WO2007065451A1

WO2007065451A1 - Means for preventing and treating rhinitis of different ethiology, method for the production thereof, a pharmaceutical composition based thereon and a preventing and treating method

Info

Publication number: WO2007065451A1
Application number: PCT/EA2006/000014
Authority: WO
Inventors: Leonid Andreevich Kozhemyakin; Olga Sergeevna Ketlinskaya
Original assignee: Leonid Andreevich Kozhemyakin
Priority date: 2005-12-07
Filing date: 2006-09-11
Publication date: 2007-06-14
Also published as: EA009610B1; EA200600141A1

Abstract

The invention relates to the chemico-pharmaceutical industry and can be used for medical practice in a monotherapeutic mode and for the multimodality treatment of rhinitis of different ethiology. Said invention also relates to a disulphide tetralithium salt and to a method for the production thereof consisting in oxidising a starting compound, embodied in the form of a reduced glutathione (η-L-glutamyl-L-cysteinyl-glutaminyl-glicine), by a hydrogen dioxide in the presence of aqueous ammonia, in releasing a free hexapeptide (bis((η-L-glutamyl)- L-cysteinil-bis-glicine) by acidifying the solution with a glacial acetic acid, in converting the acidified form of free hexapeptide in a tetralithium salt, in filtering the solution in vacuum, in stabilising the hexapeptide disulphide bond by exposing the reaction solution to an ultraviolet radiation and in releasing a final product by a liophylic drying method. The inventive means can be used in the form of a medicinal preparation for preventing and treating rhinits of different ethiology, including vasomotor rhinitis. A pharmacological composition containing a disulphide glutathione tetralithium salt, zinc sulphate, hydrogen dioxide, adrenaline and water for injections, dosage forms based on said medicinal composition, for example nasal drops and spray, and a method for preventing and treating nosological forms of acute and chronic infection and allergic diseases of nasal mucous are also disclosed.

Description

Means for the prevention and treatment of rhinitis

of various etiologies, a method for its preparation, a pharmaceutical composition based on it,

method of prevention and treatment

The invention relates to the pharmaceutical industry and can be used in medical practice in monotherapy mode and in the complex treatment of rhinitis of various etiologies.

The international classification of rhinitis [1] provides for their division into acute and chronic forms, and in the chronic form, the inflammatory process can be caused by a nonspecific or specific pathogen. Specific forms of infectious rhinitis (with diphtheria, scleroma, tuberculosis, syphilis, etc.) are quite rare. Acute infectious rhinitis (OIR) is more often caused by viruses, chronic - by bacteria, less often - by fungi. Bacteria are usually the causative agents of inflammation accompanying atrophic processes, as well as complicating the course of the postoperative period. The atrophic form of rhinitis is noteworthy, a significant pathogenesis of which is played by a specific pathogen - Klebsiella. Infectious rhinitis can be caused by a number of damaging factors: surgical trauma, exposure to chemical and physical factors, and in these cases, bacteria are also a secondary etiological factor that affects the damaged mucous membrane, devoid of its protective mechanisms.

The most typical pathogen of OIR are rhinoviruses [2]. Individual susceptibility to viral infection depends on many factors: age, physical development, sanitary conditions, degree of adaptation to meteorological shifts and hardening of the body surface to the effects of low or contrast temperatures, the presence of intercurrent diseases. Children suffer from acute runny nose more often than adults. Unlike influenza viruses and adenoviruses, which infect wide areas of the epithelium of the nasal cavity, rhinovirus infection is characterized by isolated foci of the affected epithelium, while most of the mucous membrane remains intact. Viral infection is usually only the first phase of the disease, and it “creeps up” the bacterial infection. Under the conditions of a normally functioning mechanism of mucociliary transport, bacteria are not able to contact the epithelial cells of the nasal cavity for a sufficiently long time. Such conditions are created when the cells of the ciliated epithelium affected by the virus temporarily do not function. This prolongs the contact time of pathogenic bacteria with cells and makes secondary bacterial infection possible. The entire cycle of the disease ends in 7-10 days, but may have a shorter abortive course or, under adverse conditions, drag on and lead to the development of complications — sinusitis, otitis media, tracheobronchitis, etc. Acute rhinitis can become more severe in young children. Due to the lack of nasal breathing, the child becomes irritable, screams, refuses to breast, throws his head back (symptom of false opisthotonus).

Chronic infectious rhinitis (CID) is traditionally divided into simple (catarrhal), hypertrophic and atrophic forms [3]. These forms rarely exist separately, in their pure form. Under various conditions, chronic catarrh of the nasal cavity can transform into subatrophic (atrophic) or hypertrophic forms. In addition, hyperplasia of the submucous layer of the mucous membrane is often combined with atrophic changes in the epithelium or, conversely, with an increase in the number of glands and hyperproduction of mucus. Hypertrophic processes often concern the lower nasal concha, which increase in size, gradually occupying all the lower parts of the nasal cavity. Hypertrophy can be local and capture mainly the posterior ends of the shells, which in this case take the form of a polyp. A separate form of CHR is dry anterior rhinitis. The etiology and pathogenesis of this disease remain to the end not clarified. It can occur during prolonged exposure to dust and other harmful production factors - hot dry air, chemicals (ammonia, formalin, hydrochloric, nitric, sulfuric acids and so on). The main symptom among the clinical manifestations of HIR is difficulty in nasal breathing. It can be unstable, accompanied by periodic stuffiness of one or the other half of the nose, amplified at night, almost always progresses gradually, which ultimately leads the patient to the doctor. Nasal breathing can be difficult for various reasons: as a result of swelling or hypertrophy of the nasal concha or due to the accumulation of secretions or crusts in the general nasal passage [4]. Discharges with CHR are usually thick, colorless or yellowish, greenish or gray, can leave the nose in the form of crusts, sometimes there is an admixture of blood in the secretions. The goals that, depending on the specific clinical manifestations of rhinitis, can be pursued to varying degrees when prescribing topical preparations, are: elimination of microbial pathogens, treatment of the inflammatory process, restoration of nasal breathing, decreased secretion, stimulation of reparative and trophic processes in the mucous membrane, normalization functions of protective barriers: mucociliary transport and local mucosal immunity.

