RU2493861C1 - Biologically active complex possessing anti-allergic action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents a complex of biologically active substance for treating allergic diseases of various genesis, characterised by the fact that it has been recovered from cod liver oil by gradual fractionation from ballast lipids by extraction in a two-phase oil and water extractant, centrifugation and ultrafiltration or diafiltration through a material with separation limit 25 kDa, and contains peptides 30-55%, amino acids 40-65%, carbohydrates 2-8%, micro and macroelements 2-13%.

EFFECT: invention provides the drug spectrum broadening.

8 ex, 3 tbl

Description

The invention relates to medicine, namely to dermatology and allergology, and can be used to treat acute and chronic allergic diseases of various origins.

Allergic diseases associated with a distortion of the immune response are becoming a social problem today. In different countries and individual regions, allergic manifestations are recorded with a frequency of 1 to 50% or more. On average, according to WHO, allergic diseases cover about 10% of the population. In the structure of allergic diseases, the first place is occupied by various forms of skin allergies - up to 66%.

For the treatment of allergic diseases, various pharmacotherapeutic agents are used, which can be divided into the following groups: agents that suppress allergic inflammation (glucocorticosteroids, cromons), anti-mediators (antihistamines, antileukotrienes) and drugs that prevent the development of pathophysiological effects (decongestants, bronchodilators, expectorants).

Glucocorticoids are in many cases very valuable therapeutic agents. However, they cause a number of side effects, including slowing down the process of tissue regeneration with the possible formation of common ulcers, the appearance of edema, delaying synthesis and accelerating the process of protein breakdown. Due to side effects, glucocorticoid drugs are used only with clear indications and under close medical supervision, which severely limits the possibility of their use as part of OTC drugs.

In allergological and dermatological practice, the most studied are antihistamines that block the H ₁ receptors of histamine, the first generation, which have established themselves as universal and safe means [Luss L.V. The choice of antihistamines in the treatment of allergic and pseudo-allergic reactions // Ros. allergological magazine. - 2009. - No. 1. - S.1-7.]. Drugs of this group reduce the body's response to histamine, prevent the development and facilitate the course of allergic reactions. Along with the antihistamine action, drugs of this group have other pharmacological properties. Some of them have a sedative effect, which limits in some cases the possibility of their use on an outpatient basis in patients whose profession requires a quick mental or physical reaction.

A whole arsenal of drugs based on a complex of biologically active substances obtained from extracts, extracts and lysates of animal organs and tissues (raveron, rumalon, solcoseryl, cerebrolysin, etc.) has been developed and is successfully used in clinical practice. In our country, the production of such preparations is still being mastered, and extraction of crushed organs of slaughter cattle is used to obtain preparations from animal raw materials. However, the preparations obtained in this way contain a significant amount of high molecular weight protein impurities, which determine their high reactogenicity.

World and domestic pharmaceutical experience testifies to the huge potential of marine aquatic organisms as raw materials for creating original pharmaceutical substances and medicines. Unlike indicators of species abundance, phylogenetic (macrotaxonomic) diversity in the sea is much higher. Apparently, due to adaptation to various environmental factors, a number of marine animals and plants have developed the ability to produce unique secondary metabolites, many of which have high pharmacological activity (Bhakuni DS, Rawat DS Bioactive marine natural products: Anamaya Publishers, New Delhi, India. 2005.382 p.). Biologically active substances of marine origin can be used as pharmaceutical substances and serve as synthons for drugs with new or improved pharmacological characteristics. These substances are secondary metabolites, that is, natural compounds that are not widely distributed and are present only in representatives of individual taxa.

One of the main components of the system of internal organs, rich in high-quality proteins, is the liver. The protein content in the liver, depending on the species of fish, can reach 20% (Bechtel et al. Chemical characterization of liver lipid and protein from cold-water fish species. J. Food Sci., 2006, 71 (6), 480-485 ) The liver of various fish can serve as a source of a large number of both low molecular weight substances and a whole set of peptides that can be used to obtain drugs.

As a prototype, an agent in the form of a gel having anti-inflammatory, immunotropic, anti-allergic and wound healing effects containing peptides associated with cod liver phospholipids was selected (Pat. RU 2457864 C1, publ. 10.08.2012).

The disadvantage of this tool is that its associated phospholipids, not stabilized by antioxidants, will contribute to spoilage of the finished product, as well as the narrowness of use - gel for external use.

The aim of the invention is to expand the range of drugs used in dermatovenereology and allergology.

The essence of the invention consists in the use of a new complex preparation containing peptides and such biologically active components - amino acids, carbohydrates, microelements for the treatment of allergic diseases of various origins, which have antihistamines and do not have a sedative effect. The entire listed complex of substances in the composition of the preparation is represented by natural biologically active substances isolated from the liver of fish of cod species. The liver is extracted from ballast lipids by extraction with a two-phase extractant based on oil and water, the aqueous extract is separated by centrifugation and the target product is isolated by ultrafiltration or diafiltration through a material with a separation limit of 25 kDa and subsequent drying.

