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 10.sup.7 pfu/g of gel and crude/purified Cutibacterium acnes phage lysates with a titer of at least 10.sup.5 pfu/g of gel.

Claims

1.-8. (canceled)

9. A carbomer hydrogel for maintaining natural skin microflora and suppressing pathogenic bacteria comprising crude/purified phage lysates effective against Staphylococcus aureus bacteria with a titer of at least 10.sup.7 pfu/g of gel and/or phage lysates effective against Cutibacterium acnes bacteria with a titer of at least 10.sup.5 pfu/g of gel, with a broad spectrum of activity, both sterilized by filtration through a 0.22 m filter, wherein a ratio of the stock phage lysates and the gel is at most 1:100 parts by weight of the mixture, and a lytic enzyme of SEQ ID NO: 1 and/or SEQ ID NO: 2 at a concentration of 1-50 g/g of gel.

10. The carbomer hydrogel of claim 9 further comprising phages and preservatives.

11. The carbomer hydrogel of claim 10 further comprising 2-phenoxyethanol at a concentration of 0.5-1 wt.-%.

12. The carbomer hydrogel of claim 9 further comprising antioxidants.

13. The carbomer hydrogel of claim 10 further comprising antioxidants.

14. The carbomer hydrogel of claim 12 further comprising 2-phenoxyethanol at a concentration of 0.5-1 wt.-%, -tocopherol at a concentration of 0.05-2 wt.-% and EDTA at a concentration of 0.005-0.1 wt.-%.

15. The carbomer hydrogel of claim 13 further comprising 2-phenoxyethanol at a concentration of 0.5-1 wt.-%, -tocopherol at a concentration of 0.05-2 wt.-% and EDTA at a concentration of 0.005-0.1 wt.-%.

16. The carbomer hydrogel of claim 9 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

17. The carbomer hydrogel of claim 10 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

18. The carbomer hydrogel of claim 11 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

19. The carbomer hydrogel of claim 12 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

20. The carbomer hydrogel of claim 13 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

21. The carbomer hydrogel of claim 14 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

22. The carbomer hydrogel of claim 15 further comprising phages deposited in the patent collection of microorganisms in Leibniz Institute DSMZ, Munich under accession numbers DSM 33473, DSM 33474, DSM 33475, DSM 34313, and DSM 34314.

Description

EXAMPLES

[0021] 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.

Example 1

[0022] One hundred parts by weight of a tris buffer (composition: 50 mM Tris; 10 mM NaCl; 10 mM CaCl.sub.2; 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 10.sup.9 pfu/ml for S. aureus and at least 10.sup.7 pfu/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-US-00002 TABLE 2 describes the preservatives and their percentages that the gel may contain. Concentration in the Preservative name final product in wt.-% 2-phenoxyethanol 0.5-1 Chlorohexidine digluconate solution 0.002-0.01 Benzalkonium chloride 0.002-0.01 Potassium sorbate 0.1-0.2 Euxyl PE9010 0.5-1

Example 2

[0023] 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 CaCl.sub.2; 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 0.22 m filter. The titer of the stock crude/purified lysate is at least 10.sup.9 pfu/ml for S. aureus phages and at least 10.sup.7 pfu/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 10.sup.7 pfu/g of gel for the phages effective against Staphylococcus aureus and at least 10.sup.5 pfu/g of gel for the phages effective against Cutibacterium acnes. Finally, the gel is thoroughly mixed.

Example 3

[0024] 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 0.22 m filter. The enzyme concentration in the gel is 1-50 g/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

[0025] 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 0.22 m filter. The enzyme concentration in the gel is 1-50 g/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

[0026] Table 3 presents a possible composition of the gel. The amounts of ingredients are presented as percentage concentrations.

TABLE-US-00003 TABLE 3 Substance (constituent) name Content in wt.-% Tris buffer (composition: 50 mM Tris; 10 mM Up to 100% NaCl; 10 mM CaCl.sub.2; pH = 7.5) Carbomerum 980 1 2-phenoxyethanol 0.5-1 Mixture of crude/purified phage lysates 1 effective against S. aureus bacteria in a concentration of at least 10.sup.9 pfu/ml Mixture of crude/purified phage lysates 1 effective against C. acnes bacteria in a concentration of at least 10.sup.7 pfu/ml Endolysin LysF1 at a concentration of 1 0.1-5 mg/ml Endolysin LysSA1 at a concentration of 1 0.1-5 mg/ml

Example 6

[0027] One hundred parts by weight of a tris buffer (composition: 50 mM Tris; 10 mM NaCl; 10 mM CaCl.sub.2; 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.

[0028] Besides the biologically active ingredients, the carbomer gel may contain EDTA and antioxidants as required, e.g. -tocopherol, allantoin, herb extracts, essential oils etc., as specified in Table 4.

TABLE-US-00004 TABLE 4 Other possible constituents of the carbomer hydrogel and their percentage concentrations Substance (constituent) name Content in wt.-% EDTA 0.005-0.1 -tocopherol 0.05-2 Allantoin 0.2-0.5 Arnica Montana extract 0.5-1 Matricaria chamomilla extract 0.1-2 Calendula officinalis extract 0.1-0.3 Humulus lupul extract 0.1-2 Rosmarinus Officinalis Leaf extract 0.05-0.1 Citrus Grandis Seed extract 0.1-2 Lemongrass oil 0.2-1.5 Hydrolysed collagen 0.2-2

Industrial Applicability

[0029] 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

[0030] 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. [0031] RAMASAMY, S., et al. The role of the skin microbiota in acne pathophysiology. British Journal of Dermatology, 2019, 181.4: 691-699.