Compositions Comprising Thrombin-Derived Peptides and Uses Thereof

Abstract

The present invention relates to a composition comprising a) a thrombin-derived peptide, b) a non-ionic polymer capable of forming a hydrogel, and c) an aqueous solution. The present invention also provides a composition comprising a) a thrombin-derived peptide, b) EDTA, and c) an aqueous buffer. The present invention also provides a composition comprising a) a high concentration of the thrombin-derived peptide. Articles of manufacture comprising the compositions, and compositions or articles of manufacture for use in methods of treatment are also disclosed.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the field of topical treatment of disorders, particularly disorders associated with, or at risk of becoming associated with, infection and / or inflammation. The present invention also relates to compositions useful for such treatment. In one embodiment, the present invention relates to a composition based on a non-ionic hydrogel polymer containing a thrombin-derived peptide with antibacterial and anti-inflammatory functions. [Background technology]

[0002] Background of the Invention Various types of wounds have a significant and significant impact on patients, health care, and society. Wound types include acute postoperative wounds and burns, and a large patient population has non-healing ulcers resulting from diabetes or cardiovascular disorders. The cost of chronic wounds, with a point prevalence of approximately 2 cases per 1,000, is substantial, accounting for 1–3% of total health care system costs in developed countries. Considering burns, 67 million injuries were reported in 2015, resulting in approximately 2.9 million hospitalizations and 176,000 deaths. A study published in 2014 estimated the average total cost of burn wound healing in high-income countries to be approximately US$88,000 per patient.

[0003] From a physiological perspective, wound healing is an evolutionarily conserved physiological chain of biologically linked events. An initial phase of homeostasis is followed by phases of inflammation, proliferation, and tissue remodeling. Early surveillance mediated by human innate immunity aids in bacterial control during wound formation, and lipopolysaccharide sensing by Toll-like receptors (TLRs) is essential for the early response to infection. However, excessive TLR responses can cause localized and sometimes excessive inflammation, as observed in postoperative infections, infected burn wounds, or non-healing ulcers. All of these wound complications delay proper healing, increase the risk of severe infection, and potentially lead to scar formation. Protective use of systemic antibiotics can reduce the incidence of wound and surgical infections. However, this antibiotic use poses a significant challenge, driving the development of resistance and infections caused by antibiotic-resistant strains of Staphylococcus aureus and Pseudomonas aeruginosa, bacteria that cause postoperative infections and infections in chronic wounds and burns. For example, in European hospitals, the overall rate of surgical site infections (SSIs) ranges between 3% and 4% of patients undergoing surgery. Depending on the nature of the surgery in question, the incidence of SSIs ranges from <1% to >10%. Because the incidence of SSIs is age-related, with rates doubling in patients older than 64 years, a sharp increase in the incidence of SSIs is expected in the future as the population ages. In addition to antibiotic treatment, current strategies for preventing wound infections include functionalizing gels, dressings, or biomaterials with various anti-infective components. Common additives used in clinics include silver and polyhexanide (polyhexamethylene biguanide, PHMB), which are used in acute wounds and burns, as well as non-healing ulcers. While such treatments may kill bacteria, they do not address the associated inflammatory components. In contrast, treatments that address inflammation primarily aim to inactivate and scavenge proteases, such as gelatin-based wound dressings. Thus, wound healing today only addresses one problem (infection or protease action) and there are no currently available therapeutic modalities to both control bacteria in the wound or surgical environment and target the origin and cause of excessive infection, i.e., inflammation.

[0004] Because wound healing is critical for survival, it is not surprising that multiple natural host defense systems are activated during early homeostasis and injury, including clot formation, and that proteins and peptides are activated in our innate immune system. In humans, examples of such host defense systems include the proteolytic degradation products of plasma proteins such as neutrophil-derived α-defensins and cathelicidin LL-37 and thrombin. Thrombin is initially formed by selective proteolysis by coagulation factor X and mediates fibrinogen degradation and clot formation during the acute wound phase. However, subsequent proteolysis results in the formation of an approximately 11 kDa fragment that mediates lipopolysaccharide (LPS) and bacterial aggregation, facilitating endotoxin clearance and microbial killing. Further proteolysis leads to the formation of smaller thrombin-derived C-terminal peptides (TCPs) of approximately 2 kDa, such as FYT21 (FYTHVFRLKKWIQKVIDQFGE) (SEQ ID NO:2) and HVF18 (HVFRLKKWIQKVIDQFGE) (SEQ ID NO:4), which are present in human wound fluid and have been demonstrated to exert antiendotoxin functions in vitro and in vivo. The peptide TCP-25 (GKYGFYTHVFRLKKWIQKVIDQFGE) SEQ ID NO:1, which contains these endogenous sequences, is antimicrobial, binds to and neutralizes bacterial LPS, and protects against Pseudomonas aeruginosa-induced sepsis and LPS-mediated shock in experimental animal models, primarily through the reduction of systemic cytokine responses. Furthermore, the peptide directly interacts with monocytes and macrophages and inhibits TLR4- and TLR2-induced NF-kB activation in response to multiple microbial agonists. In addition, the peptide reduces the inflammatory response to intact bacteria during phagocytosis and inhibits neutrophil responses to LPS in vitro and in vivo.

[0005] European Patent No. 1987056 discloses TCP-25 peptide and various variants thereof.

[0006] European Patent No. 2480567 discloses the use of TCP-25 peptide and various variants thereof. Summary of the Invention [Problem to be solved by the invention]

[0007] Summary of the Invention However, there is a need for suitable, stable, and effective formulations and compositions for the delivery of TCP-25 peptide and similar peptides. In particular, there is a need for pharmaceutical formulations that are useful for local treatments, such as topical drug treatments. There is also a need for pharmaceutical formulations containing TCP-25 peptides that have high stability. There is also a need for pharmaceutical formulations containing TCP-25 peptides that have high efficacy, in particular high antibacterial and / or anti-inflammatory efficacy. [Means for solving the problem]

[0008] Interestingly, the present invention provides pharmaceutical formulations containing TCP peptides that are capable of retaining significant amounts of TCP peptides at the site of topical application, while at the same time not negatively interfering with the anti-inflammatory and antibacterial effects of the TCP peptides.

[0009] The actions of TCP-25 and other TCP peptides involve structural transitions, such as the formation of C-turns and helical structures upon LSP binding, and depend in part on their ability to interact with both bacterial membranes and CD14. Unfortunately, some compounding agents induce structural changes in TCP-25, resulting in loss of activity. Interestingly, the compounding agents provided by the present invention do not interfere with the TCP peptide structure and support the function of the TCP peptide.

[0010] The present invention also provides pharmaceutical formulations containing TCP peptides with high stability. Interestingly, the present invention shows that compositions containing high concentrations of TCP peptides are more stable. Such compositions are, for example, more resistant to denaturation. The present invention shows that TCP peptides oligomerize at high concentrations in a reversible manner. Without being bound by theory, it is believed that oligomerization may help stabilize TCP peptides.

[0011] The present invention also provides pharmaceutical formulations containing TCP peptides with high antibacterial efficacy. Interestingly, the present invention shows that the antibacterial efficacy of TCP peptides can be significantly increased in the presence of EDTA.

[0012] Therefore, it is an object of the present invention to provide a composition suitable for containing TCP-25 peptide and / or other TCP peptides. It is also an object of the present invention to provide a stable composition containing TCP-25 peptide and / or other TCP peptides. It is also an object of the present invention to provide a composition containing TCP-25 peptide and / or other TCP peptides that has high antibacterial activity.

[0013] The means by which each of the above objects and others are accomplished will become apparent from the description of the invention set forth hereinafter.

[0014] The present invention discloses that nonionic hydrogels containing TCP peptides provide a local delivery scaffold that can mimic the endogenous effects of wound-derived host defense peptides (HDPs) found in biological matrices such as fibrin. As further demonstrated in the Examples section, the inventors demonstrate that hydrogels containing TCP peptides can act as "dual-function" local therapeutic agents that target both bacteria and the associated inflammatory response in experimental wound models. However, these therapeutic effects are contingent on the proper composition of the hydrogel, specifically, the hydrogel must contain a nonionic polymer capable of forming a hydrogel. Such hydrogels are believed to provide the TCP peptides with a local environment that is supportive of their therapeutic effect. As demonstrated herein, formulations according to the present invention possess antibacterial activity.

[0015] In addition, it is shown that the combination according to the present invention is able to reduce inflammation.

[0016] Therefore, the present invention provides: a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; b) a non-ionic polymer capable of forming a hydrogel when mixed with an aqueous solution; aqueous solution, A composition comprising:

[0017] Formulations containing nonionic polymers capable of forming hydrogels support the antibacterial and / or anti-inflammatory activity of TCP peptides. Without wishing to be bound by theory, it is contemplated that this is linked to the action of TCP peptides, including structural transitions such as the formation of C-turns and helical structures upon LPS binding, which are required for the ability to both interact with bacterial membranes and CD14.

[0018] a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; b) EDTA, c) aqueous buffers; and having a pH of at most 7.

[0019] Formulations containing EDTA support the antibacterial activity of TCP peptides.

[0020] Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A composition comprising a compound comprising a peptide comprising or consisting of: The peptide has a length of 10 to 100 amino acid residues, The concentration of the peptide in the composition is at least 0.01 wt%, preferably at least 0.08 wt%, at least 0.1 wt%, for example, in the range of 0.08 to 3 wt%; It is also an aspect of the present invention to provide a composition.

[0021] Such compositions generally have high stability and the TCP peptides contained therein are generally more resistant to denaturation.

[0022] It is also an aspect of the present invention to provide an article of manufacture comprising the composition of the present invention.

[0023] Additionally, it is an aspect to provide a composition of the present invention for use in a method of treating a disorder in an individual in need thereof, wherein the composition is formulated for topical administration. [Brief explanation of the drawings]