The complex of biologically active substances isolated from cod liver contains natural biologically active substances in the following amounts (wt.% Of dry matter):

- peptides 30-55,

- amino acids 40-65,

- carbohydrates 2-8,

- micro and macrocells 2-13.

The complex of biologically active substances that make up the drug ensures its high efficiency: peptides act as bioregulators, amino acids stimulate metabolic processes as a plastic material for protein synthesis, carbohydrates, the main component of which is hyaluronic acid, have immunomodulating properties, and macro and microelements enhance antioxidant properties and activity.

Significant differences of the complex drug from the prototype is the following:

1) the complex is represented by biologically active substances isolated from natural raw materials by the original method;

2) with the exception of peptides, all other ingredients do not repeat the composition of the prototype;

3) the complex can be used for the preparation of other dosage forms - solutions for inhalation and parenteral use.

Advantages over the prototype are as follows:

1) the complex is more stable due to the absence of phospholipids that are easily susceptible to oxidation:

2) the complex can be used to prepare a solution for inhalation use and parenteral administration, which has greater bioavailability;

3) the complex of natural substances is balanced and provides a synergy of their action;

4) the complex has a directed action and is intended for the treatment of allergic diseases.

The technical result of the invention is to obtain a safe tool in the form of various finished dosage forms with a pronounced therapeutic effect for the treatment of allergic reactions of various origins.

As an antiallergic agent, a complex of biologically active substances obtained from cod liver can be used for the prevention and treatment of various diseases, such as bronchial asthma, allergic bronchitis, allergic rhinitis, pollinosis, atopic dermatitis, allergic conjunctivitis, urticaria.

Thus, the proposed technical solution meets the criteria of the invention, namely, “novelty”, “inventive step” and “industrial applicability”.

The possibility of implementing the claimed invention is shown, but not limited, in examples of specific performance.

Example 1. The solution for inhalation use (in g per 100 g).

Biologically active complex - 0.01

Preservatives - q.s.

Water for injection - up to 100

Example 2. A solution for parenteral use (in g per 100 g).

Biologically active complex - 0.1

Sodium Chloride - 0.7

Preservatives - q.s.

Water for injection - up to 100

Example 3. Gel for external use (in g per 100 g).

Biologically active complex - 0.02

Hydroxyethyl cellulose - 0.65

Triethanolamine - 0.55

Polyethylene glycol - 12.00

Preservatives - q.s.

Purified water - up to 100

When applied topically, the biologically active complex of cod liver provides transport of biologically active peptides to the deeper layers of the dermis without additional use of transport structures such as liposomes, nanosomes or microcapsules.

When applied to the skin, the preparation in the form of a gel remains on its surface for a long time, on the one hand, forming a protective film, on the other hand, a depot from which the active ingredients gradually enter the skin. The film creates an occlusion effect, improving skin hydration and increasing the permeability of the stratum corneum for active components.

Example 4. The study of toxicity

In the study of acute toxicity of the preparations prepared according to examples 1-3, in mice and rats with intramuscular, intravenous, endotracheal and local routes of administration, it was impossible to establish the LD ₅₀ values, due to the absence of animal death. By the degree of exposure to the body, a complex of biologically active substances from cod liver belongs to hazard class 4 according to GOST 12.1.007 (low-hazard substances). In recommended doses, it does not have a local irritant, embryotoxic and carcinogenic effect.

Example 5. Antiallergic effect on a model of experimental contact allergic dermatitis in outbred male mice.

,As an allergen, 1-chloro-2,4-dinitrobenzene (DNCB, Sigma, USA) in 95% ethanol was used. On the 1st, 7th and 13th day of the experiment, the mice were sensitized by a single application of 100 μl of 2% DNCB solution on the shaved areas of the back. On the 17th day of the experiment, twice with an interval of 1 hour, the animals were given a resolving dose of allergen of 20 μl of a 2% solution of DNCB on the outer and inner surfaces of the “experienced” ear. On the 20th day of the experiment, the animals were euthanized in a CO ₂ -chamber and blood was taken from the heart to evaluate IgE by enzyme-linked immunosorbent assay. An “experienced” ear was taken for histological assessment of the lesion and calculation of the reaction index (IR) according to the formula $$\mathrm{AND}R=\frac{M\mathrm{about}P-M\mathrm{to}}{M\mathrm{to}}\times \mathrm{one\; hundred}\%$$

,

where M _op and M _to - the mass of the "experienced" and "control" ear, respectively.

