Novel compositions and methods for the treatment of acne vulgaris

Abstract

Biofilm formation is thought to be an important factor in the pathogenesis of acne. The present invention relates to the use of proteins, more particularly P. granulosum DNase, for the treatment and prevention of infectious skin diseases, and more particularly for the treatment and prevention of acne vulgaris. This protein has been demonstrated to be able to destroy biofilms formed by pathogenic bacteria, such as biofilms formed by P. acnes.

Description

Technical Field

[0001] Technical Field The present invention relates to the treatment and prevention of infectious skin diseases, and more specifically, to the treatment and prevention of acne vulgaris.

Background Art

[0002] Background of the Invention Acne vulgaris is a common inflammatory disease of the sebaceous follicles, which affects more than 80% of adolescents in their youth and may persist into adulthood. Propionibacterium acnes, which is sometimes also called Cutibacterium acnes, is a Gram-positive polymorphic bacillus and has conventionally been regarded as part of the normal human skin microbiota and is essentially present in the pilosebaceous system. Propionibacterium acnes plays an important role in the development of acne vulgaris together with the sebaceous glands.

[0003] P. acnes secretes lipase, chemotactic factors, metalloproteases and porphyrins. All of these interact with molecular oxygen, which generates toxic reactive oxygen species and free radicals that cause damage and inflammation of keratinocytes (Bruggemann. 2005. Insights in the pathogenic potential of Propionibacterium acnes from its complete genome. Semin Cutan Med Surg 24: 67-72).

[0004] Biofilm formation is the process by which microorganisms irreversibly attach to a surface and grow there, producing extracellular polymers that facilitate adhesion and matrix formation. This process results in changes in the phenotype of the organism with respect to its growth rate and gene transcription.

[0005] Biofilm formation is thought to be an important factor in the etiology of acne (Burkhart & Burkhart. 2007. Expanding the microcomedone theory and acne therapeutics: Propionibacterium acnes biofilm produces biological glue that holds corneocytes together to form plug. J Am Acad Dermatol 57: 722-724.). The biofilm produced by P. acnes contributes to the formation of an adhesive that results in the binding of corneocytes, causing microcomedones. Acne is a clogged hair follicle or skin pore in the skin. Keratin, or skin debris, combines with oil to block the hair follicle or pore. Acne can be open, also called a blackhead, or closed within the skin, also called a whitehead, and can occur with or without acne. The chronic inflammatory condition that usually includes both acne and inflammatory papules and pustules, or pimples, is called acne.

[0006] Cells covered by P. acnes biofilms have been demonstrated to produce more extracellular lipase and be more resistant to antimicrobial agents compared to planktonic cells. (Coenye et al. 2007. Biofilm formation by Propionibacterium acnes is associated with increased resistance to antimicrobial agents and increased production of putative virulence factors. Res Microbiol 158: 386-392). This finding can explain, to some extent, the failure of a certain number of antibiotic therapies. Other studies have shown that biofilm formation by P. acnes is less when isolated from healthy skin compared to biomaterial-related infections. (Holmberg et al. 2009. Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates. Clin Microbiol Infect 15: 787-795).

[0007] A recent case-control study investigated the presence and localization of P. acnes on the face by biopsy of acne lesions in vivo and characterized the phylotypes of P. acnes in 38 acne patients and matched controls: P. acnes within biofilms was significantly more frequent in acne patients (37% in acne patients versus 13% in control samples) (Jahns et al. 2012. An increased incidence of Propionibacterium acnes biofilms in acne vulgaris: a case-control study. Br J Dermatol 167: 50-58).

[0008] Biofilm formation has also been observed in many other skin diseases such as atopic dermatitis, candidiasis, bullous impetigo, and pemphigus foliaceus (Nusbaum et al. 2012. Biofilms in Dermatology. Skin Therapy Letter 17: 7).

[0009] As described by Rumbaugh, et al. (D. Fleming, K.P. Rumbaugh, Approaches to Dispersing Medical Biofilms, Microorganisms 5(2) (2017)), biofilm-related infectious diseases pose complex problems in the medical community in that the protection of pathogens from antibiotics and antimicrobial substances and from the host immune response is significantly increased by remaining within the protection of the biofilm. Since as many as 80% of human bacterial infections are biofilm-related, many researchers have initiated investigations into therapeutic methods that specifically target the biofilm structure and thereby disperse microbial cells into a more vulnerable planktonic ecology.

[0010] Conventionally, infectious diseases have been treated by directly targeting the causative pathogen. However, biofilms have significantly transformed the situation by providing microorganisms with greatly increased protection from antimicrobial substances, raising effective concentrations to dangerous levels. Therefore, some researchers have shifted their focus to anti-biofilm agent testing of dispersal events, i.e., compounds and strategies leading to dispersants.

[0011] Clinically, dispersal can be achieved by using enzymes, small molecules, or any other means to induce either passive or active large-scale dispersal events that release biofilm-related microorganisms into a more vulnerable planktonic state.

[0012] As further described by Rumbaugh, et al. (D. Fleming, K.P. Rumbaugh, Approaches to Dispersing Medical Biofilms, Microorganisms 5(2) (2017)), in many biofilms, extracellular DNA (eDNA) functions as a structural scaffold within the EPS and can help promote bacterial adhesion, aggregation, and horizontal gene transfer. Initially, the DNA found within biofilms was assumed to be merely the remnants of lysed cells, and the first study to show that eDNA could be a critically important contributing component of bacterial biofilms was conducted by Whitchurch et al. in 2002 (Whitchurch C.B., Tolker-Nielsen T., Ragas P.C., Mattick J.S. Extracellular DNA required for bacterial biofilm formation. Science. 2002;295:1487. doi:10.1126 / science.295.5559.1487.). The authors showed that exogenously added deoxyribonuclease (DNase I) could inhibit the formation of P. aeruginosa biofilms in vitro without significantly affecting bacterial viability. Furthermore, they found that treatment of pre-formed P. aeruginosa biofilms with DNase I for up to 60 hours resulted in dispersion. This finding has led to an increase in research targeting eDNA with various DNases as a means to eradicate biofilm infections. Table 1 summarizes many of the DNases that have been shown to have biofilm-disrupting activity to date.

