Carbomer hydrogel containing phage lysate for maintaining the natural skin microbiota and suppressing pathogenic bacteria

Abstract

A carbomer hydrogel for maintaining the natural skin microbiota and suppressing pathogenic bacteria, comprising lysozyme and / or a very stable crude / purified Staphylococcus aureus phage lysate having a titer of at least 10 7 pfu / g gel and a crude / purified Cutibacterium acnes phage lysate having a titer of at least 10 5 pfu / g gel.

Description

Technical Field

[0001] The present invention relates to a composition and production of a carbomer hydrogel containing a phage lysate effective against Staphylococcus aureus and Cutibacterium acnes (formerly Propionibacterium acne). The present invention relates to a carbomer hydrogel composition which may also contain a lytic enzyme (endolysin) encoded by a bacteriophage in addition to the bacteriophage itself. The hydrogel is suitable for maintaining the natural skin microflora and suppressing pathogenic bacteria.

Background Art

[0002] Acne vulgaris, one of the most common chronic skin diseases, is caused by the disruption of the natural skin microflora or its partial replacement by pathogenic bacteria. Although still controversial, an important role is attributed to Cutibacterium acnes (formerly Propionibacterium acne), Staphylococcus aureus, and representative microfungi of the genus Malassezia (Ramasamy et al., 2019). The above-mentioned bacteria often show antibiotic resistance. With regard to resistance and to limit the overuse of antibiotics, efforts have been made to find alternative approaches for preventing infection and suppressing pathogenic bacteria. Bacterial viruses, so-called bacteriophages, may be an alternative to antibiotics. Unlike antibiotics, they act selectively and thus do not disrupt the natural flora and are effective as a preventive or antibacterial agent against selected pathogens.

[0003] The topic of bacteriophages as antibacterial agents is not new. Their discovery dates back to the 1920s. With the discovery of antibiotics, phage research in Western European countries, especially in the United States, was pushed aside, and other types of prevention and treatment of bacterial infections (phage therapy) were supported. However, the interest in bacteriophages and their use for phage therapy continued mainly in the countries of the former Eastern Bloc (Czechoslovakia, Poland, Georgia, and the countries of the former Soviet Union). As the number of bacteria showing antibiotic resistance has been increasing, bacteriophages have started to attract attention again (Lin et al., 2017). Their advantages are high specificity for the host strain, the ability to grow at the site of infection, or the natural ability to overcome the host's defense mechanisms.

[0004] Not only bacteriophages but also lytic enzymes (endolysins) encoded by bacteriophages may be alternatives to antibiotics. These enzymes produced by bacteriophages decompose the bacterial cell membrane and promote the release of the propagated bacteriophages from the host cells. Endolysins can be used mainly for Gram-positive bacteria even when administered externally. Furthermore, synergistic effects may be obtained by using them in combination with phages.

[0005] Preparations for acne-prone skin mainly include cleansing gels and lotions, cleansing lotions and beauty essences, peeling agents containing glycolic acid, or sticks containing zinc oxide. After washing the skin, it is recommended to perform aftercare for hydration using various beauty essences, gels, and creams containing vitamin C, E, A, B3, urea, collagen, ceramide, aloe vera, or herb extracts that may have antibacterial or soothing effects, such as chamomile, calendula officinalis, sage, or green tea. Snail extracts are famous not only in Korea but also for their vitamin E, A, C, and collagen contents. Therapeutic preparations for acne-prone skin contain antibiotics, benzoyl peroxide, azelaic acid, or ichthammol.

[0006] Parabens, which have a role as preservatives, are very often included in preparations designed for irritated skin, despite the fact that they can irritate the skin, and the use of some of them in no-rinse cosmetics is prohibited in the EU. The current trend in the cosmetics industry is to eliminate parabens and use natural or less problematic preservatives.

[0007] Only sporadic products containing bacteriophages have appeared on the cosmetics market. Preparations that have been known and designed to maintain the natural skin microbiota and at the same time fight acne vulgaris often do not contain bacteriophages against Cutibacterium acnes, one of the main causative agents of this disease. Most preparations often contain bacteriophages at an often unknown concentration (titer) that is not precisely defined, or contain only lytic enzymes. Preparations containing crude / unpurified phage lysates are also known.

[0008] The effects of the above preparations are chemically based. The advantages compared to them are that the phages are of natural origin and phages are generally available from natural sources. Phages are a common component in all environments and therefore do not impose a burden on organisms or the environment. On the contrary, they are a normal part of the skin and provide a natural antibacterial effect (and help maintain the natural skin microbiota). Unlike the above available preparations containing phages, the present invention contains precisely defined phages at an exact concentration that are effective against C. acnes and S. aureus. Furthermore, their effects can be synergistically supported by lytic enzymes. The composition and preservation method of the hydrogel ensure the stability of the phages for a specified period. The stability of the phages is also supported by a suitable preparation method, preservation conditions, and specific composition. It has been shown that the individual components and processes do not adversely affect the biological components (phages, enzymes) of the gel. Summary of the Invention

[0009] The present invention consists of a hydrogel composition and preparation, which contains an aqueous component, a gel-forming substance (preferably Carbomerum 980), a very stable crude / purified Staphylococcus aureus phage lysate having a titer of at least 10 7 pfu / g gel, and a crude / purified Cutibacterium acnes phage lysate having a titer of at least 10 5 pfu / g gel, and / or a lytic enzyme (e.g., LysF1, LysSA1).