[0024] [Figure 1A]Figures 1A-D show the antibacterial and antiendotoxin effects of TCP-25 in various formulations. Figure 1A shows the peptide activity and release profile of TCP-25 formulations. The activity of TCP-25 in various formulations (HPC, CMC, and Pluronic) was determined by assessing antimicrobial activity against E. coli, P. aeruginosa, and S. aureus using RDA. The bar graphs show measurements of the resulting zone of clearance, which correspond to the inhibitory effect of the released peptide. Data are expressed as means (n=3). Figure 1B shows a bar graph showing the antimicrobial effect of TCP-25 in various formulations (HPC, CMC, and Pluronic) as assessed by viable count assay (VCA). E. coli, P. aeruginosa, and S. aureus were incubated with the formulations with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates and incubated overnight at 37°C, and the number of CFUs was determined. Data are presented as means (n ​​= 3). To examine whether TCP-25 formulations block endotoxin-induced pro-inflammatory responses, THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations (HPC, CMC, and Pluronic) with and without TCP-25. The bar graph in Figure 1C shows NF-kB activation determined by measuring SEAP production. Values ​​represent means (n ​​= 3). The MTT assay was used to assess the cell viability of TCP-25 formulations. The bar graph in Figure 1D shows the percentage of viable cells quantified using the MTT assay. Values ​​are shown relative to untreated viable cells (100%, dotted line). Data are presented as means ± SEM (n = 3). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. *P ≤ 0.05; NS not significant. [Figure 1B]Figures 1A-D show the antibacterial and antiendotoxin effects of TCP-25 in various formulations. Figure 1A shows the peptide activity and release profile of TCP-25 formulations. The activity of TCP-25 in various formulations (HPC, CMC, and Pluronic) was determined by assessing antimicrobial activity against E. coli, P. aeruginosa, and S. aureus using RDA. The bar graphs show measurements of the resulting zone of clearance, which correspond to the inhibitory effect of the released peptide. Data are expressed as means (n=3). Figure 1B shows a bar graph showing the antimicrobial effect of TCP-25 in various formulations (HPC, CMC, and Pluronic) as assessed by viable count assay (VCA). E. coli, P. aeruginosa, and S. aureus were incubated with the formulations with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates and incubated overnight at 37°C, and the number of CFUs was determined. Data are presented as means (n ​​= 3). To examine whether TCP-25 formulations block endotoxin-induced pro-inflammatory responses, THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations (HPC, CMC, and Pluronic) with and without TCP-25. The bar graph in Figure 1C shows NF-kB activation determined by measuring SEAP production. Values ​​represent means (n ​​= 3). The MTT assay was used to assess the cell viability of TCP-25 formulations. The bar graph in Figure 1D shows the percentage of viable cells quantified using the MTT assay. Values ​​are shown relative to untreated viable cells (100%, dotted line). Data are presented as means ± SEM (n = 3). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. *P ≤ 0.05; NS not significant. [Figure 1C]Figures 1A-D show the antibacterial and antiendotoxin effects of TCP-25 in various formulations. Figure 1A shows the peptide activity and release profile of TCP-25 formulations. The activity of TCP-25 in various formulations (HPC, CMC, and Pluronic) was determined by assessing antimicrobial activity against E. coli, P. aeruginosa, and S. aureus using RDA. The bar graphs show measurements of the resulting zone of clearance, which correspond to the inhibitory effect of the released peptide. Data are expressed as means (n=3). Figure 1B shows a bar graph showing the antimicrobial effect of TCP-25 in various formulations (HPC, CMC, and Pluronic) as assessed by viable count assay (VCA). E. coli, P. aeruginosa, and S. aureus were incubated with the formulations with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates and incubated overnight at 37°C, and the number of CFUs was determined. Data are presented as means (n ​​= 3). To examine whether TCP-25 formulations block endotoxin-induced pro-inflammatory responses, THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations (HPC, CMC, and Pluronic) with and without TCP-25. The bar graph in Figure 1C shows NF-kB activation determined by measuring SEAP production. Values ​​represent means (n ​​= 3). The MTT assay was used to assess the cell viability of TCP-25 formulations. The bar graph in Figure 1D shows the percentage of viable cells quantified using the MTT assay. Values ​​are shown relative to untreated viable cells (100%, dotted line). Data are presented as means ± SEM (n = 3). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. *P ≤ 0.05; NS not significant. [Figure 1D]Figures 1A-D show the antibacterial and antiendotoxin effects of TCP-25 in various formulations. Figure 1A shows the peptide activity and release profile of TCP-25 formulations. The activity of TCP-25 in various formulations (HPC, CMC, and Pluronic) was determined by assessing antimicrobial activity against E. coli, P. aeruginosa, and S. aureus using RDA. The bar graphs show measurements of the resulting zone of clearance, which correspond to the inhibitory effect of the released peptide. Data are expressed as means (n=3). Figure 1B shows a bar graph showing the antimicrobial effect of TCP-25 in various formulations (HPC, CMC, and Pluronic) as assessed by viable count assay (VCA). E. coli, P. aeruginosa, and S. aureus were incubated with the formulations with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates and incubated overnight at 37°C, and the number of CFUs was determined. Data are presented as means (n ​​= 3). To examine whether TCP-25 formulations block endotoxin-induced pro-inflammatory responses, THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations (HPC, CMC, and Pluronic) with and without TCP-25. The bar graph in Figure 1C shows NF-kB activation determined by measuring SEAP production. Values ​​represent means (n ​​= 3). The MTT assay was used to assess the cell viability of TCP-25 formulations. The bar graph in Figure 1D shows the percentage of viable cells quantified using the MTT assay. Values ​​are shown relative to untreated viable cells (100%, dotted line). Data are presented as means ± SEM (n = 3). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. *P ≤ 0.05; NS not significant. [Figure 1E]Figures 1E–H further compare the TCP-25 formulation in HPC with the related polymer hydroxyethyl cellulose (HEC). Figure E shows the peptide activity and release profile of TCP-25 in HPC and HEC. TCP-25 release and activity were determined by assessing its antimicrobial activity against Escherichia coli in RDA. This figure shows measurements of the resulting zone of clearance. Data are presented as mean ± SEM (n = 6). P values ​​were determined using the Mann-Whitney U test. Figure 1F shows a VCA demonstrating the antimicrobial effect of TCP-25 formulation in HPC and HEC. E. coli was incubated with formulation gel with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). Figure 1G shows a comparison of the antiendotoxin effects of TCP-25 formulations in HPC and HEC, in which THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations with and without TCP-25. The bar graph shows NF-kB activation determined by measuring SEAP production. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 1H shows that simultaneous analysis of the toxic effects of formulation components alone and in combination with TCP-25 was performed. The histogram shows the percentage of viable cells quantified using the MTT assay. Lysed cells were used as a positive control. Values ​​are shown relative to untreated viable cells (100%). Data are presented as mean ± SEM (n = 3). ***P ≤ 0.001; ****P ≤ 0.0001; NS not significant. [Figure 1F]Figures 1E–H further compare the TCP-25 formulation in HPC with the related polymer hydroxyethyl cellulose (HEC). Figure E shows the peptide activity and release profile of TCP-25 in HPC and HEC. TCP-25 release and activity were determined by assessing its antimicrobial activity against Escherichia coli in RDA. This figure shows measurements of the resulting zone of clearance. Data are presented as mean ± SEM (n = 6). P values ​​were determined using the Mann-Whitney U test. Figure 1F shows a VCA demonstrating the antimicrobial effect of TCP-25 formulation in HPC and HEC. E. coli was incubated with formulation gel with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). Figure 1G shows a comparison of the antiendotoxin effects of TCP-25 formulations in HPC and HEC, in which THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations with and without TCP-25. The bar graph shows NF-kB activation determined by measuring SEAP production. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 1H shows that simultaneous analysis of the toxic effects of formulation components alone and in combination with TCP-25 was performed. The histogram shows the percentage of viable cells quantified using the MTT assay. Lysed cells were used as a positive control. Values ​​are shown relative to untreated viable cells (100%). Data are presented as mean ± SEM (n = 3). ***P ≤ 0.001; ****P ≤ 0.0001; NS not significant. [Figure 1G]Figures 1E–H further compare the TCP-25 formulation in HPC with the related polymer hydroxyethyl cellulose (HEC). Figure E shows the peptide activity and release profile of TCP-25 in HPC and HEC. TCP-25 release and activity were determined by assessing its antimicrobial activity against Escherichia coli in RDA. This figure shows measurements of the resulting zone of clearance. Data are presented as mean ± SEM (n = 6). P values ​​were determined using the Mann-Whitney U test. Figure 1F shows a VCA demonstrating the antimicrobial effect of TCP-25 formulation in HPC and HEC. E. coli was incubated with formulation gel with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). Figure 1G shows a comparison of the antiendotoxin effects of TCP-25 formulations in HPC and HEC, in which THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations with and without TCP-25. The bar graph shows NF-kB activation determined by measuring SEAP production. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 1H shows that simultaneous analysis of the toxic effects of formulation components alone and in combination with TCP-25 was performed. The histogram shows the percentage of viable cells quantified using the MTT assay. Lysed cells were used as a positive control. Values ​​are shown relative to untreated viable cells (100%). Data are presented as mean ± SEM (n = 3). ***P ≤ 0.001; ****P ≤ 0.0001; NS not significant. [Figure 1H]Figures 1E–H further compare the TCP-25 formulation in HPC with the related polymer hydroxyethyl cellulose (HEC). Figure E shows the peptide activity and release profile of TCP-25 in HPC and HEC. TCP-25 release and activity were determined by assessing its antimicrobial activity against Escherichia coli in RDA. This figure shows measurements of the resulting zone of clearance. Data are presented as mean ± SEM (n = 6). P values ​​were determined using the Mann-Whitney U test. Figure 1F shows a VCA demonstrating the antimicrobial effect of TCP-25 formulation in HPC and HEC. E. coli was incubated with formulation gel with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar plates, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). Figure 1G shows a comparison of the antiendotoxin effects of TCP-25 formulations in HPC and HEC, in which THP-1-XBlue™-CD14 cells were stimulated with E. coli LPS in the presence of various formulations with and without TCP-25. The bar graph shows NF-kB activation determined by measuring SEAP production. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 1H shows that simultaneous analysis of the toxic effects of formulation components alone and in combination with TCP-25 was performed. The histogram shows the percentage of viable cells quantified using the MTT assay. Lysed cells were used as a positive control. Values ​​are shown relative to untreated viable cells (100%). Data are presented as mean ± SEM (n = 3). ***P ≤ 0.001; ****P ≤ 0.0001; NS not significant. [Figure 2A]Figures 2A–C show the secondary structural changes of TCP-25 determined by CD spectroscopy. Figure 2A shows the CD spectra of TCP-25 measured after incubation with Tris buffer, LPS, HPC, HEC, CMC, or Pluronic (TCP-25 to polymer ratios of 1:1 and 1:5). Figure 2B shows the α-helical content of TCP-25 calculated from molar ellipsometry at 222 nm in the presence of Tris buffer, LPS, and polymer (1:5 ratio). Data are presented as mean ± SEM (n = 3). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; NS = not significant. Figure 2C shows the VCA demonstrating the antimicrobial effect of various concentrations of TCP-25 in HEC gel formulations. Staphylococcus aureus and Pseudomonas aeruginosa were incubated with the hydrogels with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). [Figure 2B] Figures 2A–C show the secondary structural changes of TCP-25 determined by CD spectroscopy. Figure 2A shows the CD spectra of TCP-25 measured after incubation with Tris buffer, LPS, HPC, HEC, CMC, or Pluronic (TCP-25 to polymer ratios of 1:1 and 1:5). Figure 2B shows the α-helical content of TCP-25 calculated from molar ellipsometry at 222 nm in the presence of Tris buffer, LPS, and polymer (1:5 ratio). Data are presented as mean ± SEM (n = 3). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; NS = not significant. Figure 2C shows the VCA demonstrating the antimicrobial effect of various concentrations of TCP-25 in HEC gel formulations. Staphylococcus aureus and Pseudomonas aeruginosa were incubated with the hydrogels with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). [Figure 2C] Figures 2A–C show the secondary structural changes of TCP-25 determined by CD spectroscopy. Figure 2A shows the CD spectra of TCP-25 measured after incubation with Tris buffer, LPS, HPC, HEC, CMC, or Pluronic (TCP-25 to polymer ratios of 1:1 and 1:5). Figure 2B shows the α-helical content of TCP-25 calculated from molar ellipsometry at 222 nm in the presence of Tris buffer, LPS, and polymer (1:5 ratio). Data are presented as mean ± SEM (n = 3). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; NS = not significant. Figure 2C shows the VCA demonstrating the antimicrobial effect of various concentrations of TCP-25 in HEC gel formulations. Staphylococcus aureus and Pseudomonas aeruginosa were incubated with the hydrogels with or without TCP-25. To quantify antimicrobial activity, appropriate dilutions of the reaction mixture were plated onto TH broth agar, incubated overnight at 37°C, and the number of CFU was determined. Data are presented as mean ± SEM (n = 3). [Figure 3A] Figures 3A-B show the in vitro antibacterial effect of TCP-25 formulated in HEC gel (TCP-25 Gel #1). Figure 3A shows bacterial bioluminescence measurements after treatment with TCP-25 Gel #1. Bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa (107 / mL CFU) were treated with TCP-25 Gel #1. The bioluminescence released from the bacteria was measured using a luminescence plate reader. The line graph shows the total bioluminescence counts at the indicated time points. Data are presented as mean ± SEM (n = 3). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 3B shows the VCA demonstrating the antimicrobial effect of TCP-25 HEC formulation against Staphylococcus aureus or Pseudomonas aeruginosa. Data are presented as mean ± SEM (n = 3). P values ​​were determined using the Mann-Whitney U test. P values ​​were determined using an unpaired t-test. Comparisons were made with the respective gel controls. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 3B] Figures 3A-B show the in vitro antibacterial effect of TCP-25 formulated in HEC gel (TCP-25 Gel #1). Figure 3A shows bacterial bioluminescence measurements after treatment with TCP-25 Gel #1. Bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa (107 / mL CFU) were treated with TCP-25 Gel #1. The bioluminescence released from the bacteria was measured using a luminescence plate reader. The line graph shows the total bioluminescence counts at the indicated time points. Data are presented as mean ± SEM (n = 3). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 3B shows the VCA demonstrating the antimicrobial effect of TCP-25 HEC formulation against Staphylococcus aureus or Pseudomonas aeruginosa. Data are presented as mean ± SEM (n = 3). P values ​​were determined using the Mann-Whitney U test. P values ​​were determined using an unpaired t-test. Comparisons were made with the respective gel controls. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 4A]Figures 4A-E show the antibacterial and anti-inflammatory effects of the TCP-25 Gel #1 formulation in a mouse model of subcutaneous infection and inflammation. Figure 4A shows in vivo infection imaging by IVIS in a mouse model of subcutaneous infection. Control HEC gel and TCP-25 Gel #1 were deposited subcutaneously on the backs of SKH1 mice after inoculation with 10 CFU of bioluminescent Pseudomonas aeruginosa or Staphylococcus aureus. To visualize in vivo drug localization, the TCP-25 formulation was spiked with Cy5-labeled TCP-25. At different time points, bacterial bioluminescence intensity and TCP-25 Cy5 fluorescence were noninvasively analyzed using the IVIS bioimaging system. Representative images show bacterial luminescence (lum) and TCP-25 Cy5 fluorescence (flu) 6 hours after infection. The bar graph shows the measured bioluminescence intensity emitted by bacteria at 6 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group and n = 7 mice in the TCP-25 gel #1 group for each bacterial infection). P values ​​were determined using the Mann-Whitney U test. Figure 4B shows representative images of H&E staining of mouse skin tissue from the site of gel deposition. Arrows indicate tissue destruction and hyperinflammation. Figure 4C shows in vivo inflammation imaging using IVIS in NF-kB reporter mice. LPS in HEC gel or TCP-25 HEC formulations was subcutaneously deposited on the back of transgenic BALB / cTg(NF-kB-RE-luc)-Xen reporter mice. In vivo bioimaging of NF-kB reporter gene expression was performed using the IVIS Spectrum system. To image in vivo drug localization, TCP-25 was spiked with Cy5-labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 Cy5 fluorescence (flu) at 6 hours. Bar graphs show the measured light intensity emitted from these reporter mice. Data are expressed as mean ± SEM (n = 7 mice for the gel group and n = 5 mice for the TCP-25 gel #1 group). P values ​​were determined using the Mann-Whitney U test.Figure 4D shows cytokine analysis from wound fluid extracted from implanted PU discs. Data are presented as mean ± SEM (n = 4 mice in the Gel group, n = 4 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01; ***P ≤ 0.001. Figure 4E further shows microbial analysis of tissues 24 h postinfection. Data are presented as mean ± SEM (n = 7 mice in the Gel group, n = 7 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01. [Figure 4B]Figures 4A-E show the antibacterial and anti-inflammatory effects of the TCP-25 Gel #1 formulation in a mouse model of subcutaneous infection and inflammation. Figure 4A shows in vivo infection imaging by IVIS in a mouse model of subcutaneous infection. Control HEC gel and TCP-25 Gel #1 were deposited subcutaneously on the backs of SKH1 mice after inoculation with 10 CFU of bioluminescent Pseudomonas aeruginosa or Staphylococcus aureus. To visualize in vivo drug localization, the TCP-25 formulation was spiked with Cy5-labeled TCP-25. At different time points, bacterial bioluminescence intensity and TCP-25 Cy5 fluorescence were noninvasively analyzed using the IVIS bioimaging system. Representative images show bacterial luminescence (lum) and TCP-25 Cy5 fluorescence (flu) 6 hours after infection. The bar graph shows the measured bioluminescence intensity emitted by bacteria at 6 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group and n = 7 mice in the TCP-25 gel #1 group for each bacterial infection). P values ​​were determined using the Mann-Whitney U test. Figure 4B shows representative images of H&E staining of mouse skin tissue from the site of gel deposition. Arrows indicate tissue destruction and hyperinflammation. Figure 4C shows in vivo inflammation imaging using IVIS in NF-kB reporter mice. LPS in HEC gel or TCP-25 HEC formulations was subcutaneously deposited in the dorsum of transgenic BALB / cTg(NF-kB-RE-luc)-Xen reporter mice. In vivo bioimaging of NF-kB reporter gene expression was performed using the IVIS Spectrum system. To image in vivo drug localization, TCP-25 was spiked with Cy5-labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 Cy5 fluorescence (flu) at 6 hours. Bar graphs show the measured light intensity emitted from these reporter mice. Data are expressed as mean ± SEM (n = 7 mice in the gel group and n = 5 mice in the TCP-25 gel #1 group). P values ​​were determined using the Mann-Whitney U test. Figure 4D shows cytokine analysis from wound fluid extracted from implanted PU discs.Data are presented as mean ± SEM (n = 4 mice in the gel group, n = 4 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01; ***P ≤ 0.001. Figure 4E further shows the microbial analysis of tissues 24 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group, n = 7 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01. [Figure 4C]Figures 4A-E show the antibacterial and anti-inflammatory effects of the TCP-25 Gel #1 formulation in a mouse model of subcutaneous infection and inflammation. Figure 4A shows in vivo infection imaging by IVIS in a mouse model of subcutaneous infection. Control HEC gel and TCP-25 Gel #1 were deposited subcutaneously on the backs of SKH1 mice after inoculation with 10 CFU of bioluminescent Pseudomonas aeruginosa or Staphylococcus aureus. To visualize in vivo drug localization, the TCP-25 formulation was spiked with Cy5-labeled TCP-25. At different time points, bacterial bioluminescence intensity and TCP-25 Cy5 fluorescence were noninvasively analyzed using the IVIS bioimaging system. Representative images show bacterial luminescence (lum) and TCP-25 Cy5 fluorescence (flu) 6 hours after infection. The bar graph shows the measured bioluminescence intensity emitted by bacteria at 6 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group and n = 7 mice in the TCP-25 gel #1 group for each bacterial infection). P values ​​were determined using the Mann-Whitney U test. Figure 4B shows representative images of H&E staining of mouse skin tissue from the site of gel deposition. Arrows indicate tissue destruction and hyperinflammation. Figure 4C shows in vivo inflammation imaging using IVIS in NF-kB reporter mice. LPS in HEC gel or TCP-25 HEC formulations was subcutaneously deposited in the dorsum of transgenic BALB / cTg(NF-kB-RE-luc)-Xen reporter mice. In vivo bioimaging of NF-kB reporter gene expression was performed using the IVIS Spectrum system. To image in vivo drug localization, TCP-25 was spiked with Cy5-labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 Cy5 fluorescence (flu) at 6 hours. Bar graphs show the measured light intensity emitted from these reporter mice. Data are expressed as mean ± SEM (n = 7 mice in the gel group and n = 5 mice in the TCP-25 gel #1 group). P values ​​were determined using the Mann-Whitney U test. Figure 4D shows cytokine analysis from wound fluid extracted from implanted PU discs.Data are presented as mean ± SEM (n = 4 mice in the gel group, n = 4 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01; ***P ≤ 0.001. Figure 4E further shows the microbial analysis of tissues 24 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group, n = 7 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01. [Figure 4D]Figures 4A-E show the antibacterial and anti-inflammatory effects of the TCP-25 Gel #1 formulation in a mouse model of subcutaneous infection and inflammation. Figure 4A shows in vivo infection imaging by IVIS in a mouse model of subcutaneous infection. Control HEC gel and TCP-25 Gel #1 were deposited subcutaneously on the backs of SKH1 mice after inoculation with 10 CFU of bioluminescent Pseudomonas aeruginosa or Staphylococcus aureus. To visualize in vivo drug localization, the TCP-25 formulation was spiked with Cy5-labeled TCP-25. At different time points, bacterial bioluminescence intensity and TCP-25 Cy5 fluorescence were noninvasively analyzed using the IVIS bioimaging system. Representative images show bacterial luminescence (lum) and TCP-25 Cy5 fluorescence (flu) 6 hours after infection. The bar graph shows the measured bioluminescence intensity emitted by bacteria at 6 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group and n = 7 mice in the TCP-25 gel #1 group for each bacterial infection). P values ​​were determined using the Mann-Whitney U test. Figure 4B shows representative images of H&E staining of mouse skin tissue from the site of gel deposition. Arrows indicate tissue destruction and hyperinflammation. Figure 4C shows in vivo inflammation imaging using IVIS in NF-kB reporter mice. LPS in HEC gel or TCP-25 HEC formulations was subcutaneously deposited in the dorsum of transgenic BALB / cTg(NF-kB-RE-luc)-Xen reporter mice. In vivo bioimaging of NF-kB reporter gene expression was performed using the IVIS Spectrum system. To image in vivo drug localization, TCP-25 was spiked with Cy5-labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 Cy5 fluorescence (flu) at 6 hours. Bar graphs show the measured light intensity emitted from these reporter mice. Data are expressed as mean ± SEM (n = 7 mice in the gel group and n = 5 mice in the TCP-25 gel #1 group). P values ​​were determined using the Mann-Whitney U test. Figure 4D shows cytokine analysis from wound fluid extracted from implanted PU discs.Data are presented as mean ± SEM (n = 4 mice in the gel group, n = 4 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01; ***P ≤ 0.001. Figure 4E further shows the microbial analysis of tissues 24 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group, n = 7 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01. [Figure 4E]Figures 4A-E show the antibacterial and anti-inflammatory effects of the TCP-25 Gel #1 formulation in a mouse model of subcutaneous infection and inflammation. Figure 4A shows in vivo infection imaging by IVIS in a mouse model of subcutaneous infection. Control HEC gel and TCP-25 Gel #1 were deposited subcutaneously on the backs of SKH1 mice after inoculation with 10 CFU of bioluminescent Pseudomonas aeruginosa or Staphylococcus aureus. To visualize in vivo drug localization, the TCP-25 formulation was spiked with Cy5-labeled TCP-25. At different time points, bacterial bioluminescence intensity and TCP-25 Cy5 fluorescence were noninvasively analyzed using the IVIS bioimaging system. Representative images show bacterial luminescence (lum) and TCP-25 Cy5 fluorescence (flu) 6 hours after infection. The bar graph shows the measured bioluminescence intensity emitted by bacteria at 6 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group and n = 7 mice in the TCP-25 gel #1 group for each bacterial infection). P values ​​were determined using the Mann-Whitney U test. Figure 4B shows representative images of H&E staining of mouse skin tissue from the site of gel deposition. Arrows indicate tissue destruction and hyperinflammation. Figure 4C shows in vivo inflammation imaging using IVIS in NF-kB reporter mice. LPS in HEC gel or TCP-25 HEC formulations was subcutaneously deposited in the dorsum of transgenic BALB / cTg(NF-kB-RE-luc)-Xen reporter mice. In vivo bioimaging of NF-kB reporter gene expression was performed using the IVIS Spectrum system. To image in vivo drug localization, TCP-25 was spiked with Cy5-labeled TCP-25. Representative images show bioluminescence (lum) and TCP-25 Cy5 fluorescence (flu) at 6 hours. Bar graphs show the measured light intensity emitted from these reporter mice. Data are expressed as mean ± SEM (n = 7 mice in the gel group and n = 5 mice in the TCP-25 gel #1 group). P values ​​were determined using the Mann-Whitney U test. Figure 4D shows cytokine analysis from wound fluid extracted from implanted PU discs.Data are presented as mean ± SEM (n = 4 mice in the gel group, n = 4 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01; ***P ≤ 0.001. Figure 4E further shows the microbial analysis of tissues 24 hours postinfection. Data are presented as mean ± SEM (n = 7 mice in the gel group, n = 7 mice in the TCP-25 Gel #1 group). P values ​​were determined using the Mann-Whitney U test. **P ≤ 0.01. [Figure 5A]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5B]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5C]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5D]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5E]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5F]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5G]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 5H]Figures 5A–H show the effect of TCP-25 gel in a porcine partial-thickness wound model. Figure 5A shows the wound creation plan in minipigs. Twelve partial-thickness wounds, six on each side, were created on the back of Göttingen minipigs using an electric dermatome and infected with Staphylococcus aureus. Each wound was infected with 10 CFU of S. aureus. This figure also shows the wound dressing plan. Briefly, after infection and gel application, the wound was covered with a primary polyurethane dressing, followed by a transparent, breathable, fixative dressing. For better fixation, the dressing was then secured with skin staples. The wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a layer of flexible, self-adhesive bandage was used to support and protect the underlying dressing. In addition, these figures describe the two therapeutic approaches used in the minipigs study: short-term and long-term. Figure 5B shows representative photographic images of miniature pig wounds after the short-term treatment regimen. Wounds with either S. aureus or mixed infection (S. aureus and P. aeruginosa co-infection) were treated daily with TCP-25 gel or without. Uninfected control wounds were treated with TCP-25 gel (scale bar 1 cm). Figure 5C shows clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 4 pigs; mixed infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds in the uninfected control, from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5D shows the microbial analysis of wounds from days 2, 3, and 4. Data are expressed as mean ± SEM (S. aureus-infected group: n = 10 wounds with gel, n = 9 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 4 pigs; mixed-infection group: n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected control from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test.Figure 5E shows analysis of wound fluid cytokines collected on days 2, 3, and 4. Data are presented as mean ± SEM (n = 8–10 wounds with gel, n = 7–9 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from four pigs in the S. aureus-infected group; n = 4 wounds with gel, n = 5 wounds with TCP-25 gel, and n = 3 wounds with uninfected controls from two pigs in the mixed-infection group). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 5F shows representative images showing H&E staining of wound biopsies 4 days after treatment. Arrows indicate multiple tissue destruction and hyperinflammatory conditions in the wounds. Arrowheads indicate wound re-epithelialization. Bar graphs show histological analysis of wound tissue. Data are presented as mean ± SEM (n = 12 with gel, n = 12 with TCP-25 gel). P values ​​were determined using the Mann-Whitney U test. Figure 5G shows representative photographic images of minipig wounds after the long-term treatment regimen. Wounds were infected with Staphylococcus aureus and treated with TCP-25 gel on days 1, 2, 3, 5, 7, and 9. In the lower panel, images show H&E staining of wound biopsies. Dot plots show microbial analysis of wounds on days 2, 5, and 7. Data are expressed as mean ± SEM (n = 10 for gel and n = 10 for TCP-25 gel from 4 pigs). P values ​​were determined using the Mann-Whitney U test. Figure 5H shows the effect of TCP-25 gel treatment on wound healing (non-infected wounds) in minipig animals. Partial-thickness wounds were created in minipig animals and treated with TCP-25 gel. Representative photographic images of wounds and H&E-stained wound biopsies are shown. Bar graphs show histological analysis of wound tissue. Data are expressed as mean ± SEM (n = 10 for gels and n = 9 for TCP-25 gels from 4 pigs). P values ​​were determined using the Mann-Whitney U test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; NS: not significant. [Figure 6A]Figures 6A-D show the degradation of TCP-25 by human neutrophil elastase in vitro and a comparison with proteolytic thrombin fragments generated in vitro and in vivo. Figure 6A shows the digestion pattern of TCP-25 after treatment with HNE. Enzymatic digestion was performed for different time periods and analyzed by mass spectrometry. The table shows the sequences of the major peptides and the number successfully identified by mass spectrometry at 10, 30, 60, and 180 minutes. Figure 6B shows a graphical representation of the major peptides obtained after digestion and a comparison with those found after thrombin digestion and peptides detected in wounds in vivo. *Peptides reported to exhibit antibacterial effects. Figure 6C shows a representative high-resolution MALDI mass spectrum of HNE-digested TCP-25. The same peptide fragments were detected in buffer solution and gel. After 180 minutes, intact TCP-25 could not be detected in the solution or gel samples. The identified peptide sequences are shown in the lower panel. Figure 6D shows the release and activity of TCP-25 degradation products was determined by assessing antimicrobial activity against E. coli by RDA in 10 mM Tris pH 7.4 with or without 0.15 M NaCl. The bar graph shows the resulting zone of clearance measurements. Data are presented as mean ± SEM (n = 3). [Figure 6B]Figures 6A-D show the degradation of TCP-25 by human neutrophil elastase in vitro and a comparison with proteolytic thrombin fragments generated in vitro and in vivo. Figure 6A shows the digestion pattern of TCP-25 after treatment with HNE. Enzymatic digestion was performed for different time periods and analyzed by mass spectrometry. The table shows the sequences of the major peptides and the number successfully identified by mass spectrometry at 10, 30, 60, and 180 minutes. Figure 6B shows a graphical representation of the major peptides obtained after digestion and a comparison with those found after thrombin digestion and peptides detected in wounds in vivo. *Peptides reported to exhibit antibacterial effects. Figure 6C shows a representative high-resolution MALDI mass spectrum of HNE-digested TCP-25. The same peptide fragments were detected in buffer solution and gel. After 180 minutes, intact TCP-25 could not be detected in the solution or gel samples. The identified peptide sequences are shown in the lower panel. Figure 6D shows the release and activity of TCP-25 degradation products was determined by assessing antimicrobial activity against E. coli by RDA in 10 mM Tris pH 7.4 with or without 0.15 M NaCl. The bar graph shows the resulting zone of clearance measurements. Data are presented as mean ± SEM (n = 3). [Figure 6C]Figures 6A-D show the degradation of TCP-25 by human neutrophil elastase in vitro and a comparison with proteolytic thrombin fragments generated in vitro and in vivo. Figure 6A shows the digestion pattern of TCP-25 after treatment with HNE. Enzymatic digestion was performed for different time periods and analyzed by mass spectrometry. The table shows the sequences of the major peptides and the number successfully identified by mass spectrometry at 10, 30, 60, and 180 minutes. Figure 6B shows a graphical representation of the major peptides obtained after digestion and a comparison with those found after thrombin digestion and peptides detected in wounds in vivo. *Peptides reported to exhibit antibacterial effects. Figure 6C shows a representative high-resolution MALDI mass spectrum of HNE-digested TCP-25. The same peptide fragments were detected in buffer solution and gel. After 180 minutes, intact TCP-25 could not be detected in the solution or gel samples. The identified peptide sequences are shown in the lower panel. Figure 6D shows the release and activity of TCP-25 degradation products was determined by assessing antimicrobial activity against E. coli by RDA in 10 mM Tris pH 7.4 with or without 0.15 M NaCl. The bar graph shows the resulting zone of clearance measurements. Data are presented as mean ± SEM (n = 3). [Figure 6D]Figures 6A-D show the degradation of TCP-25 by human neutrophil elastase in vitro and a comparison with proteolytic thrombin fragments generated in vitro and in vivo. Figure 6A shows the digestion pattern of TCP-25 after treatment with HNE. Enzymatic digestion was performed for different time periods and analyzed by mass spectrometry. The table shows the sequences of the major peptides and the number successfully identified by mass spectrometry at 10, 30, 60, and 180 minutes. Figure 6B shows a graphical representation of the major peptides obtained after digestion and a comparison with those found after thrombin digestion and peptides detected in wounds in vivo. *Peptides reported to exhibit antibacterial effects. Figure 6C shows a representative high-resolution MALDI mass spectrum of HNE-digested TCP-25. The same peptide fragments were detected in buffer solution and gel. After 180 minutes, intact TCP-25 could not be detected in the solution or gel samples. The identified peptide sequences are shown in the lower panel. Figure 6D shows the release and activity of TCP-25 degradation products was determined by assessing antimicrobial activity against E. coli by RDA in 10 mM Tris pH 7.4 with or without 0.15 M NaCl. The bar graph shows the resulting zone of clearance measurements. Data are presented as mean ± SEM (n = 3). [Figure 7A]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7B]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7C]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Prontosan Ag). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7D]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after the establishment of infection (scale bar 1 cm). Figure 7G shows the microbial analysis of wounds (from the established infection model) from days 2, 3, 5, 9, and 10. Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows the analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7E]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7F]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7G]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7H]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7I]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7J]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7K]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7L]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with MepilexAg, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 7M]Figures 7A-M show a comparison of TCP-25 gel with wound treatment benchmarks. TCP-25 gel was compared with two current standard benchmarks for wound healing: Mepilex Ag and Prontosan. Figure 7A shows representative photographic images of miniature pig wounds after a short-term treatment regimen. Wounds were infected with 10 CFU of Staphylococcus aureus and treated once daily with TCP-25 gel, Mepilex Ag, or Prontosan. Figure 7B shows microbial analysis of wounds from days 2, 3, and 4. Swab samples were collected from the wounds, and appropriate dilutions were plated on TH broth agar to determine the number of CFU. Data are presented as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Mepilex Ag, n = 6 wounds for Prontosan from 3 pigs). Comparisons are shown relative to the "gel" group; P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7C shows the clinical scoring of wounds after the short-term treatment regimen. Data are expressed as medians with 95% confidence intervals (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, and n = 6 wounds with Prontosan from 3 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7D shows representative images showing H&E staining of wound biopsies. Arrows indicate severe tissue destruction and inflammatory infiltrates in the wounds. Arrowheads indicate areas of wound re-epithelialization. Figure 7E shows the experimental design of the established minipig infection model. Figure 7F shows representative photographs of minipig wounds on days 2 and 10 of the established infection treatment regimen. Wounds were infected with S. aureus and treated with control gel, TCP-25, or Prontosan on days 2, 3, 5, 7, and 9 after confirmation of infection (scale bar 1 cm). Figure 7G shows microbial analysis of wounds from days 2, 3, 5, 9, and 10 (from the established infection model). Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7H shows analysis of TNF-α in wound fluid collected on days 2, 3, and 5.Data are expressed as mean ± SEM (n = 7 wounds with gel, n = 7 wounds with TCP-25 gel, n = 7 wounds with Prontosan from 2 pigs). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 7I shows in vivo infection imaging with IVIS in a mouse model of subcutaneous infection. TCP-25 gel formulations were subcutaneously deposited on the backs of SKH1 mice after addition of bioluminescent Staphylococcus aureus or Pseudomonas aeruginosa. Bacterial bioluminescence intensity at different time points was noninvasively analyzed with the IVIS bioimaging system. Representative images show bacterial luminescence at 6 hours post-infection (n = 6 in each group). Figure 7J shows in vivo inflammation imaging with IVIS in NF-kB reporter mice. Prontosan or TCP-25 gel was mixed with LPS and deposited subcutaneously on the left and right sides of the backs of transgenic BALB / c Tg(NF-kB-RE-luc)-Xen reporter mice, respectively. In vivo imaging of NF-kB reporter gene expression was achieved using an IVIS Spectrum bioimaging system. Representative images show bioluminescence 6 hours after subcutaneous deposition. Bar graphs show the measured bioluminescence intensity emitted from these mice. Data are presented as mean ± SEM (n = 5 per group). P values ​​were determined using the Mann-Whitney U test. Figure 7K shows a comparison of the anti-inflammatory potential of TCP-25 with PHMB, the anti-sepsis component of Prontosan. THP1-XBlue™-CD14 reporter cells were stimulated with E. coli LPS in the presence of PHMB and TCP-25. Data are presented as mean ± SEM (n = 6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. NS: Not significant. Figure 7L further shows cytokine analysis of wound fluid collected on days 2 and 3. Data are expressed as mean ± SEM (n = 6 wounds with gel, n = 6 wounds with TCP-25 gel, n = 6 wounds with Mepilex Ag, n = 6 wounds with Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by a Dunn post-hoc test. *P ≤ 0.05; **P ≤ 0.01.Figure 7M further shows IL-1β analysis of wound fluid collected from the established infection model on days 2, 3, and 5. Data are expressed as mean ± SEM (n = 6 wounds for gel, n = 6 wounds for TCP-25 gel, n = 6 wounds for Prontosan). P values ​​were determined using the Kruskal-Wallis test followed by Dunn's post-hoc test. **P ≤ 0.01; NS not significant. [Figure 8A] Figures 8A-C demonstrate that TCP-25 targets inflammation in wounds. Figure 8A shows NF-kB activation in THP-1-XBlue™-CD14 reporter cells in response to stimulation with wound fluid from infected minipig wounds and minipig wounds treated with TCP-25 gel. Data are presented as mean ± SEM (n = 6). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 8B demonstrates that TCP-25 reduces the ability of minipig wound fluid to activate inflammation. THP1-XBlue™-CD14 reporter cells were stimulated with wound fluid from infected minipig wounds from days 1, 2, and 3 in the presence of TCP-25. Data are presented as mean ± SEM (n = 4). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 8C demonstrates that TCP-25 reduces the ability of human wound fluid to activate inflammation. THP-1-XBlue™-CD14 reporter cells were stimulated with chronic wound fluid (CWF) from infected wounds of patients in the presence of TCP-25. CWFs 1-5 represent five human patients. Data are presented as mean ± SEM (n=6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001; NS not significant. [Figure 8B]Figures 8A-C demonstrate that TCP-25 targets inflammation in wounds. Figure 8A shows NF-kB activation in THP-1-XBlue™-CD14 reporter cells in response to stimulation with wound fluid from infected minipig wounds and minipig wounds treated with TCP-25 gel. Data are presented as mean ± SEM (n = 6). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 8B demonstrates that TCP-25 reduces the ability of minipig wound fluid to activate inflammation. THP1-XBlue™-CD14 reporter cells were stimulated with wound fluid from infected minipig wounds from days 1, 2, and 3 in the presence of TCP-25. Data are presented as mean ± SEM (n = 4). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 8C demonstrates that TCP-25 reduces the ability of human wound fluid to activate inflammation. THP-1-XBlue™-CD14 reporter cells were stimulated with chronic wound fluid (CWF) from infected wounds of patients in the presence of TCP-25. CWFs 1-5 represent five human patients. Data are presented as mean ± SEM (n=6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001; NS not significant. [Figure 8C]Figures 8A-C demonstrate that TCP-25 targets inflammation in wounds. Figure 8A shows NF-kB activation in THP-1-XBlue™-CD14 reporter cells in response to stimulation with wound fluid from infected minipig wounds and minipig wounds treated with TCP-25 gel. Data are presented as mean ± SEM (n = 6). P values ​​were determined using a Kruskal-Wallis test followed by a Dunn post-hoc test. Figure 8B demonstrates that TCP-25 reduces the ability of minipig wound fluid to activate inflammation. THP1-XBlue™-CD14 reporter cells were stimulated with wound fluid from infected minipig wounds from days 1, 2, and 3 in the presence of TCP-25. Data are presented as mean ± SEM (n = 4). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. Figure 8C demonstrates that TCP-25 reduces the ability of human wound fluid to activate inflammation. THP-1-XBlue™-CD14 reporter cells were stimulated with chronic wound fluid (CWF) from infected wounds of patients in the presence of TCP-25. CWFs 1-5 represent five human patients. Data are presented as mean ± SEM (n=6). P values ​​were determined using one-way ANOVA with Tukey's post-hoc test. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001; NS not significant. [Figure 9A] Figures 9A-B show the rheological properties of TCP-25 gels. The gel strength of 2% HEC gels without or with 0.1% or 1% TCP-25 was analyzed on a Kinexus Pro rheometer. Figure 9A shows the pour point (strain) as a measure of gel strength. Data are expressed as means with 95% confidence intervals (n=3). NS: not significant. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. Figure 9B shows representative elastic modulus (G') and viscous modulus (G") plotted against strain (n=3). [Figure 9B]Figures 9A-B show the rheological properties of TCP-25 gels. The gel strength of 2% HEC gels without or with 0.1% or 1% TCP-25 was analyzed on a Kinexus Pro rheometer. Figure 9A shows the pour point (strain) as a measure of gel strength. Data are expressed as means with 95% confidence intervals (n=3). NS: not significant. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. Figure 9B shows representative elastic modulus (G') and viscous modulus (G") plotted against strain (n=3). [Figure 10A]Figures 10A-E show the in vitro release and in vivo pharmacokinetics of TCP-25 gel. (A) In vitro diffusion of TCP-25 from the gel into buffer. TCP-25 gel #1 was prepared with TAMRA-labeled TCP-25 and loaded into the apical compartment of a transwell insert. Buffer from the basolateral compartment was collected at various time points, and cumulative fluorescence was measured to assess the diffusion of TCP-25 from the gel into the buffer. The control was a 0.1% solution of TAMRA-TCP-25. Data are presented as mean ± SEM (n = 3). (B) Pharmacokinetics of TCP-25 Cy5-spiked subcutaneously deposited TCP-25 gel #1 and the effect of LPS. To image the in vivo pharmacokinetics of the gel, TCP-25 was spiked with Cy5-labeled TCP-25 and subcutaneously deposited on the back of SKH1 hairless mice. In some mice, LPS was added to the gel before injection. In vivo fluorescence imaging was performed using IVIS Spectrum. Representative images show the distribution of TCP-25 Cy5 at 1, 6, and 24 hours after gel deposition. Brighter colors indicate higher signal intensity. Bar graphs show fluorescence measured locally around the gel deposition site (local) and throughout the body (body). Data are presented as mean ± SEM (n = 3). (C) In vivo tissue uptake of TCP-25 in minipigs. TCP-25 gel #2 or #3 spiked with TCP-25 Cy3 was applied topically to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (examples of specific staining identified by white arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n = 3). (D) TCP-25 uptake in a minipig ex vivo skin model. TCP-25 gel #4 spiked with Cy3-TCP-25 was topically applied to either intact or wounded ex vivo skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (white, examples of specific staining identified by arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n=3). (E) In vivo stability and systemic uptake of TCP-25 in wound dressings after topical application of TCP-25 gel to minipig wounds.In a minipig model of partial-thickness wounds, TCP-25 Gel #2 was applied topically, and wound fluid from the dressing was collected 24 hours after application and analyzed using mass spectrometry. Control wounds were treated with gel without TCP-25. To test the systemic uptake of TCP-25 after topical application to wounds, plasma from minipigs was collected and analyzed by mass spectrometry (n = 4-5). The LOQ for the assay was 100 nM. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. *P ≤ 0.05; **P ≤ 0.01. [Figure 10B]Figures 10A-E show the in vitro release and in vivo pharmacokinetics of TCP-25 gel. (A) In vitro diffusion of TCP-25 from the gel into buffer. TCP-25 gel #1 was prepared with TAMRA-labeled TCP-25 and loaded into the apical compartment of a transwell insert. Buffer from the basolateral compartment was collected at various time points, and cumulative fluorescence was measured to assess the diffusion of TCP-25 from the gel into the buffer. The control was a 0.1% solution of TAMRA-TCP-25. Data are presented as mean ± SEM (n = 3). (B) Pharmacokinetics of TCP-25 Cy5-spiked subcutaneously deposited TCP-25 gel #1 and the effect of LPS. To image the in vivo pharmacokinetics of the gel, TCP-25 was spiked with Cy5-labeled TCP-25 and subcutaneously deposited on the back of SKH1 hairless mice. In some mice, LPS was added to the gel before injection. In vivo fluorescence imaging was performed using IVIS Spectrum. Representative images show the distribution of TCP-25 Cy5 at 1, 6, and 24 hours after gel deposition. Brighter colors indicate higher signal intensity. Bar graphs show fluorescence measured locally around the gel deposition site (local) and systemic fluorescence (body). Data are presented as mean ± SEM (n = 3). (C) In vivo tissue uptake of TCP-25 in minipigs. TCP-25 gel #2 or #3 spiked with TCP-25 Cy3 was applied topically to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (examples of specific staining identified by white arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n = 3). (D) TCP-25 uptake in a minipig ex vivo skin model. TCP-25 gel #4 spiked with Cy3-TCP-25 was topically applied to either intact or wounded ex vivo skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (white, examples of specific staining identified by arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n=3). (E) In vivo stability and systemic uptake of TCP-25 in wound dressings after topical application of TCP-25 gel to minipig wounds.In a minipig model of partial-thickness wounds, TCP-25 Gel #2 was applied topically, and wound fluid from the dressing was collected 24 hours after application and analyzed using mass spectrometry. Control wounds were treated with gel without TCP-25. To test the systemic uptake of TCP-25 after topical application to wounds, plasma from minipigs was collected and analyzed by mass spectrometry (n = 4-5). The LOQ for the assay was 100 nM. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. *P ≤ 0.05; **P ≤ 0.01. [Figure 10C]Figures 10A-E show the in vitro release and in vivo pharmacokinetics of TCP-25 gel. (A) In vitro diffusion of TCP-25 from the gel into buffer. TCP-25 gel #1 was prepared with TAMRA-labeled TCP-25 and loaded into the apical compartment of a transwell insert. Buffer from the basolateral compartment was collected at various time points, and cumulative fluorescence was measured to assess the diffusion of TCP-25 from the gel into the buffer. The control was a 0.1% solution of TAMRA-TCP-25. Data are presented as mean ± SEM (n = 3). (B) Pharmacokinetics of TCP-25 Cy5-spiked subcutaneously deposited TCP-25 gel #1 and the effect of LPS. To image the in vivo pharmacokinetics of the gel, TCP-25 was spiked with Cy5-labeled TCP-25 and subcutaneously deposited on the back of SKH1 hairless mice. In some mice, LPS was added to the gel before injection. In vivo fluorescence imaging was performed using IVIS Spectrum. Representative images show the distribution of TCP-25 Cy5 at 1, 6, and 24 hours after gel deposition. Brighter colors indicate higher signal intensity. Bar graphs show fluorescence measured locally around the gel deposition site (local) and throughout the body (body). Data are presented as mean ± SEM (n = 3). (C) In vivo tissue uptake of TCP-25 in minipigs. TCP-25 gel #2 or #3 spiked with TCP-25 Cy3 was applied topically to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (examples of specific staining identified by white arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n = 3). (D) TCP-25 uptake in a minipig ex vivo skin model. TCP-25 gel #4 spiked with Cy3-TCP-25 was topically applied to either intact or wounded ex vivo skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (white, examples of specific staining identified by arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n=3). (E) In vivo stability and systemic uptake of TCP-25 in wound dressings after topical application of TCP-25 gel to minipig wounds.In a minipig model of partial-thickness wounds, TCP-25 Gel #2 was applied topically, and wound fluid from the dressing was collected 24 hours after application and analyzed using mass spectrometry. Control wounds were treated with gel without TCP-25. To test the systemic uptake of TCP-25 after topical application to wounds, plasma from minipigs was collected and analyzed by mass spectrometry (n = 4-5). The LOQ for the assay was 100 nM. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. *P ≤ 0.05; **P ≤ 0.01. [Figure 10D]Figures 10A-E show the in vitro release and in vivo pharmacokinetics of TCP-25 gel. (A) In vitro diffusion of TCP-25 from the gel into buffer. TCP-25 gel #1 was prepared with TAMRA-labeled TCP-25 and loaded into the apical compartment of a transwell insert. Buffer from the basolateral compartment was collected at various time points, and cumulative fluorescence was measured to assess the diffusion of TCP-25 from the gel into the buffer. The control was a 0.1% solution of TAMRA-TCP-25. Data are presented as mean ± SEM (n = 3). (B) Pharmacokinetics of TCP-25 Cy5-spiked subcutaneously deposited TCP-25 gel #1 and the effect of LPS. To image the in vivo pharmacokinetics of the gel, TCP-25 was spiked with Cy5-labeled TCP-25 and subcutaneously deposited on the back of SKH1 hairless mice. In some mice, LPS was added to the gel before injection. In vivo fluorescence imaging was performed using IVIS Spectrum. Representative images show the distribution of TCP-25 Cy5 at 1, 6, and 24 hours after gel deposition. Brighter colors indicate higher signal intensity. Bar graphs show fluorescence measured locally around the gel deposition site (local) and throughout the body (body). Data are presented as mean ± SEM (n = 3). (C) In vivo tissue uptake of TCP-25 in minipigs. TCP-25 gel #2 or #3 spiked with TCP-25 Cy3 was applied topically to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (examples of specific staining identified by white arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n = 3). (D) TCP-25 uptake in a minipig ex vivo skin model. TCP-25 gel #4 spiked with Cy3-TCP-25 was topically applied to either intact or wounded ex vivo skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (white, examples of specific staining identified by arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n=3). (E) In vivo stability and systemic uptake of TCP-25 in wound dressings after topical application of TCP-25 gel to minipig wounds.In a minipig model of partial-thickness wounds, TCP-25 Gel #2 was applied topically, and wound fluid from the dressing was collected 24 hours after application and analyzed using mass spectrometry. Control wounds were treated with gel without TCP-25. To test the systemic uptake of TCP-25 after topical application to wounds, plasma from minipigs was collected and analyzed by mass spectrometry (n = 4-5). The LOQ for the assay was 100 nM. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. *P ≤ 0.05; **P ≤ 0.01. [Figure 10E]Figures 10A-E show the in vitro release and in vivo pharmacokinetics of TCP-25 gel. (A) In vitro diffusion of TCP-25 from the gel into buffer. TCP-25 gel #1 was prepared with TAMRA-labeled TCP-25 and loaded into the apical compartment of a transwell insert. Buffer from the basolateral compartment was collected at various time points, and cumulative fluorescence was measured to assess the diffusion of TCP-25 from the gel into the buffer. The control was a 0.1% solution of TAMRA-TCP-25. Data are presented as mean ± SEM (n = 3). (B) Pharmacokinetics of TCP-25 Cy5-spiked subcutaneously deposited TCP-25 gel #1 and the effect of LPS. To image the in vivo pharmacokinetics of the gel, TCP-25 was spiked with Cy5-labeled TCP-25 and subcutaneously deposited on the back of SKH1 hairless mice. In some mice, LPS was added to the gel before injection. In vivo fluorescence imaging was performed using IVIS Spectrum. Representative images show the distribution of TCP-25 Cy5 at 1, 6, and 24 hours after gel deposition. Brighter colors indicate higher signal intensity. Bar graphs show fluorescence measured locally around the gel deposition site (local) and throughout the body (body). Data are presented as mean ± SEM (n = 3). (C) In vivo tissue uptake of TCP-25 in minipigs. TCP-25 gel #2 or #3 spiked with TCP-25 Cy3 was applied topically to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (examples of specific staining identified by white arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n = 3). (D) TCP-25 uptake in a minipig ex vivo skin model. TCP-25 gel #4 spiked with Cy3-TCP-25 was topically applied to either intact or wounded ex vivo skin (2 and 24 hours). Fluorescence imaging of cryosections was used to detect Cy3-TCP-25 (white, examples of specific staining identified by arrows). Nuclei (gray) were counterstained with DAPI nuclear stain (n=3). (E) In vivo stability and systemic uptake of TCP-25 in wound dressings after topical application of TCP-25 gel to minipig wounds.In a minipig model of partial-thickness wounds, TCP-25 Gel #2 was applied topically, and wound fluid from the dressing was collected 24 hours after application and analyzed using mass spectrometry. Control wounds were treated with gel without TCP-25. To test the systemic uptake of TCP-25 after topical application to wounds, plasma from minipigs was collected and analyzed by mass spectrometry (n = 4-5). The LOQ for the assay was 100 nM. P values ​​were determined using the Kruskal-Wallis test with Dunn's post-hoc test. *P ≤ 0.05; **P ≤ 0.01. [Figure 11A] Figures 11A-B show the solubility of TCP-25 at pH 7.4 and pH 5. The solubility of 0.1% TCP-25 in Tris buffer (10 mM or 25 mM Tris) containing 2% or 1.9% glycerol and EDTA (2.5 mM) was assessed (A), and the solubility of 0.1% TCP-25 in acetate buffer (10 mM or 25 mM) containing 2% and 1.9% glycerol and 2.5 mM EDTA (B). Photographs of each buffer without TCP-25 are shown for comparison. [Figure 11B] Figures 11A-B show the solubility of TCP-25 at pH 7.4 and pH 5. The solubility of 0.1% TCP-25 in Tris buffer (10 mM or 25 mM Tris) containing 2% or 1.9% glycerol and EDTA (2.5 mM) was assessed (A), and the solubility of 0.1% TCP-25 in acetate buffer (10 mM or 25 mM) containing 2% and 1.9% glycerol and 2.5 mM EDTA (B). Photographs of each buffer without TCP-25 are shown for comparison. [Figure 12A]Figure 12 shows the effectiveness of Tris- and acetate-based gels containing 0.1% TCP-25 and 2.5 mM EDTA against S. aureus biofilms. A) Shows the effect on biofilms of Tris-buffered gel formulations (10 mM and 25 mM Tris) containing TCP-25 alone or in combination with EDTA. B) Shows the effect on biofilms of acetate-buffered gel formulations (10 mM and 25 mM acetate) containing TCP-25 alone or in combination with 2.5 mM EDTA. [Figure 12B] Figure 12 shows the effectiveness of Tris- and acetate-based gels containing 0.1% TCP-25 and 2.5 mM EDTA against S. aureus biofilms. A) Shows the effect on biofilms of Tris-buffered gel formulations (10 mM and 25 mM Tris) containing TCP-25 alone or in combination with EDTA. B) Shows the effect on biofilms of acetate-buffered gel formulations (10 mM and 25 mM acetate) containing TCP-25 alone or in combination with 2.5 mM EDTA. [Figure 13A] Figure 13 shows the effectiveness of a combination of 0.1% TCP-25 and EDTA in Tris- and acetate-based gels against Pseudomonas aeruginosa biofilms. A) Demonstrates the effect on biofilms with Tris-buffer-based gel formulations (10 mM and 25 mM Tris) and with 2.5 mM EDTA and TCP-25. B) Demonstrates the effect on biofilms with acetate-buffer-based gel formulations (10 mM and 25 mM acetic acid) and with 2.5 mM EDTA and TCP-25. [Figure 13B]Figure 13 shows the effectiveness of a combination of 0.1% TCP-25 and EDTA in Tris- and acetate-based gels against Pseudomonas aeruginosa biofilms. A) Demonstrates the effect on biofilms with Tris-buffer-based gel formulations (10 mM and 25 mM Tris) and with 2.5 mM EDTA and TCP-25. B) Demonstrates the effect on biofilms with acetate-buffer-based gel formulations (10 mM and 25 mM acetic acid) and with 2.5 mM EDTA and TCP-25. [Figure 14A] Figure 14 shows the antibacterial effect of a gel containing a combination of TCP-25 and EDTA in a pig skin ex vivo model. A) shows the bacterial count (CFU) on the surface of the burn wound. B) shows the bacterial count (CFU) found in the tissue after treatment. [Figure 14B] Figure 14 shows the antibacterial effect of a gel containing a combination of TCP-25 and EDTA in a pig skin ex vivo model. A) shows the bacterial count (CFU) on the surface of the burn wound. B) shows the bacterial count (CFU) found in the tissue after treatment. [Figure 15A]Figure 15 shows the effect of pH and concentration on TCP-25 oligomerization. Panel A) shows representative photographs of cuvettes containing 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM acetate pH 5 immediately after storage at 4 °C (0 min) and at the indicated time points after incubation at RT. Panel B) shows absorbance and transmittance values ​​at 405 nm for 10–300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0. Panel C) shows 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0, centrifuged, and the pellet and supernatant analyzed by SDS-PAGE. The graph shows the TCP-25 concentration ± SD after centrifugation. Panel D) TEM images demonstrating that oligomerization is pH- and concentration-dependent. TCP-25 was dissolved in pH 7.4 and 5.0 buffers at the indicated concentrations and analyzed by TEM. All experiments were performed in triplicate (n=3). * indicates p<0.05. P values ​​were determined using one-way ANOVA with Dunnett's multiple comparison test. [Figure 15B]Figure 15 shows the effect of pH and concentration on TCP-25 oligomerization. Panel A) shows representative photographs of cuvettes containing 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM acetate pH 5 immediately after storage at 4 °C (0 min) and at the indicated time points after incubation at RT. Panel B) shows absorbance and transmittance values ​​at 405 nm for 10–300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0. Panel C) shows 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0, centrifuged, and the pellet and supernatant analyzed by SDS-PAGE. The graph shows the TCP-25 concentration ± SD after centrifugation. Panel D) TEM images demonstrating that oligomerization is pH- and concentration-dependent. TCP-25 was dissolved in pH 7.4 and 5.0 buffers at the indicated concentrations and analyzed by TEM. All experiments were performed in triplicate (n=3). * indicates p<0.05. P values ​​were determined using one-way ANOVA with Dunnett's multiple comparison test. [Figure 15C]Figure 15 shows the effect of pH and concentration on TCP-25 oligomerization. Panel A) shows representative photographs of cuvettes containing 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM acetate pH 5 immediately after storage at 4 °C (0 min) and at the indicated time points after incubation at RT. Panel B) shows absorbance and transmittance values ​​at 405 nm for 10–300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0. Panel C) shows 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0, centrifuged, and the pellet and supernatant analyzed by SDS-PAGE. The graph shows the TCP-25 concentration ± SD after centrifugation. Panel D) TEM images demonstrating that oligomerization is pH- and concentration-dependent. TCP-25 was dissolved in pH 7.4 and 5.0 buffers at the indicated concentrations and analyzed by TEM. All experiments were performed in triplicate (n=3). * indicates p<0.05. P values ​​were determined using one-way ANOVA with Dunnett's multiple comparison test. [Figure 15D]Figure 15 shows the effect of pH and concentration on TCP-25 oligomerization. Panel A) shows representative photographs of cuvettes containing 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM acetate pH 5 immediately after storage at 4 °C (0 min) and at the indicated time points after incubation at RT. Panel B) shows absorbance and transmittance values ​​at 405 nm for 10–300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0. Panel C) shows 300 μM TCP-25 dissolved in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.8 and 5.0, centrifuged, and the pellet and supernatant analyzed by SDS-PAGE. The graph shows the TCP-25 concentration ± SD after centrifugation. Panel D) TEM images demonstrating that oligomerization is pH- and concentration-dependent. TCP-25 was dissolved in pH 7.4 and 5.0 buffers at the indicated concentrations and analyzed by TEM. All experiments were performed in triplicate (n=3). * indicates p<0.05. P values ​​were determined using one-way ANOVA with Dunnett's multiple comparison test. [Figure 16A]Figure 16 shows the structural analysis of TCP-25 oligomers. Panel A shows the α-helical content ± SD, calculated from CD spectra acquired at 222 nm. A significant increase in α-helical content was observed at 300 μM TCP-25 in 10 mM Tris at pH 7.4. * indicates P<0.05, calculated using one-way analysis of variance and Dunnett's multiple comparison test (n=3). Panel B shows that separation on 4-16% (w / v) BN-PAGE followed by Western blot analysis indicates increased oligomerization of TCP-25 at higher concentrations. One representative image from three independent experiments is shown (n=3). In Panel C, TCP-25 was cross-linked with different concentrations of BS3 for 30 min and then analyzed on a 10-20% Tris-Tricine gel followed by Coomassie staining. Higher concentrations of cross-linker resulted in higher molecular weight TCP-25 oligomers. A representative image of one of three independent experiments is shown (n=3). D) Reverse-phase C18 chromatography of TCP-25 in the absence of BS3 (black) or in the presence of 145 μM (dashed black line) or 540 μM (gray line) BS3 shows an altered elution profile. [Figure 16B]Figure 16 shows the structural analysis of TCP-25 oligomers. Panel A shows the α-helical content ± SD, calculated from CD spectra acquired at 222 nm. A significant increase in α-helical content was observed at 300 μM TCP-25 in 10 mM Tris at pH 7.4. * indicates P<0.05, calculated using one-way analysis of variance and Dunnett's multiple comparison test (n=3). Panel B shows that separation on 4-16% (w / v) BN-PAGE followed by Western blot analysis indicates increased oligomerization of TCP-25 at higher concentrations. One representative image from three independent experiments is shown (n=3). In Panel C, TCP-25 was cross-linked with different concentrations of BS3 for 30 min and then analyzed on a 10-20% Tris-Tricine gel followed by Coomassie staining. Higher concentrations of cross-linker resulted in higher molecular weight TCP-25 oligomers. A representative image of one of three independent experiments is shown (n=3). D) Reverse-phase C18 chromatography of TCP-25 in the absence of BS3 (black) or in the presence of 145 μM (dashed black line) or 540 μM (gray line) BS3 shows an altered elution profile. [Figure 16C]Figure 16 shows the structural analysis of TCP-25 oligomers. Panel A shows the α-helical content ± SD, calculated from CD spectra acquired at 222 nm. A significant increase in α-helical content was observed at 300 μM TCP-25 in 10 mM Tris at pH 7.4. * indicates P<0.05, calculated using one-way analysis of variance and Dunnett's multiple comparison test (n=3). Panel B shows that separation on 4-16% (w / v) BN-PAGE followed by Western blot analysis indicates increased oligomerization of TCP-25 at higher concentrations. One representative image from three independent experiments is shown (n=3). In Panel C, TCP-25 was cross-linked with different concentrations of BS3 for 30 min and then analyzed on a 10-20% Tris-Tricine gel followed by Coomassie staining. Higher concentrations of cross-linker resulted in higher molecular weight TCP-25 oligomers. A representative image of one of three independent experiments is shown (n=3). D) Reverse-phase C18 chromatography of TCP-25 in the absence of BS3 (black) or in the presence of 145 μM (dashed black line) or 540 μM (gray line) BS3 shows an altered elution profile. [Figure 16D]Figure 16 shows the structural analysis of TCP-25 oligomers. Panel A shows the α-helical content ± SD, calculated from CD spectra acquired at 222 nm. A significant increase in α-helical content was observed at 300 μM TCP-25 