[0013]

Table 1

[0014] As described in Kuehnast, et al. (T. Kuehnast, F. Cakar, T. Weinhaupl, A. Pilz, S. Selak, M.A. Schmidt, C. Ruter, S. Schild, Comparative analyses of biofilm formation among different Cutibacterium acnes isolates, Int J Med Microbiol 308(8) (2018) 1027-1035), it is becoming increasingly clear that biofilm formation is an important feature in the etiology of P. acnes in skin diseases and implant-related infections. P. acnes isolates are characterized by high genetic heterogeneity, which allows classification into different phylotypes and subtypes. Kuehnast et al. used an extensive collection of P. acnes isolates, including specimens classified by phylotype (IA1, IA2, IB, IC, II, and III), IA1 SLST subtypes, and anatomical isolation sites (skin and implant), to provide the first comparative analysis of in vitro biofilm-forming ability. In microtiter plate assays using a more stringent washing procedure, skin-derived and implant-derived IA1 isolates showed a 2- to 8-fold higher biofilm-forming ability compared to other phylotypes. In particular, SLST subtypes A1 and A2 showed high biofilm-forming ability, which is an interesting finding considering that these subtypes have been shown to have a stronger association with mild to severe acne. Microscopic analysis of biofilm morphology enabled visualization and evaluation of the three-dimensional biofilm structure. This allowed for a more rigorous assessment of biofilm formation by a diverse range of P. acnes isolates with well-structured mature biofilms formed by phylotypes IA1, IB, and IC. Consistently, these isolates also showed the highest attachment rates to abiotic surfaces. Overall, no consistent differences in biofilm formation were observed between skin-derived and implant-derived isolates of the same phylotype.The notable exception is the IA1 strain type, where the biofilm values of implant-derived isolates are slightly higher compared to skin-derived isolates in both assays. The proteinase K susceptibility assay and the DNase I susceptibility assay revealed that both eDNA and protein are important for the initial attachment to abiotic surfaces, and that protein is an important constituent of the mature biofilms formed by all strain types. In contrast, strain-type dependent differences in the DNase I susceptibility of mature P. acnes biofilms could be observed. Taken together, the results indicated that biofilm formation by P. acnes is primarily determined by the strain type and, to a much lesser extent, by the anatomical site of isolation.

[0015] The effects of DNase I treatment and proteinase K treatment were also evaluated by Kuehnast et al. in a microtiter plate biofilm assay. Using this high-throughput assay, they were able to test the effects of several different enzyme concentrations. However, the assay was limited to the IA1 isolate as only the IA1 isolate showed proper biofilm formation in the microtiter plate. In contrast to the flow cell-based assay, the IA1 biofilm in the microtiter plate was susceptible to the effects of both DNase I treatment and proteinase K treatment. A significant reduction in biofilm amount was observed compared to the mock-treated control, with the proteinase K concentration decreasing to 1.9 μg / ml and the DNase I concentration decreasing to 1.9 ng / ml. Therefore, Kuehnast et al. speculated that DNase I treatment combined with a more vigorous washing step during the microtiter plate assay has a stronger negative impact on the adhesion properties of the biofilm compared to the same treatment performed under the assay conditions inside the microscope chamber. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0016] Summary of the Invention The present inventors have discovered that the ability of P. acnes to form biofilms is negatively affected in the presence of Propionibacterium granulosum. The present inventors have further been able to demonstrate that P. granulosum secretes a protein that disrupts P. acnes biofilms. The P. granulosum protein has been isolated and identified as having deoxyribonuclease activity.

[0017] Accordingly, one aspect of the present invention provides an isolated protein having an amino acid sequence according to SEQ ID NO: 2 for use in medicine, and a functional variant thereof having at least 50% amino acid sequence identity with SEQ ID NO: 2 and having at least 80% of the DNase activity of the protein according to SEQ ID NO: 2 in a quantitative assay of deoxyribonuclease activity at pH 7 and 32°C.

[0018] This protein may further be for use in a method for the treatment and / or prevention of diseases caused by and / or associated with an infection with one or more biofilm-forming bacteria and / or fungi.

[0019] The protein can be for use according to the above, and the diseases are Propionibacterium acnes, P. aeruginosa, Vibrio cholerae, E. coli, S. pyogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Shewanella oneidensis, S. heamolyticus, Bordetella pertussis, Bordetella bronchiseptica, Campylobacter jejuni, H. influenza, B. bacteriovorus, S. aureus, Enterococcus faecalis, Listeria monocytogenes, Candida albicans, Aspergillus fumigatus.It is caused by or complicated with the infection of Streptococcus pneumonia, B. licheniformis, S. epidermidis, Staphylococcus salivarius, Staphylococcus constellatus, Staphylococcus lugdunesis, Staphylococcus anginosus, E. coli, Streptococcus intermedius, Micrococcus luteus, and Bacillus subtilis.

[0020] The protein can be for the use according to the above, and the disease is a skin disease.

[0021] The protein can be for the use according to the above, and the skin disease is selected from the group consisting of acne vulgaris, candidiasis, bullous impetigo, rosacea, and pemphigus foliaceus.

[0022] The protein can be for the use according to the above, and the protein is for use in a method for promoting wound healing.

[0023] The protein can be for the use according to the above, and the wound is selected from diabetic foot ulcers, pressure ulcers, vascular ulcers, ischemic wounds, burn wounds, and surgical wounds.

[0024] Furthermore, the present disclosure provides a pharmaceutical composition comprising the protein according to the above and optionally a pharmaceutically acceptable excipient.

[0025] The pharmaceutical composition according to the above may further comprise a lipid carrier system and / or an aqueous pH buffer.

[0026] According to one embodiment of the pharmaceutical composition according to the above, the lipid carrier system comprises a lipid in solid form or crystalline form.

[0027] Further provided herein is a method for the treatment and / or prevention of a disease caused by and / or complicated with an infection of one or more biofilm-forming bacteria and / or fungi, the method comprising administering the protein or pharmaceutical composition according to the above to a subject suffering from said infection. The protein or pharmaceutical composition is preferably administered in an amount effective to reduce the biofilm to the site of the biofilm-forming bacteria and / or fungi.

