5 2 2 10 one 0 8 one one 0 physical rr one 9 0 0 0 one hundred St. aureus 3 2 0 0 fifty 10 5 2 one 2 0 one 5 four one 0 0 physical rr 8 one 0 one 0

Example 6 PM induces the production of chemokines, which are antagonists of the human immunodeficiency virus (HIV).

To infect human immunocompetent cells (T cells, monocytes, DC, MF), HIV uses a receptor (CD4 molecule) and two coreceptors, which are the chemokine receptors CCR5 and CXCR4. In particular, the CCR5 receptor is used by the so-called lymphotropic strains of HIV, which predominantly infect T cells. Individuals who lack CCR5 as a result of mutations are relatively resistant to HIV. Therefore, HIV treatment and prevention methods are being developed to prevent the binding of HIV to CCR5. There are basically two types of such methods: 1) the use of synthetic ligands CCR5 [Westby M, van der Ryst E. CCR5 antagonists: host-targeted antivirals for the treatment of HIV infection. Antivir Chem Chemother 2005; 16: 339-54]; 2) the induction of the production of chemokines, the natural ligands of CCR5, which, by binding to CCR5, block the interaction of HIV with this receptor [Verani A, Lusso P. Chemokines as natural HIV antagonists. Curr Mol Med 2002; 2: 691-702]. We showed that PM induces the production of three natural CCR5 ligands — the chemokines MIP-1α, MIP-1β and RANTES — by human dendritic cells (Table 6). The experiments were carried out as in Example 2.

Donors who received 400 μg PM intramuscularly (see Example 3) showed a statistically significant increase in serum MIP-1β 4 hours after injection (table 7).

Thus, PM induces the production of natural HIV antagonists both in vitro and in vivo, which is the rationale for the use of PM for the prevention of HIV infection, as well as in the framework of complex treatment of HIV infection.

Example 7 PM inhibits the growth of a malignant tumor (B16 melanoma) in mice.

Inhibition of the growth of B16 melanoma after replanting it to C57BL / 6 mice is a classic model for studying the effectiveness of various methods of chemo- and immunotherapy of malignant neoplasms. The therapeutic efficacy of PM was investigated in prophylaxis and treatment regimens. In the prophylaxis mode, groups of mice received a single subcutaneous injection (at the withers) of 100 μg or 10 μg of PM per 50 μl of physiological saline, or 50 μl of physiological saline without preparation, and after 24 hours 5 × 10 ⁵ suspended B16 cells were injected into all paw pads . On day 14, the mice were sacrificed, the primary tumor was removed, and its volume was evaluated. In the treatment regimen, the mice were first injected with tumor cells in the amount indicated above, and on the 3rd day the indicated doses of PM or physiological saline were injected subcutaneously. Tumor volume was evaluated at the same time. The experimental results shown in table 8 show that PM at a dose of 100 μg / mouse in the prophylactic mode led to a significant decrease in tumor volume by about 30%, and in the treatment mode by about 50% compared with the groups that received saline. At a dose of 10 μg / mouse, the effect was less pronounced. Thus, PM dose-dependently inhibits the growth of a malignant tumor in model animals.

Thus, the information presented proves the possibility of a new use of PM in the treatment and prevention of a number of disorders, diseases and conditions that are associated with the mechanisms described in the above examples.

Thus, the examples prove the therapeutic applicability of PM preparations. The choice of specific treatment regimens is the responsibility of the attending physician and cannot be limited to the specific options described in the text of this application.

The scope of claims within the framework of the present invention is determined solely by the claims.

Claims (16)