in 10 mM Tris at pH 7.4. * indicates P<0.05, calculated using one-way analysis of variance and Dunnett's multiple comparison test (n=3). Panel B shows that separation on 4-16% (w / v) BN-PAGE followed by Western blot analysis indicates increased oligomerization of TCP-25 at higher concentrations. One representative image from three independent experiments is shown (n=3). In Panel C, TCP-25 was cross-linked with different concentrations of BS3 for 30 min and then analyzed on a 10-20% Tris-Tricine gel followed by Coomassie staining. Higher concentrations of cross-linker resulted in higher molecular weight TCP-25 oligomers. A representative image of one of three independent experiments is shown (n=3). D) Reverse-phase C18 chromatography of TCP-25 in the absence of BS3 (black) or in the presence of 145 μM (dashed black line) or 540 μM (gray line) BS3 shows an altered elution profile. [Figure 17A]Figure 17 shows the thermal and chemical denaturation of TCP-25. TCP-25 (10 and 300 μM) in 10 mM Tris at pH 7.4 or 10 mM NaOAc at pH 5.0 was denatured by increasing the temperature (A) or by adding increasing amounts of urea (B) or Gdn-HCl (C). The unfolding process was analyzed by recording emission spectra between 300 and 450 nm with excitation at 280 nm. Representative emission spectra are shown for 300 μM TCP-25 dissolved at pH 7.4 or 5.0 using different denaturation methods (n = 3). The denaturation curves are reported below. For thermal denaturation, data were obtained by fitting the normalized maximum emission fluorescence as a function of temperature. For chemical denaturation, results were obtained using the fluorescence ratio (F337 / F350) as a function of the concentration of the chemical agent. Each data point represents the mean ± SEM (n = 3). (D) Table showing Tm and Cm ± SEM calculated from denaturation curves obtained from three independent experiments performed in duplicate (n = 3). → indicates a shift in maximum fluorescence intensity (λmax), while ↑Imax and ↓Imax indicate an increase and decrease in maximum fluorescence intensity, respectively. [Figure 17B]Figure 17 shows the thermal and chemical denaturation of TCP-25. TCP-25 (10 and 300 μM) in 10 mM Tris at pH 7.4 or 10 mM NaOAc at pH 5.0 was denatured by increasing the temperature (A) or by adding increasing amounts of urea (B) or Gdn-HCl (C). The unfolding process was analyzed by recording emission spectra between 300 and 450 nm with excitation at 280 nm. Representative emission spectra are shown for 300 μM TCP-25 dissolved at pH 7.4 or 5.0 using different denaturation methods (n = 3). The denaturation curves are reported below. For thermal denaturation, data were obtained by fitting the normalized maximum emission fluorescence as a function of temperature. For chemical denaturation, results were obtained using the fluorescence ratio (F337 / F350) as a function of the concentration of the chemical agent. Each data point represents the mean ± SEM (n = 3). (D) Table showing Tm and Cm ± SEM calculated from denaturation curves obtained from three independent experiments performed in duplicate (n = 3). → indicates a shift in maximum fluorescence intensity (λmax), while ↑Imax and ↓Imax indicate an increase and decrease in maximum fluorescence intensity, respectively. [Figure 17C]Figure 17 shows the thermal and chemical denaturation of TCP-25. TCP-25 (10 and 300 μM) in 10 mM Tris at pH 7.4 or 10 mM NaOAc at pH 5.0 was denatured by increasing the temperature (A) or by adding increasing amounts of urea (B) or Gdn-HCl (C). The unfolding process was analyzed by recording emission spectra between 300 and 450 nm with excitation at 280 nm. Representative emission spectra are shown for 300 μM TCP-25 dissolved at pH 7.4 or 5.0 using different denaturation methods (n = 3). The denaturation curves are reported below. For thermal denaturation, data were obtained by fitting the normalized maximum emission fluorescence as a function of temperature. For chemical denaturation, results were obtained using the fluorescence ratio (F337 / F350) as a function of the concentration of the chemical agent. Each data point represents the mean ± SEM (n = 3). (D) Table showing Tm and Cm ± SEM calculated from denaturation curves obtained from three independent experiments performed in duplicate (n = 3). → indicates a shift in maximum fluorescence intensity (λmax), while ↑Imax and ↓Imax indicate an increase and decrease in maximum fluorescence intensity, respectively. [Figure 17D]Figure 17 shows the thermal and chemical denaturation of TCP-25. TCP-25 (10 and 300 μM) in 10 mM Tris at pH 7.4 or 10 mM NaOAc at pH 5.0 was denatured by increasing the temperature (A) or by adding increasing amounts of urea (B) or Gdn-HCl (C). The unfolding process was analyzed by recording emission spectra between 300 and 450 nm with excitation at 280 nm. Representative emission spectra are shown for 300 μM TCP-25 dissolved at pH 7.4 or 5.0 using different denaturation methods (n = 3). The denaturation curves are reported below. For thermal denaturation, data were obtained by fitting the normalized maximum emission fluorescence as a function of temperature. For chemical denaturation, results were obtained using the fluorescence ratio (F337 / F350) as a function of the concentration of the chemical agent. Each data point represents the mean ± SEM (n = 3). (D) Table showing Tm and Cm ± SEM calculated from denaturation curves obtained from three independent experiments performed in duplicate (n = 3). → indicates a shift in maximum fluorescence intensity (λmax), while ↑Imax and ↓Imax indicate an increase and decrease in maximum fluorescence intensity, respectively. [Figure 18A] Figure 18 shows the reversibility of thermal denaturation of TCP-25 at pH 7.4 and pH 5.0. 10 and 300 μM TCP-25 in 10 mM Tris (A) or 10 mM NaOAc (B) at pH 7.4 by exposing the peptide to 100°C and returning the temperature to 20°C. Refolding was analyzed by recording the intrinsic fluorescence of the peptide. Spectra were taken at 20°C (black line), 100°C (dashed line), and 20°C (dotted line) after denaturation at 100°C. Each graph is a representative result of three independent experiments (n=3). [Figure 18B]Figure 18 shows the reversibility of thermal denaturation of TCP-25 at pH 7.4 and pH 5.0. 10 and 300 μM TCP-25 in 10 mM Tris (A) or 10 mM NaOAc (B) at pH 7.4 by exposing the peptide to 100°C and returning the temperature to 20°C. Refolding was analyzed by recording the intrinsic fluorescence of the peptide. Spectra were taken at 20°C (black line), 100°C (dashed line), and 20°C (dotted line) after denaturation at 100°C. Each graph is a representative result of three independent experiments (n=3). [Figure 19A] Figure 19 shows the size and distribution of oligomers. (A-B) Representative graphs obtained from DLS analysis of 300 μM TCP-25 in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.0. Oligomer size at pH 7.4 (C) and pH 5.0 (D) is shown, as well as the distribution after up to 24 hours of storage at RT, 4°C, or -20°C (C-D) or after 1 week of storage (E). Oligomers were classified into four families: small (0.4-5 nm, black bars), medium (20-150 nm, light gray bars), large (200-950 nm, dark gray bars), and giant (1 × 103-5 × 103 nm, white bars). For each sample, spectra were recorded three times, including 11 subruns, using multimodal mode. In the graph, the concentrations of oligomers belonging to different families are reported as mean ± SD (n=2). [Figure 19B]Figure 19 shows the size and distribution of oligomers. (A-B) Representative graphs obtained from DLS analysis of 300 μM TCP-25 in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.0. Oligomer size at pH 7.4 (C) and pH 5.0 (D) is shown, as well as the distribution after up to 24 hours of storage at RT, 4°C, or -20°C (C-D) or after 1 week of storage (E). Oligomers were classified into four families: small (0.4-5 nm, black bars), medium (20-150 nm, light gray bars), large (200-950 nm, dark gray bars), and giant (1 × 103-5 × 103 nm, white bars). For each sample, spectra were recorded three times, including 11 subruns, using multimodal mode. In the graph, the concentrations of oligomers belonging to different families are reported as mean ± SD (n=2). [Figure 19C] Figure 19 shows the size and distribution of oligomers. (A-B) Representative graphs obtained from DLS analysis of 300 μM TCP-25 in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.0. Oligomer size at pH 7.4 (C) and pH 5.0 (D) is shown, as well as the distribution after up to 24 hours of storage at RT, 4°C, or -20°C (C-D) or after 1 week of storage (E). Oligomers were classified into four families: small (0.4-5 nm, black bars), medium (20-150 nm, light gray bars), large (200-950 nm, dark gray bars), and giant (1 × 103-5 × 103 nm, white bars). For each sample, spectra were recorded three times, including 11 subruns, using multimodal mode. In the graph, the concentrations of oligomers belonging to different families are reported as mean ± SD (n=2). [Figure 19D]Figure 19 shows the size and distribution of oligomers. (A-B) Representative graphs obtained from DLS analysis of 300 μM TCP-25 in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.0. Oligomer size at pH 7.4 (C) and pH 5.0 (D) is shown, as well as the distribution after up to 24 hours of storage at RT, 4°C, or -20°C (C-D) or after 1 week of storage (E). Oligomers were classified into four families: small (0.4-5 nm, black bars), medium (20-150 nm, light gray bars), large (200-950 nm, dark gray bars), and giant (1 × 103-5 × 103 nm, white bars). For each sample, spectra were recorded three times, including 11 subruns, using multimodal mode. In the graph, the concentrations of oligomers belonging to different families are reported as mean ± SD (n=2). [Figure 19E] Figure 19 shows the size and distribution of oligomers. (A-B) Representative graphs obtained from DLS analysis of 300 μM TCP-25 in 10 mM Tris pH 7.4 or 10 mM NaOAc pH 5.0. Oligomer size at pH 7.4 (C) and pH 5.0 (D) is shown, as well as the distribution after up to 24 hours of storage at RT, 4°C, or -20°C (C-D) or after 1 week of storage (E). Oligomers were classified into four families: small (0.4-5 nm, black bars), medium (20-150 nm, light gray bars), large (200-950 nm, dark gray bars), and giant (1 × 103-5 × 103 nm, white bars). For each sample, spectra were recorded three times, including 11 subruns, using multimodal mode. In the graph, the concentrations of oligomers belonging to different families are reported as mean ± SD (n=2). [Figure 20A]Figure 20. Inhibitory and bactericidal effects of TCP-25 of SEQ ID NO:1 in various formulations. (A) Representative photographs of test tubes containing 1% TCP-25 and 1.5% HEC gel formulations made in Tris or acetate buffer (supplemented with 2 or 1.9% glycerol to make isotonic, respectively) with or without 2.5 mM EDTA. (B-C) Schematic representations of MIC (B) and MBC (C) values ​​obtained for Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli after treatment with TCP-25 in Tris or acetate buffer supplemented with various concentrations of EDTA. [Figure 20B] Figure 20. Inhibitory and bactericidal effects of TCP-25 of SEQ ID NO:1 in various formulations. (A) Representative photographs of test tubes containing 1% TCP-25 and 1.5% HEC gel formulations made in Tris or acetate buffer (supplemented with 2 or 1.9% glycerol to make isotonic, respectively) with or without 2.5 mM EDTA. (B-C) Schematic representations of MIC (B) and MBC (C) values ​​obtained for Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli after treatment with TCP-25 in Tris or acetate buffer supplemented with various concentrations of EDTA. [Figure 20C] Figure 20. Inhibitory and bactericidal effects of TCP-25 of SEQ ID NO:1 in various formulations. (A) Representative photographs of test tubes containing 1% TCP-25 and 1.5% HEC gel formulations made in Tris or acetate buffer (supplemented with 2 or 1.9% glycerol to make isotonic, respectively) with or without 2.5 mM EDTA. (B-C) Schematic representations of MIC (B) and MBC (C) values ​​obtained for Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli after treatment with TCP-25 in Tris or acetate buffer supplemented with various concentrations of EDTA. [Figure 21]Figure 21. Antibacterial efficacy of TCP-25 in various formulations. Bar graph demonstrating the bactericidal efficacy of 80 μM TCP-25 alone or in combination with 2.5 mM EDTA in either Tris or acetate buffer. CFU / ml of P. aeruginosa was determined using the VCA assay and a 1-hour treatment time (n=4). Data are presented as mean ± SEM. One-way ANOVA with multiple comparisons was used to determine p-values. **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 22] Figure 22. Bacterial cell aggregation when treated with TCP-25 and / or EDTA. Heatmap demonstrating the distribution of aggregated bacterial cells according to area. Distribution is expressed as a percentage of total aggregated amount. Single cells or aggregates smaller than 20 μm2 are not represented here. n=3. 10 images from each treatment for each replicate. [Figure 23A] Figure 23. Antibacterial effect of TCP-25 against Staphylococcus aureus in a time-kill assay. A) The graph shows bacterial growth over a 24-hour period. Formulations contained 80 μM TCP-25 in either Tris or acetate buffer with or without 2.5 mM EDTA. Samples were taken at 5, 15, and 30 minutes and at 1, 3, 6, and 24 hours. Results are expressed as CFU / ml. B) The size of bacterial aggregates in the Live / Dead assay is shown as a heat map. The relative abundance of aggregates for each size class is expressed as a percentage of the total number of aggregates. Single cells or aggregates smaller than 20 μm were excluded. Aggregates are representative of 10 images taken from each replicate sample (n=3). [Figure 23B]Figure 23. Antibacterial effect of TCP-25 against Staphylococcus aureus in a time-kill assay. A) The graph shows bacterial growth over a 24-hour period. Formulations contained 80 μM TCP-25 in either Tris or acetate buffer with or without 2.5 mM EDTA. Samples were taken at 5, 15, and 30 minutes and at 1, 3, 6, and 24 hours. Results are expressed as CFU / ml. B) The size of bacterial aggregates in the Live / Dead assay is shown as a heat map. The relative abundance of aggregates for each size class is expressed as a percentage of the total number of aggregates. Single cells or aggregates smaller than 20 μm were excluded. Aggregates are representative of 10 images taken from each replicate sample (n=3). [Figure 24A] Figure 24 shows the antibacterial effect of TCP-25 against Pseudomonas aeruginosa in a time-kill assay. A) The graph shows bacterial growth over a 24-hour period. Formulations contained 80 μM TCP-25 in either Tris or acetate buffer with or without 2.5 mM EDTA. Samples were taken at 5, 15, and 30 minutes and at 1, 3, 6, and 24 hours. Results are expressed as CFU / ml. B) Bacterial aggregates imaged in the live / dead assay are presented in a heat map showing the percentage of aggregates of a particular size found in the sample. Single cells or aggregates smaller than 20 μm are not represented. Aggregates are representative of 10 images taken from each replicate sample (n=3). [Figure 24B]Figure 24 shows the antibacterial effect of TCP-25 against Pseudomonas aeruginosa in a time-kill assay. A) The graph shows bacterial growth over a 24-hour period. Formulations contained 80 μM TCP-25 in either Tris or acetate buffer with or without 2.5 mM EDTA. Samples were taken at 5, 15, and 30 minutes and at 1, 3, 6, and 24 hours. Results are expressed as CFU / ml. B) Bacterial aggregates imaged in the live / dead assay are presented in a heat map showing the percentage of aggregates of a particular size found in the sample. Single cells or aggregates smaller than 20 μm are not represented. Aggregates are representative of 10 images taken from each replicate sample (n=3). [Figure 25A] Figure 25. EDTA enhances TCP-25-mediated reduction of biofilm-associated bacteria. A-B) Bar graphs demonstrating the reduction of bacterial load within 48-hour mature biofilms. Biofilms were exposed to solution (A) or gel formulation (B) containing 0.1% TCP-25 in 10 mM Tris, pH 7.4 or 10 mM Tris, pH 5, with or without 2.5 mM EDTA. C) CFU / ml from 48-hour mature biofilms was counted after treatment with the formulation in solution form. D) CFU / ml from 48-hour mature biofilms treated with TCP-25 and EDTA in 1.5% HEC gel in either 25 mM Tris or 25 mM acetate with 1.9% glycerol. Data are presented as mean ± SEM. One-way analysis of variance with Tukey's post hoc multiple comparisons was used to determine p-values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. [Figure 25B]Figure 25. EDTA enhances TCP-25-mediated reduction of biofilm-associated bacteria. A-B) Bar graphs demonstrating the reduction of bacterial load within 48-hour mature biofilms. Biofilms were exposed to solution (A) or gel formulation (B) containing 0.1% TCP-25 in 10 mM Tris, pH 7.4, or 10 mM Tris, pH 5.0, with or without 2.5 mM EDTA. C) CFU / ml from 48-hour mature biofilms was counted after treatment with the formulation in solution form. D) CFU / ml from 48-hour mature biofilms treated with TCP-25 and EDTA in 1.5% HEC gel in either 25 mM Tris or 25 mM acetate with 1.9% glycerol. Data are presented as mean ± SEM. One-way analysis of variance with Tukey's post hoc multiple comparisons was used to determine p-values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. [Figure 25C] Figure 25. EDTA enhances TCP-25-mediated reduction of biofilm-associated bacteria. A-B) Bar graphs demonstrating the reduction of bacterial load within 48-hour mature biofilms. Biofilms were exposed to solution (A) or gel formulation (B) containing 0.1% TCP-25 in 10 mM Tris, pH 7.4, or 10 mM Tris, pH 5.0, with or without 2.5 mM EDTA. C) CFU / ml from 48-hour mature biofilms was counted after treatment with the formulation in solution form. D) CFU / ml from 48-hour mature biofilms treated with TCP-25 and EDTA in 1.5% HEC gel in either 25 mM Tris or 25 mM acetate with 1.9% glycerol. Data are presented as mean ± SEM. One-way analysis of variance with Tukey's post hoc multiple comparisons was used to determine p-values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. [Figure 25D]Figure 25. EDTA enhances TCP-25-mediated reduction of biofilm-associated bacteria. A-B) Bar graphs demonstrating the reduction of bacterial load within 48-hour mature biofilms. Biofilms were exposed to solution (A) or gel formulation (B) containing 0.1% TCP-25 in 10 mM Tris, pH 7.4, or 10 mM Tris, pH 5.0, with or without 2.5 mM EDTA. C) CFU / ml from 48-hour mature biofilms was counted after treatment with the formulation in solution form. D) CFU / ml from 48-hour mature biofilms treated with TCP-25 and EDTA in 1.5% HEC gel in either 25 mM Tris or 25 mM acetate with 1.9% glycerol. Data are presented as mean ± SEM. One-way analysis of variance with Tukey's post hoc multiple comparisons was used to determine p-values. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. [Figure 26A] Figure 26. Effect of TCP-25 formulations in a porcine skin wound infection model. A) Dose-dependent antimicrobial effect of TCP-25 on Pseudomonas aeruginosa CFU / ml of infected ex vivo pig skin at the wound surface and in the tissue after treatment with increasing doses (0.1, 0.5, or 1%) of TCP-25 in a Tris-based hydrogel (1.5% HEC and 2% glycerol). B) The indicated concentrations of EDTA were added to 0.1% TCP-25 formulated in acetate buffer (1.5% HEC and 2% glycerol) at pH 5.0. The Pseudomonas aeruginosa CFU / ml on the surface and in the tissue after treatment was determined. A 0.1% TCP-25 hydrogel at pH 7.4 was used for comparison. C) Dose-dependent effect of TCP-25 in the presence of 10 mM EDTA. The CFU / ml of Pseudomonas aeruginosa and Staphylococcus aureus from surface and tissue samples 2 hours after treatment was determined. Data are presented as mean ± SEM. One-way analysis of variance with Tukey post hoc multiple comparisons was used to determine p-values. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 26B]Figure 26. Effect of TCP-25 formulations in a porcine skin wound infection model. A) Dose-dependent antimicrobial effect of TCP-25 on Pseudomonas aeruginosa CFU / ml of infected ex vivo pig skin at the wound surface and in the tissue after treatment with increasing doses (0.1, 0.5, or 1%) of TCP-25 in a Tris-based hydrogel (1.5% HEC and 2% glycerol). B) The indicated concentrations of EDTA were added to 0.1% TCP-25 formulated in a pH 5.0 acetate buffer (1.5% HEC and 2% glycerol). The Pseudomonas aeruginosa CFU / ml in the surface and tissue after treatment was determined. A 0.1% TCP-25 hydrogel at pH 7.4 was used for comparison. C) Dose-dependent effect of TCP-25 in the presence of 10 mM EDTA. The CFU / ml of Pseudomonas aeruginosa and Staphylococcus aureus on the surface and from tissue samples 2 hours after treatment was determined. Data are presented as mean ± SEM. One-way analysis of variance with Tukey post hoc multiple comparisons was used to determine p-values. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 26C] Figure 26. Effect of TCP-25 formulations in a porcine skin wound infection model. A) Dose-dependent antimicrobial effect of TCP-25 on Pseudomonas aeruginosa CFU / ml of infected ex vivo pig skin at the wound surface and in the tissue after treatment with increasing doses (0.1, 0.5, or 1%) of TCP-25 in a Tris-based hydrogel (1.5% HEC and 2% glycerol). B) The indicated concentrations of EDTA were added to 0.1% TCP-25 formulated in a pH 5.0 acetate buffer (1.5% HEC and 2% glycerol). The Pseudomonas aeruginosa CFU / ml in the surface and tissue after treatment was determined. A 0.1% TCP-25 hydrogel at pH 7.4 was used for comparison. C) Dose-dependent effect of TCP-25 in the presence of 10 mM EDTA. The CFU / ml of Pseudomonas aeruginosa and Staphylococcus aureus on the surface and from tissue samples 2 hours after treatment was determined. Data are presented as mean ± SEM. One-way analysis of variance with Tukey post hoc multiple comparisons was used to determine p-values. *P≦0.05; **P≦0.01; ***P≦0.001; ****P≦0.0001. [Figure 27]Figure 27. Stability of TCP-25 in acetate buffer with or without EDTA. The peptide was dissolved at 0.1% in acetate buffer (pH 5) with or without EDTA and analyzed by reverse-phase C18 chromatography after storage at RT, 4, or 37°C. Data are expressed as percentages of the total area (100%), which corresponds to the sum of the areas of all eluted peaks. The amount of TCP-25 after storage is represented by black bars, and degradation products are represented by white bars. na not analyzed; w weeks; ms months. [Figure 28] Figure 28. Stability of TCP-25 at different pH. The peptide was dissolved at 0.1% in distilled water, and then the pH was adjusted by adding NaOH or HCl to reach the indicated pH. Samples were then analyzed by reverse-phase C18 chromatography after storage at RT, 4, 37, or 70°C. Data are expressed as percentages of the total area (100%), which corresponds to the sum of the areas of all eluted peaks. The amount of TCP-25 after storage is represented by the black bars, and degradation products are represented by the white bars. d days; w weeks; ms months. [Figure 29] Figure 29 shows a summary of the stability and antimicrobial activity of TCP-25 at different pH and concentrations in the presence and absence of EDTA. At pH 7.4, concentrations of TCP-25 greater than 0.1% increase stability, likely due to oligomerization. At pH 5, EDTA significantly amplifies antimicrobial activity. At pH 5, EDTA increases stability, likely due to oligomer formation with EDTA. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention definition As used herein, unless otherwise specified, "a" or "an" means "one or more."

