RU2493852C1 - Composition containing enzyme deoxyribonuclease and/or stearyl glycyrrhetinate or glycyrrhetinic acid or salt thereof: ammonium or dipotassium or trisodium glycyrrhizinate - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and concerns a composition for local application containing the enzyme deoxyribonuclease and stearyl glycyrrhetinate or a glycyrrhetinic acid or a salt thereof: ammonium or dipotassium or trisodium glycyrrhizinate which may be used in medicine for treating and preventing viral infections caused by DNA-containing virals, such as herpes, herpes zoster, human papilloma, adenovirus and others.

EFFECT: invention provides preparing the stable composition with maintaining the enzymatic activity for five years.

1 cl, 5 ex, 1 tbl

Description

The invention relates to medicine, namely to a composition for topical use, containing an enzyme deoxyribonuclease and stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate, or dipotassium, or trisodium, which can be used in medicine for the treatment and prevention of viral infections caused by DNA- containing viruses such as herpes, shingles, human papilloma, adenoviruses and others.

There are known topical medicines used for the treatment and prevention of herpes virus infections, containing antiviral components that are not enzymes, for example Zovirax ™ cream (acyclovir) (http://www.vidal.ru/poisk_preparatov/zovirax~27174.htm); capsules and lyophilisate for the preparation of a solution for intravenous administration of Phosphogliv ™ (phospholipids and trisodium salt of glycyrrhizic acid) (http://www.vidal.ru/poisk_preparatov/phosphogliv.htm); Epigen-intim ™ spray (glycyrrhizic acid) (http: //www.vidal. ru / poisk_preparatov / epigen-intim.htm); ointments: alpi-sarin (http://alpizarin.ru/), helepin (http://www.vidal.ru/poisk preparatov / helepin-d20830.htm), flacoside (http://ru.wikipedia.org/ wiki /% D4% EB% E0% EA% EE% E7% E8% E4) containing plant extracts.

A topical composition is known from RU 2410123 C2 containing, as the active ingredient, deoxyribonuclease (DNase) 0.001-0.5 wt.% And acceptable carriers and excipients (Abstract, page 4). The claimed composition differs from RU 2410123 C2 in that it contains stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium.

A topical composition is known from US 2009/0220440 A1, containing as an antiviral ingredient a glycyrrhizic acid salt, in particular ammonium glycyrrhizinate, 0.25-0.5 wt.% And acceptable carriers and excipients ([0013], [0024] ], the formula).

The claimed composition differs from US 2009/0220440 A1 in that it contains deoxyribonuclease.

The difference between the claimed composition from RU 2410123 C2 and US 2009/0220440 A1 also lies in the complex antiviral effect: stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium induces the formation of interferon and interrupts the replication of viruses in the early stages from a capsid, thereby preventing its penetration into cells, and deoxyribonuclease hydrolyzes the DNA of the capsid-free virus.

A topical agent is known from RU 2411039 C2, containing glycyrrhizic acid or its salts and acceptable carriers and excipients, in particular a hydrophobic base, tocopherol acetate as an antiviral ingredient (page 8, example 7, formula).

From US 2010/0080768 A1, the use of bisabolol in a topical composition is known (claims 1, 5, 6).

Known La-Cree ™ lip balm cosmetic product containing licorice extract and bisabolol used to soften the skin of the lips (http://vertex.spb.ru/catalog/68/34/la-kri_balyzam_dlya_gub.html).

Known lip balm Uriage Barierderm, containing shea butter, borage oil, unsaponifiable fractions of avocado oil, stearyl glycyrrhetinate, ascorbyl palmitate, tocopherol, used to repair damaged lip skin (http://www.krason.ru/catalog-bariederm/1740-3620- balzam-dlya-gub.aspx? perf = 1).

Known drug Glycyram (ammonium glycyrrrhizinate), used in the form of tablets for the treatment of bronchial asthma and adrenal hypofunction (http: //www.vida! .Ru / poisk_preparatov / glycyram-tablets-0.05_27575.htm).