1. The use of a composition comprising three components of a bacterial nature, namely
(1) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri),
(2) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GM), and
(3) GMetter dimer (diHMetter),
where the connection between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetter residue and the ω-amino group of the mesodiaminopimelic acid of another GMetter residue,
or their salts,
for the manufacture of a medicament intended for the prophylaxis or treatment of conditions and diseases caused by bacteria, which require an increase in the nonspecific resistance of the organism to bacteria, diseases caused by the human immunodeficiency virus (HIV) or herpes simplex virus, or diseases and conditions requiring an increase in the body's ability to rejection of a malignant tumor, or stimulation of myelopoiesis. 2. The use according to claim 1, where the disease caused by bacteria is selected from the group including:
diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract, female genital organs and prostate gland;
purulent complications of surgical operations;
contagious infectious diseases, manifested in the form of epidemics. 3. The use according to claim 1, where the disease caused by the human immunodeficiency virus (HIV) or herpes simplex virus is selected from the group including:
chronic recurrent herpes virus infection (HVHVI) caused by herpes simplex virus types 1 and 2;
acquired immunodeficiency syndrome caused by HIV. 4. The use according to claim 1, where the disease or condition requiring strengthening the body's ability to reject a malignant tumor is associated with the presence of a solid tumor in the patient. 5. The use according to claim 1, where the disease or condition requiring increased myelopoiesis is acute or chronic radiation sickness, poisoning with myelotoxic substances, conditions during or after radiation therapy or chemotherapy of malignant neoplasms. 6. The pharmaceutical composition for the treatment and prevention of diseases and conditions caused by bacteria, which require an increase in the nonspecific resistance of the body to bacteria, diseases caused by human immunodeficiency virus (HIV) or herpes simplex virus, or diseases and conditions requiring an increase in the body's ability to reject a malignant tumor, or stimulation of myelopoiesis, containing an effective amount of a mixture consisting of
(1) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri),
(2) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GM), and
(3) GMetter dimer (diHMetter),
where the connection between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetter residue and the ω-amino group of the mesodiaminopimelic acid of another GMetter residue,
or their salts,
and a pharmaceutically acceptable carrier, preservative and isotonic agent. 7. The pharmaceutical composition according to claim 6, where the disease caused by bacteria is selected from the group including:
diseases of the skin, soft tissues, respiratory tract and lungs, gastrointestinal tract, kidneys, urinary tract, female genital organs and prostate gland;
purulent complications of surgical operations;
contagious infectious diseases, manifested in the form of epidemics. 8. The pharmaceutical composition according to claim 6 in the form of a solution, ready for use. 9. The pharmaceutical composition according to claim 6 in the form of a suppository. 10. The pharmaceutical composition according to claim 9, where the disease is a disease of the female genital organs or rectum caused by bacteria, or hrgvi with damage to the external genital organs, or HIV infection. 11. The pharmaceutical composition according to claim 6 in solid form, selected from the group consisting of tablets, pellets, dragees and capsules, intended for oral or oral administration. 12. The pharmaceutical composition according to claim 6 in the form of a gel, cream, ointment, patch for external use. 13. The pharmaceutical composition according to item 12, where the disease is a skin disease caused by bacteria, or hrhVI with damage to the skin. 14. The pharmaceutical composition according to claim 6 in the form of a medicine or syrup for oral administration. 15. A kit used to treat or prevent diseases and conditions caused by bacteria, which require an increase in the body's non-specific resistance to bacteria, diseases caused by human immunodeficiency virus (HIV) or herpes simplex virus, or diseases and conditions that require an increase in the body's ability to rejection of a malignant tumor, or stimulation of myelopoiesis, containing the pharmaceutical composition according to any one of claims 8, 9, 11, 12 or 13 in a dosage form in an amount of 1-20 units; instructions for use in the treatment or prevention of these diseases and conditions; and packaging. 16. A liquid pharmaceutical composition for the treatment and prevention of diseases and conditions caused by bacteria, which require an increase in the nonspecific resistance of the organism to bacteria, diseases caused by the human immunodeficiency virus (HIV) or herpes simplex virus, or diseases and conditions requiring an increase in the body's ability to rejection of a malignant tumor, or stimulation of myelopoiesis, containing an effective amount of a mixture consisting of
(1) β-N-acetyl-D-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimelic acid (GMtri),
(2) β-T-acetyl-B-glucosaminyl- (1 → 4) -N-acetyl-D-muramoyl-L-alanyl-D-isoglutaminyl-meso-diaminopimeloyl-D-alanine (GM), and
(3) GMetter dimer (diHMetter),
where the connection between the monomer residues of the GMetter is due to the carboxyl group of the terminal D-alanine of one GMetter residue and the ω-amino group of the mesodiaminopimelic acid of another GMetter residue,
or their salts,
and a pharmaceutically acceptable carrier, preservative and isotonic agent,
where the specified composition is included in the injection device, which is a single injector, ready for use.