[0028] The diseases are Propionibacterium acnes, P. aeruginosa, Vibrio cholerae, E. coli, S. pyogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Shewanella oneidensis, S. heamolyticus, Bordetella pertussis, Bordetella bronchiseptica, Campylobacter jejuni, H. influenza, B. bacteriovorus, S. aureus, Enterococcus faecalis, Listeria monocytogenes, Candida albicans, Aspergillus fumigatus.It can be caused by or complicated with the infection of Streptococcus pneumonia, B. licheniformis, S. epidermidis, Staphylococcus salivarius, Staphylococcus constellatus, Staphylococcus lugdunesis, Staphylococcus anginosus, E. coli, Streptococcus intermedius, Micrococcus luteus, and Bacillus subtilis.

[0029] According to one embodiment of the method, the disease is a skin disease.

[0030] According to one embodiment of the method, the skin disease is selected from the group consisting of acne vulgaris, candidiasis, bullous impetigo, rosacea, and pemphigus foliaceus.

[0031] According to one embodiment of the method, this method is for promoting the healing of wounds. According to a further embodiment, the wound is selected from diabetic foot ulcers, pressure ulcers, vascular ulcers, ischemic wounds, burn wounds, and surgical wounds.

[0032] The protein according to the above may be P. granulosum DNase PG_1116 having the sequence of SEQ ID NO: 2, a homologous DNase derived from P. granulosum DSM20700 strain (GenBank accession number WP_021104654), or a homologous DNase derived from P. granulosum TM11 strain (GenBank accession number ERF66724). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Brief Description of the Drawings

Figure 1A

Figure 1B

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0034] Sequence Listing The following sequences are included in the sequence listing. SEQ ID NO: 1: DNA sequence encoding an isolated protein according to SEQ ID NO: 2. Accession No. 2: An isolated protein having DNase activity, derived from Propionibacterium granulosum. This protein is also referred to as "PG_1116".

[0035] Detailed Description of the Invention The protein having DNase activity according to the present invention can be isolated from bacteria of the species Propionibacterium granulosum and / or can be produced by recombinant DNA techniques well known in the art. As used herein, the term "isolated" reflects that the protein is isolated from its natural environment.

[0036] The present invention relates to an isolated protein having an amino acid sequence according to SEQ ID NO: 2 and to functional variants of this protein that retain, or essentially have, the same DNase activity as the protein of SEQ ID NO: 2, i.e., the ability to degrade deoxyribonucleic acid (DNA). Functional variants are proteins in which one or more amino acid insertions, deletions, or substitutions, either conservative or non-conservative, have been made at one or more positions (provided that such changes result in a protein in which the function associated with DNase activity is significantly retained). In this context, "significantly" means that the functional variant has at least 80%, such as 85%, 90%, 95%, 100% or more of the DNase activity of the protein according to SEQ ID NO: 2 in a quantitative assay for deoxyribonuclease I (EC 3.1.21.1) activity. The functional variant can be evaluated for the retained DNase activity, for example, at pH 7 and 32 °C or at pH 6 and 25 °C. Such quantitative assays are known in the art and are also described in the following Examples section. Functional variants preferably have at least 50%, such as 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity with SEQ ID NO: 2.

[0037] Thus, a functional variant of an isolated protein having the amino acid sequence according to SEQ ID NO: 2 retains its DNase activity and its ability to disrupt biofilms.

[0038] “Conservative substitutions” are intended to mean substitutions within the groups Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

[0039] Such variants can be made using methods of protein engineering and site-directed mutagenesis well known in the art.

[0040] When used in medicine, the DNase of the present invention can be administered in the form of conventional pharmaceutical compositions.

[0041] The pharmaceutical composition can be in the form of an aqueous solution. An aqueous solution refers to a solution having physiologically or pharmaceutically acceptable properties with respect to pH, ionic strength, isotonicity, etc. Examples include isotonic solutions of water and other biocompatible solvents, aqueous solutions (e.g., physiological saline and glucose solutions), and hydrogel-forming materials. The aqueous solution can be buffered with, for example, phosphate-buffered saline (PBS).

[0042] The pharmaceutical composition can further contain pharmaceutically acceptable excipients such as preservatives, antioxidants, isotonic agents, coloring agents, etc. to prevent the growth of microorganisms in the composition. In an aqueous suspension, the composition can be combined with suspending agents and stabilizers. The pharmaceutical composition can further contain additional pharmaceutically active compounds such as antibiotics.

[0043] Due to the colloidal nature of the composition, the composition can be aseptically prepared by using a final aseptic filtration step.

[0044] To form a gel, the protein can preferably be formulated with a hydrogel-forming material. Examples of hydrogel-forming materials include synthetic polymers such as polyvinyl alcohol, polyvinylpyrolidone, polyacrylic acid, polyethylene glycol, poloxamer block copolymers; semi-synthetic polymers such as cellulose ethers including carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, methylhydroxypropylcelltalose and ethylhydroxyethyl cellulose; and natural gums such as acacia, carragenan, chitosan, pectin, starch, xanthan gum.

[0045] It is advantageous to use a hydrogel that is mucoadhesive. In this regard, it is particularly useful to use hyaluronic acid and its derivatives, carbomer-type and polycarbophil-type crosslinked polyacrylic acids, and polymers that readily form gels, which are known to strongly adhere to mucous membranes.

[0046] It is also advantageous to use a poloxamer-type block copolymer, i.e., a polymer consisting of blocks of polyethylene glycol and polypropylene glycol. Certain poloxamers dispersed in water are thermoreversible, having low viscosity at room temperature but showing a marked increase in viscosity at high temperature and forming a gel at body temperature. This can extend the contact time of a pharmaceutical formulation administered to relatively warm skin, and thus improve the effectiveness of the introduced DNase.

[0047] The pharmaceutical composition of the present invention can be formulated for topical administration or enteral administration, i.e., oral administration, buccal administration, sublingual administration, mucosal administration, nasal administration, bronchial administration, rectal administration, and intravaginal administration.

[0048] In a preferred embodiment of the present invention, the route of administration can be topical.

[0049] Non-limiting examples of pharmaceutical compositions for topical administration are solutions, sprays, suspensions, emulsions, gels, and films. Optionally, bandages, plasters, or patches to which the pharmaceutical composition has been added can be used. Tablets, capsules, solutions, or suspensions can be used for enteral administration.