The therapeutic effect of the drugs was evaluated after the development of an allergic reaction, i.e. after the resolution stage. For this purpose, drug treatment was carried out from the 7th day and the next 12 days of the experiment.

The agent of Example 3 was administered intramuscularly at a dose of 2 mg / kg and 8 mg / kg. Suprastin, injection solution was used as a comparison drug. Suprastin was administered intramuscularly at a dose of 22 mg / kg. Data on the evaluation of antiallergic action on the model of contact dermatitis are shown in table 1.

Table 1 Evaluation of antiallergic action on the model of contact dermatitis (M ± m, N = 10) Group number Group Dose mg / kg The reaction index,% Serum IgE level, ng / ml one. Intact - 7.2 ± 1.8 1325 ± 275 2. The control - 51.6 ± 8.0 * 2425 ± 350 * 3. Placebo - 49.5 ± 10.0 * 1800 ± 200 four. The tool for pr 4 0.004 34.8 ± 7.8 * ** 2250 ± 250 * 5. 0.008 21.8 ± 7.5 * ** 925 ± 200 ** 6. Suprastin 22.0 11.4 ± 8.4 ** 750 ± 350 ** * differences are statistically significant compared with intact animals according to student criterion, at p <0.05; ** - differences are statistically significant compared with control animals according to student criterion, with

As a result of the study, it was found that a product containing a biologically active complex from cod liver has a dose-dependent antiallergic effect - a statistically significant decrease in IR and IgE concentration compared to the control animals.

Example 6. Sedative effect Sedative effect was evaluated in the test "open field" and "elevated cross-shaped labyrinth" taking into account the orientational research emotional, stereotypical and motor components of the behavioral atlas for rodents [Poshivalov, V.P. Pharmacoetology (collection of articles) - St. Petersburg: Izd. SPGMU, 1997. - 140 s]. The study was conducted on 60 outbred rats weighing 180-220 g (Laboratory animal nursery RAMS "Rappolovo", Leningrad region). The biologically active complex was administered intragastrically through an atraumatic probe in a wide dose range from 10,000 to 60,000 mg / kg. The drug for administration was dissolved in water. Water was used as a control.

When tested in the “open field” and “elevated cruciform labyrinth”, animals after a single intragastric administration of the studied biologically active complex from the cod liver showed a predominantly balanced type of behavior, with moderate locomotor and search activity without signs of anxiety. The biologically active complex from cod liver is highly soluble in water and does not dissolve in lipophilic solvents. Thus, without lipophilicity, it does not cross the blood-brain barrier and does not block the H ₁ histamine receptors of the brain, as well as central serotonin and m-cholinergic receptors, i.e. does not have the sedative effect characteristic of 1st generation antihistamines.

Example 7. The effect of funds on the sensitivity of H ₁ histamine receptors in situ.

The ability of the agent to bind to H1-histamine receptors was evaluated by histamine-induced guinea pig ileum contraction.

Experiments with isolated tissues make it possible to obtain complete and accurate data on the directivity and mechanism of action of various mediators directly on the receptor apparatus of tissues and organs.

The ileum was removed from the abdominal cavity of anesthetized animals and placed in a container with Krebs-Henseleit buffer cooled to + 4 ° C. Then, the ileum was cut into fragments about 1 cm long, the fragments were fixed in flow chambers and perfused with oxygenated Krebs-Henseleit buffer at + 30 ° C for 60 minutes, after which the flow chambers were filled with 80 mM KCl, which caused a strong reduction in bowel segments. The results obtained were subsequently taken as a 100% reduction.

Perfusion was resumed with oxygenated Krebs-Henseleit buffer at a temperature of + 30 ° C to wash the tissues and restore their ability to contract. After 60 minutes of washing, perfusion was performed with a solution of the agent in Krebs-Henseleit buffer. Pyrilamine maleate (0.005 μg / ml) was used as a comparison drug. After a thirty-minute incubation period, the solutions were drained, and the flow chambers were filled with an aqueous solution of histamine (10 ^-6 M). Within 10 minutes, the contraction force was recorded, and the amplitude and time of the onset of the maximum response were estimated (Table 2).

table 2 Amplitude of histamine-induced contraction and time of onset of maximum response (Me ± Q, N = 8) Group Concentration Indicators Amplitude (% of 80 mM KCl) Time (fractions from 80 mM KCl) Histamine 10 ^-6 M 100.8 ± 10.3 1.0 ± 0.2 Pyrilamine maleate 0.005 mcg / ml 5.7 ± 4.8 * 57.4 ± 17.1 * 4 mcg / ml 101.6 ± 7.6 ** 1.0 ± 0.1 ** 8 mcg / ml 94.5 ± 5.2 ** 2.7 ± 0.8 * ** The tool for pr.2 16 mcg / ml 54.5 ± 7.6 * ** 15.5 ± 3.0 * ** 32 mcg / ml 40.8 ± 13.6 *** 26.3 ± 4.2 * ** 64 mcg / ml 25.0 ± 9.1 * ** 36.9 ± 6.0 * ** 128 mcg / ml 6.5 ± 4.5 * 56.5 ± 15.0 * Note: * statistically significant difference from the control group (histamine), with p <0.05 ** statistically significant difference from the indicators of the group of pyrilamine maleate, with p <0.05