There are known topical cosmetics used for the prevention of herpes, containing antiviral components that are not enzymes, for example, extracts of medicinal plants and germanium-organic compounds (Antiherpetic preventative lipstick (P16 9158 915854 71.100.70) 292472. TU 9124-001-00358210 -96), glycyrrhizic acid ointment "Herpinat" ointment; ban. 20 ml; No. 77.01.12.915.P.02970.02.00 from the Research Institute of Atherosclerosis of the Russian Academy of Natural Sciences (Russia), INAT-PHARMA (Russia) (http://www.biomedservice.ru/publish/pub16.htm).

Known cosmetic lip balm Aven with cold cream to protect the skin of the lips from UV radiation and chapping (http://www.krason.ru/catalog-cold-cream/2471-10585-kold-krem-balzam-dlya-gub. aspx).

A cosmetic enzyme-containing composition is known where the enzyme is selected from the group of proteinases, including trypsin, chymotrypsin, bromelain, papain and ficin, as well as lysozyme, elastase, α-lipase and α-amylase, which is used to treat acne, care for skin and skin cleansing (DE 4305460). The described composition does not contain nuclease enzymes with direct antiviral activity, and therefore cannot be used to prevent viral infections.

Known pharmaceutical preparation "Deoxyribonuclease", produced in the form of lyophilized powder in hermetically sealed vials and ampoules of 0.005; 0.01; 0.025 and 0.05 g. This drug is prescribed for herpetic keratitis, adenoviral conjunctivitis, to reduce viscosity and improve the evacuation of sputum and pus in bronchiectasis and is used topically in the form of a 0.2% solution on isotonic sodium chloride solution (Reference "Medicines" edited by M.A. Klyuyev 2001).

Known pharmaceutical drug "Ribonuclease", produced in the form of lyophilized powder in hermetically sealed vials of 0.01 g. This drug is prescribed for diseases of the respiratory tract with difficult to separate sputum, periodontal disease, gingivitis, purulent wounds, trophic ulcers, tick-borne encephalitis, thrombophlebitis. topical application is sprinkled on the wound surface in the amount of 0.025-0.05 g (Reference "Medicines" edited by MA Klyuev 2001).

Compositions containing the enzyme DNase and stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate, or dipotassium, or trisodium, have not been described for topical use in the prevention of viral infections.

Thus, it is an object of the present invention to provide a topical composition comprising a DNAase enzyme and stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium, which can be used to prevent viral infections caused by DNA-containing viruses, such as herpes, herpes zoster, human papilloma, adenoviruses, etc.

The present invention provides a topical composition containing, as an active ingredient, 0.001-0.5 wt.% Deoxyribonuclease (DNase) 0.001-0.5 wt.% And stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium 0.001-0.5 wt.% and acceptable carriers and excipients.

As a preferred embodiment of the claimed invention, there is provided a composition as defined above, further comprising one or more than one vitamin and / or anti-inflammatory ingredient.

Most preferred is a composition containing DNAase and stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium as the active ingredient, bisabolol, panthenol triacetate, ascorbyl palmitate or tocopherol acetate and acceptable as additional active ingredients and , in the following ratio of components, wt.%:

DNAase 0.001-0.5 stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium 0.001-0.5 hydrogenated lecithin 0.001-5.0 preservative 0.5-1.0 bisabolol 0.1-0.5 panthenol triacetate 1.0-5.0 ascorbyl palmitate or tocopherol acetate 0.001-0.1 carrier 0.05-1.0 pH regulator 0.05-1.0 demineralized water 1.00-5.00 hydrophobic base 80,40-97,386