[0050] Depending on the mode of administration, the pharmaceutical composition, according to one embodiment of the invention, comprises from 0.05% by weight (weight percent) to 99% by weight of the protein of the invention, and according to an alternative embodiment, from 0.10 to 50% by weight (all weight percents are based on the total composition).

[0051] A therapeutically effective amount for the practice of the invention can be determined by using known criteria such as the age, weight, and response of the individual patient and can be interpreted by one of ordinary skill in the art within the context of the disease being treated or prevented.

[0052] The protein for use according to the invention can be produced by recombinant DNA technology.

[0053] Techniques for the construction of plasmids, vectors, and expression systems and for the transfection of cells are well known in the art, and one of ordinary skill in the art is familiar with standard resource materials that describe specific conditions and procedures.

[0054] The construction of the plasmids, vectors, and expression systems of the present invention employs standard ligation and restriction techniques well known in the art (generally, for example, see Ausubel, et al, Current Protocols in Molecular Biology, Wiley Interscience, 1989; Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd ed. 2001). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, adjusted, and sorted into the desired form. The sequence of a DNA construct can be confirmed, for example, using standard methods for DNA sequence analysis (see, for example, Sanger et al. (1977) Proc. Natl. Acad. Sci., 74, 5463-5467).

[0055] Yet another convenient method for isolating a particular nucleic acid molecule is by polymerase chain reaction (PCR) (Mullis et al. Methods Enzymol 155:335-350, 1987) or reverse transcription PCR (RT-PCR). A particular nucleic acid sequence can be isolated from RNA by RT-PCR. RNA is isolated from, for example, cells, tissues, or whole organisms by techniques known to those skilled in the art. Complementary DNA (cDNA) is then generated using a poly-dT primer or random hexamer primer, deoxynucleotides, and an appropriate reverse transcriptase. The desired polynucleotide can then be amplified from the generated cDNA by PCR. Alternatively, the polynucleotide of interest can be amplified directly from an appropriate cDNA library. Primers that hybridize to both the 5' and 3' ends of the polynucleotide sequence of interest are synthesized and used for PCR. The primers can also contain specific restriction enzyme sites at the 5' end for easy digestion and for ligation of the amplified sequence into a similarly restriction-digested plasmid vector.

[0056] As is clear from the following examples, the inventors have shown that P. granulosum DNase PG_1116, a protein having the amino acid sequence according to SEQ ID NO: 2, is significantly more effective than NucB in degrading the biofilm produced by P. acnes at pH 7. The pH on the surface of normal skin ranges from 4 to 5.5. However, acne-affected skin usually has a higher pH than non-affected skin, and the average value for acne patients is pH 6.4, although some patients reach pH levels of 10 or higher (Prakash, C. et al 2017 Skin Surface pH in Acne Vulgaris: Insights from an Observational Study and Review of the Literature. J Clin Aesthet Dermatol. 10: 33-39). As a result, PG_1116 is more efficient than other enzymes, such as NucB, used for the same purpose when treating acne-affected skin to degrade the biofilm produced by P. acnes.

[0057] Accordingly, the present disclosure provides an isolated protein having the amino acid sequence according to SEQ ID NO: 2, and functional variants thereof that retain DNase activity.

[0058] The isolated protein according to the above may be for use in medicine. This protein may further be for use in the treatment and / or prevention of diseases caused by and / or complicated by infections with one or more biofilm-forming bacteria and / or fungi.

[0059] The diseases are Propionibacterium acnes, P. aeruginosa, Vibrio cholerae, E. coli, S. pyogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Shewanella oneidensis, S. heamolyticus, Bordetella pertussis, Bordetella bronchiseptica, Campylobacter jejuni, H. influenza, B. bacteriovorus, S. aureus, Enterococcus faecalis, Listeria monocytogenes, Candida albicans, Aspergillus fumigatus.It can be caused by or complicated with the infection of Streptococcus pneumonia, B. licheniformis, S. epidermidis, Staphylococcus salivarius, Staphylococcus constellatus, Staphylococcus lugdunesis, Staphylococcus anginosus, E. coli, Streptococcus intermedius, Micrococcus luteus, and Bacillus subtilis. All of these are biofilm-forming bacteria or fungi.

[0060] The protein can be for use as described above, and the disease is a skin disease. The skin disease can be selected from the group consisting of acne vulgaris, candidiasis, bullous impetigo, rosacea, and pemphigus foliaceus.

[0061] Preferably, the protein can be for use in the treatment and / or prevention of acne vulgaris. Furthermore, the protein can be for use in the degradation of biofilms formed by Propionibacterium acnes (P. acnes). The protein can further be for use in the degradation of biofilms formed by P. acnes of subtype I1A.

[0062] Biofilms formed on implants such as pacemaker devices have been shown to cause biofilm-related infections. These infections can be difficult to address and can lead to surgical wounds that do not heal properly. In general, biofilms are difficult to break down by the immune system, which can prevent any wound from healing properly. Okuda et al. (K.I. Okuda, R. Nagahori, S. Yamada, S. Sugimoto, C. Sato, M. Sato, T. Iwase, K. Hashimoto, Y. Mizunoe, The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices, Front Microbiol 9 (2018) 182) investigated the effectiveness of enzymes targeting P. acnes biofilm matrix components against biofilm formation by five isolates. They used DNase I, Proteinase K, and Dispase B, which digest DNA, protein, and poly-N-acetylglucosamine (poly-GlcNAc), respectively, and showed that DNase I significantly inhibits biofilm formation for strains isolated from cardiac pacemaker devices. Therefore, the protein according to the present invention may be for use according to the above, and the protein is for use in promoting wound healing. The wound may be selected from diabetic foot ulcers, pressure ulcers, vascular ulcers, ischemic wounds, burn wounds, and surgical wounds.

[0063] Furthermore, the present disclosure provides a pharmaceutical composition comprising the protein according to the above and optionally a pharmaceutically acceptable excipient.

[0064] The pharmaceutical composition according to the above may further comprise a lipid delivery system and / or an aqueous pH buffer.

[0065] According to one embodiment of the pharmaceutical composition according to the above, the lipid carrier system comprises a lipid in solid form or crystalline form.