[0026] As used herein, the term "approximately" when used in connection with a numerical value refers to ±10%, preferably ±5%, and more preferably ±1%.

[0027] As used herein, the term "amino acid" includes the twenty standard amino acids and their corresponding stereoisomers in the "D" form (as compared to the naturally occurring "L" form), other naturally occurring amino acids of the omega amino acids, unconventional amino acids (e.g., α,α-disubstituted amino acids, N-alkyl amino acids, etc.), and chemically derivatized amino acids (see below).

[0028] The term "standard amino acid" refers to any of the 20 genetically encoded amino acids commonly found in naturally occurring peptides. Standard amino acids are referred to herein by both the IUPAC single-letter and three-letter codes. The term "standard amino acid" is used to refer to both free standard amino acids and standard amino acids incorporated into peptides. In the peptides shown, each coded amino acid residue is represented by a single-letter designation, where appropriate.

[0029] As used herein, the term "EDTA" refers to ethylenediaminetetraacetic acid.

[0030] The term "pour point" as used herein refers to the value of the shear stress at the crossover point G' = G", where G' is the storage modulus and G" is the loss modulus at 1 Hz frequency and 25°C. For example, the pour point can be determined using a Kinexus Pro rheometer (Malvern Panalytical Ltd., Malvern, UK) equipped with a plate-plate geometry and a 1 mm gap. A shear strain of 0.001 to 10 strain is applied to determine the linear viscoelastic region (LVR) and the pour point (shear stress at the G' and G" crossovers) at 1 Hz and 25°C. The pour point is determined directly by rheometry. In some examples, the pour point is provided as the strain at the G' and G" crossovers, but unless otherwise indicated, the pour point is typically the shear stress at the G' and G" crossovers, in Pa.

[0031] As used herein, the term "hydrogel" refers to a continuous phase of an aqueous solution and a hydrophilic polymer that can swell upon contact with water. A "hydrogel" comprises nanostructures formed from the polymer and water, typically containing more than 90% water. Hydrogels are typically transparent or translucent regardless of the degree of hydration. Hydrogels are generally distinguishable from hydrocolloids, which typically comprise a hydrophobic matrix containing dispersed hydrophilic particles. Hydrogels typically have a flow point of at least 10 Pa, at least 15 Pa, for example, in the range of 10-80 Pa, or in the range of 40-60 Pa.

[0032] As used herein, the term "hydrophilic polymer" refers to a polymer characterized by being soluble and compatible in water. Typically, hydrophilic polymers possess a polymer backbone composed of carbon and hydrogen, and generally possess a high percentage of oxygen either in the main polymer backbone or in pendant groups substituted along the polymer backbone.

[0033] As used herein, the term " local administration " refers to any form of administration of the composition of the present invention directly to the intended area of ​​the body to be treated.In many cases, the local administration will be the external administration directly to the site of injury.For example, when the injury is a wound, the local administration implies that the composition is directly applied to the wound.

[0034] As used herein, the term "nonionic polymer" refers to a polymer that, in a protic solvent at room temperature and under 1 atm pressure, is substantially free of structural units having cationic or anionic groups that must be counterbalanced by counterions to maintain electroneutrality. Specifically, a "nonionic polymer" according to the present invention may be a hydrophilic polymer that does not contain monomer units having ionizable functional groups, such as acidic or basic groups. Such a polymer will not be charged in aqueous solution.

[0035] As used herein, the term "polymer capable of forming a hydrogel" refers to a hydrophilic polymer that can swell upon contact with water. Useful polymers will absorb at least 10 times, preferably at least 50 times, such as in the range of 50-200 times, the weight of the polymer in anhydrous form.

[0036] As used herein, the term "sequence identity" refers to the percentage of identical amino acids or nucleotides between a candidate sequence and a reference sequence after alignment. Thus, for a candidate sequence to share 80% amino acid identity with a reference sequence, after alignment, 80% of the amino acids in the candidate sequence must be identical to the corresponding amino acids in the reference sequence. Identity according to the present invention is determined with the aid of computer analysis, including but not limited to, the Clustal Omega computer alignment program for aligning polypeptide sequences (Sievers et al. (2011 October 11) Molecular Systems Biology 7:539, PMID:21988835; Li et al. (2015 April 06) Nucleic Acids Research 43(W1):W580-4 PMID:25845596; McWilliam et al., (2013 May 13) Nucleic Acids Research 41(Web Server issue):W597-600 PMID:23671338) and the default parameters suggested therein. The Clustal Omega software is available from EMBL-EBI at https: / / www.ebi.ac.uk / Tools / msa / clustalo / . Using this program with default settings, the mature (biologically active) portion of the query and the reference polypeptide are aligned. The number of completely conserved residues is counted and divided by the length of the reference polypeptide. The MUSCLE or MAFFT algorithm may be used for aligning nucleotide sequences. Sequence identity may be calculated in a similar manner to that shown for amino acid sequences. The sequence identity provided herein is therefore calculated over the entire length of the reference sequence.

[0037] As used herein, the term "topical administration" or "administering topically" refers to the application of a composition to an external surface of a patient, particularly to the skin or mucous membrane. Preferably, the external surface is skin, and topical administration includes application of the composition to intact skin, broken skin, raw skin, or an incised skin wound.

[0038] As used herein, the term "treatment" refers to any type of treatment or prevention of a disorder, including ameliorating a subject's disorder (e.g., one or more symptoms), delaying the progression of a disorder, delaying the onset of symptoms, or slowing the progression of symptoms. Treatment may also be ameliorative or curative treatment. Thus, the term "treatment" encompasses prophylactic treatment of an individual to prevent the onset of symptoms.

[0039] The term "denaturation" as used herein refers to a process of partial or total alteration of the natural secondary, and / or tertiary, and / or quaternary structure of a protein or nucleic acid, resulting in loss of biological activity. Denaturation can be induced by several factors, such as by application of external stress, for example, by heating or radiation, and / or by incubation with chemical denaturant(s), such as strong acid or base, concentrated inorganic salt, organic solvent (e.g., alcohol or chloroform). Examples of chemical denaturants include urea or guanidinium chloride (Gnd-HCl). The term "thermal denaturation" refers to denaturation induced by increasing the temperature. Chemical denaturation refers to incubating a peptide with a chemical denaturant, such as high concentrations of urea or guanidinium chloride (Gnd-HCl).

[0040] As used herein, the terms "Tm" and "Cm" refer to the midpoint of unfolding of a given peptide. It is the temperature at which both the folded and unfolded states exist equally in equilibrium (T m ) or chemical denaturant concentration (C m ) Tm and Cm may be determined, for example, as described in Example 7.

[0041] composition The present invention relates to a composition comprising a compound containing a TCP peptide, a non-ionic polymer capable of forming a hydrogel, and an aqueous solution. Examples of useful compounds containing a TCP peptide, non-ionic polymers, and aqueous solutions are described herein below.

[0042] The composition may preferably be in the form of a hydrogel or a viscous solution. The form of the composition depends on the intended use or application area. Preferably, the composition is a hydrogel. Hydrogels are useful for topical administration and may be readily useful for external administration. Furthermore, hydrogels, due to their high water content, are particularly suitable for use in the treatment methods of the present invention. If the composition is a viscous solution, it may be suitable for eye, ear, or nose drops or sprays. If the composition is a viscous solution, it may, for example, be applied to a product or absorbed by a product.

[0043] In embodiments of the invention, when the composition is a hydrogel, the hydrogel preferably has a flow point of at least 15 Pa, more preferably at least 25 Pa, such as in the range of 40-60 Pa. It is advantageous for hydrogels to have an appropriate flow point to be particularly useful for topical administration. Thus, in many cases, it is preferred that the hydrogel be sufficiently thick so that it remains largely at the site of administration.

[0044] It is preferred that the TCP peptide diffuses very slowly from the composition of the present invention. For example, it is preferred that the diffusion rate of the TCP peptide from the composition of the present invention into the adjacent buffer solution is so slow that at most 20%, for example at most 10%, of the TCP peptide diffuses into the buffer solution within 2 hours. This may be particularly true for embodiments of the present invention in which the composition is a hydrogel. The diffusion rate may be determined, for example, as described in the "TCP-25 Gel Diffusion" section of Example 1 below.

[0045] It is also preferred that the TCP peptide diffuses only slowly from the compositions of the invention when administered to an individual. Thus, upon topical administration of the composition, for example to a wound, it is preferred that less than 100 nM of TCP peptide be detectable in the plasma of the individual. This may be particularly true for embodiments of the invention in which the composition is a hydrogel. Furthermore, this may be particularly true for embodiments of the invention in which the composition contains TCP peptide in the range of 0.08-3 wt%, such as in the range of 0.1-2%.

[0046] The compositions may be used as is, for example, by topical administration directly to the site of the disorder to be treated. In particular, the compositions may be administered topically. Alternatively, the compositions may be used in conjunction with a product.

[0047] The composition should preferably be pharmaceutically acceptable, i.e., not toxic, and therefore may be provided as a pharmaceutical composition, although it is contemplated that the composition may not be pharmaceutically acceptable if it is to be used in a manner that does not involve contact with human or animal tissue, such as to disinfect an object.

[0048] The compositions may be subjected to conventional pharmaceutical operations, such as sterilization, and / or may contain conventional adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

[0049] Those skilled in the art will appreciate that the compositions of the present invention may be administered topically. Routes of administration include topical, ophthalmic, nasal, buccal, oral, vaginal, and rectal administration. In a preferred embodiment, the compositions of the present invention are for use in a method of treatment by topical administration.

[0050] The compositions are preferably administered to a patient in a pharmaceutically effective amount, by which is meant an amount sufficient to produce the desired effect in relation to the condition for which it is administered, i.e., an amount sufficient to provide the desired wound healing, antibacterial and / or anti-inflammatory effect.

[0051] Typically, the TCP peptide is present in the composition at a concentration of at least 0.01 wt%, more preferably in the range 0.01-5 wt%, such as 0.08-3 wt%, for example 0.1-2%.

[0052] The compositions of the present invention may have any desired pH, for example, in the range of 7-8, such as in the range of 4-8, such as in the range of 5-8. Compositions having a pH greater than 6, such as a pH greater than 7, such as in the range of 6-8, for example, in the range of 7-8, may be particularly stable even in the absence of EDTA. The compositions may be administered in a single dose or multiple doses. The compositions may be administered alone or in combination with other therapeutic agents.

[0053] Compositions containing EDTA In one embodiment, the present invention provides a composition comprising a compound comprising a TCP peptide, EDTA, and preferably also an aqueous buffer. Useful TCP peptides and aqueous buffers are described below.

[0054] In some embodiments, compositions comprising TCP peptides and EDTA preferably have a pH of at most 7, although in other embodiments, compositions comprising TCP peptides and EDTA may have any useful pH, such as a pH of at most 8, or in the range of 3-10, such as in the range of 3-8, such as in the range of 3.5-8, for example, in the range of 5-8.

[0055] The composition may also include a non-ionic polymer capable of forming a hydrogel. Useful non-ionic polymers are described below. In such embodiments, the composition will typically be in the form of a hydrogel.

[0056] In some embodiments, compositions containing EDTA also preferably contain TCP peptide at a high concentration, i.e., at least 0.08 wt%, such as at least 0.1 wt%, such as in the range of 0.08-3 wt%.

[0057] The composition may include any useful amount of EDTA, preferably at a concentration of at least 1 mM, such as in the range of 1-100 mM, preferably at least 1.5 mM, such as at least 2 mM, for example, in the range of 2-100 mM, such as in the range of 2-50 mM, such as in the range of 2-25 mM.

[0058] In embodiments of the invention in which the composition comprises a non-ionic polymer capable of forming a hydrogel, the composition may comprise at least 2 mM, such as at least 10 mM, e.g., at least 15 mM, such as in the range of 15-100 mM, e.g., in the range of 15-50 mM, etc. Preferably, the composition comprises at least 2 mM, such as in the range of 2-100 mM, e.g., in the range of 2-50 mM, of EDTA.

[0059] This is advantageous because it has been surprisingly shown that EDTA, which has essentially no or very limited antibacterial effect by itself, provides a synergistic effect in significantly improving the antibacterial effect of the composition comprising TCP peptide.Therefore, adding EDTA to the composition comprising TCP peptide leads to improved antimicrobial activity, particularly improved antibacterial effect against different types of bacteria and improved antibacterial effect against biofilm.For example, the bacteria can be gram-negative bacteria.

[0060] In some embodiments, it is preferred that compositions containing EDTA have a relatively low pH, as the synergistic antimicrobial effect may be more pronounced at low pH.

[0061] In one embodiment, the pH of the composition comprising EDTA and TCP peptide is preferably below 7, preferably below 6, and not greater than 5.5. The pH may also preferably be higher than 3, such as at least 3.5. The pH may therefore be in the range of 3 to 6, such as approximately 5. The desired pH may be obtained by using a suitable aqueous buffer, such as an acetate buffer, having the desired pH, as discussed below.

[0062] In some embodiments, it is preferred that the composition containing EDTA have a high concentration of TCP-25 peptide, for example, a concentration of at least 0.08 wt %, because the synergistic antimicrobial effect may be more pronounced in such a composition.

[0063] As noted above, the addition of EDTA to a composition containing TCP peptides results in synergistically improved antimicrobial activity. However, the addition of EDTA may also have other beneficial effects. For example, the addition of EDTA may significantly improve the stability of TCP peptides, especially at low pH.

[0064] Thus, in some embodiments of the present invention, compositions preferably contain a compound comprising a TCP peptide, as described below, and EDTA at a concentration of at least 1 mM, such as in the range of 1-100 mM, preferably at least 1.5 mM, such as at least 2 mM, for example, in the range of 2-100 mM, such as in the range of 2-50 mM, such as in the range of 2-25 mM. The formulation may have any useful pH, such as a pH of at most 8, for example, a pH of at most 7, such as a pH of less than 6, or a pH of less than 5.5. The pH may also be preferably greater than 3, but at least 3.5. Such compositions are particularly stable.

[0065] Thus, in one embodiment, the composition of the invention still contains at least 90%, such as at least 95%, of the original content of TCP peptide compounds after storage at 37°C for 2 months.

[0066] Thus, in one embodiment, the composition of the invention still contains at least 75%, such as at least 80%, such as at least 90%, at least 95%, etc. of the original content of TCP peptide compounds after storage at 37°C for 4 months.

[0067] Thus, in one embodiment, the composition of the invention still contains at least 70%, such as at least 80%, such as at least 90%, at least 95%, etc. of the original content of TCP peptide compounds after storage at 37°C for 6 months.

[0068] Thus, in one embodiment, the compositions of the invention still contain at least 90%, such as at least 95%, of the original content of TCP peptide compounds after 8 months of storage at room temperature.

[0069] Composition containing high concentrations of TCP peptides In one embodiment, the present invention provides a composition comprising a compound containing a high concentration of TCP peptides. Useful compounds and useful TCP peptides are described below.

[0070] The compound containing the TCP peptide at a high concentration may specifically contain the compound or the TCP peptide at a concentration of at least 0.08 wt%, for example, at least 0.1 wt%. Thus, the TCP peptide may be present in the composition at a concentration in the range of 0.08 to 3 wt%, for example, 0.1 to 2%.

[0071] The concentration of the TCP peptide may also be provided as a molar concentration. The conversion from wt% to molar concentration depends on the specific composition and the specific TCP peptide. A concentration of 0.1 wt% of the TCP-25 peptide of SEQ ID NO:1 corresponds to a concentration of 300 μM in most aqueous solutions. Thus, a compound containing such a high concentration of TCP peptide may specifically comprise a composition containing the compound or TCP peptide at a concentration of at least 0.2 mM, such as at least 0.25 mM, or at least 0.3 mM. Thus, the compositions of the present invention may contain a compound containing a TCP peptide in the range of 0.2 mM to 100 mM, such as in the range of 0.25 mM to 100 mM, or in the range of 0.3 mM to 100 mM.