According to the international consensus on the treatment of allergic rhinitis [5], published in 2000, allergic rhinitis is a disease of the nasal mucosa, which is based on allergic inflammation caused by causative allergens, clinically manifested by rhinorrhea, nasal congestion, itching in the nasal cavity, repeated sneezing. Additional symptoms may include headache, impaired sense of smell, and conjunctivitis.

Depending on the characteristics of the course and exacerbations of allergic rhinitis associated with the time of year, year-round and seasonal forms of the disease are distinguished. Seasonal Allergic Rhinitis (CAP) is associated with exposure to pollen allergens. The incidence of CAP significantly depends on the geographical area, local climate and time of flowering plants. All this must be taken into account when interpreting the symptoms of the disease and making a diagnosis. A feature of CAP is the frequency of exacerbations. The clinical symptoms of the disease recur from year to year at the same time and have a clear relationship with the flowering period of certain plant species. Symptoms of CAP in most patients disappear immediately after the flowering period of allergenic plants.

Year-round allergic rhinitis (CAR) is caused by allergens from house dust, house dust mites, pets, feather pillows, cockroaches, mice, rats, and some types of mold fungi. For year-round allergic rhinitis is characterized by constant clinical symptoms throughout the year. The most common and typical clinical symptom in chronic CAR is nasal congestion. Sneezing attacks, colorless discharge from the nose, itching in the nose, eyes, ears, throat can confirm the allergic nature of rhinitis. A characteristic symptom of CAR is a paroxysmal sneezing on waking up or in the early morning hours. In some patients, nasal congestion is more pronounced at night, which adversely affects sleep. The course of CAR is aggravated by the influence of nonspecific factors (cold air, tobacco smoke, changes in weather conditions), as well as various infectious agents (viruses, bacteria, fungi).

The chronic course of CAR in children can contribute to the development of serous otitis media, nosebleeds, pain in the frontal sinuses, decreased sense of smell, sleep disturbance, as well as coughing or dry coughing. With prolonged current CAR, children can develop polyps, malocclusion, gingival hypertrophy.

In recent years, non-allergic rhinitis with eosinophilia syndrome (NARSE) has been isolated - a disease similar in clinical symptoms with allergic rhinitis, but not based on immunological (allergic) mechanisms of development. Most often, the cause of NARSE are medications, occupational hazards, foreign bodies.

Topical preparations for the treatment of rhinitis are known [6, 7, 8]. They can be divided into the following groups: antibacterial, antiviral, vasoconstrictor, anticholinergic blockers, mucoactive, vaccines, moisturizers and hygiene products, drugs with a complex effect, combined and phytopreparations [9].

Antibacterial drugs

Mupirocin - a nasal ointment, an antibiotic with a bactericidal effect - has almost 100% effectiveness against Staphylococcus aureus and epidermal Staphylococcus aureus, including methicillin-resistant and other multi-resistant S. aires. The standard course of treatment consists in daily two-, three-fold application of ointment on the mucous membrane of the nasal cavity for 5 days.

Fyuzafyunzhin - an inhaled antibiotic, available in the form of a metered-dose aerosol. Fyusafyunzhin effective against bacteria, most often the causative agents of rhinitis. Its distinguishing feature is the stability of the spectrum of antibacterial action. In addition to antibacterial properties, Fyuzafyunzhin has its own anti-inflammatory effect.

Framycetin is an antibiotic, due to its antibacterial effect it can be used in the treatment of acute and chronic rhinitis. Available in the form of nasal drops and nasal spray.

Octenisept, an antiseptic for the treatment of mucous membranes, has an exceptionally wide spectrum of antimicrobial activity, which encompasses gram-positive and gram-negative bacteria, chlamydia, mycoplasma, fungi, protozoa, and viruses herpes simplex and AIDS. The main disadvantage of the drug is that it is not available in forms convenient for independent use (drops, aerosol), therefore its use is limited so far mainly by the ENT doctors' offices.

There is a group of combined drugs, which in addition to antibacterial agents include corticosteroids and / or vasoconstrictor drugs: Polydex with phenylephrine (polymyxin + neomycin + dexamethasone + phenylephrine), Gikomycin-Teva (neomycin + hydrocortisone), Dexarinosprey (neomycin + dexazase ) and others. In addition to the direct bactericidal action, these drugs also have anti-inflammatory and desensitizing effects, however, when they are prescribed, one should remember about 100% of the bioavailability of their tav corticosteroid drugs.

