RU2488402C1 - Agent possessing anti-inflammatory and antiallergic action - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmacy, medicine and veterinary science, particularly new drugs for inflammatory and allergic diseases. The assigned problem preparing a non-toxic, easy-to-use and storage-stable drug in the form of eye drops, intranasal drops and spray and causing anti-inflammatory and antiallergic action is solved by creating a composition containing a pigment-mineral complex of sea-urchin shell (0.05-2.0%) containing spinochromes B, D, dimer polyhydroxynaphthoquinone and a mineral ingredient (calcium, magnesium, phosphor, sodium, potassium) in the form of water-soluble salts, a co-solvent (0.01-10%), a preserving agent (0.01-0.2%), an antioxidant (0.01-0.2%), an acidity regulator (to pH 6.0-8.0) and water (to 100%).

EFFECT: what is described is the experimentally specified method for preparing the agent according to the invention eliminating the oxidation of the pigment-mineral complex as early as the stage of preparing.

3 cl, 3 tbl, 1 dwg, 6 ex

Description

The invention relates to the field of pharmacy, medicine and veterinary medicine, and in particular to new drugs for the treatment of inflammatory and allergic diseases.

Edible sea urchins, in addition to a fishing facility for the food industry, are a rich source of unique biologically active substances. Such substances include polyhydroxynaphthoquinone pigments of the shells and needles of sea urchins - spinochromes. Unlike endogenous antioxidants, such as vitamin E and ubiquinone, naphthoquinones are able to neutralize the action of the main initiators of the non-enzymatic process of oxidation of membrane lipids - iron cations that accumulate in the area of ischemic tissue damage. Such features of the mechanism of action distinguish spinochromes from a number of well-known bioantioxidants and open the prospect for creating new-generation drugs on their basis.

A known composition containing derivatives of natural or synthetic polyhydroxynaphthoquinones (alkyl, hydroxyalkyl, acyl, nitro, amino or halogen derivatives) with fungic, algo, herbicidal and bactericidal properties (Pat. Appl. GB2159056A, publ. 27.11. 1985).

Known pharmaceutical composition containing as an active ingredient an ethyl acetate extract of the shell of sea urchins, having antithrombotic action and recommended for hypertension and diseases of the cardiovascular system associated with increased thrombosis, such as myocardial infarction, stroke, etc. (Pat. Appl. KR20020091909A, published on December 11, 2002).

Acetone extract of the shells of sea urchins Strongylocentrotus nudus is part of the pharmaceutical composition, the use of which is recommended to reduce the level of total cholesterol in hypertension, diseases of the cardiovascular system and cerebrovascular diseases (Pat. Appl. KR 20020085543 A, publ. 16.11.2002).

Known biologically active food supplement (BAA) with a lipid-lowering and antioxidant effect, containing in its composition an extract of flat sea urchins, containing 0.75-0.85% echinochrome A (Pat. RU 2340216 C1, publ. 10.12.2008).

A known composition containing an extract of gonads, intestines or shells of sea urchins (Anthocidaris crassispina, Hemicentrotus pulcherrimus, Pseudocentrotus depressus or Strongylocentrotus internmedius} for the prevention and treatment of liver diseases with antioxidant and detoxifying effects (Pat. Appl. KR20110001.011 A, 20112011 011.2011 A. )

An alcohol solution containing echinochrome A in combination with chitosan, ascorbic, lipoic, citric and succinic acids is used to correct pathological disorders of the carbohydrate, lipid and antioxidant status of the body (Pat RU2360683 C1, publ. 04/16/2008).

Closest to the present invention is the drug Histochrome, used for the treatment of inflammatory diseases of the retina and cornea of the eyes, which is a 0.02% isotonic solution of di- and trisodium salts of the pigment from the group of spinochromes - echinochrome A (Pat. RU 2134107 C1, publ. 10.08. 1999).