[0072] Interestingly, the present invention demonstrates that TCP peptides may be more stable at higher concentrations. Without being bound by theory, it is believed that TCP peptides oligomerize at higher concentrations, such as at least 0.08 wt%, at least 0.1 wt%, at least 0.2 mM, at least 0.25 mM, at least 0.3 mM, etc., resulting in higher stability. The TCP peptide oligomers may have hydrodynamic diameters in the range of 0.2 nm to 10,000 nm, such as in the range of 0.4 nm to 8,000 nm, such as in the range of 5 nm to 6,000 nm, such as in the range of 20 nm to 5,000 nm, or in the range of 0.4 nm to 2,000 nm. The TCP peptide oligomers may have an increased α-helical structure.

[0073] Oligomerization of the TCP peptides may increase the antibacterial and / or anti-inflammatory activity of the composition.

[0074] Interestingly, TCP peptides, or compounds containing TCP peptides, may be more stable at higher concentrations, particularly they may be more stable to denaturation, e.g., they may be more stable to exposure to high temperatures, such as temperatures in the range of 20-100°C, e.g., 30-50°C, and / or incubation with high concentrations of denaturing agents.

[0075] The stability of the TCP peptides may be determined by any useful method, for example, the stability of the TCP peptides may be determined by measuring the T of the TCP peptides by measuring the intrinsic fluorescence of tryptophan. m This may be done, for example, as described in Example 7 hereinbelow. Alternatively, the stability of a TCP peptide may be measured by determining the C of the TCP peptide with respect to one or more chemical denaturants by measuring the intrinsic fluorescence of tryptophan. m The chemical denaturant may be, for example, urea and / or guanidinium chloride (Gnd-HCl). This may be performed, for example, as described in Example 7 herein below.

[0076] High T m and / or a high Cm indicates high stability. Thus, in some embodiments, the compositions of the present invention have a T for TCP peptide of at least 30°C, preferably at least 35°C, and more preferably at least 40°C. m wherein the Tm is preferably determined as described in Example 7 below. In some embodiments, the compositions of the invention have a C for TCP peptide of at least 0.8 M, preferably at least 1.0 M, more preferably at least 1.1 M. m urea and C above m urea is determined as described in Example 7 below. In some embodiments, the compositions of the invention have a C for TCP peptide of at least 0.8M, preferably at least 0.9M. m Gnd-HCI and C above m Gnd-HCI is determined as described in Example 7 below.

[0077] In one embodiment, the compositions of the invention still contain at least 90%, such as at least 95%, of the original content of TCP peptide compounds after storage at 37° C. for 2 months.

[0078] In one embodiment, the composition of the invention still contains at least 75%, such as at least 80%, for example at least 90% of the original content of TCP peptide compound after storage at 37°C for 4 months.

[0079] Thus, in one embodiment, the composition of the invention still contains at least 70%, such as at least 80%, for example at least 85% of its original content of TCP peptide compound after storage at 37°C for 6 months.

[0080] Thus, in one embodiment, the compositions of the invention still contain at least 90%, such as at least 95%, of the original content of TCP peptide compounds after 8 months of storage at room temperature.

[0081] Compositions containing high concentrations of compounds containing TCP peptides may also contain EDTA, and preferably an aqueous buffer, for example, as described herein above in the section "Compositions containing EDTA."

[0082] A composition containing a compound comprising a high concentration of TCP peptide may have any suitable pH, for example, a pH range of 4 to 8, a pH range of 5 to 8, for example, a pH range of 7 to 8. Compositions containing a compound comprising a high concentration of TCP peptide and having a pH greater than 6, such as a pH greater than 7, such as a pH range of 6 to 8, for example, a pH range of 7 to 8, may be particularly stable even in the absence of EDTA.

[0083] The composition containing a compound containing a high concentration of TCP peptide may also contain a non-ionic polymer capable of forming a hydrogel. Useful non-ionic polymers are described below. In some embodiments, the composition will typically be in the form of a hydrogel.

[0084] Nonionic polymers capable of forming hydrogels The present invention provides a composition comprising a non-ionic polymer capable of forming a hydrogel.

[0085] In the context of the present invention, nonionic polymers are understood to include polymers that do not substantially contain structural units with anionic or cationic groups that must be counterbalanced by counterions to maintain electroneutrality in protic solvents at room temperature and under a pressure of 1 atm. Cationic groups include, for example, quaternary ammonium groups and protonated amines. Anionic groups include, for example, carboxyl and sulfonic acid groups.

[0086] Nonionic polymers, also called nonionic hydrogel polymers, are polymers that can form hydrogels when mixed with an aqueous solution or aqueous buffer. Depending on the concentration of the polymer, the composition may be in the form of a viscous liquid or a hydrogel, i.e., a gel.

[0087] The non-ionic polymer must be hydrophilic, and therefore the non-ionic polymer is preferably hydroxylated.

[0088] Examples of suitable nonionic polymers for use in the method of the present invention are polyallyl alcohol, polyvinyl alcohol, polyacrylamide, polyethylene glycol (PEG), polyvinylpyrrolidone, starches such as corn starch and hydroxypropyl starch, alkylcelluloses such as C1-C6-alkylcelluloses including methylcellulose, ethylcellulose, and n-propylcellulose, hydroxyalkylcelluloses, preferably substituted alkylcelluloses including hydroxy-C1-C6-alkylcelluloses and hydroxy-C1-C6-alkyl-C1-C6-alkylcelluloses, such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxypropylmethylcellulose, and ethylhydroxyethylcellulose. Mixtures of the foregoing may also be used.

[0089] In one embodiment, the non-ionic polymer is selected from the group consisting of hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), poly(vinyl) alcohol (PVA), polyacrylamide (PA), polyethylene glycol (PEG) and polyvinylpyrrolidone, and mixtures thereof.

[0090] In one embodiment, the non-ionic polymer is selected from the group consisting of hydroxyalkylcelluloses, preferably hydroxy-C1-C6-alkylcelluloses or hydroxy-C1-C6-alkyl-C1-C6-alkylcelluloses.

[0091] Of the non-ionic polymers, hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC) are preferred.

[0092] In preferred embodiments, the concentration of nonionic polymer in the composition of the present invention is sufficient to obtain a composition having a pour point of at least 10 Pa, such as at least 15 Pa, for example in the range of 10 to 80 Pa, such as in the range of 40 to 60 Pa.

[0093] Pour point may be measured, for example, as detailed in the Examples section.

[0094] The composition of the present invention may in particular comprise the non-ionic polymer at a concentration of at least 0.05 wt%, such as at least 0.09 wt%, such as at least 0.1 wt%, for example at least 0.5 wt%, such as at least 0.8 wt%, for example at least 1.0 wt%, for example in the range of 0.09-4 wt%, such as in the range of 0.1-3%, more preferably in the range of 1.0-2.5 wt%. This may be the case, for example, when the non-ionic polymer is HEC.

[0095] In one embodiment, the concentration of nonionic polymer is sufficient to obtain a viscosity of the composition of at least 10 mPas, and preferably no more than 100,000, such as 14,000 mPas. At 10 mPas, the composition is typically in the form of a viscous solution, while at 100,000 mPas, the composition may be in the form of a thick gel.

[0096] aqueous solution The composition of the present invention may comprise an aqueous solution. In particular, the aqueous solution may be an aqueous buffer.

[0097] Aqueous solutions or aqueous buffers contain water. When the aqueous solution is an aqueous buffer, it also contains buffer system components, i.e., weak bases or acids, and their conjugated acids and bases, to obtain an aqueous buffer that can provide a generally stable pH. Examples of buffers include Trizma, bicine, tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazole lactate, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO, and TES.

[0098] In one embodiment, the pH of the composition is preferably less than 7, preferably less than 6, more preferably less than 5.5, and preferably greater than 3, such as at least 3.5. Thus, the pH may range from 3 to 6, such as approximately 5. This is advantageous because, surprisingly, lower pH has been shown to significantly increase the antimicrobial efficacy of hydrogel compositions, particularly in the presence of EDTA. Furthermore, a pH below 7 also increases the solubility of the TCP peptide. Solubility may be determined by visual inspection, for example, as described in Example 4. The desired pH may be achieved by using an appropriate aqueous buffer having the desired pH, as discussed above.

[0099] In such embodiments, the aqueous solution or buffer may be an acetate buffer containing acetate, preferably at a concentration of 5 to 50 mM, more preferably at a concentration in the range of 10 to 30 mM, and may have a pH of, for example, about 5, such as in the range of 3 to 6, such as in the range of 3.6 to 5.8.

[0100] Acetate buffers, such as sodium acetate buffer, are particularly suitable for achieving a pH of 3.6 to 5.8. Typically, a 10 mM sodium acetate buffer will achieve a pH of 5; see Example 2. However, as explained above, other aqueous buffers can be used to achieve the desired pH.

[0101] In another embodiment, the composition has a pH between 7 and 8, preferably pH 7.4.

[0102] A neutral pH may be preferred in some instances as it provides the composition with a pH that is closer to that of the human or animal body, thereby reducing the risk of irritation from the composition when administered to a human or animal.

[0103] In such embodiments, the aqueous solution or aqueous buffer may be a trisaminomethane (Tris) buffer containing trisaminomethane, preferably at a concentration in the range of 5-50 mM, such as 10-30 mM.

[0104] Tris buffer may be used, for example, to obtain a pH of approximately 7.4, however, as explained above, other aqueous buffers may be used to obtain the desired pH.

[0105] The aqueous solution or aqueous buffer may additionally or alternatively contain further components such as diluents, adjuvants, tonicity adjusters and / or excipients. Generally, such further components should be pharmaceutically acceptable.

[0106] The term "diluent" is intended to mean an aqueous or non-aqueous solution having the purpose of diluting the peptide in the composition. The diluent may be one or more of saline, polyethylene glycol, propylene glycol, ethanol or oil (such as sunflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

[0107] The term "adjuvant" is intended to mean any compound added to a formulation to increase the biological effect of a peptide.The adjuvant can be colloidal silver, or zinc, copper, or silver salts with different anions, such as, but not limited to, one or more of fluoride, chloride, bromide, iodide, thiocyanide, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetate with different acyl compositions.The adjuvant can also be a compound with antibacterial and / or anti-inflammatory properties.

[0108] In one embodiment, it is preferred that the composition of the present invention does not include any ionic polymers. In particular, it would be preferred that the composition does not include any cationic polymers.

[0109] The "excipient" may be any useful excipient such as one or more of a polymer, lipid, and mineral.

[0110] The term "tonicity modifier" refers to a compound capable of adjusting the tonicity of a composition. Generally, it is preferred that the compositions of the present invention are either isotonic or slightly hypotonic. A hypotonic composition can have a tonicity that is, for example, 50-99% of isotonic. A tonicity modifier can be, for example, a salt or glycerol.

[0111] In one embodiment, the composition further comprises glycerol, preferably at a concentration of 1-2.5%, preferably 1.2-2.2 vol%. The concentration is provided as the concentration in the composition, e.g., in the hydrogel. Generally, a composition containing 2% glycerol is isotonic, and thus, in some embodiments, the composition may comprise approximately 2% glycerol. However, in other embodiments, the composition may be hypotonic, in which case it may comprise glycerol in the range of 1.2-1.9%.

[0112] TCP peptide-containing compounds The present invention relates to compositions comprising compounds containing TCP peptides. Useful TCP peptides are described in the TCP peptides section below. In some embodiments, the compounds may consist of the TCP peptides, such as TCP-25. However, in other embodiments, the compounds may comprise TCP peptides conjugated to one or more additional moieties. For example, the TCP peptides may comprise one or more amino acids that have been modified or derivatized, for example, by PEGylation, amidation, esterification, acylation, acetylation, and / or alkylation.

[0113] For example, TCP peptides (eg, TCP-25) may be modified or derivatized as described in International Patent Application WO2011 / 036442, pages 11, I.1 to 15, I.14.

[0114] The compounds may also be pharmaceutically acceptable acid or base addition salts of TCP peptides. The acids used to prepare pharmaceutically acceptable acid addition salts of TCP peptides are particularly those that form non-toxic acid addition salts, i.e., salts containing pharmaceutically acceptable anions, such as hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, acid, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthoate) salts).

[0115] TCP peptide The present invention relates to compositions comprising thrombin-derived C-terminal (TCP) peptides. As used herein, the term "TCP peptide" refers to Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A peptide comprising or consisting of It refers to a peptide having a length of 10 to 100, for example 20 to 100, 18 to 35, etc., for example 18 to 25 amino acid residues.

[0116] In one embodiment, the TCP peptide is Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 -X 11 -X 12 -X 13 (where, X4, 6, 9、11 is any standard amino acid, X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H, X 12 is I, M or T, X 13 is D, K, Q or R) comprising or consisting of It has a length of 20 to 100, 18 to 35, for example, 18 to 25 amino acid residues.

[0117] In one embodiment, the TCP peptide has the amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 -X 11 -X12 -X 13 -X 14 -X 15 -X 16 -X 17 (where, X4, 6, 9、11、14、15 is any standard amino acid, X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H, X 12 is I, M or T, X 13 is D, K, Q or R, X 16 is G or D, X 17 is E, L, G, R or K) comprising or consisting of It has a length of 20 to 100, 18 to 35, for example, 18 to 25 amino acid residues.

[0118] In one embodiment, the TCP peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE(SEQ ID NO1), FYTHVFRLKKWIQKVIDQFGE(SEQ ID NO2), GKYGFYTHVFRLKKWIQKVI(SEQ ID NO3), HVFRLKKWIQKVIDQFGE(SEQ ID NO4), KYGFYTHVFRLKKWIQKVIDQFGE(SEQ ID NO:5), GKYGFYTHVFRLKKWIQKVIDQF(SEQ ID NO:6), GKYGFYTHVFRLKKWIQKV(SEQ ID NO:7) It comprises or consists of any of the following:

[0119] In one embodiment, the TCP peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE(SEQ ID NO1), FYTHVFRLKKWIQKVIDQFGE(SEQ ID NO2), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO3), or HVFRLKKWIQKVIDQFGE(SEQ ID NO4) It comprises or consists of any of the following:

[0120] In one preferred embodiment, the TCP peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), or GKYGFYTHVFRLKKWIQKVI(SEQ ID NO:3) It comprises or consists of any of the following:

[0121] It is preferred that the TCP peptide be capable of simultaneously binding to both lipopolysaccharide and the LPS-binding hydrophobic pocket of CD14.

[0122] The Examples section demonstrates the antibacterial effects of several TCP peptides, including TCP-25. As used herein, the term "TCP-25" refers to a peptide consisting of the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1). The Examples section further demonstrates that TCP-25 can be cleaved into several peptides, including FYT21, GKY20, and HVF18, i.e., SEQ ID NOs: 2, 3, and 4, and that these peptides similar to TCP-25 are also antibacterial. The above peptides also contain amino acid sequences required for both lipopolysaccharide (LPS) binding and CD14 binding. Thus, in some embodiments, a TCP peptide may comprise or consist of any of these peptides (TCP-25, FYT21, GKY20, and HVF18). Preferably, the peptides are 18 to 25 amino acids in length, but as long as the peptide is based on any of these peptides, the peptide may be up to 35 amino acids in length.

[0123] In another preferred embodiment, the peptide has at least 90% sequence identity with the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO. 1), preferably the TCP-25 peptide has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO. 1).

[0124] Because different amino acids may have side chains that provide similar properties, and the biological effects of a peptide are caused by the side chains of multiple amino acids, e.g., hydrophobic side chains, cooperating to provide a specific conformation or local chemical / electronic environment, a peptide may alternatively have at least 90% sequence identity with the TCP-25 sequence. Thus, a peptide may correspond to a TCP-25 peptide in which one or more, up to 1 / 10, i.e., up to three, amino acids in the TCP-25 peptide have been replaced by other amino acids. Despite such replacements, the general activity of the peptide will be similar to that of the TCP-25 peptide (SEQ ID NO: 1).

[0125] It is preferred that the TCP peptide be capable of simultaneously binding to both lipopolysaccharide and the LPS-binding hydrophobic pocket of CD14.

[0126] Treatment method The compositions of the present invention may be for use in a method of treatment. In particular, the compositions may be for use in a method of local treatment of a disorder. The disorder may be any disorder for which local treatment is appropriate. Thus, the method may involve topical administration of the compositions of the present invention directly to the local area affected by the disorder.

[0127] For example, the disorder may be a disorder of the skin, ear, eye or nose, and the method of treatment may therefore involve topical administration to the affected area of ​​the skin, ear, eye or nose.

[0128] In one embodiment, the method of treatment involves topical administration, for example, topical administration to the skin.

[0129] In a preferred embodiment, the composition of the present invention is for use in a method for the treatment of skin disorders. In particular, the composition may be for a method for treating wounds.

[0130] The compositions or products of the present invention may be applied directly to the skin or wound. As shown in the examples, the compositions of the present invention are antibacterial, reduce inflammation, and provide faster and better wound healing compared to prior art techniques and products.

[0131] In a preferred embodiment, the method of wound treatment includes methods of treating burn wounds and non-healing ulcers, hi another embodiment, the method of wound treatment includes methods of treating surgical wounds.

[0132] These types of wounds may require specialized measures, and the compositions of the present invention are particularly useful in treating such wounds because they provide both antibacterial and anti-inflammatory effects.

[0133] The composition or product may, for example, be applied directly to the wound or may be applied in the form of any of the products described herein, for example, as a bandage or suture to treat a surgical wound, etc.

[0134] The disorder to be treated may in particular be a disorder involving inflammation, or a disorder associated with inflammation, or a disorder at risk of suffering from inflammation. In particular, the disorder may be a disorder involving or associated with local inflammation.

[0135] The disorder to be treated may in particular be a disorder involving an infection, or a disorder associated with an infection, or a disorder at risk of contracting an infection. In particular, the disorder may be a disorder involving or associated with a local infection. The infection may in particular be a bacterial infection, i.e., a bacterial infection.

[0136] The bacterium may be any infectious bacterium. For example, the bacterium may be a gram-negative or gram-positive bacterium. Thus, the bacterium may belong to a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter, Haemophilus, Pseudomonas, and Citrobacter. In one embodiment, the bacterium is selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. In another embodiment, the bacterium is a gram-negative bacterium.

[0137] The bacteria may be multidrug-resistant. Surprisingly, the compositions (and thus products) of the present invention can provide antibacterial effects against multiple multidrug-resistant bacteria, i.e., bacteria that are resistant to multiple known antibiotics. The compositions therefore provide additional methods of treating these bacteria, including treating wounds infected by these bacteria.