Antiviral drugs. Interferon α is actually the only antiviral drug that has been used for insertion into the nasal cavity. It is believed that the interferon solution should be used at the very early stages of the development of infectious rhinitis, therefore, in the first hours of the disease, it is recommended that the interferon solution be instilled into both halves of the nose every 20-30 minutes, then 4-5 times a day. The effectiveness of intranasal administration of interferons for the prevention of influenza and acute respiratory viral infections began to be studied in the 1960s, however, according to some studies, it turned out to be quite low.

Topical vasoconstrictor drugs (decongestants). The vasoconstrictors used in the form of drops and aerosols act on the regulation of the tone of the blood vessels of the nasal cavity. By activating adrenergic receptors, they cause a reduction in the cavernous tissue of the nasal concha and, as a result, the expansion of the nasal passages and the improvement of nasal breathing. According to the mechanism of action, most topical decongestants are α-adrenomimetics, and they can selectively act test on ar or α ₂ receptors. Adrenaline itself stands out, which is able to stimulate both the α _b and α ₂ adrenergic receptors, as well as ephedrine, which promotes the release of norepinephrine, and cocaine, which prevents its utilization by tissues.

Decongestants differ in their pharmacodynamic characteristics, severity and duration of action and side effects. All of these drugs, with prolonged use, cause the development of ricochet syndrome (as a variant of withdrawal syndrome). Long-term (over 10 days) use of these drugs can cause severe swelling in the nasal cavity, nasal hyperreactivity, changes in the histological structure (remodeling) of the mucous membrane, i.e., the development of drug rhinitis. You should also be aware of the possible side effects of benzalkonium chloride, a preservative that is part of most topical decongestants. Decongestants should be prescribed with caution in the presence of atrophic and subatrophic changes in the mucous membrane, as well as in children under 2 years of age, because the existing interval between the therapeutic and toxic dose is small. Decongestants include: Naphthyzine drops 0.05% and 0.1%, Farmazolin drops 0.05% and 0.1%. One of the known effective topical decongestants is the drug Evkazolin. Its main components: 0.1% solution of xylometazoline hydrochloride and eucalyptus oil. It is used for acute rhinitis and rhinosinusitis, hay fever, otitis media to reduce swelling of the mucous membrane of the nasopharynx, while preparing the patient for diagnostic and therapeutic procedures in the nasal passages.

Anticholinergic drugs. The classic representative of this group is ipratropium bromide, which, blocking the muscarinic receptors of the glands of the mucous membrane, interrupts parasympathetic stimulation mediated by the classical mediator acetylcholine, and reduces the amount of watery secretion intensely produced in the nasal cavity, for example, at an early stage of development of OID.

Bacterial vaccines. An example of the use of bacterial lysates for intranasal use is the drug IPC-19, containing lysates of 19 different bacteria - the most common pathogens of respiratory infections. The specific action of IPC-19 is associated with increased synthesis of secretory IgA.

Moisturizing and hygiene products. This group of drugs is not a medicine. They are prepared either from sea water, sterilizing it and bringing the salt content to an isotonic concentration (Aqua Maris, Salin, etc.), or from mineral water with medicinal properties (for example, Ermers Salz).

In the treatment of rhinitis, drugs with a complex effect are used, combined and phytopreparations, in particular those that include essential oils, peppermint oil (menthol): Pinosol, Cameton, etc.

Thus, the arsenal of drugs for the treatment of OIR and HIR is currently quite diverse. However, the disadvantages of these drugs include the fact that they do not affect the mechanisms underlying the chronization of the disease, the fundamental processes that form the imbalance of the local immunity system in the nasal and nasopharynx mucosa. Therefore, the entire level of the aforementioned previous biotechnology for the treatment of rhinitis is essentially symptomatic, solving only short-term tasks, forming a vicious circle of chronicity of the pathological process and, consequently, the gradual death of the epithelium of the nasopharynx.

Thus, it should be emphasized that there are practically no means to: - restore basic violations in the relationship of immunological and neurotrophic processes,

- improve cell metabolism,

- to provide a cytoprotective effect, and thus prevent the formation of atrophic processes in the nasal mucosa and correct the local immunity system.

Correction of the local immunity system is especially important and necessary, since the imbalance of the local immunity system is the fundamental mechanism for the formation of chronic, especially vasomotor rhinitis. The system of local immunity, being initially a weak point in diseases of an infectious-allergic nature, is more and more destroyed during treatment with antibacterial drugs and hormonal agents. Remissions are becoming less frequent, susceptibility to infection is increasing and the process becomes chronic with the development of metabolic disorders in the epithelial cells of the nasopharynx. At a certain stage, destructive processes become prevailing, determining the nature and severity of the chronic infectious and inflammatory process, in this case, the clinical course of chronic rhinitis.

An object of the present invention is to provide a pharmaceutical composition having antibacterial, antiviral, immunomodulatory, cytoprotective activity, anti-inflammatory and anti-destructive action.