[0010] Therefore, the present invention provides a carbomer hydrogel for maintaining the natural skin microbiota and suppressing pathogenic bacteria, which contains a Staphylococcus aureus lysate and a Cutibacterium acnes phage lysate having a broad spectrum of activity, and both are sterilized by filtration through a 0.22 μm filter. The ratio of the stock phage lysate to the gel is at most 1 part:100 parts based on the weight of the mixture.

Mode for Carrying Out the Invention

[0011] The main active components of the gel are a phage cocktail consisting of crude / purified Staphylococcus aureus phages (deposited under accession numbers DSMZ33473, DSMZ33474, and DSMZ33475) and Cutibacterium acnes phages (deposited under accession numbers DSMZ34313 and DSMZ34314) as specified in Table 1.

Table 1

[0012] The advantage of this gel is that the gel contains a very stable bacteriophage cocktail against S. aureus and C. acnes, ensuring a wide range of activities. The stability of the phage is guaranteed over a minimum storage period under appropriate storage conditions. Carbomerum 980 is preferably used as the gelling substance. It is a high molecular weight polymer of acrylic acid cross-linked with polyalkenyl ethers of saccharides or polyhydric alcohols. It contains 56.0% - 68.0% carboxyl groups (-COOH) with respect to the dry matter.

[0013] Other auxiliary features of the gel are improved by adding healing substances, such as herb extracts and essential oils, allantoin, chelating substances, such as EDTA, antioxidants (see Table 4), such as α-tocopherol, and preservatives (see Table 2).

[0014] According to the present invention, another advantage of the gel is its excellent spreadability and absorbability on the skin, as well as the gradual release of the active ingredient throughout the gel absorption period.

[0015] The (carbomer) gel containing phage and / or lytic enzyme is applied as a layer 1 - 4 times a day to be absorbed by the skin.

[0016] The hydrogel protects from light and needs to be stored at a temperature of 2°C - 8°C. In case of improper storage, the optimal activity can be impaired by the degradation of the bacteriophage.

[0017] The protein sequences of LysF1 (SEQ ID NO: 1) and LysSA1 (SEQ ID NO: 2) are shown in a separate sequence listing. The sequence listing also includes the sequences of the bacteriophages used, namely SEQ ID NO: 3 for phage DSMZ33473, SEQ ID NO: 4 for phage DSMZ33474, SEQ ID NO: 5 for phage DSMZ33475, SEQ ID NO: 6 for phage DSMZ34313, and SEQ ID NO: 7 for phage DSMZ34314.

Example

[0018] Gel compositions containing bacteriophages and / or lytic proteins are shown in the following examples, which are merely illustrative and do not limit the scope of the subject matter of the present invention in any way.

[0019] Example 1 100 parts by weight of Tris buffer (composition: 50 mM Tris, 10 mM NaCl, 10 mM CaCl2, pH = 7.5) and 1 part by weight of carbomer (trade name Carbomerum 980) are placed in a mortar for grinding, left immersed for 15 - 30 minutes, and mixed thoroughly. Using a 1 - 10 M sodium hydroxide solution, adjust the pH to a value of pH 7 (+ / - 0.3) while stirring continuously. Place the resulting carbomer gel in a suitable container in a steam autoclave and sterilize at 121 °C for 15 minutes. After cooling to room temperature, add a preservative (see Table 2), a crude / purified phage lysate having a titer of at least 10 9 pfu / ml for S. aureus and at least 10 7 pfu / ml for C. acnes to the gel. Add the phage to the gel at a ratio of up to 1 part:100 parts based on the weight of the mixture. Finally, mix the gel thoroughly.

Table 2

[0020] Example 2 The method for manufacturing a larger volume of carbomer gel consists of putting 100 parts by weight of Tris buffer (composition: 50 mM Tris, 10 mM NaCl, 10 mM CaCl2, pH = 7.5) into a container and evenly covering the surface of the liquid with 1 part by weight of carbomer (trade name Carbomerum 980). Keep this immersed for 15 - 30 minutes and then mix well using a shaft stirrer. After adding the carbomer, acidify the mixture. To achieve the gel structure, it is necessary to neutralize the solution with 1 - 10 M sodium hydroxide while continuously stirring. The pH value of the resulting gel is 7 (+ / - 0.3). Put the obtained carbomer gel into a suitable container in a steam autoclave and sterilize it at 121 °C for 15 minutes. After cooling to room temperature, add a preservative (see Table 2), a crude / purified phage lysate effective against S. aureus and C. acnes bacteria (both sterilized by filtration through a 0.22 μm filter) aseptically to the gel. The titer of the stock crude / purified lysate is at least 10 9 pfu / ml for S. aureus phage and at least 10 7 pfu / ml for C. acnes phage. Add the phage to the gel at a ratio of up to 1 part:100 parts based on the weight of the mixture. Then, the total phage titer in the final carbomer gel is at least 10 7 pfu / g gel for phage effective against Staphylococcus aureus and at least 10 5 pfu / g gel for phage effective against Cutibacterium acnes. Finally, mix the gel well.

