RU2117482C1 - Agent showing antiasthmatic and antiadhesion effect - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: agent is an oligomeric product of chitin deacetylation or their pharmaceutically acceptable salts. Preferable polymerization degree of oligomeric products is 4-8. Agent can contain additionally pharmaceutically acceptable special additions and biologically active substances of other effect. Agent shows effective and nontoxic product producing from natural raw and can be used for treatment of patients with bronchial asthma and diseases where leukocytes take part an essential role in pathogenesis process. EFFECT: enhanced effectiveness of an agent. 4 cl, 1 tbl, 1 dwg, 7 ex

Description

 The invention relates to medicine, specifically to a tool that can find application for the treatment of bronchial asthma and other diseases, in the pathogenesis of which a significant role is played by processes occurring on the surface of leukocytes (cell adhesion, interaction with antigens, etc.).

 The problem of creating highly effective pharmacotherapeutic agents for the treatment of bronchial asthma as one of the most common and serious human diseases is very urgent throughout the world. Unfavorable trends were revealed in relation to the increase in the incidence of bronchial asthma. Despite the creation of a number of bronchodilators and the use of corticosteroids, it is not yet possible to change the tendency to spread and worsen the course of the disease / 1 /.

 One of the possible explanations for this situation is that the pathogenesis of bronchial asthma, like any systemic human pathology, is a complex set of biochemical and pathophysiological processes. These include the interaction and mutual regulation of immune systems, metabolism of exogenous and endogenous compounds, genetically determined features of signaling, receptor systems of the cell, etc.

 Now the concept of bronchial asthma as a chronic immune (allergic) inflammation of the airways is clearly formulated; revealed many of the leading links in the pathogenesis of bronchial asthma; the role of zosinophils and eosinophilic proteins in damage to the bronchial mucosa was determined, which regulates the role of T-lymphocytes and the cytokines produced by them; established the value of leukotrienes in bronchial inflammation / 1 /.

 Successes in the field of pharmacotherapy of bronchial asthma are reduced mainly to the development of rational dosage forms - inhaled (beta-2-adrenergic agonists, M-anticholinergics, corticosteroids, intal) and sustained-release oral drug forms (beta-2-adrenergic agonists, theophylline) / 1 /. According to pulmonologists, the biggest achievement in this area is the creation of inhaled corticosteroids and highly effective long-acting beta-2-adrenergic agonists-formoterol and salmeterol. However, there is still no convincing evidence that with the help of these drugs it is possible to solve the main issue of therapeutic tactics in AD - the irreversible suppression of the inflammatory process in the airways. Even corticosteroids only inhibit but do not completely eliminate immune inflammation in the lungs, and asthma mortality remains high. In addition, the listed anti-asthma drugs cause a wide range of side effects: among them are the cardiotoxic effects of theophylline and beta-2-adrenergic agonists and the well-known negative effects of corticosteroids (osteomalacia, electrolyte imbalance, etc.).

 This invention is directed to the creation of new anti-asthma drugs from the class of oligosaccharides (chitin products), the effect of which is due to the effect on cell adhesion.

 Known substances that are chitin deacetylation products (molecular weight 300,000 - 1,000,000), with anti-inflammatory effect / 2 /.

 However, there is no information about the possibility of their use in bronchial asthma.

 The closest analogue of the claimed drug with anti-asthma and andiadhesion action is the drug intal (cromolyn sodium) / 3 /.

 The objective of the invention was the creation of a new highly effective non-toxic drug from natural raw materials for the treatment of bronchial asthma and other diseases, in the pathogenesis of which an important role is played by increased cell adhesion.

 This problem was solved by the development of an agent representing oligosaccharides that are products of the deacetylation of a natural product of chitin, namely, chitosan oligomers or their pharmaceutically acceptable salts. Preferably, the degree of polymerization of the oligomers is 4-8.

 The agent may further comprise pharmaceutically acceptable target additives, such as solvents, excipients, dispersing agents, emulsifiers, and the like.

Oligomers with a polymerization degree of 4-8 can be obtained, for example, according to the method described in / 4 /, by carrying out the process of deacetylation of chitin in a concentrated solution of sulfuric acid (65%) at 40-50 ^o C, for 20-120 minutes.

 However, chitosan oligomers obtained by any known method can be used.

 Pharmaceutically acceptable salts of chitosan oligomers may be acetates, hydrochlorides, nitrates or phosphates.

 The tool can be made in the form of tablets, granules, capsules, powder, inhalation forms, suspensions, solutions, ointments or creams. These forms can be obtained by known methods.

 Other active ingredients, such as antibacterial agents, may be added to the agent.

 The amount of oligomeric product in dosage form can vary widely, for example, from 0.3 to 20% by weight.

The research results of the proposed drug
Example 1

 1. In vitro experiments.

 1.1. Investigation of the effect of the claimed drug on potential target cells iv vitro.

 Using oligosaccharides labeled with a fluorescent substance fluoresceinisothiocyanate (FITC) and appropriate microscopy, it was found that oligosaccharides bind to mononuclear blood cells, and this binding is observed only on the cell surface. Oligomers do not penetrate the cell itself.

 Surveys were performed on 200-300 cells using a fluorescence microscope. As a control, we used cells fixed on glasses that were incubated with oligomers and then with oligomers labeled with FITC: no luminescence was detected.

 Thus, the authors of the present invention found that chitosan oligomers interact with molecules of the plasma membrane of cells that are directly related to inflammatory and immunological processes, including allergic rhinitis.