The objective of the invention is to provide a tool containing as an active component a biologically active complex obtained from the shells and needles of green sea urchins Strongylocentrotus droebachiensis, containing in its composition from 0.02% to 10% of the pigment complex containing spinochromes B and D, as well as dimeric polyhydroxynaphthoquinones and from 7.5% to 20% of the mineral component of the shell of sea urchins (calcium, magnesium, phosphorus, sodium, potassium) in the form of water-soluble salts, for topical use in the form of eye drops, intranasal drops and spray, rendering present anti-inflammatory and antiallergic action, non-toxic, easy to use and stable during storage.

The problem is solved by creating a tool with the following components in g per 100 g:

Pigment-mineral complex - 0.05-2.0;

Co-solvent - 0.01-10;

Preservative - 0.01-0.2

Antioxidant - 0.01-0.2

Acidity regulator - up to pH 6.0-8.0

Water - up to 100.

The method of obtaining funds is implemented as follows: the preservative is mixed with water for injection at a temperature of 80-90 ° C and mixed until completely dissolved. After cooling to a temperature of 35-40 ° C, the calculated amount of a co-solvent, antioxidant is added to the solution and stirred until a clear solution is obtained. Then a pigment-mineral complex is introduced into the solution and the pH of the solution is adjusted to 6.0-8.0 using an acidity regulator. The solution is filtered and poured into vials. If necessary, sterilize.

In particular, sterile filtration or thermal steam sterilization under pressure can be used for sterilization.

We found that the order of mixing of the ingredients is essential to ensure the stability of the pigment-mineral complex. It has a high redox potential and is easily oxidized. To ensure the pharmacological activity of the pigment-mineral complex, such as inhibiting the free radical oxidation of lipids in cell membranes, modulating prostaglandin synthesis, increasing the activity of antioxidant enzymes in the body, etc., it is necessary to protect it from oxidation during preparation and storage. It has been experimentally established that an essential factor for ensuring the stability of a composition during storage is the introduction of an antioxidant into its composition, which are sodium metabisulfite, sodium sulfite, sodium thiosulfate or ascorbic acid.

The use of a pigment-mineral complex in the composition at a concentration of 0.05-2.0 wt.% Provides therapeutic activity and good tolerance to the drug. An increase in its concentration over the specified limit is undesirable and leads to a change in the isotonicity and isohydricity of the solution, and is also not economically justified.

We have experimentally established that the introduction of a preservative in the composition of the agent is necessary to create a pigment-mineral complex composition that is stable for a long time (at least 2 years) in the form of a solution, as well as taking into account their repeated use after opening the package, which promotes microbial contamination. As a preservative, pharmaceutically acceptable preservatives such as lower alkyl p-hydroxybenzoates (e.g. nipagin, nipazole), chlorhexidine in the form of acetate or gluconate and pharmaceutically applicable quaternary ammonium compounds (benzalkonium chloride and / or benzhexonium) can be used.

A cosolvent is introduced into the composition of the agent, which makes it possible to increase, if necessary, the concentration of the pigment-mineral complex in the composition of the agent, and improves the bioavailability of the active substance. As a co-solvent, pharmaceutically acceptable solvents can be used, such as ethyl alcohol, polysorbate 80, polyethylene glycol, propylene glycol, glycerin, polyoxyethylene-40-glycerol-hydroxystearate, diethylene glycol monoethyl ether, polyvinylpyrrolidone, polyvinyl alcohol.

The introduction of an acidity regulator was carried out in order to create a medium comfortable for the cells, without causing damage or burning sensations. Sodium salts of carbonic acid, ethylenediaminetetraacetic acid in the form of tri-, tetrasodium salts, sodium hydroxide are used as the most suitable for body fluids.

The technical result provided by the present invention is the use of a pigment-mineral complex obtained from the shells and needles of sea urchins as an agent that has anti-inflammatory and anti-allergic effects by blocking the H1 histamine receptors.

The above set of distinctive features of the proposed method is not described in the literature, and their influence on the achievement of a technical result does not follow from a known level of knowledge in the field of pharmaceutical production technology.