[0066] As described above, the above composition may comprise a pH buffer, preferably a water-based pH buffer. As described above, the skin affected by acne has a higher pH than the non-affected skin. By including a pH buffer in the composition, the pH on the skin affected by acne can be buffered to a pH that is disadvantageous for P. acnes, thus further improving the result of the treatment of acne with such a composition. However, as will be apparent from the following example section, care must be taken not to lower the pH to a level at which the efficiency of the protein of the present invention decreases.

[0067] Thus, the protein can degrade the biofilm, while the pH buffer can buffer the pH to a level that can help cure the bacterial infection of P. acnes that causes acne. Therefore, the composition according to the present disclosure can have two mechanisms of action. The primary mechanism is to efficiently degrade the biofilm at a relatively high pH that is typical of acne-affected skin, thereby allowing the composition to penetrate the pustules associated with acne. The secondary mechanism is to buffer the pH, thereby making the growth conditions of P. acnes less than optimal. Therefore, the composition according to the above can be provided for use in medicine. The composition according to the above can be for use in the treatment and / or prevention of diseases caused by and / or complicated by infections of one or more biofilm-forming bacteria and / or fungi.Many of the compositions according to the above are for use, and the diseases are Propionibacterium acnes, P. aeruginosa, Vibrio cholerae, Escherichia coli, S. pyogenes, Klebsiella pneumoniae, Acinetobacter baumannii, Aggregatibacter actinomycetemcomitans, Shewanella oneidensis, S. heamolyticus, Bordetella pertussis, Bordetella bronchiseptica, Campylobacter jejuni, H. influenza, B. bacteriovorus, S. aureus, Enterococcus faecalis, Listeria monocytogenes, Candida albicans, Aspergillus fumigatus.It is caused by, or complicated with, infection by Streptococcus pneumonia, B. licheniformis, S. epidermidis, Staphylococcus salivarius, Staphylococcus constellatus, Staphylococcus lugdunesis, Staphylococcus anginosus, E. coli, Streptococcus intermedius, Micrococcus luteus, and Bacillus subtilis.

[0068] The composition according to the above may be intended for use in the degradation of biofilms formed by Propionibacterium acnes (P. acnes). The composition according to the above may further be intended for use in the degradation of biofilms formed by P. acnes of subtype I1A.

[0069] The composition according to the above may be for use, and the disease is a skin disease. The skin disease may be selected from the group consisting of acne vulgaris, candidiasis, bullous impetigo, rosacea, and pemphigus foliaceus.

[0070] The composition according to the above may further be for use in promoting the healing of wounds. The wounds may be selected from diabetic foot ulcers, pressure ulcers, vascular ulcers, ischemic wounds, burn wounds, and surgical wounds.

[0071] The lipid carrier system can contain lipids in solid or crystalline form. Preferably, the lipid is in crystalline form. The crystalline form of the lipid can be, for example, monoglyceride. In general, it has already been observed that enzyme activity is at least partially inhibited by the presence of lipids. Many of the enzymes according to the prior art are highly sensitive to both pH and the presence of lipids because the enzymes are inactivated. This is also a problem because sebum is present on the skin of patients with acne skin. However, by using the protein of the present invention, this problem is overcome. Since the protein is not inactivated by the presence of lipids, it can be active in formulations together with lipids and on the skin even in the presence of sebum.

[0072] Amino acid sequence identity The percent identity between two amino acid sequences is determined as follows. First, the amino acid sequences are compared to SEQ ID NO:2 using the BLAST2 sequence (Bl2seq) program from the stand-alone version of BLASTZ, including, for example, BLASTN version 2.0.14 and BLASTP version 2.0.14. The stand-alone version of BLASTZ can be obtained from the website of the National Center for Biotechnology Information of the US government (ncbi.nlm.nih.gov). Instructions for using the Bl2seq program can be found in the readme file attached to BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the options for Bl2seq are set as follows: -i is set to the file containing the first amino acid sequence to be compared (for example, C:\seq1.txt); -j is set to the file containing the second amino acid sequence to be compared (for example, C:\seq2.txt); -p is set to blastp; -o is set to any desired file name (for example, C:\output.txt); all other options remain at their default settings. For example, the following command: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. can be used to generate an output file containing the comparison between two amino acid sequences. If the two compared sequences share homology, the specified output file presents the regions of their homology as the aligned sequences. If the two compared sequences do not share homology, the specified output file does not present the aligned sequences. Once aligned, the number of matches is determined by counting the number of positions at which the same nucleotide or amino acid residue is presented in both sequences.

[0073] The percent identity is determined by dividing the number of matches by the length of the sequence shown in the identified array and then multiplying the resulting value by 100. For example, if an array is compared to the array shown in SEQ ID NO: A (the length of the array shown in SEQ ID NO: A is 10) and the number of matches is 9, the array has 90% identity (i.e., 9 / 10 × 100 = 90) to the array shown in SEQ ID NO: A.

Example

[0074] Example Stock used and culture conditions Propionibacterium granulosum DSM 20700 was obtained from the German collection of microorganisms. This strain was anaerobically grown at 37°C either on solid medium on blood agar Petri dishes or in liquid medium in BHI supplemented with 2 g / L glucose. The suspension culture was grown with shaking (200 rpm), while the biofilm was grown in a static flask with medium changed every other day.

