A carbomer hydrogel comprising a phage lysate for maintaining natural skin microflora and suppressing pathogenic bacteria

Abstract

A carbomer hydrogel for maintaining the natural skin microflora and suppressing pathogenic bacteria that contains lytic enzymes and / or highly stable crude / purified Staphylococcus aureus phage lysates with a titer of at least 107 pfu / g of gel and crude / purified Cutibacterium acnes phage lysates with a titer of at least 105 pfu / g of gel.

Description

[0001]A carbomer hydrogel for maintaining natural skin microflora and suppressing pathogenic bacteria Technical Field The invention relates to the composition and preparation of a carbomer hydrogel containing phage lysates effective against Staphylococcus aureus and Cutibacterium acnes (formerly Propionibacterium acnes) bacteria. The invention relates to the composition of a carbomer hydrogel that may, besides the bacteriophages themselves, also contain bacteriophage-encoded lytic enzymes (endolysins). The hydrogel is suitable for maintaining the natural skin microflora and suppressing pathogenic bacteria. Background Art Acne vulgaris, one of the most common chronic skin diseases, occurs due to disruption of the natural skin microflora or its partial replacement by pathogenic bacteria. A significant role, though still controversial, is ascribed to the bacteria Cutibacterium acnes (formerly Propionibacterium acnes), Staphylococcus aureus, as well as to a representative of micromycetes of the genus Malassezia (Ramasamy et al., 2019). The bacteria mentioned above frequently show antibiotic resistance. With respect to the resistance as well as to restrict excessive use of antibiotics, there is an effort to find alternative approaches to prevent infections and suppress pathogenic bacteria. The viruses of bacteria, so-called bacteriophages, represent a possible alternative to antibiotics. Unlike antibiotics, they act selectively, so they do not disrupt the natural bacterial microflora and are effective as prevention or an antimicrobial agent against selected pathogens. The topic of bacteriophages as antimicrobial agents is not new. Their discovery dates back to the 1920s. With the discovery of antibiotics, in the Western counties, especially in the U.S., the research of bacteriophages was put aside in favour of other kinds of prevention and treatment of bacterial infections (phage therapy). However, the interest in bacteriophages and their use for phage therapy continued mainly in countries of the former Eastern Block (Czechoslovakia, Poland, Georgia and countries of the former Soviet Union). With the ever-growing occurrence of bacteria showing antibiotic resistance, bacteriophages started to come to the fore again (Lin et al., 2017). Their advantage is high specificity for the host strain, the capability of multiplication at the site of infection or their natural ability to overcome the host’s defence mechanisms. It is not only bacteriophages but also bacteriophage-encoded lytic enzymes (endolysins) that represent a possible alternative to antibiotics. These enzymes produced by bacteriophages degrade the cellular membrane of bacteria to facilitate releasing of multiplied bacteriophages from the host cells. Endolysins can be mainly used with gram-positive bacteria, even though they are administered from the outside. Moreover, their use together with phages may have a synergistic effect. Preparations for skin prone to acne mainly comprise washing gels and emulsions, cleansing lotions and serums, peeling agents containing glycolic acid, or sticks containing zinc oxide. After cleaning the skin, hydration after-care is recommended, using various serums, gels and creams containing vitamins C, E, A, B3, urea, collagen, ceramides, aloe vera or herb extracts that may have antibacterial or calming effects, e.g. camomile, calendula officinalis, sage or green tea. A snail extract is famous for its vitamins E, A, C and collagen content, not only in Korea. Treatment preparations for acne-prone skin contain antibiotics, benzoyl peroxide, azelaic acid or ichthamol. Parabens in the role of preservatives very often appear in preparations designed for irritated skin, even though they may irritate the skin, and using some of them in no-rinse cosmetics is prohibited in the EU. A current trend in cosmetics is to eliminate parabens and use natural or less controversial preservatives. Only sporadic products containing bacteriophages appear in the cosmetic market. Preparations that are known so far and designed to maintain the natural skin microflora and fight acne vulgaris at the same time often do not contain bacteriophages against Cutibacterium acnes, one of the main causative agents of this disease. Most preparations contain not precisely defined bacteriophages, often at an unknown concentration (titer), or a lytic enzyme only. Preparations that contain crude / not purified phage lysate are also known. The effect of the preparations mentioned above is chemically based. An advantage compared to them is the natural origin of phages, which are commonly available from natural resources. Phages are a common constituent of every environment and therefore do not represent any load for the organism or environment. Conversely, they are its normal part and provide a natural antimicrobial effect (and help preserve the natural skin microflora). Unlike the above-mentioned available preparations with phages, the present invention contains well-defined phages at a precise concentration effective against C. acnes and S. aureus. In addition, their effect may be synergistically supported by the lytic enzymes. The hydrogel's composition and preservation method ensure phage stability for a specified period. The phage stability is also supported by suitable method of preparation, storage conditions and specific composition. It has been demonstrated that individual components and processes do not negatively impact the gel's biological constituents (phages, enzymes). Summary of Invention The invention consists in the composition and preparation of a hydrogel that contains an aqueous constituent, a gel-forming substance (preferably Carbomerum 980), highly stable crude / purified Staphylococcus aureus phage lysates with a titer of at least 107pfu / g of gel and crude / purified Cutibacterium acnes phage lysates with a titer of at least 105pfu / g of gel and / or lytic enzymes (e.g. LysF1, LysSA1). Accordingly, the invention provides a carbomer hydrogel to maintain the natural skin microflora and to suppress pathogenic bacteria, characterized in that it contains Staphylococcus aureus and Cutibacterium acnes phage lysates with a broad spectrum of activity, both sterilized by filtration through a 0.22µm filter. The ratio of the stock phage lysates to the gel is max.1:100 parts by weight of the mixture. The main active ingredient of the gel is a phage cocktail consisting of crude / purified Staphylococcus aureus phages (deposited under accession numbers DSMZ 33473, DSMZ 33474 and DSMZ 33475) and Cutibacterium acnes phages (deposited under accession numbers DSMZ 34313 and DSMZ 34314) specified in Table 1. Table 1: Bacteriophages used in the carbomer hydrogel. An advantage of the gel is that it contains a highly stable cocktail of bacteriophages against S. aureus and C. acnes bacteria, which