To solve this problem, a group of inventions is proposed, united by a single inventive concept, namely:

- the use of bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate lithium (tetralitium salt of disulfide glutathione) with stabilized disulfide bond as a means for the prevention and treatment of rhinitis of various etiologies; - a method of obtaining the specified funds, including the oxidation of the starting compound - reduced glutathione (γ-L-glutamyl-L-cysteinyl-glycine) - hydrogen peroxide in the presence of aqueous ammonia by stirring an aqueous solution of the starting material and these reagents for 20-35 minutes at 18-25 ^° C and pH 8; isolation of free hexapeptide (bis- (γ-L-glutamyl) -L-cystinyl-bis-glycine) by acidifying the solution with glacial acetic acid to pH 5; the conversion of the oxidized form of the free hexapeptide to the tetralithium salt by treating its aqueous solution with an aqueous solution of LiOH; filtering the solution obtained after the oxidation of water with vacuum; stabilization of the disulfide bond of the hexapeptide due to its energy; isolation of the final product by freeze drying;

- stabilization of the disulfide bond of bis- (γ-L-glutamyl) -L-cystinyl-lithium bis-glycinate by irradiating the reaction solution with an ultraviolet irradiation apparatus;

- a pharmaceutical composition for the prevention and treatment of rhinitis of various etiologies, including bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate (tetralithium salt of disulfide glutathione), zinc sulfate, hydrogen peroxide, adrenaline and water for injection with their following ratio,% macc:

Bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate

(tetralithium salt of disulfide glutathione) 0.5-7.0

Zinc Sulfate 0.02-0.8

Hydrogen peroxide 0.01-0.5

Adrenaline 0.001-0.1

Water for injection rest;

- the pharmaceutical composition may be in the form of drops for the nose or a spray for the nose;

- a method for the prevention and treatment of rhinitis of various etiologies, characterized in that the pharmaceutical composition is used 2-6 times per day, as the inflammatory process is stopped, the number of injections of the drug is reduced to 2-3 times a day, the course of treatment is continued until the symptoms of the disease disappear.

The essence of the invention is to develop a pharmaceutical composition based on bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate lithium (tetralithium salt of disulfide glutathione), which has unique properties: the ability to induce apoptosis of virus-infected cells and increase the resistance of uninfected cells to infection by bacteria, viruses and other agents. It is a similar set of properties of the tetralitic salt of disulfide glutathione and, therefore, the claimed pharmaceutical composition that allows a new solution to the problem of prevention and treatment of acute and chronic rhinitis.

A preferred embodiment of the idea of the invention was the development of an original method for the synthesis of tetralithium salt of disulfide glutathione with a stabilized disulfide bond and the inclusion of lithium on all carboxyl groups of disulfide glutathione. Moreover, a feature of the method lies in the fact that during the synthesis process, the reaction solution containing the named compound is exposed to ultraviolet radiation, thereby achieving the stability of the final product and, therefore, its inherent biological and pharmacological effects.

The formulation of the pharmaceutical composition is developed, i.e., a strictly defined composition and ratio of its components (ingredients). The optimal composition of the pharmaceutical composition is the basis of its biological and pharmacological efficacy, which is established in clinical and experimental studies and is presented in the implementation examples.

The integrative result of the biological and pharmacological effects of the claimed pharmacological composition as a means of implementing a method for the prevention and treatment of chronic rhinitis of various etiologies is its ability to regulate the ratio of humoral and cellular

and immunity, as well as metabolic processes underlying the proliferation, differentiation and apoptosis of the cells of the nasal mucosa and nasopharynx.

For the prevention and treatment of rhinitis of various etiologies, a tool is proposed - a tetralithium salt of disulfide glutathione with a stabilized disulfide bond, having the structural formula:

Characteristics of the substance:

1. Chemical name: Bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate lithium.

2. Gross formula: C _2O H _2S N ₆ Oi ₂ Li ₄ S ₂ .

3. Molecular mass: 636.37.

4. Description: odorless white powder.

5. Solubility: soluble in water, 0.9% isotonic sodium chloride solution for injection; insoluble in 95% alcohol, chloroform, ether and other organic solvents.

6. The transparency and color of the solution: a solution of 0.05 g of the drug in 10 ml of water is transparent and colorless.

7. pH of 0.1% solution: 5.0-6.0 potentiometrically.

8. Authenticity: - amino acid analysis (6 N. HCl ₅ PO ⁰ C ₅ 20 h): glycine-2.0 (2.0); glutamic acid - 1.9 (2.0); cysteine - 1.9 (2.0); cysteine - 1.7 (2.0);

- NMR (^^ spectroscopy: CH ₂ (δ 2.05; 2.40; 3.00; 3.80); CH (δ 3.72; 4.65);

- HPLC: according to the output time corresponds to the standard.

9. Quantification: HPLC:> 95%,

10. Amino acid analysis:> 85%,

11. TCX: homogeneous,

12. The content of lithium (Li) by emission spectral method: (4.4 ± 0, l)%.

The proposed method for the production of tetralithium salt of disulfide glutathione (bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate) includes:

- oxidation of the starting compound (I) - reduced glutathione (γ-L-glutamyl-L-cysteinylglycine) - hydrogen peroxide in the presence of aqueous ammonia by stirring an aqueous solution of the starting material and these reagents for 20-35 minutes at 18-25 ⁰ C and pH 8;

- isolation of free hexapeptide (bis- (γ-L-glutamyl) -L-cystinyl-bis-glycine) (II)

H-γ-L-Glu-L-Sus-Gl-OH-H-γ-L-Glu-L-Sus-Gl-OH

by acidifying the solution with glacial acetic acid to pH 5;

- the conversion of the oxidized form of the free hexapeptide to the tetralitic salt (III) by titration of an aqueous solution of compound (III) with four equivalents of an aqueous LiOH solution; solution filtration; stabilization of the disulfide bond of the tetralithium salt of disulfide glutathione by irradiating the reaction solution with an Isolda type ultraviolet irradiation (UV) apparatus;

- selection of the final product - tetralithium salt of disulfide glutathione by freeze drying.