It was found that the agent of example 2 dose-dependently blocked the H ₁ histamine receptors of the ileum smooth muscle sarcolemma. The effective dose (ED ₅₀ ) was 24.73 μg / ml. Thus, the tool is an effective blocker of H ₁ -histamine receptors.

Example 8. Antiallergic effect on the model of allergic bronchial asthma in guinea pigs.

Sensitization was carried out by a single intraperitoneal administration of ovalbumin 100 μg / ml in 0.5 ml of physiological saline containing 100 mg / ml Al (OH) ₃ . Airway hyperreactivity was provoked for 3 consecutive days 44-46 by inhalation of an ovalbumin solution with an increasing concentration of 0.1; 0.3 and 0.5 mg / ml using an ultrasonic inhaler "Monsoon 2" - 03 (Kinematica, Switzerland). Inhalation was performed for 5 minutes or until signs of asphyxiation appeared (sneezing, coughing, falling on its side). On day 47, permissive inhalation of an ovalbumin solution with a concentration of 1 mg / ml was performed.

The administration of the test agent of Example 2, the comparison drugs and placebo began 3 weeks after sensitization and ended the day before the inhalation of the resolving dose of ovalbumin. During the experiment, the number of leukocytes and the blood formula and histological changes in the lung tissue were studied (Table 3).

Table 3 Evaluation of the results of a histological examination of the lungs of guinea pigs on a model of experimental bronchial asthma (n = 6) Group Dose mg / kg The number of mucous plugs in the lumens of the bronchi (by 3 bronchi in a guinea pig), Me (Q ₁ ; Q ₂ ) The number of eosinophils in the wall of the bronchus per 1 mm, M ± m The number of goblet cells per 1 mm of the bronchial mucosa, M ± m No traffic jams Plugs fill <½ of the lumen Tubes fill> ½ of the lumen Intact - 3 (3; 3) 0 (0; 0) 0 (0; 0) 0.3 ± 0.3 13.2 ± 1.2 The control - 0 (0; 1) * 1 (1; 2) * 2 (1; 2) * 32.2 ± 1.4 * 40.3 ± 1.1 * Cromohexal 6.9 1 (1; 1) * 1.5 (1; 2) * 0 (0; 1) ** 11.3 ± 0.8 * ** 28.7 ± 1.3 * ** Dexamethasone 0.7 2 (1; 3) * ** 1 (0; 1) * 0 (0; 0) ** 15.7 ± 1.4 * ** k 29.8 ± 1.3 * ** Wed on pr.2 0.5 1 (0; 1) * 1 (1; 2) * 1 (1; 1) * 19.7 ± 1.2 * ** c 32.8 ± 1.1 * ** Wed on pr.2 1,0 1 (1; 2) * 1 (1; 2) * 0.5 (0; 1) 18.0 ± 1.1 * ** c 31.2 ± 1.6 * ** Wed on pr.2 2.0 1.5 (1; 2) * ** 1 (1; 1) * 0.5 (0; 1) ** 19.2 ± 0.9 * ** c 30.7 ± 1.2 * ** Note: * - the differences are statistically significant according to the criteria of Mann-Whitney and Student in comparison with the intact group, at (p <0.05) * - differences are statistically significant according to the criteria of Mann-Whitney and Student in comparison with the control group, at (p <0.05); to - the differences are statistically significant according to the student criterion compared with the cromohexal group, at (p <0.05).

It was found that the agent prepared according to example 2 in all studied doses of 0.5; 1.0 and 2.0 mg / kg has an antiallergic effect due to its ability to reduce the number of major effector cells of allergic inflammation - eosinophils, as well as the number of mucous plugs in the lumens of the bronchi.

Thus, as a result of tests of a drug containing a complex of biologically active substances from cod liver, it was found that the drug is promising in the treatment of allergic diseases of various genesis: along with high efficiency, it has good tolerance and safety and does not have a sedative effect.

Claims (1)

A complex of biologically active substances for the treatment of allergic diseases of various origins, characterized in that it is isolated from the liver of cod fish by sequential fractionation from ballast lipids by extraction with a two-phase extractant based on oil and water, centrifugation and ultrafiltration or diafiltration through a material with a separation limit of 25 kDa and includes peptides 30-55%, amino acids 40-65%, carbohydrates 2-8%, micro and macro elements 2-13%.