ensures a broad spectrum of activity. Stability of phages is guaranteed for the minimum shelf-life period on condition of proper storage. Carbomerum 980 is preferably used as the gel forming substance. It is a high molecular-weight polymer of acrylic acid cross-linked by polyalkenyl ethers of saccharides or polyalcohols. In relation to the dried substance, they contain 56.0 % to 68.0 % of carboxylic groups (-COOH). Other auxiliary characteristics of the gel are improved by the addition of healing substances such as herb extracts and essential oils, allantoin, chelating substances, e.g. EDTA, antioxidants like α- tocopherol (see Table 4) and preservatives (see Table 2). According to the invention, another advantage of the gel is its outstanding spreadability and absorbability on the skin and the gradual release of active ingredients throughout the gel absorption period. The (carbomer) gel containing phages and / or lytic enzymes is applied 1-4 times a day in a layer that is left to be absorbed by the skin. The hydrogel must be protected from light and stored at a temperature from 2°C to 8°C. In case of improper storage, the optimum activity may be affected due to bacteriophage degradation. Protein sequences of LysF1 (SEQ ID NO: 1) and LysSA1 SEQ ID NO: 2) are included in a separate Sequence Listing. The sequence listing further comprises the sequences of the used bacteriophages, namely SEQ ID NO: 3 for the phage DSMZ33473, SEQ ID NO: 4 for DSMZ33474, SEQ ID NO: 5 for DSMZ33475, SEQ ID NO: 6 for DSMZ34313 and SEQ ID NO: 7 for DSMZ34314. The composition of the gel containing bacteriophages and / or lytic proteins is presented in the examples below, which are only illustrative and do not restrict the scope of the subject matter of the invention in any way. One hundred parts by weight of a tris buffer (composition: 50 mM Tris; 10 mM NaCl; 10 mM CaCl2; pH = 7.5) and 1 part by weight of the carbomer (trade name Carbomerum 980) are dosed into a grinding mortar, left to soak for 15–30 minutes and mixed thoroughly. A 1-10 M sodium hydroxide solution is used to adjust pH to the value of pH 7 (+ / - 0,3) under continuous stirring. The obtained carbomer gel is sterilized in a steam autoclave at 121°C for 15 minutes in a suitable vessel. After cooling down to room temperature, preservatives (see Table 2), crude / purified phage lysates with a titer of at least 109pfu / ml for S. aureus and at least 107pfu / ml for C. acnes are added to the gel. The phages are added to the gel at the ratio of max. 1:100 parts by weight of the mixture. Finally, the gel is mixed thoroughly. Table 2 describes the preservatives and their percentages that the gel may contain. Example 2 The production method of the carbomer gel in bigger volumes consists in dosing 100 parts by weight of a tris buffer (composition: 50 mM Tris; 10 mM NaCl; 10 mM CaCl2; pH = 7.5) into a vessel and the surface of the fluid is equally covered with 1 part by weight of the carbomer (trade name Carbomerum 980). It is left to soak for 15-30 minutes and then mixed thoroughly using a shaft stirrer. After the addition of the carbomer, the mixture is acidified. The solution must be neutralized with 1- 10 M sodium hydroxide under continuous stirring to achieve a gel structure. The pH value of the resulting gel is 7 (+ / - 0.3). The obtained carbomer gel is sterilized in a suitable vessel in a steam autoclave at 121°C for 15 minutes. After cooling to room temperature, preservatives (see Table 2), phage crude / purified lysates effective against the bacteria S. aureus and C. acnes are admixed to the gel sterilely, both sterilized by filtration through a 0.22µm filter. The titer of the stock crude / purified lysate is at least 109pfu / ml for S. aureus phages and at least 107pfu / ml for C. acnes phages. The phages are added to the gel at the ratio of max.1:100 parts by weight of the mixture. The total titer of phages in the final carbomer gel is then at least 107pfu / g of gel for the phages effective against Staphylococcus aureus and at least 105pfu / g of gel for the phages effective against Cutibacterium acnes. Finally, the gel is thoroughly mixed. Example 3 A lytic enzyme (LysF1, LysSA1) is additionally admixed to the gel containing phages according to Example 1 or 2. In the same way as the bacteriophages, the enzyme is sterilized by filtration through a 0.22µm filter. The enzyme concentration in the gel is 1-50 µg / g of gel. The amount of the enzyme in the gel results from the concentration of the prepared batch. The enzyme is added in such a way that the gel is not diluted more than 1:100 parts by weight of the mixture. After the addition of the enzyme, the gel is thoroughly mixed. Example 4 The preparation procedure of the gel base is equal to Example 1 or 2. In this case, the bacteriophages are not added. After cooling the gel to room temperature, preservatives, see Table 2, and a lytic enzyme (LysF1, LysSA1) are admixed sterilely. Before the addition, the enzyme is sterilized by filtration through a 0.22µm filter. The enzyme concentration in the gel is 1-50 µg / g of gel. The amount of the enzyme in the gel results from the concentration of the prepared batch and is added in such a way that the gel is not diluted more than 1:100 parts by weight of the mixture. After the addition of the enzyme, the gel is thoroughly mixed. Example 5 Table 3 presents a possible composition of the gel. The amounts of ingredients are presented as percentage concentrations. Table 3 Example 6 One hundred parts by weight of a tris buffer (composition: 50 mM Tris; 10 mM NaCl; 10 mM CaCl2; pH = 7.5) are dosed into a vessel, and the surface of the fluid is equally covered with 1 part by weight of the carbomer (trade name Carbomerum 980). It is left to soak for 15-30 minutes and then mixed thoroughly using a shaft stirrer. After the addition of the carbomer, the mixture is acidified. The solution must be neutralized with 1-10 M sodium hydroxide under continuous stirring to achieve a gel structure. The pH value of the resulting gel is 7 (+ / - 0.3). A preservative, see Table 2, is admixed into the obtained gel, and the gel is sterilized in a suitable vessel in a steam autoclave at 121 °C for 15 minutes. After cooling to room temperature, the crude / purified phage lysates and / or lytic enzymes are admixed to the gel sterilely, as specified in Examples 2, 3 or 4. Besides the biologically active ingredients, the carbomer gel may contain EDTA and antioxidants as required, e.g. α-tocopherol, allantoin, herb extracts, essential oils etc., as specified in Table 4. Table 4: Other possible constituents of the carbomer hydrogel and their percentage concentrations Industrial applicability The carbomer gel containing highly stable bacteriophages with a broad spectrum of activity and / or lytic enzymes is applicable in the cosmetic or pharmaceutical industry. The preparation is suitable for maintaining the natural skin microflora and suppressing pathogenic bacteria S. aureus and C. acnes. References LIN, Derek M.; KOSKELLA, Britt; LIN, Henry C. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World journal of gastrointestinal pharmacology and therapeutics, 2017, 8.3: 162. RAMASAMY, S., et al. The role of the skin microbiota in acne pathophysiology. British Journal of Dermatology, 2019, 181.4: 691-699.