The method is illustrated by the following example: Example 1

Restored glutathione in an amount of 170 g (0.55 mol) was placed in a flask equipped with a stirrer and a thermometer, suspended in 200 ml of water and 269 ml of 1N was added with stirring. LiOH solution. The resulting clear, slightly yellowish solution is cooled to a temperature of 18-20 ^° C and 283 ml of a 3% hydrogen peroxide solution are added in small portions over 5 minutes so that the temperature of the reaction mixture does not exceed 22-25 ^° C. The reaction mixture is stirred for 35 minutes at the indicated temperature, while acidifying the solution with glacial acetic acid to pH 5. Then, the potentiometer electrode is installed in the flask, the pH of the solution is determined and 1N is added dropwise. LiOH solution to pH 5.8-6.0, while the temperature should not exceed 22-25 ⁰ C. Then the cooling is removed and the reaction mixture is stirred for 30 minutes at room temperature.

The completeness of the oxidation reaction is monitored by HPLC. If, after standard integration of the chromatogram, the content of the oxidized form of glutathione is less than 97%, stirring of the reaction mixture is continued for another 30 minutes under the same mode and the chromatographic control is repeated.

If the content of the oxidized form of glutathione is at least 97%, the reaction is considered complete. The solution obtained after the oxidation reaction is filtered under vacuum through a filter with a pore size of not more than 0.7 μm, avoiding foaming of the solution. The flask and filter are washed with distilled water (2x20 ml), the washing is attached to the filtrate.

The filtered solution is subjected to ultraviolet irradiation on an Isolda-type apparatus, with a radiation source power of 8 watts.

The selection of the final product is carried out by freeze drying.

The proposed pharmaceutical composition includes bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate lithium as the main active substances, zinc sulfate, hydrogen peroxide, adrenaline as additional active ingredients and water for injection in the following ratio,% of the mass:

Bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate

(tetralithium salt of disulfide glutathione) 0.5-7

Zinc Sulfate 0.02-0.8

Hydrogen peroxide 0.01-0.5

Adrenaline 0.001-0.1

Water for injection rest.

Example 2

Obtaining a pharmaceutical composition in the form of a droplet for noca dosage form

The composition of the drops, g:

Bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate

(tetralithium salt of disulfide glutathione) 20.0

Zinc Sulphate 2.5

Hydrogen peroxide 1, 0

Adrenaline 0, 1

Water for injection up to 1 liter.

To prepare the drops, a 0.1% hydrogen peroxide solution is prepared, 20.0 g of the tetralithium salt of disulfide glutathione, 2.5 g of zinc sulfate and 0.1 g of adrenaline are successively dissolved in it. The volume of the resulting solution was adjusted to 1 liter with water for injection. The resulting solution is subjected to sterile filtration and poured into vials. Bottles are hermetically sealed.

This pharmaceutical composition can be used in two dosage forms: in addition to nasal drops, a nasal spray is proposed that provides a polydisperse aerosol with a microparticle diameter of 150-300 microns. It should be noted that the proposed original pharmaceutical composition was developed for the first time and has not previously been used in clinical practice for the prevention and treatment of rhinitis of various etiologies.

The results of preclinical studies of the general toxic effect of the ingredients of the composition make it possible to classify the main component of the claimed pharmaceutical composition as class 5 — practically non-toxic medicinal substances [10, 11].

As a result of preclinical studies, it was found that the claimed pharmaceutical composition has unique biological and pharmacological effects, providing immunomodulatory effects, including activation of anti-infection immunity, normalization of metabolic processes in the tissues of the tissues of the nasal mucosa and nasopharynx, and therefore, activation of reparative processes, i.e. epithelization of lesion sites along with the production of anti-inflammatory cytokines.

The above biological and pharmacological effects are mainly due to the mechanisms of biochemical and immunological activity of the tetralithium salt of disulfide glutathione due to its positive effect on metabolism and, hence, on the bioenergetics of nasopharyngeal epithelial cells and immunocompetent cells.

The tetralithium salt of disulfide glutathione along with pronounced metabolic effects regulates the endogenous production of cytokines, including interferons (IFN) alpha and gamma, which determines the immunorehabilitation and antiviral effects of the pharmaceutical composition.

In addition, due to the stable form of the tetralithium salt of disulfide glutathione obtained by the original synthesis method, the correlation of the production of Thl / Th2 class cytokines with the predominant activation of ThI cells is carried out due to stimulation of the endogenous production of IL-12, which ensures a stable level of cell-mediated a given immune response, including the regulated functional capacity of resident macrophages. A remarkable property of the pharmacological activity of the energetic form of the tetralithium salt of disulfide glutathione is its neurobiochemical effects, which include the restoration of the metabolism of nerve cell cells, and therefore, the provision of nervous trophism of the nasal mucosa and nasopharynx.

The remaining components of the pharmaceutical composition complement the effects of the tetralithium salt of disulfide glutathione:

- hydrogen peroxide provides the functions of a pharmaceutically acceptable carrier, and also has a bactericidal effect due to the release of active oxygen;

- Zinc sulfate has an antiseptic, astringent, drying and local anti-inflammatory effect;

- adrenaline increases the content of cAMP in the cells of the nasal mucosa and therefore has a vasoconstrictive effect, and also normalizes the activity of resident macrophages.