The technical result of the invention also lies in obtaining a safe agent based on a pigment-mineral complex in the form of a finished dosage form for topical application (drops or spray) with a pronounced therapeutic effect. The new property of the pigment-mineral complex of shells and needles of sea urchins was established by the authors for the first time experimentally.

The composition of the pigment-mineral complex is directly dependent on the method of preparation (Pat. RU No. 2441661 (C1), publ. 02/10/2012).

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. Eye drops (in g per 100 g). In a suitable vessel with a stirrer, thermostatically controlled at 80-90 ° C, 0.1 g of nipagin is dissolved in 91.9 ml of water for injection. The resulting solution is cooled to a temperature of 35 ° C, add 0.1 g of ascorbic acid, mix, add 3.5 g of ethyl alcohol, mix until a clear solution is obtained. 0.05 g of a pigment-mineral complex is introduced into the solution and the pH level is adjusted by adding 4.35 g of sodium bicarbonate. After filtration, a solution with a pH of about 6.2 is obtained. Bottling is carried out in 5 ml glass vials followed by autoclaving at a temperature of 100 ° C for 30 minutes. Get 19 vials (including losses) of 5 ml of eye drops containing 0.05% pigment-mineral complex.

Example 2. Nasal drops, spray (in g per 100 g). 0.05 g of benzalkonium chloride is dissolved in 93.4 ml of water for injection in a suitable vessel with a stirrer, thermostatically controlled at 80-90 ° C. The resulting solution is cooled to a temperature of 35 ° C, add 0.15 g of sodium metabisulfite, 2.5 g of polyvinylpyrrolidone, mix until a clear solution is obtained. Into the resulting solution add 1.0 g of the pigment-mineral complex and adjust the pH of the resulting solution to 7.0 by adding 2.9 g of ethylenediaminetetraacetic acid in the form of disodium salt dihydrate. After filtering, a solution with a pH of about 7 is obtained. The finished solution is filtered, subjected to sterilizing filtration in a stream of nitrogen, and then poured and sealed under aseptic conditions into dark glass penicillin bottles equipped with dropper dispensers or a spray nozzle for spray, with a volume of 5 ml. Get 19 vials (including losses) of 5 ml, containing 1.0% pigment-mineral complex.

Example 3. Antiallergic activity in vivo in a model of allergic conjunctivitis.

Allergic conjunctivitis (AK) was induced by topical application of 0.1 ml of Compound 48/80 solution (10 mg / ml) to the rabbit eye conjunctiva (gray chinchilla). The tool according to example 1 with the content of the pigment-mineral complex in an amount of 0.05% and comparison preparations Opatanol (0.1% solution of olopatadine hydrochloride) and Histochrome were injected intraconjunctively at 0.05 ml according to the treatment-and-prophylactic regimen: 1 time per day 3 days before the induction of pathology and 10, 30, 45 and 100 minutes after the induction of pathology.

The condition of the surface structures and the anterior part of the eye was assessed by the point system (Miyazaki et at. Conjunctival mast cell as a mediator of eosinophilic response in ocular allergy // Mol. Vis. 2008. 14. 1525-1532) by indicators: conjunctival edema, eyelid edema , lacrimation, conjunctival injection before application and after 5, 15 minutes, 1, 2 and 24 hours after application of Compound 48/80 solution (Figure 1).

Antiallergic activity of the agent according to example 1 in all investigated parameters exceeded the activity of the comparison drugs. In the total assessment of scores for the entire observation period, it was found that the antiallergic activity of the agent according to example 1 with respect to the external signs of allergic conjunctivitis is almost 3 times higher than that of Histochrom and exceeds the antiallergic activity of Opatanol by 50%.

Example 4. The effect of funds on the sensitivity of H1-histamine receptors ex vivo

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 ability of the agent of Example 1 with a pigment-mineral complex content of 0.05% to bind to H1-histamine receptors was evaluated by histamine-induced guinea pig ileum reduction.