[0075] Genome sequencing of P. granulosum Genomic DNA of P. granulosum was isolated from liquid cultures using the GenElute™ Bacterial Genomic DNA Kit (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Sequencing of P. granulosum was performed on an Illumina HiSeq 2000 as 2×100 bp paired-end, yielding approximately 100-fold raw base pair overall coverage. Raw sequencing data was quality controlled using FastQC version 0.10.1 (http: / / www.bioinformatics.bbsrc.ac.uk / projects / fastqc). De novo assembly using the raw reads was performed using SOAPDenovo version 1.05 (Li et al. 2010. De novo assembly of human genomes with massively parallel short read sequencing.” Genome Res 20(2): 265-272). Standard parameters for paired-end reads were used. The K-mer setting that generated the scaffold sequence with the largest N50 was 77. For gap closure, K-mer error correction on the raw reads was performed using Quake version 0.3.4 (Kelley et al. 2010. Quake: quality-aware detection and correction of sequencing errors.” Genome Biol 11(11): R116). The total genome size was 2,488,918 bp, the longest sequence was 702,365 bp, and the N50 was 359,503 bp. Prior to annotation, the raw contig sequences were trimmed (≥300 bp) and reverse sorted according to sequence length.Gene annotation was performed using the CloVR pipeline version 1.0-RC4 (Angiuoli et al. 2011. CloVR: A virtual machine for automated and portable sequence analysis from the desktop using cloud computing. Bmc Bioinformatics 12). Specifically, rRNA annotation was performed using RNAmmer (Lagesen et al. 2007. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Research 35(9): 3100-3108). tRNA annotation was performed using tRNAscan-SE (Lowe et al. 1997. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res 25(5): 955-964), and prediction of protein-coding regions (CDS) was performed using Glimmer (Delcher et al. 2007. Identifying bacterial genes and endosymbiont DNA with Glimmer. Bioinformatics 23(6): 673-679). Functional annotation of CDS was performed using the IGS annotation engine (http: / / ae.igs.umaryland.edu / cgi / ae_pipeline_outline.cgi). PG_1116 is a predicted protein of 939 amino acids (SEQ ID NO: 2) with a molecular weight of 96 kDa and was assigned to COG2347, which contains a predicted extracellular nuclease, by BLAST homology. PG_1116 contains a DNase I domain, which contains a putative catalytically active DNA binding site, phosphate binding site, and Mg binding site at its C-terminus, as well as a Lamin-tail domain at its N-terminus.The TAT peptide for protein secretion via the Sec pathway was also identified at its distal N-terminus. There are also two further domains that are still not fully elucidated, namely, the YhcR-OBF domain corresponding to a subfamily of the OB fold domain that may be important for the recognition of a specific pattern, and a non-specific fungal domain of unknown function.

[0076] Further BLAST analysis revealed that the PG_1116 sequence has 97% identity with the corresponding published genomic sequence of P. granulosum DSM20700 and 94% identity with P. granulosum TM11. This protein is also conserved as P. avidum in other Propionibacterium species (61% identity with 87% coverage, the fungal domain is missing). Interestingly, this protein has no annotation for all P. acnes present on the NCBI database, the maximum coverage is 35%, and it always contains only one of the described domains. Therefore, this specific domain arrangement may be important for the potential anti-P. acnes biofilm activity of the protein.

[0077] Overexpression and purification of PG_1116 The PG_1116 gene was amplified by PCR and cloned into pET-ZZ1a using the restriction sites corresponding to NcoI and HindIII. PG_1116 was overexpressed and purified using NiNTA and Q-Sepharose (cleaved with TEV) columns and eluted in buffer 50 mM NaP 8.0, 500 mM NaCl, 20 mM imidazole. An aliquot of the protein at a concentration of 0.5 to 1 mg / mL was frozen at -80 °C.

[0078] DNase activity assay P. acnes DNA (1 μg) or plasmid DNA (1.5 μg) was added to 2 mM CaCl 2 and 2 mM MgCl 2It was diluted in 20 mM TrisHCl at pH 7.4 supplemented with [substance not specified in the original], and the rest was as described. Incubation was carried out at 37 °C in a water bath with 6 μg of the appropriate enzyme, namely DNase I, PG_1116 or PG_1116 heat-inactivated at 95 °C for 10 minutes, with or without them. The reaction was stopped at the appropriate time by adding 6× DNA Loading Dye buffer (Thermo Scientific™) containing EDTA. Aliquots (5 μL) of different samples and a molecular weight marker (GeneRuler 1 kb DNA Ladder, Thermo Scientific™) were electrophoresed on a 1% agarose mini-gel under a constant current of 100 V for 40 minutes. DNA was visualized using GelRed™ Nucleic Acid Gel Stain (Biotium) and a Gel Doc™ imager (BioRad).

[0079] Culture and disruption tests of P. acnes biofilms A 48-hour pre-culture of P. acnes IB in suspension was diluted to 5% v / v in BHI supplemented with 2 g / L glucose, and 2 mL was dispensed into each well of a 24-well plate (Thermo Scientific™ Nunc™ Non-Treated Multidishes 144530). The plates were incubated at 37 °C under static anaerobic conditions. The medium was renewed every other day. The effect of different substances was tested on the existing 6-day-old biofilms by replacing the medium with appropriate dilutions of the substances on day 6. All subsequent incubations were carried out under semi-aerobic conditions.

[0080] To test whether the PG_1116 DNase is an effector protein of the P. granulosum supernatant that can actually disrupt the P. acnes biofilm, first, the biofilm was grown for 6 days and then incubated with PG_1116 under different conditions. The biofilm was incubated with PBS, DNase I, PG_1116, or protein buffer for 1 hour or 2 hours. PG_1116 and DNase I were able to disrupt the 6-day-old biofilm of P. acnes, and this activity was also impaired by the addition of EDTA. The biofilm incubated with PG_1116 was more highly disrupted than the biofilm incubated with DNase I at both time points tested, especially 2 hours later when the biofilm incubated with PG_1116 was barely detectable.

[0081] Interestingly, incubation of the P. granulosum conditioned medium showed DNase-like activity and DNA degradation (Figure 1B). Similarly, co-incubation of the P. granulosum conditioned medium with the P. acnes biofilm disrupted the biofilm.

[0082] PG_1116 inhibits biofilm formation from P. acnes To test whether exposure to the PG_1116 protein prevents biofilm formation by P. acnes cells, a suspension culture of P. acnes was grown, exposed to PG_1116 for different times (30 minutes, 1 hour, and 2 hours), washed, and then processed as a pre-culture for biofilm formation. All bacteria were then able to form a thin biofilm on the bottom of the flask, but the biofilms formed by bacteria exposed to PG_1116 were much more fragile than those formed by bacteria pre-exposed to PBS or buffer, even at the shortest time (30 minutes). Furthermore, when the cells were not washed and only diluted after exposure and before biofilm formation, no biofilm formation was seen from cells exposed to PG_1116, while cells exposed only to buffer formed adjacent to a normal biofilm.