Therefore, the preferred embodiment of the invention is the pharmacologically and pharmaceutically optimal combination of the substances listed and their ratio in the composition of the claimed pharmaceutical composition intended for the prevention and treatment of rhinitis of various etiologies by its use in monotherapy and / or as part of complex therapy with traditional drugs to achieve preventive or therapeutic effect due to the regulatory effect on the pathogenetic mechanisms of development and course of named x diseases.

According to the invention, pharmaceutically acceptable preparations are provided in the form of “drops for noca” and “spray for noca” based on the formulation of the claimed pharmaceutical composition for the treatment of the following nosological forms:

S acute infectious, seasonal or perennial allergic rhinitis, including sub- and atrophic rhinitis; •

S exacerbation of chronic rhinitis in the postoperative period with adenectomy, polypectomy, septoplasty, turbinectomy;

S prevention of inflammatory diseases of the sinuses of the skull (sinusitis, frontal sinusitis, sinusitis) and otitis media;

S damage to the nasal mucosa and nasopharynx by dust, paint, flour and other occupational hazards.

As a result of clinical trials of the pharmaceutical composition, the method of application and dose of the drug were determined.

Method of application: Intranasal.

Doses:

Adults: Instill at least 3-5 times a day in both nasal passages for 5-7 days. In the treatment of chronic rhinitis, these courses of treatment must be repeated at intervals of 10 days between courses until a therapeutic effect is obtained.

Children: For diseases of the nose and nasopharynx, acute respiratory viral infections: for children from 2 weeks and older - 2-4 instillations in each nasal passage daily (if necessary, more). The use of the drug is possible for a long time (from 7 days to several months).

• For hygiene of the nasal mucosa and prevention of diseases of the nose and nasopharynx: for children older than 2 years and adults — 1 instillation in each nasal passage daily (if necessary, more).

• When caring for the nasal mucosa in the postoperative period: children 6 years of age and older and adults — 4–6 instillations in each nasal passage daily (if necessary, more). The use of the drug is possible for a long time - from 7 days to several months.

• Each instillation involves the introduction of 5-10 drops of the drug into the nasal passages.

Precautions: When used in the postoperative period, a doctor's consultation is necessary. According to the invention, a method of treating rhinitis consists in the use by a patient in need of this of the proposed pharmaceutical composition, and its use is carried out at least twice a day for the period necessary to achieve a therapeutic effect.

According to the invention, the proposed original pharmaceutical composition is used in the form of a spray or drops for the nose 2-6 times a day, as the inflammatory process is stopped, the number of instillations is reduced to 2-3 times a day, the treatment is continued until the symptoms of the disease disappear.

A method of treating rhinitis using the proposed pharmaceutical composition is illustrated by the following examples.

Example 3

A study of inflammatory cells and cytokines

in patients with seasonal allergic rhinitis

A placebo-controlled, randomized trial was conducted during the flowering season. The subjects participated in the study for 14 days and visited the clinic twice: at the beginning of the study and after 14 days.

The study included only patients who met the following criteria: men and women over 18 years of age; with seasonal allergic rhinitis (CAP) due to contact with seasonal allergens for the previous 2 weeks; with symptoms of rhinitis in the previous 2 weeks; with a minimum total symptom score (OOS)> 6 of the initial examination (see below) that gave informed consent before being included in the study. Exclusion criteria: acute and chronic infections of the upper respiratory tract within 30 days before the start of the study; anatomical disorders of the nose (for example, curvature of the nasal septum), nasal polyps, the use of antibiotics, corticosteroids orally or intranasally during the previous 4 weeks and change in antihistamines in the previous one week. All patients were divided into 2 groups (10 patients per group) and received a pharmaceutical composition (instillation 5 times a day) or placebo (instillation 5 times a day) for 2 weeks. Other pharmacological treatments during the study period were not allowed. In an extreme case, washing of the nose with saline was allowed. All subjects had nasal lavage before and after treatment, and nasal scrapings were obtained.

Nasal cytology

Cytological samples from the nasal cavity were obtained by scraping from the head of the lower nasal concha with a cotton swab. After scraping, the tip of a cotton swab was immersed in a plastic tray with a phosphate buffer solution (FBI) and transferred to a 10 ml polypropylene tube. The resulting liquids were centrifuged for 10 minutes at 500 rpm and the pellet was resuspended in PBS (2 ml). Cell suspensions were filtered to reduce mucus, and microscopic preparations were prepared using a cytocentrifuge using standard techniques.

The smears were stained with Diff Quik and examined under an optical microscope (Olympus U-SPT, Olympus-Italy, Rome, Italy). The number of inflammatory cells was expressed as an average of 10 fields of view at a 100-fold increase. The samples were studied using the “blind ohm method”.