Claims

Claims 1. A carbomer hydrogel for maintaining natural skin microflora and suppressing pathogenic bacteria, characterized in that it contains crude / purified phage lysates effective against Staphylococcus aureus bacteria with a titer of at least 107pfu / g of gel and / or phage lysates effective against Cutibacterium acnes bacteria with a titer of at least 105pfu / g of gel, with a broad spectrum of activity, both sterilized by filtration through a 0.22µm filter, wherein the ratio of the stock phage lysates and the gel is at most 1:100 parts by weight of the mixture.

2. The carbomer hydrogel for maintaining natural skin microflora and suppressing pathogenic bacteria, characterized in that it contains a lytic enzyme of SEQ ID NO: 1 and / or SEQ ID NO: 2 at a concentration of 1-50 µg / g of gel.

3. The carbomer hydrogel of claim 1, that besides the phage lysates also contains a lytic enzyme of SEQ ID NO: 1 and / or SEQ ID NO: 2 at a concentration of 1-50 mg / g of gel.

4. The carbomer hydrogel of claim 1 or 2, which contains preservatives in addition to phages.

5. The carbomer hydrogel of claim 4, characterized in that it contains 2-phenoxyethanol at a concentration of 0.5-1 wt.-%.

6. The carbomer hydrogel of claims 1, 2, 3 or 4, characterized in that it also contains additional constituents from the group of antioxidants.

7. The carbomer hydrogel of claim 6, characterized in that in its base contains 2-phenoxyethanol at a concentration of 0.5-1 wt.-%, alpha-tocopherol at a concentration of 0.5-2 wt.-% and EDTA at a concentration of 0.005-0.1 wt.-%.

8. A carbomer hydrogel for maintaining natural skin microflora and suppressing pathogenic bacteria, characterized in that it contains phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSMZ 33473, DSMZ 33474, DSMZ 33475, DSMZ 34313 and DSMZ 34314.

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