The ileum was removed from the abdominal cavity of anesthetized animals and placed in a container with Krebs-Henseleit buffer (BH) 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 BKH at a temperature of + 30 ° С 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 BKH at a temperature of + 30 ° C to wash the tissues and restore their ability to contract. After 60 minutes of washing, perfusion was carried out with a solution of the agent of Example 1 in BCH. The concentration of the agent of Example 1 in the final solution ranged from 1 to 10 μl / ml. Pyrilamine maleate (10 μM) 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 (1 μM). Within 10 minutes, the contraction force was recorded, the amplitude and time of the onset of the maximum response were estimated (table 1).

Table 1 Amplitude of histamine-induced contraction and time of onset of maximal response (Me ± Q, n = 8) Group Dose Amplitude (% of contraction at 80 mM KCl) Time (fractions of the contraction time at 80 mM KCl) Control (histamine) 1 μM 100.81 ± 10.35 ** 1.32 ± 0.38 ** Pyrilamine maleate 10 μM 5.72 ± 4.79 * 10.22 ± 3.96 * The tool according to example 1 1 μl / ml 69.10 ± 20.22 *, ** 3.45 ± 2.54 *, ** The tool according to example 1 5 μl / ml 42.15 ± 11.57 *, ** 5.19 ± 3.02 * The tool according to example 1 10 μl / ml 26.49 ± 8.33 *, ** 10.09 ± 2.71 * * values are statistically significant compared with the control group, at p <0.05 ** values are statistically significant compared with the group of pyrilamine maleate, at p <0.05

The agent according to example 1 in all proposed concentrations blocked the H1-histamine receptors of the ileum smooth muscle sarcolemma. The effective dose (ED ₅₀ ) of the preparation of Example 1 was 2.97 μl / ml.

Thus, the agent of Example 1 is an effective blocker of H1-histamine receptors.

Example 5. Anti-inflammatory activity in vivo in a model of acute rhinosinusitis (ORS) in rats.

The experiment was carried out on male Wistar rats weighing 220-270 grams of body contained in a standard diet. Pathology was induced by intranasal administration of 7.5% formalin of 20 μl in each nasal passage. The pathology was characterized by plethora, hyperplasia, and focal necrosis of the mucous membrane of the nasal passages, an increase in the number of goblet cells, infiltration by monononuclear cells and leukocytes, and hyperproduction of mucus by the glands of the submucosa.

The tool according to example 2 with a pigment-mineral complex content of 1% was administered according to the treatment regimen for 7 days once a day, intranasally at a dose of 0.025 ml in each nasal passage. Comparison drugs Vibrocil and Aqua Maris were administered in the same way at doses of 0.1 ml in each nasal passage. On the 8th day after the induction of pathology, the animals were euthanized and comparative macroscopic and morphological studies of the mucous and submucous membranes of the nasal passages were performed (Table 2).

Pathology caused by intranasal administration of 7.5% formalin was characterized by a catarrhal form of rhinosinusitis. When using the funds of example 2, a cure of 30% of the experimental animals was observed. Comparison preparations in this model were not effective and against the background of their use, serous rhinosinusitis developed in 70-80% of animals and serous-purulent rhinosinusitis in 20% of animals. The use of the agent according to example 2 prevented the development of severe forms of inflammation of the mucous membrane of the nasal passages.

Against the background of the use of the agent of Example 2, the number of goblet cells per 1 mm of the mucous membrane of the nasal passages was significantly reduced, and leukocyte infiltration of the submucosa was at the level of the intact group of animals.

Thus, the pharmacological activity of the agent according to example 2 in the model of acute rhinosinusitis exceeds the activity of both the local anti-inflammatory agent Aqua Maris and the anti-congestive, vasoconstrictor and anti-allergic agent Vibrocil.