Rinse nose

The nasal cavity was washed with 5 ml of physiological saline, in accordance with standard methods. During the wash, the patient sat with his head bowed back, with a closed soft palate. The resulting fluid was comparable in all groups; it was obtained in order to assess the level of cytokines and stored at -20 ⁰ C. Cytokine study

Cytokines were measured by ELISA (Solid State Immunoassay) (R&D Systems, Charleston, South Carolina, USA). During the analysis, a quantitative immunoenzymatic sandwich assay was used. Monoclonal antibodies specific for each cytokine were added to the microplate. Analysis of the test samples and standard dilutions of each specific cytokine was performed separately, in duplicate, in accordance with the instructions of the manufacturer of the ELISA kit. Standard and test samples were pipetted into the wells of the plate and each cytokine bound to an immobilized antibody. After removal of unbound substances, polyclonal antibodies specific for each cytokine were added to the wells in the wells. After removal of the unbound antibody-enzyme reagent, a substrate solution was added to the wells, followed by color development, the intensity of which was proportional to the amount of cytokine bound in the first stage. Then the color development was stopped and the color intensity was measured. Color intensity was measured on a spectrophotometer at a wavelength of 490 nm (Molecular Device, Denver, Colorado, USA). A calibration curve was constructed from the average optical density and the amount of each cytokine in all samples was estimated by interpolation using a standard curve. The results were the average of two replicates.

Research results

The results of the study are presented in Fig. 1/2, 2/2 and in table 1.

The content of cytokines in the flushing fluid

patients before and after treatment (pg / ml)

Table 1

Conclusion

A study showed that the claimed pharmaceutical composition has immunomodulating activity, which is manifested in a decrease in the level of inflammatory cells and proinflammatory cytokines ip vivo, which provides convincing evidence of the effectiveness of this pharmaceutical composition for the treatment of rhinitis of various etiologies, including vasomotor rhinitis.

Example 4

Patient K. Anatoly Viktorovich, 22 years old, outpatient card N ° 1015. Diagnosis: Exacerbation of vasomotor rhinitis.

Complaints during examination: On holding nasal breathing, mucous membranes from the nose.

Anamnesis of the disease: Sick a week ago, when he noticed nasal congestion, watery discharge from the nose.

Previous treatment: Naphthyzine eye drops, 2 times a day for a week, without therapeutic effect.

Objective examination: Near anterior rhinoscopy: the nasal mucosa is edematous, its dryness is noted, the nasal concha is edematous, the mucous membrane is separated in the lumen of the nasal passage on the right and left, and when sown, eosinophilia. Nasal breathing is absent.

The course of therapy is pharmaceutical. Pharmaceutical composition, 3 times in the composition: day, nasal spray, 7 days. Concomitant treatment has not been performed.

Conclusion Conducting a course of therapy with the pharmaceutical composition provided a positive trend, which was expressed in the absence of signs of inflammation of the nasal mucosa: the nasal mucosa is pale pink, smooth, there is no dryness, nasal breathing has been restored. Example 5

Patient P. Polina Arkadyevna, 18 years old, outpatient card Ne 1311.

Diagnosis: Chronic infectious rhinitis.

Complaints during examination: On mucopurulent discharge from the nose, a marked decrease in the function of nasal breathing.

Anamnesis of the disease: Sick for about 7 years, frequent relapses of rhinitis, periodic lack of nasal breathing.

Objective examination: With anterior rhinoscopy: nasal septum along the midline, mucous membrane hyperemic, purulent discharge in the lumen of the nasal passage on the left and right. When sowing - Staphylococcus aureus.

The course of therapy is pharmaceutical. Pharmaceutical composition, 3 times in the composition: day, nasal drops, 5 days. Three repeated courses with breaks of 7 days. Concomitant treatment has not been performed.

The condition of the patient after the end of the course of treatment: therapy using pharmaceutical compositions provided:

- lack of patient complaints;

- the absence of purulent discharge from the nasal cavity;

- significant improvement in nasal breathing;

- the absence of recurrence of rhinitis within 6 months after the end of treatment.

Example 6

Patient H. Ludmila Igorevna, 49 years old, outpatient card N ° 1043. Diagnosis: Dry anterior rhinitis.

Complaints during examination: Difficulty in nasal breathing, periodic nasal congestion on one side and the other, intensifying at night. Anamnesis of the disease: Works at a chemical plant for about 20 years, the work is associated with the effects of occupational hazards: ammonia, hydrochloric and nitric acid. Complaints about delayed nasal breathing for about 15 years.

The preceding le periodically applies vasoconstrictors, for example: tibacterial nasal drops or sprays, sea buckthorn or menthol oil, antibacterial ointments.

Objective examination: With anterior rhinoscopy: the nasal septum in the midline, the nasal mucosa is edematous, dry, the nasal concha are edematous, and there is no discharge in the nasal passage.

The course of therapy is Pharma-Pharmaceutical Composition, 3 times a day, cevtic composition-spray in the nose, 15 days, then 1 time per day: for 15 days. Concomitant treatment has not been performed.

The condition of the patient after completion of the course of treatment:

Objective examination: At the time of examination: the nasal septum in the midline, the mucous membrane of the nose on the left and right is not changed, the nasal concha is within normal limits, there is no discharge in the nasal passage. Respiratory function restored.

Example 7

Patient 3. Victor Ivanovich, 37 years old.

Diagnosis: Vasomotor rhinitis, hypertrophy of the posterior ends of the lower nasal concha.

Complaints: On the absence of nasal breathing for 18 years, sneezing in the morning, light-watery discharge from the nose (hydraea).

Previous treatment: Used by Naphthyzine 15 years 2-3 times a day; 7-8 bottles per month. Objective examination: Rhinoscopically: nasal septum along the midline, congestion in the anterior ends of the lower nasal concha, edema, cyanosis, raspberry-shaped concha, the posterior ends are bullishly thickened, cover the choanae by 1/3, nasal breathing is absent.