[0021] Example 3 Further mix a lytic enzyme (LysF1, LysSA1) into the gel containing the phage according to Example 1 or 2. Similar to the bacteriophage, sterilize the enzyme by filtration through a 0.22 μm filter. The concentration of the enzyme in the gel is 1 - 50 μg / g gel. The amount of the enzyme in the gel is determined from the concentration of the prepared batch. Add the enzyme so that the gel is not diluted more than 1 part:100 parts based on the weight of the mixture. After adding the enzyme, mix the gel well.

[0022] Example 4 The preparation procedure of the gel base is the same as that in Example 1 or 2. In this case, bacteriophage is not added. After cooling the gel to room temperature, a preservative (see Table 2) and lytic enzymes (LysF1, LysSA1) are aseptically mixed. Before addition, the enzymes are sterilized by filtration through a 0.22 μm filter. The concentration of the enzymes in the gel is 1 - 50 μg / g gel. The amount of the enzymes in the gel is determined from the concentration of the prepared batch and is added so that the gel is not diluted to more than 1 part:100 parts based on the weight of the mixture. After adding the enzymes, the gel is thoroughly mixed.

[0023] Example 5 Table 3 shows the possible compositions of the gel. The amounts of the components are shown as percent concentrations.

Table 3

[0024] Example 6 Put 100 parts by weight of Tris buffer (composition: 50 mM Tris, 10 mM NaCl, 10 mM CaCl2, pH = 7.5) into a container and evenly cover the surface of the liquid with 1 part by weight of carbomer (trade name Carbomerum 980). Keep it immersed for 15 - 30 minutes and then mix thoroughly using a shaft stirrer. After adding the carbomer, acidify the mixture. To achieve the gel structure, it is necessary to neutralize the solution with 1 - 10 M sodium hydroxide while continuously stirring. The pH value of the resulting gel is 7 (+ / - 0.3). Mix a preservative (see Table 2) into the obtained gel, put the gel into a suitable container in a steam autoclave and sterilize it at 121 °C for 15 minutes. After cooling to room temperature, mix the crude / purified phage lysate and / or lytic enzymes specified in Example 2, 3, or 4 into the gel aseptically.

[0025] In addition to the biologically active components, the carbomer gel may optionally contain EDTA and antioxidants specified in Table 4, such as α - tocopherol, allantoin, herb extracts, and essential oils.

Table 4

Industrial Applicability

[0026] A carbomer gel containing a highly stable bacteriophage and / or lysozyme having a broad activity spectrum can be applied in the cosmetics and pharmaceutical industries. The preparation is suitable for maintaining the natural skin microbiota and suppressing the pathogenic bacteria S. aureus and C. acnes.

[0027] 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

1. A carbomer hydrogel for maintaining the natural skin microbiome and suppressing pathogenic bacteria, wherein the gel has a broad activity spectrum and at least 10 ７ Crude / purified phage lysates and / or at least 10 pfu / g gel titers effective against Staphylococcus aureus. ５ It contains a phage lysate effective against Cutibacterium acnes having a pfu / g gel titer, both of which are sterilized by filtration through a 0.22 μm filter, and the ratio of the stock phage lysate to the gel is at most 1 part:100 parts based on the weight of the mixture. The carbomer hydrogel is further characterized in that, in addition to the phage lysate, it also contains the lytic enzyme of SEQ ID NO: 1 and / or SEQ ID NO: 2 at a concentration of 1 to 50 μg / g gel.

2. The carbomer hydrogel according to claim 1, comprising a preservative in addition to a phage.

3. The carbomer hydrogel according to claim 2, characterized by containing 2-phenoxyethanol at a concentration of 0.5 to 1% by weight.

4. The carbomer hydrogel according to claim 1 or 2, characterized in that it also includes additional components from the group of antioxidants.

5. The carbomer hydrogel according to claim 4, characterized in that the base of the carbomer hydrogel contains 2-phenoxyethanol at a concentration of 0.5 to 1% by weight, α-tocopherol at a concentration of 0.5 to 2% by weight, and EDTA at a concentration of 0.005 to 0.1% by weight.

6. A carbomer hydrogel for maintaining the natural skin microbiome and suppressing pathogenic bacteria, as described in claim 1, characterized in that it contains phages deposited in the patent microbiological collection of the Leibniz Institute DSMZ in Munich under acceptance numbers DSMZ33473, DSMZ33474, DSMZ33475, DSMZ34313, and DSMZ34314.