The active ingredients of the claimed pharmaceutical composition are an enzyme: DNAase and stearyl glycyrrhetinate and glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium. DNAase has the ability to inhibit the development of herpes viruses, adenoviruses and other DNA-containing viruses without damaging the DNA of macroorganism cells (Mashkovsky M.D. Medicines. Volume 2. M.: New Wave, 2000, pp. 111-112 ) Stearyl glycyrrhetinate is a fat-soluble form of glycyrrhizic acid and retains all its properties. Glycyrrhizic acid and its salts are used as an antiviral agent for external and local use. Glycyrrhizic acid is active against DNA and RNA-containing viruses, including various strains of Herpes simplex, Varicella zoster, human papilloma viruses, cytomegaloviruses. The antiviral effect is apparently associated with the induction of the formation of interferon. Interrupts the replication of viruses in the early stages, causes the virion to exit the capsid, thereby preventing its penetration into the cells. This is due to selective dose-dependent inhibition of phosphorylating kinase R. It interacts with the structures of the virus, changing various phases of the viral cycle, which is accompanied by irreversible inactivation of viral particles (which are in a free state outside the cells), blocking the introduction of active viral particles through the cell membrane into the cell, as well as impaired ability viruses to the synthesis of new structural components. Inhibits viruses in concentrations that are non-toxic to normally functioning cells. Virus strains resistant to acyclovir and iodouridine are highly sensitive to glycyrrhizic acid. It also has anti-inflammatory, analgesic and tissue regeneration-enhancing effects both in the early manifestations of viral infection and in ulcer forms (http://www.vidal.ru/poisk preparatov / act 1347.htm).

Tocopherol acetate - Vitamin E. It is an active antioxidant, inhibits lipid peroxidation, intensified in many diseases, prevents damage to cellular structures by free radicals. It takes part in the processes of tissue respiration, biosynthesis of heme and proteins, metabolism of fats and carbohydrates, cell proliferation and other metabolic processes. Vitamin E is present in many products, especially some fats and oils (argan, olive) are rich in them (http://ru.wikipedia.org/wiki/%D0%92%D0%B8%D1%82%D0%B0%D0 % BC% D0% B8% D0% BD_% D0% 95).

Bisabolol is a monocyclic sesquiterpene alcohol. The source of this bis-bolol is the bark of the Candean tree (Vanillosmopsis Erythropappa), from which essential oil containing up to 85% bisabolol is obtained, has the following properties:

anti-inflammatory: reduces redness, irritation (ammonia was used as an irritant in the experiments), promotes resorption of inflammations, prevents the appearance of erythema from UV radiation, and in case of its occurrence promotes faster restoration of the skin (Leite et al., 2010; Baylac et al. , 2003; Yakovlev et al., 1979);

- reduces the formation of melanin in cells, makes the skin lighter and more homogeneous in color (Saebom et al., 2008; Lee et al., 2010);

- wound healing: accelerates healing of burns by 25%;

- antibacterial, especially against gram-positive bacteria, fungi and Candida albicans, according to other sources, bisabolol showed low activity against Staphylococcus aureus, Bacillus cereus and Escherichia coli, but activity against Candida albicans was significant and comparable in effectiveness to linalool (Seatlholo et al. , 2006);

- increases the susceptibility of Staphylococcus aureus to antibiotics, presumably by violating the barrier function of the bacterial membrane, this allows exogenous substances (antibiotics) to penetrate the bacteria, this effect is more pronounced in gram-positive bacteria (Brehm-Stecher et al., 2003);

- deodorizing effect in combination with antibacterial essential oils;

- antifungal: inhibits the growth of mycelium Botrytis cinerea (Nunez et al., 2007);

- inhibits the parasite causing leishmaniasis in vitro (Morales-Yusfe et al., 2010);

- antioxidant: helps to restore the redox balance and thus counteracts oxidative stress (Braga et al., 2009), in vitro showed low activity against DPPH radicals (Seatlholo et al., 2006);

- deeply penetrates the skin (Hahn B, Holzl J., 1987);

- enhancer: improves the penetration of other substances from the product into the skin (Monti et al., 1995);

- insecticidal activity against Argentifolii Bemisia.

Dexpanthenol or its fat-soluble form of panthenol triacetate belongs to the B vitamins, is a derivative of pantothenic acid. It plays an important role in the processes of acetylation and oxidation, participates in carbohydrate and fat metabolism, in the synthesis of acetylcholine, corticosteroids, porphyrins. It has a pronounced effect on the formation and function of epithelial tissue, has some anti-inflammatory activity (http://www.vidal.ru/poisk preparatov / act 303. htm)

Escin is a triterpene glycoside (saponin) from the fruits (seeds) of horse chestnut (in the form of sodium salt). It has a pronounced capillaroprotective activity, has an antiexudative effect (http://www.vidal.ru/poisk preparatov / act 23.htm)

Ascorbyl palmitate is a fat-soluble form of ascorbic acid, necessary for the formation of collagen, the normalization of redox reactions.