Pharmaceutical therapy 09/20/2000 prescribed a course of therapy with the composition: pharmaceutical composition 3 times a day, spray in the nose, 14 days, then 1 time per day for 14 days, then 3 times a week for a month. Concomitant treatment has not been performed.

After completion of the course of therapy: On 11/24/2000, with anterior rhinoscopy: pink mucosa, nasal passages are free, nasal breathing is not difficult.

The pharmaceutical composition in the conditions of preclinical and clinical studies has demonstrated a high therapeutic effect due to:

* ^* * immunocorrective activity due to the regulation of local immunity, including the regulation of the function of resident macrophages; *> cytoprotective activity due to activation of the metabolism and bioenergy of the cells of the nasal mucosa and nasopharynx;

*> antibacterial and antiviral activity due to immunocorrective effects, primarily due to an increase in the level of interferons (IFNα - Table 1);

* l * anti-inflammatory and reparative action of drugs due to the regulation of the level of proinflammatory cytokines;

* l * induction of apoptosis of infected cells, which provides potentiation of the effects of traditional antibacterial and antiviral chemotherapy; *> desensitizing effect due to the regulation of the immunological and biochemical phases of the allergic reaction in the nasal and nasopharynx mucosa.

Sources of information

1. International consensus reports on diagnosis and management of rhypitis. Interpatiophyl rhypitis management gr. Allergu 1994; 49 (suprl. 19): 1-34.

2. Vap Sauwepberge P. V., vap Kemrep M. J., Vashert S. Theme somopod attе turp оf thе milleppшm // Am. J. Rhypol. - 2000. — VoI. 14.— JUfe 5.— P. 339-343.

3. Hodyakov H. D. Acute and chronic nonspecific inflammatory diseases of the nasal cavity. A multivolume guide to otorhinolaryngology; 3: 51-84.

4. Richelmann G., Lopatin A.S. Grew up. rhinol., 1994; 4: 33-47.

5. Vap Sauwepberge R., Washert C ₅ Passalasqua C, Wousquet Y., Sapopis G.

M. et al. Consensus statistics op tе triatmеpt оf allеrgiс rhiпitis // Аllеrgu apd сliпiсl immuпологу. -2000. -VoI. 55.-N 01. -P. 116-134.

6. Encyclopedia of medicines, annual collection, vol. 10, 2003, Moscow, LLC “PLC-2003”.

7. Vidal Handbook “Medicines in Russia”, ed. 8,

2002, Moscow, “Accelerator”.

8. Radar-Pharmacist, annual collection, vol. 5, 2003, Moscow, LLC “PLC-2003”.

9. Kryukov A. I., Turovsky A. B. Symptomatic therapy for rhinitis and pharyngitis // Consilium rovisost, 2001, N ° 5, v. 1.

10. Rules for preclinical pharmacological safety assessment

funds (GLP). RD 64-126-91, Moscow, FC, 1992.

11. Problems of the norm in toxicology (under the editorship of prof. IM Trakhtenberg), Moscow, "Medicine", 1991.

Claims

Claim

1. The use of bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate lithium (tetralithium salt of disulfide glutathione) with stabilized disulfide bond as a means for the prevention and treatment of rhinitis of various etiologies.

2. The method of obtaining funds according to claim 1, including the oxidation of the starting compound - reduced glutathione (γ-L-glutamyl-L-cysteinyl-glycine) - with hydrogen peroxide in the presence of aqueous ammonia by stirring an aqueous solution of the starting material and these reagents for 20- 35 min at 18-25 ^° C and pH 8; isolation of free hexapeptide (bis- (γ-L-glutamyl) -L-cystinyl-bis-glycine) by acidifying the solution with glacial acetic acid to pH 5; the conversion of the oxidized form of the free hexapeptide to the tetralithium salt by treating its aqueous solution with an aqueous solution of LiOH; filtering the solution obtained after the oxidation of water with vacuum; stabilization of the disulfide bond of the hexapeptide due to its energy; isolation of the final product by freeze drying.

3. The method according to claim 2, wherein the stabilization of the disulfide bond of lithium bis- (γ-L-glutamyl) -L-cystinyl-bis-glycinate is carried out by irradiating the reaction solution with an ultraviolet irradiation apparatus.

4. A pharmaceutical composition for the prevention and treatment of rhinitis of various etiologies, including bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate (tetralithium salt of disulfide glutathione), zinc sulfate, hydrogen peroxide, adrenaline and water for injection in the following ratio,% macc:

Bis- (γ-L-glutamyl) -L-cystinyl-bis-lithium glycinate

(tetralithium salt of disulfide glutathione) 0.5-7.0

Zinc Sulfate 0.02-0.8

Hydrogen peroxide 0.01-0.5

Adrenaline 0.001-0.1 Water for injection rest.

5. The pharmaceutical composition of claim 4 may be in the form of nasal drops or a nasal spray.

6. A method for the prevention and treatment of rhinitis of various etiologies, characterized in that the pharmaceutical composition according to paragraphs. 4 and 5 are used 2-6 times a day, as the inflammatory process is stopped, the number of injections of the drug is reduced to 2-3 times a day, the course of treatment is continued until the symptoms of the disease disappear.