Example 6. Antioxidant activity

It is known that during allergic processes the rate of free radical oxidation increases (Simonyan A.V. Antioxidants in modern healthcare. // Medical Herald. - 2008. - No. 16. - 443). Thus, the antioxidant effect of the antiallergic drug is its additional advantage, ensuring the systemic effect of the drug.

The effect of the agent of Example 2 on the antioxidant status of the organism was investigated in a model of streptozotocin-induced type 2 diabetes.

The experiment was carried out on male outbred mice. Pathology was induced by a single injection of streptozotocin (60 mg / kg). In the period from 11 to 20 days, the intraperitoneal administration of the agent according to Example 2 was carried out with a pigment-mineral complex content of 1% in an amount ensuring the administration of the complex in an amount of 1.8 mg / kg, which corresponds to a dose of 10 mg for humans. The effect of the agent according to example 2 on the state of the antioxidant status of the organism was evaluated by the change in the concentration of reduced glutathione and malondialdehyde in the peripheral blood (table 3).

Table 3 The effect of the means of example 2 on the antioxidant status of the body (M ± m) Group The concentration of reduced glutathione, mmol / ml The concentration of malondialdehyde, µmol / l Intact (n = 10) 0.60 ± 0.04 11.1 ± 0.4 Control (pathology + placebo) ¹ (n = 9) 0.26 ± 0.06 * 19.0 ± 1.3 * The tool according to example 2 (n = 10) 0.66 ± 0.05 ** 9.4 ± 0.6 *, ** ¹ - placebo - water * - differences are statistically significant compared with the intact group at p <0.05 ** - differences are statistically significant compared with the control group at p <0.05;

An increase in the concentration of malondialdehyde and a decrease in the concentration of reduced glutathione in the peripheral blood of experimental animals of the control group indicates the development of oxidative stress with the introduction of streptozotocin.

The tool according to example 2 had a pronounced antioxidant effect, consisting in increasing the concentration of reduced glutathione and lowering the concentration of malondialdehyde and restoring them to the level of intact animals.

Thus, the agents of examples 1 and 2 expand the range of effective, safe anti-inflammatory and anti-allergic agents from natural sources.

Claims (3)

1. An agent having anti-inflammatory and anti-allergic effect, in the form of eye drops, intranasal drops or spray, containing an active substance, a cosolvent, a preservative, an acidity regulator and water, characterized in that it further comprises an antioxidant selected from the group of sodium metabisulfite, sodium sulfite, sodium thiosulfate or ascorbic acid, a co-solvent selected from the group of ethyl alcohol, polysorbate 80, polyethylene glycol, propylene glycol, glycerin, polyoxyethylene-40-glycerol-hydroxystear , diethylene glycol monoethyl ether, polyvinylpyrrolidone, polyvinyl alcohol, as an active substance contains the pigment complex of sea urchin shells Strongylocentrotus droebachiensis (0.02-10%), containing spinochroms B and D, as well as dimeric polyhydroxynaphthoquinones, calcium mineral , magnesium, phosphorus, sodium, potassium) in the form of water-soluble salts, and an acidity regulator in the following ratio of components, g per 100 g:
Pigment and mineral complex 0.05-2.0 Co-solvent 0.01-10 Preservative 0.01-0.2 Antioxidant 0.01-0.2 Acidity regulator to pH of the solution 6.0-8.0 Water up to 100. 2. The method of obtaining funds according to claim 1, characterized in that it includes
The following stages:
a) the preparation of a preservative solution in water at a temperature of 80-90 ° C, cooling the resulting solution to 35-40 ° C;
b) dissolving in a cooled solution of a co-solvent and antioxidant to obtain a clear solution;
c) the introduction of a pigment-mineral complex in the resulting solution of excipients with constant stirring;
g) the introduction of an acidity regulator and adjusting the pH of the solution to 6.0-8.0;
d) filtering the resulting solution and pouring into bottles. 3. The production method according to claim 2, characterized in that sterilization can be used for sterilization or thermal sterilization with steam under pressure.

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