[0083] Comparison of the biofilm-degrading activities of PG_1116 and NucB in different culture environments The P. acnes strain KPA171202, a skin isolate, was used as the reference strain for all experiments (The complete genome sequence of Propionibacterium acnes, a commensal of human skin. Brueggemann H, Henne A, Hoster F, Liesegang H, Wiezer A, Strittmatter A, Hujer S, Duerre P, Gottschalk G. Science. 2004 Jul 30;305(5684):671-3). First, the bacteria were cultured on anaerobic blood agar plates under anaerobic conditions. The bacteria grown on the plates were further grown as liquid cultures in brain heart infusion broth (BHI). These precultures were used as inoculum for main cultures grown anaerobically for 24 or 48 h. Biofilm cultures were grown with 10 ml broth in T-25 cell culture flasks (Sarstedt, Nuembrecht, Germany) and incubated for 7 days with medium changes every other day (Transcriptomic analysis of Propionibacterium acnes biofilms in vitro. Jahns AC, Eilers H, Alexeyev OA. Anaerobe. 2016 Dec;42:111-118).

[0084] Comparison of the biofilm-degrading / dispersing activities of NucB and PG_1116 in the medium After 7 days of incubation, PG_1116 protein and NucB protein at a concentration of 0.1 mg / mL (equimolar ratio) were added and incubated for an additional 24 h. The effects of PG_1116 and NucB on P. acnes biofilms in the medium were tested after 24 h. The biofilm incubated with PG_1116 was estimated to be three times smaller compared to NucB.

[0085] Comparison of the biofilm-degrading / dispersing activities of NucB and PG_1116 in medium supplemented with artificial sebum After 7 days of incubation, a 5% sebum emulsion consisting of 150 mg of sebum (Pickering Laboratories, Inc., Mountain View, CA, USA), 10% gum arabic, and 20 mM Tris-HCL was added to the flask for the purpose of mimicking the follicular environment. After adding the sebum, PG_1116 protein and NucB protein at a concentration of 0.1 mg / mL (equimolar ratio) were added to each flask and incubated for 24 hours. After 24 hours of incubation, the biofilm degradation / dispersion activities of PG_1116 and NucB in the sebum-like environment were compared. The effects of PG_1116 and NucB incubated with P. acnes biofilm in the sebum emulsion were tested after 24 hours. The biofilm incubated with PG_1116 was estimated to be 3 to 4 times smaller compared to NucB.

[0086] DNase activity of PG_1116 against P. acnes biofilm To test whether PG_1116 DNase activity is related to the degradation of biofilm, a known enzyme inhibitor (EDTA) can be used. PG_1116 and PG_1116 supplemented with EDTA were incubated with P. acnes biofilm in the medium for 2 hours. The biofilm degradation / dispersion activity of PG_1116 was inhibited when EDTA was added, so the biofilm degradation / dispersion activity of PG_1116 is due to DNase activity.

[0087] Analytical assay for the determination of DNase activity

[0088] [Table 2]

[0089] [Table 3]

[0090]

Table 4

[0091] Based on the procedure developed by Sigma Aldrich, a simple method for measuring enzyme activity was established. In this assay, DNase catalyzes the degradation of DNA according to the following reaction:

Chemical formula

[0092] In the first experiment (Figure 3A), three different incubation buffers were prepared in Falcon tubes by changing the pH of the acetate buffer (pH 5.0, 6.0, and 7.0) while keeping all other parameters constant. The volume of each component added to the incubation buffer was as follows: · 1.25 ml of sodium acetate buffer (pH 5.0, 6.0, 7.0) (10%) · 0.625 ml of MgSO4 (5%) · 9.125 ml of purified water (73%) · 1.5 ml of DNA solution (added last after adjusting the pH of the buffer). The DNA solution was reconstituted according to the protocol (to a concentration of 0.33 mg / ml)

[0093] Sigma's DNase was reconstituted in 1 ml of 0.85% NaCl solution and further diluted 1:5 with 0.85% NaCl immediately before use. Blank samples were prepared with each incubation buffer by mixing 100 μl of 0.85% NaCl solution with 500 μl of incubation buffer (reagent cocktail according to the protocol). After zero-adjusting the UV spectrophotometer with the blank, the actual measurement of the DNase reaction was started. In this measurement, 100 μl of DNase was mixed with 500 μl of incubation buffer containing DNA (in a quartz cuvette), and the measured values were recorded every minute for 15 minutes. The experiment was carried out at room temperature (about 25 °C). For each buffer (pH 5.0 - 7.0), the measured absorbance at 260 nm was plotted against time for the graph representing enzyme kinetics (see Figure 3A). It can be seen from the graph that the substrate is consumed after 6 minutes in the buffers at pH 6 and 7 (flattened curve). The enzyme reaction is slightly slower in the buffer at pH 5 according to the visual evaluation of the curve. However, this experiment was carried out with the aim of establishing a laboratory assay that can be used for the analysis of PG_1116 and NucB. Therefore, without further data analysis, it was concluded that this assay meets its purpose of further screening for enzyme activity.

[0094] Effect of pH on enzyme activity in PG_1116 and NucB In this experiment, the activities of PG_1116 and NucB were evaluated at different pHs. The experimental work was carried out as described in the above "Analytical assay for determination of DNase activity". One of the main differences in this experiment was that due to the different concentrations of PG_1116 and NucB, the dilutions of the enzyme and the preparations of the blank samples were prepared as described below: PG_1116 blank sample - 100 μl of 0.85% NaCl + 500 μl incubation buffer The PG_1116 sample (1 mg / ml) was diluted from 1:5 to 0.2 mg / ml with 0.85% NaCl and then mixed with the incubation buffer. The final reaction buffer contained 100 μl of 0.2 mg / ml PG_1116 + 500 μl of the incubation buffer NucB blank sample (0.2 mg / ml) - 100 μl of NucB storage buffer + 500 μl of the incubation buffer NucB sample - 100 μl of 0.2 mg / ml NucB + 500 μl of the incubation buffer