 1.2. Investigation of the effect of oligomers on in vitro anaphylactic bronchoconstriction models.

The experiment used guinea pigs weighing 350-500 g, sensitized with injections of ovalbumin at a dose of 10 μg and 100 mg of Al (OH) ₃ per guinea pig according to the Andersson method. 3-4 weeks after the start of sensitization of animals, an isolated trachea preparation was prepared according to Blattner et al. / 1980 /. The trachea preparation was placed in a thermostatic chamber (37 ^° C) containing Krebs-Hensleit solution (119 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl ₂ , 1.2 mM CaCl ₂ , 2.53 mM NaHCO ₃ , 24 , 8 mM glucose, pH = 7.4). The initial load on the drug was 0.7-1 g. The study of the relaxing ability of the studied substances was carried out in isotonic mode. Tracheal contraction was recorded using the 6Mx2B Mechanotron and the KSP-4 recorder. The relaxation of the smooth muscle preparation of the trachea was expressed as a percentage of the maximum amplitude of contraction caused by the addition of a mediator to the incubation medium. The average value from measurements of 4-5 segments was determined. A dose-response curve was built. ED _{50 was} determined graphically by the method of Litchfield and Wilcoxon.

 It was established that oligomers caused the effect of inhibiting anaphylactic contracture of the isolated trachea of a sensitized guinea pig / table one/. Comparison drug cromolyn sodium in these conditions showed a weak activity.

 Thus, the anti-asthmatic effect of oligosaccharides in vitro is shown.

 Example 2

 2. In vivo experiments.

 2.1. Acute toxicity

 The toxicity of oligosaccharides upon oral administration to white non-inbred mice is practically absent - LD50 more than 25 g / kg.

LD50 with parenteral (intraperitoneal) administration to mice - 5.68 ± 1.19 g / kg;
LD50 for intraperitoneal administration to rats is 4.73 ± 1.33 g / kg.

 2.2. The study of bronchodilator activity of oligomers in experimental models in vivo.

In the experiment, guinea pigs weighing 350-500 g were used. Animals were sensitized by the administration of ovalbumin at a dose of 10 μg and 100 mg of Al (OH) ₃ per guinea pig according to the Andersson method. 3-4 weeks after the start of sensitization, the animals were anesthetized with sodium ethamine (40 mg / kg, intraperitoneally). The trachea was isolated from the anesthetized pig, a cannula was inserted into it, which was connected using a system of polyvinyl chloride tubes to the bronchospasm sensor and artificial respiration apparatus (Ugo Basile). During the experiment, a certain breathing regime was maintained: the volume (V) of respiration was 6-8 ml, the frequency per minute. The resolving dose of ovalbumin (150-200 μg / kg) was administered intravenously (V. jugularis) to obtain maximum bronchospasm that does not cause death of animals from suffocation. The test compound was administered intravenously (10 mg / kg 15 minutes before the resolution dose of ovalbumin was added. Using a recorder connected to the sensor, changes in airway resistance, i.e. the magnitude of the bronchoconstrictor reaction, were recorded. The effect of the test compound was evaluated by the degree of inhibition of bronchospasm expressed in percentage with respect to control.

 The experiments carried out allowed to establish / pic. 1 / that the oligosaccharide preparation when administered intravenously 15 minutes before OA caused a pronounced anti-asthmatic effect. The effect of sodium cromoglycate on this model was weaker than that of oligomers.

 Examples of the claimed funds.

 Example A. Oligomers of chitosan; degree of polymerization 4-8.

 1% solution (in isotonic sodium chloride solution) for aerosol inhalation administration using a nebulizer.

 Example B. 1% solution of chitosan oligomer (in isotonic sodium chloride solution) with the addition of antibiotics.

 Example B. 1% solution of chitosan oligomers (in isotonic sodium chloride solution) for topical application in the form of drops for the eyes and nose.

 Example D. Dry fine powder of chitosan oligomers with a particle size of 5-10 microns for inhalation using a metering inhaler of the type cyclohaler.

 Example D. A tablet with 0.25 g of oral chitosan oligomers containing low molecular weight medical polyvinylpyrrolidone (0.0157 g) / FS 42-1194-79 /, potato starch (9.0417 g) / GOST 7699-78 /, talc (0.0094 g) / VFS-42-2550-95 /, magnesium stearic acid (0.0032 g) / TU-6-09-129-75 /. The average tablet weight is 0.32 g.

Sources of information
1. Joint report of the National Institute of Heart, Lungs and Blood and the World Health Organization. Edition N 95-3659, January, 1995.

 2. Yasuhiro Horiuchi; Koji Kifune; Mobuyuki Tanimoto, Anti-inflammatory composition. EP. A 61 K 31/73, Publ. number: 0382 210 A1, 1990, 90/33.

 3. Mashkovsky M. D. Medicines (Manual for doctors). Medicine, Moscow, 1993, v. 1, p. 362-363.

 4. V.I. Maksimov, V.M. Denisov and N.V. Makarov. SU, Copyright certificate 1571047 A1, cl. C 08 B 37/08, 1990.

Claims (4)

 1. A means of anti-asthma and anti-adhesive action, characterized in that it represents the oligomeric products of chitin deacetylation or their pharmaceutically acceptable salts.  2. The tool according to claim 1, characterized in that the degree of polymerization of the oligomeric products is 4 to 8.  3. The tool according to p. 1, characterized in that it further comprises pharmaceutically acceptable target additives.  4. The tool according to p. 1, characterized in that it additionally contains biologically active substances of a different action.

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