Thus, a complex antiviral effect is carried out: interruption of virus replication in the early stages, stimulation of the release of the virion from the capsid, hydrolysis of viral DNA and the induction of interferon formation, as well as suppression of secondary microflora and inflammation in the affected area and acceleration of epithelization.

To prepare the composition, the enzyme preparation “Deoxyribonuclease” is used. The preparation was obtained from the pancreas of cattle by extraction from crushed raw materials, followed by purification and freeze drying. Lyophilized dried powder “Deoxyribonuclease” is used for herpetic keratitis and keratouveitis, acute catarrh of the upper respiratory tract of an adenovirus nature and pneumonia.

DNA-ase in the form of this preparation is not used for the treatment and prevention of viral infections due to its difficult absorption through intact skin and low level of consumer properties, as well as the lack of stability when the moisture content is more than 3% and in solution, with the loss of enzyme activity during the day.

Surprisingly, DNAase and hydrogenated lecithin can be included in a stable topical composition while maintaining the activity of the enzyme for a long time, despite the fact that such a composition contains a significant amount of water (up to 5%).

The composition of the present invention is usually made by conventional methods using solid or liquid acceptable carriers or excipients selected from emulsifiers, dispersants, preservatives, flavorings, pH adjusters, polymer carriers and other excipients that are suitable for the preparation of compositions for topical use. The composition of the present invention can be prepared in the form of lipstick, gel, cream, paste, ointment and the like.

Additional advantages of this composition are increased stability, potentiated complex antiviral effect, ease of use, and ease of dosing.

The claimed composition has stability with respect to the activity of the enzymes included in its composition during long-term storage, which is achieved by immobilization of enzymes on carriers contained in the specified composition. As these carriers, any lecithins, cyclodextrins and polymers capable of binding to enzymes to form enzyme-carrier complexes can be used. Examples of such polymeric carriers are gums, polyvinylpyrrolidone, carrageenans, sodium alginate, chitosan polyethylene glycol ether, sodium carboxymethyl cellulose, polyacrylate, cyclodextrins, lecithins.

The activity of the claimed composition is due to the activity of its enzyme and stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium. It was found that the composition for topical application, containing DNA ase 0.001-0.5 wt.%, Has stability with respect to the activity of the enzyme during its long-term storage, while the activity of this enzyme, prepared in the form of a solution, rapidly decreases. Stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate or dipotassium or trisodium are stable compounds and do not require stabilization. Therefore, the claimed composition can be used for the effective prevention of DNA-containing viruses, such as herpes virus, herpes zoster, human papillomas, adenoviruses and others.

The present invention is illustrated by the following examples.

EXAMPLE 1

The composition in the form of a gel based on DNAase and ammonium glycyrrhizinate has the following composition:

DNAase 0.001 Ammonium Glycyrrhizinate 0.001 β-cyclodextrin 5,0 emulsifier 0.5 preservative 1,0 dexpanthenol 5,0 escin 5,0 polymer carrier 1,0 pH regulator 1,0 demineralized water 82,498

The specified composition is prepared according to the following method. DNase, β-cyclodextrin, ammonium glycyrrhizinate are dissolved in demineralized water, dexpanthenol, escin, an emulsifier, preservative, a polymeric carrier, and a pH regulator are added. The resulting gel is stirred, vacuum and homogenized.

EXAMPLE 2

The composition in the form of a gel based on DNAase and dipotassium glycyrrhizinate has the following composition:

DNAase 0.5 Dipotassium glycyrrhizinate 0.5 β-cyclodextrin 5,0 emulsifier 0.5 preservative 1,0 tocopherol acetate 5,0 bisabolol 5,0 polymer carrier 1,0 pH regulator 1,0 demineralized water 81.5

The specified composition is prepared as in EXAMPLE 1, characterized in that instead of ammonium glycyrrhizinate, glycirrhizinatedicalium is used, and bisabolol and tocopherol acetate are used instead of escin and dexpanthenol.