[0095] For plots of the enzyme activities of both proteins, see Figures 3B and 3C. By visually observing the curve of PG_1116, it is clearly seen that the optimal activity is achieved at pH 6.0. This curve also flattens out after 8 minutes, indicating that the substrate is consumed at this stage. To more accurately evaluate the activity, the enzyme activity was also calculated according to the Sigma protocol. To exclude the portion when the substrate was consumed at pH 6.0, the graph was replotted from 0 to 8 minutes. Figures 3D and 3E show the replotted graphs, and Table 2 shows the calculated results of the enzyme activities of each enzyme (NucB and PG_1116). The plot of NucB activity appears to be slightly different as no flattening of the curve is seen. Compared to the starting absorbance in PG_1116, the enzyme reaction is clearly slower at higher pH, and the background absorbance is higher. Although there are several different parameters that can affect the enzyme rate and its measurement, no clear conclusion can be drawn at this stage regarding the higher absorbance detected at the start. One possible interpretation is that due to the relatively low purity profile of NucB (85% purity), there are process-related impurities in the sample that interfere with the measurement at 260 nm. If the process-related impurities contain high levels of plasmid DNA fragments, this can also affect the reaction as the concentration of the substrate, which is a DNA solution, subsequently increases. The starting absorbance at 0 minutes also varies between different pHs and is more obvious in Figure 3C. Due to the fact that it also takes several seconds to read the measurement values, the reaction may already have started during this time, and the actual reading value at 0 minutes may be slightly higher than the true value at this point.

[0096] Linear regression analysis was performed for each replot curve, and the slopes of the regression lines (speed / rate of the enzyme reaction) were compared. From the data analysis (Table 2) and Figures 3D to 3E, it can be seen that the highest ratio of NucB was obtained at pH 5.0, while the highest ratio of PG_1116 was obtained at pH 6.0. At pH 7.0, PG_1116 has significantly higher activity than NucB. To better overview the differences between different incubation conditions, the comparison of activities is also summarized in Table 5.

[0097]

Table 5

[0098] For more details on the calculation results of enzyme activity, please refer to the following example of calculation results. Calculation results of enzyme activity, example of NucB, 25 °C, pH 5.0 · The slope is 0.0588 at pH 5.0. Slope = ΔA260 / min · According to the Sigma procedure, 0.001 / min / ml of ΔA260 = 1 unit · The unit of our sample = 0.0588 / 0.001 = 58.8 units / ml · Since the concentration of 0.2 mg / ml is diluted 6-fold with the reagent cocktail, the final concentration in the reaction buffer is 0.033 mg / ml protein · The activity per mg is equal to 58.8 / 0.033 = 1781 units / mg

[0099] Effect of temperature on enzyme activity To investigate the effect of temperature on enzyme activity, new experiments were designed with the aim of further optimizing the conditions for optimal activity for both PG_1116 and NucB. In this case, a temperature of 32 °C was the most interesting temperature as it is the temperature of the skin surface. The experimental work was carried out as described in the above "Analysis assay for determination of DNase activity", with minor deviations described below. · Measurements were made only 6 times ·Since the cuvettes containing the final reaction buffer were incubated in a heating cabinet at 32 °C between measurements, it was more realistic to reduce the number of measurements.

[0100] The results of this experiment are shown in Table 6.

[0101]

Table 6

[0102] The effect of temperature had a positive impact on NucB activity, except at pH 7.0, which had slightly lower activity than that observed at pH 7.0, 25 °C. The activity at pH 5.0 was significantly higher at higher temperatures, being 2521 units / mg at 32 °C compared to 1781 units / mg at 25 °C. On the other hand, the activity of PG_1116 was very similar at pH 5.0, 32 °C compared to pH 5.0, 25 °C, being 327 compared to 333 units / mg. The activity at pH 6.0 and 7.0 was lower at higher temperatures. However, the purpose of this experiment was to compare the activities of NucB and PG_1116 at the skin temperature of 32 °C in the same assay. The most unexpected and interesting result of this evaluation was the approximately three-fold higher activity of PG_1116 observed at pH 7.0 compared to NucB. Since acne-affected skin has a slightly higher pH than normal skin, this data can be used as a basis for further evaluation of PG_1116 for use in acne treatment.

[0103] The purpose of this test was to evaluate the potential of PG_1116 for use in acne treatment, so not much emphasis was placed on the reproducibility of the method. Two proteins were compared in the same assay on the same day, keeping all other parameters constant, but data could vary between assays performed on different days. One parameter that could affect the measurements is the concentration of the DNA substrate, which can vary between different vials. However, since the assay is used for routine analysis and each vial is thought to contain 1 mg of DNA as labeled on the vial, the impact of this parameter is considered to be minimal. For future analysis and more thorough investigation, it is recommended to confirm the DNA concentration according to the Sigma protocol and the minimum qualification of the assay.

[0104] Conclusion The most interesting result of this evaluation test is the higher activity of PG_1116 at pH 7.0 compared to commercially available NucB. The activity was approximately 3-fold higher at both temperatures of 25 °C and 32 °C. On the other hand, at pH 5.0 and 25 °C, NucB had 5-fold higher activity than PG_1116, and at 32 °C, the activity seen in NucB was almost 8-fold higher. However, due to the fact that the pH of acne-affected skin is slightly high (>6.5), the activity at pH 7 is more appropriate for the evaluation of activity.

Claims

A composition for use in the treatment and / or prevention of a disease caused by or associated with an infection by Propionibacterium acnes, comprising: an isolated protein having the amino acid sequence set forth in SEQ ID NO: 2, or a functional variant thereof having at least 90% amino acid sequence identity with SEQ ID NO: 2 and having at least 80% of the DNase activity of the protein set forth in SEQ ID NO: 2 in a quantitative assay of deoxyribonuclease activity at pH 7 and 32°C **Claim 2** The composition according to claim 1, wherein the disease is a skin disease. **Claim 3** The composition according to claim 2, wherein the skin disease is acne vulgaris or rosacea. **Claim 4** The composition according to claim 2, wherein the skin disease is acne vulgaris. **Claim 5** The composition according to any one of claims 1 to 4, wherein the composition promotes wound healing. **Claim 6** The composition according to claim 5, wherein the wound is a surgical wound. **Claim 7** The composition according to any one of claims 1 to 6, further comprising a pharmaceutically acceptable excipient. **Claim 8** The composition according to claim 7, further comprising a lipid carrier system and / or an aqueous pH buffer. **Claim 9** The composition according to claim 8, wherein the lipid carrier system comprises a lipid in solid or crystalline form. ​

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