EXAMPLE 3

The composition in the form of a gel based on DNAase and trisodium glycyrrhizinate has the following composition:

DNAase 0.5 Glycyrrhizin sodium 0.5 β-cyclodextrin 5,0 emulsifier 0.5 preservative 1,0 ascended palmit 5,0 bisabolol 0.1 polymer carrier 1,0 pH regulator 1,0 demineralized water 86.4

The specified composition is prepared as in EXAMPLE 1, characterized in that instead of ammonium glycyrrhizinate, trisodium glycyrrhizinate is used, and ascorbyl palmitate is used instead of dexpanthenol.

EXAMPLE 4

The composition in the form of a lip balm based on DNase and stearyl glycyrrhetinate has the following composition:

DNAase 0.5 Stearyl glycyrrhetinate 0.5 hydrogenated lecithin 0.2 preservative 0.5 bisabolol 0.1 panthenol triacetate 1,0 ascorbil palmitate 0.001 carbomer 0.05 triethanolamine 0.05 demineralized water 1.00 jojoba wax / polyglyceryl-3-ether 84.90

The specified composition is prepared as follows: in a random sequence, all components are excluded, excluding ascorbyl palmitate and heated in a melter at 80 ° C, then cooled to 45 ° C and ascorbyl palmitate is added, mixed, packaged in cases and packaged.

EXAMPLE 5

Enzyme Stability Assessment

Since the main disadvantage of DNase is its instability in the presence of water from 3%, a comparative assessment was made of the conservation of enzyme activity during storage in the form of a solution and in the form of a composition for local use (for example, a gel).

DNAase activity was determined by the following procedure. Determination of DNAase activity was carried out according to the Kunitz method. For the unit of activity, DNA-basics take the smallest amount of the enzyme, which when exposed to the substrate under the conditions of the experiment releases such an amount of acid-soluble products, which leads to an increase in optical density at a wavelength of 260 nm per unit. 0.5 ml of substrate (DNA) and 0.3 ml of 0.2 M Tris buffer (pH = 7) are added to the control and two test tubes. 0.2 ml of the enzyme solution is added to the experimental, preheated tubes. Samples are thermostated for 30 minutes at a temperature of 37.5 ° C. Then, 1 ml of a 1N solution of perchloric acid (HClO ₄ ) cooled in an ice bath is added to the samples; 0.2 ml of the enzyme solution is added to the control. The tubes are shaken and kept in an ice bath for 16-25 minutes. Precipitation formed is separated by centrifugation at 4000 rpm for 10 minutes. 0.5 ml of the solution was taken from the supernatant, 2.5 ml of water was added, and the absorbance was measured at λ = 260 nm.

The results are shown in the following table.

Test samples Main ingredients Storage stability one 2 3 Gel number 1 According to Example 1 DNase activity 90% for 5 years of observation Gel number 2 According to Example 2 DNase activity 92% for 5 years of observation Native enzyme solution - 5% activity for DNAase for 1 day

As can be seen from the table, the use of polymeric carriers and stabilizers in the composition makes it possible to obtain stable compositions with preservation of β enzyme activity for 5 years of observation due to the creation of a stable conformation of the enzyme with β-cyclodextrin, lecithin and a carrier polymer. Similar data were obtained using the compositions of EXAMPLES 3, 4.

Claims (1)

A topical composition containing, as active ingredients, 0.001-0.5 wt.% Deoxyribonuclease (DNA bases) and 0.001-0.5 wt.% Stearyl glycyrrhetinate or glycyrrhizic acid or its salt: ammonium glycyrrhizinate or dipotassium or trisodium, as additional active ingredients - bisabolol, panthenol triacetate, ascorbyl palmitate or tocopherol acetate, as carriers: β-cyclodextrins or lecithins 0.001-5.0 wt.%, and acceptable carriers or excipients in the following ratio of components, wt.%:
DNAase 0.001-0.5 stearyl glycyrrhetinate or glycyrrhizic acid or its salts: ammonium glycyrrhizinate, or dipotassium or trisodium 0.001-0.5 hydrogenated lecithin 0.001-5.0 preservative 0.5-1.0 bisabolol 0.1-0.5 panthenol triacetate 1.0-5.0 ascorbyl palmitate or tocopherol acetate 0.001-0.1 carrier 0.05-1.0 pH regulator 0.05-1.0 demineralized water 1.00-5.00 hydrophobic base 80,40-97,386