[0138] The individual in need of treatment may be any individual. Typically, the individual is a mammal, and preferably, the individual is a human. In one embodiment, the individual is an individual suffering from diabetes, arterial insufficiency, or venous insufficiency. Individuals suffering from diabetes, arterial insufficiency, or venous insufficiency often also suffer from non-healing ulcers, and the disorder may therefore be a non-healing ulcer in an individual suffering from diabetes, arterial insufficiency, or venous insufficiency.

[0139] In one embodiment, the composition or product is for use in a method for treating a skin, ear, eye, or nose disorder, such as treating a wound. The disorder may be selected from the group consisting of, for example, atopic dermatitis, impetigo, chronic skin ulcers, infected acute wounds and burn wounds, acne, otitis externa, fungal infections, pneumonia, seborrheic dermatitis, candidal intertrigo, candidal vaginitis, oropharyngeal candidiasis, eye infections, and nasal infections. Furthermore, the disorder may be a burn wound, a surgical wound, or a skin trauma.

[0140] The aforementioned disorders are often accompanied by complications such as bacterial infection and / or inflammation, and therefore would benefit from the anti-infective and anti-inflammatory treatment provided by the compositions of the present invention.

[0141] The treatment may be a ameliorative treatment, a curative treatment, and / or a preventive treatment. Thus, the composition of the present invention may be used in a method for reducing the risk of infection and / or inflammation associated with a disorder. For example, the composition may be administered to a wound to reduce the risk of infection and / or inflammation in the wound. However, the composition of the present invention may also be administered to an individual who is already suffering from local infection and / or inflammation.

[0142] product The present invention also provides a product comprising a composition according to the present invention. The product may be, for example, a product that can assist in the topical administration of the composition of the present invention.

[0143] In such embodiments, the product may be, for example, selected from the group consisting of a gel, a drop, a spray, a cream, a liquid, a wound cleansing solution, a contact lens solution, an ointment, a suture, a prosthesis, an implant, a wound dressing, a plaster, a catheter, a skin graft, a skin substitute, and a bandage.

[0144] Drops and sprays may be configured (i.e., formulated) to apply the composition to, for example, the ear, eye, or nose. The composition may be formulated, for example, as a viscous liquid that can be easily applied to the eye or ear, or as a hydrogel for easy application to the ear.

[0145] Products such as hydrogels, drops, sprays, wound dressings, plasters, skin substitutes, and bandages may be configured or formulated for administration of the composition to the skin or other epithelial surface or to a wound.

[0146] Generally, compositions and products may be formulated for topical administration, particularly for external application.

[0147] In such embodiments, the composition may be coated, painted or sprayed onto the product, or the composition may be adsorbed or absorbed by the product.

[0148] In doing so, the composition may impart antibacterial and anti-inflammatory properties to the product.

[0149] As used herein, the term "coated" refers to a composition applied to the surface of an article. Thus, the article may be painted or sprayed with a solution containing the composition. Alternatively, the article may be immersed in a reservoir of the composition.

[0150] Advantageously, the product is impregnated with the composition. By "impregnated" it is meant that the composition is absorbed or adsorbed onto the product.

[0151] item The present invention may be further defined by any one of the following: 1.c) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; d) a non-ionic polymer capable of forming a hydrogel when mixed with an aqueous solution; e) aqueous solutions, A composition comprising: 2.a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; b) a non-ionic polymer capable of forming a hydrogel when mixed with an aqueous solution; c) an aqueous solution; A composition comprising: i. the concentration of the compound in the composition is at least 0.08 wt %; and / or ii. the nonionic polymer is present in the composition at a concentration of at least 0.05 wt %; composition. 3. The composition according to any one of the preceding items, which is a hydrogel or a viscous solution, preferably a hydrogel. 4. The composition of any one of the preceding items, wherein the nonionic polymer is hydroxylated. 5. The composition of any one of the preceding items, wherein the nonionic polymer is selected from the group consisting of polyallyl alcohol, polyvinyl alcohol, polyacrylamide, polyethylene glycol (PEG), polyvinylpyrrolidone, starches such as corn starch and hydroxypropyl starch, alkylcelluloses such as C1-C6-alkylcelluloses including methylcellulose, ethylcellulose and n-propylcellulose; hydroxyalkylcelluloses, preferably substituted alkylcelluloses including hydroxy-C1-C6-alkylcelluloses and hydroxy-C1-C6-alkyl-C1-C6-alkylcelluloses such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose and mixtures of the foregoing. 6. The composition of any one of the preceding items, wherein the nonionic polymer is selected from the group consisting of hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose (HPMC), poly(vinyl) alcohol (PVA), polyacrylamide (PA), polyethylene glycol (PEG) and polyvinylpyrrolidone, and mixtures thereof. 7. The composition according to any one of the preceding items, wherein the nonionic polymer is selected from the group consisting of hydroxyalkyl celluloses, preferably from the group consisting of hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC). 8. The composition according to any one of the preceding items, wherein the concentration of the nonionic polymer in the composition of the present invention is sufficient to obtain a composition having a pour point of at least 10 Pa, at least 15 Pa, such as in the range of 10 to 80 Pa, such as in the range of 40 to 60 Pa. 9. The composition according to any one of the preceding items, wherein the nonionic polymer is present in the composition at a concentration of at least 0.05 wt%, such as at least 0.09 wt%, such as at least 0.1 wt%, for example at least 0.5 wt%, such as at least 0.8 wt%, for example at least 1.0 wt%, preferably in the range of 0.09 to 4 wt%, more preferably in the range of 1 to 3 wt%. 10. The composition of any one of the preceding items, wherein the nonionic polymer is present in the composition at a concentration of at least 1 wt%. 11. The composition according to any one of the preceding items, further comprising glycerol, preferably at a concentration of 1 to 3 vol%, more preferably at a concentration of 1 to 2 vol%. 12. The composition of any one of the preceding items, wherein the aqueous solution is an aqueous buffer. 13. The composition of any one of the preceding items, further comprising EDTA. 14.d) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; e) EDTA, f) aqueous buffers; Including, The composition has a pH of at most 7. 15.a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of a compound, wherein the peptide has a length of 10 to 100 amino acid residues; b) EDTA, c) aqueous buffers; A composition comprising: i. the composition has a pH of at most 8, and / or ii. The concentration of the compound in the composition is at least 0.08 wt%; composition. 16.a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) A compound comprising a peptide comprising or consisting of A composition comprising: The peptide has a length of 10 to 100 amino acid residues, The concentration of the compound in the composition is at least 0.01 wt%, preferably at least 0.08 wt%, for example in the range of 0.08 to 3 wt%; composition. 17.a) Amino acid sequence X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 (where, X4, 6, 9 are any standard amino acid; X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H) a compound comprising a peptide comprising or consisting of A composition comprising: The peptide has a length of 10 to 100 amino acid residues, The concentration of the compound in the composition is at least 0.2 mM, such as at least 0.25 mM, such as at least 0.3 mM; composition. 18. Peptides are made up of amino acid sequences X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 -X 11 -X 12 -X 13 (where, X4, 6, 9、11 is any standard amino acid, X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H, X 12 is I, M or T, X 13 is D, K, Q or R) comprising or consisting of having a length of 20 to 100 amino acid residues, The composition according to any one of the preceding items. 19. Peptides are made up of amino acid sequences X1-X2-X3-X4-X5-X6-W-X8-X9-X 10 -X 11 -X 12 -X 13 -X 14 -X 15 -X 16 -X 17 (where, X4, 6, 9、11、14、15 is any standard amino acid, X1 is I, L or V; X2 is any standard amino acid except C; X3 is A, E, Q, R or Y; X5 is any standard amino acid other than R; X8 is I or L; X 10 is any standard amino acid other than H, X 12 is I, M or T, X 13 is D, K, Q or R, X 16 is G or D, X 17 is E, L, G, R or K) comprising or consisting of having a length of 20 to 100 amino acid residues, The composition according to any one of the preceding items. 20. The peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE(SEQ ID NO1), FYTHVFRLKKWIQKVIDQFGE(SEQ ID NO2), GKYGFYTHVFRLKKWIQKVI(SEQ ID NO3), HVFRLKKWIQKVIDQFGE(SEQ ID NO4), KYGFYTHVFRLKKWIQKVIDQFGE(SEQ ID NO:5), GKYGFYTHVFRLKKWIQKVIDQF(SEQ ID NO:6), GKYGFYTHVFRLKKWIQKV(SEQ ID NO:7) Containing or consisting of any of the following: The composition according to any one of the preceding items. 21. The peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), or GKYGFYTHVFRLKKWIQKVI(SEQ ID NO:3) Containing or consisting of any of the following: The composition according to any one of the preceding items. 22. A peptide is a compound with an amino acid sequence Item 1. The composition of any one of the preceding items, having at least 90% sequence identity with GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO. 1), preferably wherein the peptide consists of the amino acid sequence GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO. 1). 23. The composition of any one of the preceding items, wherein the peptide is capable of simultaneously binding to both lipopolysaccharide and the LPS-binding hydrophobic pocket of CD14. 24. The composition according to any one of the preceding items, wherein the peptide is present in the composition at a concentration of at least 0.08 wt%, for example at least 0.1 wt%. 25. A composition according to any one of the preceding items, wherein the peptide is present in the composition at a concentration of at least 0.01 wt%, more preferably 0.01-5 wt%, 0.08-3 wt%, etc. 26. The composition according to any one of items 13 to 25, wherein EDTA is present in said composition at a concentration of at least 1 mM, such as in the range of 1 to 100 mM, preferably at least 1.5 mM, such as at least 2 mM, for example in the range of 2 to 100 mM, such as in the range of 2 to 25 mM. 27. The composition according to any one of items 13 to 25, wherein EDTA is present in said composition at a concentration of at least 2 mM, such as at least 10 mM, for example at least 15 mM, such as in the range of 15 to 100 mM, for example in the range of 15 to 50 mM EDTA. 28. A composition according to any one of the preceding items, wherein the pH of the composition is at most 7. 29. A composition according to any one of the preceding items, wherein the pH of the composition is lower than 7, preferably lower than 6, more preferably 5.5 or lower. 30. The composition according to any one of the preceding items, wherein the pH of the composition is lower than 7, preferably lower than 6, more preferably 5.5 or less, and higher than 3, at least 3.5. 31. A composition according to any one of the preceding items, wherein the pH of the composition is in the range of 3 to 6, such as approximately 5. 32. The composition according to any one of items 12 to 31, wherein the aqueous buffer is an acetate buffer containing acetate, preferably at a concentration of 10 to 50 mM, more preferably at a concentration of 25 mM, and having a pH of 3.6 to 5, such as 5. 33. The composition according to any one of items 1 to 27, wherein the pH of the composition is at most 8, such as in the range of 3 to 8, for example in the range of 3.5 to 8, such as in the range of 5 to 8. 34. The composition according to any one of items 1 to 27, wherein the pH of the composition is between 7 and 8, preferably approximately 7.4. 35. The composition according to any one of items 1 to 27 and 33 to 34, wherein the aqueous solution or aqueous buffer is a trisaminomethane (Tris) buffer containing trisaminomethane, preferably at a concentration of 5 to 20 mM, approximately 10 mM. 36. The peptide in the composition has a T of at least 30°C, preferably at least 35°C, and more preferably at least 40°C. m Item 1. The composition of any one of the preceding items, having: 37. The compound containing the peptide in the composition has a T of at least 30°C, preferably at least 35°C, and more preferably at least 40°C. m Item 1. The composition of any one of the preceding items, having: 38. The peptide in the composition has a C of at least 0.8M, preferably at least 1.0M, more preferably at least 1.1M. m urea Item 1. The composition of any one of the preceding items, having: 39. The compound containing the peptide in the composition has a C of at least 0.8M, preferably at least 1.0M, more preferably at least 1.1M. m urea Item 1. The composition of any one of the preceding items, having: 40. The peptide in the composition has a C of at least 0.8M, preferably at least 0.9M m Gnd-HCI Item 1. The composition of any one of the preceding items, having: 41. The compound containing the peptide in the composition has a C of at least 0.8M, preferably at least 0.9M m Gnd-HCI Item 1. The composition of any one of the preceding items, having: The composition of any one of the preceding items, comprising at least 90%, at least 95%, etc., of the original content of said compound, including said peptide, after storage at 42.37°C for 2 months. A composition according to any one of the preceding items, comprising at least 75%, such as at least 80%, for example at least 90%, of the original content of said compound, including said peptide, after storage at 43.37°C for 4 months. 44. The composition of any one of the preceding items, comprising at least 70%, such as at least 80%, for example at least 85%, of the initial content of said compound, including said peptide, after storage at 37°C for 6 months. 45. The composition of any one of the preceding items, comprising at least 90%, at least 95%, etc., of the initial content of said compound, including said peptide, after storage at room temperature for 8 months. 46. ​​A product comprising the composition described in any of the above items. 47. The product according to item 46, which is selected from the group consisting of gels, drops, sprays, creams, liquids, wound cleansing solutions, contact lens solutions, ointments, sutures, prostheses, implants, wound dressings, plasters, catheters, skin grafts, skin substitutes, and bandages. 48. The product according to any one of items 46 to 47, wherein the composition is coated, painted or sprayed onto the product, or adsorbed or absorbed by the product. 49. The composition according to any of items 1 to 45, or the product according to any one of items 46 to 48, for use in a method for treating a disorder in an individual in need thereof. 50. A composition for use according to item 49, prepared for topical administration. 51. Use of a composition according to any one of items 1 to 45 for the preparation of a medicament for the treatment of a disorder in an individual in need thereof. 52. The use according to item 51, wherein the composition is prepared for topical administration. 53. A method for the treatment of a disorder in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of the composition according to any one of items 1 to 45 or the product according to any one of items 46 to 48. 54. The method according to item 53, wherein the administration is topical administration. 55. A composition for use, use or method according to any one of items 49 to 54, wherein said treatment is selected from the group consisting of ameliorative treatment, curative treatment and preventive treatment. 56. The composition for use, use or method according to any one of items 49 to 55, wherein the disorder is a disorder of the skin, ear, eye or nose. 57. A composition, use or method for use according to any one of items 49 to 56, wherein the disorder is a wound. 58. A composition for use, use or method according to item 57, wherein the wound is selected from the group consisting of burns and non-healing ulcers. 59. A composition, use or method for use according to item 57, wherein the wound is a surgical wound. 60. A composition for use, use or method according to any one of items 49 to 59, wherein the disorder comprises or is associated with inflammation. 61. A composition for use, use or method according to any one of items 49 to 60, wherein the disorder comprises or is associated with a bacterial infection. 62. The composition, use or method for use according to item 61, wherein the bacteria is gram-negative or gram-positive. 63. The composition, use or method for use according to item 61, wherein the bacterium is a gram-negative bacterium. 64. The composition, use, or method for use according to item 61, wherein the bacterium is of a genus selected from the group consisting of Staphylococcus, Enterococcus, Streptococcus, Corynebacterium, Escherichia, Klebsiella, Stenotrophomonas, Shigella, Moraxella, Acinetobacter, Haemophilus, Pseudomonas, and Citrobacter. 65. The composition, use or method for use according to item 61, wherein the bacteria is selected from the group consisting of Staphylococcus aureus and Pseudomonas aeruginosa. 66. The composition for use, use or method according to items 61 to 65, wherein the bacterium is a multidrug-resistant bacterium. 67. The composition for use, use or method according to any one of items 49 to 66, wherein said individual suffers from diabetes, arterial insufficiency or venous insufficiency. 68. A composition for use, use or method according to any one of items 49 to 67, wherein said method of treatment is a topical medicinal treatment.

[0152] Example 1 The examples describe hydrogel formulations functionalized with TCP-25. The term "TCP-25" used in these examples refers to a peptide with the following sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1). The formulations are useful for dual targeting of bacteria and PAMP-induced inflammation. Using various in vitro assays, different TCP-25 gels were actively tested for efficacy against Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa, and various other clinical bacterial isolates. The dual antimicrobial and anti-inflammatory effects of TCP-25 gel were demonstrated in experimental mouse models of subcutaneous Staphylococcus aureus and Pseudomonas aeruginosa infection and in an NF-kB reporter mouse model of endotoxin-induced inflammation. The efficacy of TCP-25 gel was demonstrated in a preclinical porcine partial-thickness wound infection model. The pharmacokinetics of TCP-25 in the hydrogel were investigated in vitro, ex vivo, and in vivo using fluorescence spectroscopy, IVIS bioimaging, and mass spectrometry. To examine the fate of the active compound in the hydrogel, TCP-25 degradation was analyzed by mass spectrometry. The bioactivity of the major TCP-25 fragments was demonstrated by in vitro assays. Additionally, the stability of TCP-25 in the gel and in plasma after long-term storage was analyzed by mass spectrometry. Finally, the efficacy of TCP-25 gel treatment was compared with clinically utilized wound treatments in a preclinical porcine partial-thickness wound infection model. To further demonstrate clinical translation, the effect of TCP-25 on the pro-inflammatory activity of wound fluid from the infected porcine wound and from patients with non-healing wounds colonized with Staphylococcus aureus and Pseudomonas aeruginosa was evaluated using a monocyte model.

[0153] Materials and Methods Ethics Statement. All animal experiments were performed in accordance with the Swedish Animal Welfare Act SFS 1988:534 and approved by the Animal Ethics Committee of Malmö / Lund, Sweden (permit numbers M252-11, M131-16, M88-91 / 14, M5934-19, 8871-19). The use of human wound material was approved by the Ethics Committee at Lund University (LU708-01 and LU509-01).

[0154] Statistical Analysis. All microbial and cell culture-based assays represented biological replicates and were repeated at least three times. Data are presented as mean ± SEM. Clinical wound scoring is presented as median. Differences in means between two groups were analyzed using Student's t-test for normally distributed data and the Mann-Whitney test for others. To compare means between more than two groups, one-way analysis of variance with post hoc test (Tukey) or Kruskal-Wallis test with post hoc test (Dunn) was used for normally distributed data. Statistical analyses, indicated in each figure legend, were performed using GraphPad Prism software v8. A P value of <0.05 was considered statistically significant.

[0155] Peptides, buffers, and gel formulations. TCP-25 (97% purity, acetate salt) was synthesized by Ambiopharm (Madrid, Spain). Tetramethylrhodamine (TAMRA), cyanine 3 (Cy3), and cyanine 5 (Cy5)-labeled TCP-25 peptides were synthesized by Biopeptide (San Diego, CA, USA). The labels were added to the N-terminus of the peptide in all cases. The purity of the labeled peptides (95%) was confirmed by mass spectrometry (MALDI-TOF, Voyager, Applied Biosystems, Framingham, MA, USA). The gel-forming substances used were hydroxypropyl cellulose (HPC, Klucel™ MF, MW 850000; Ashland Industries Europe GmbH, Schaffhausen, Switzerland), hydroxyethyl cellulose (HEC, Natrosol™ 250HX, MW 1000000; Ashland Industries Europe GmbH, Schaffhausen, Switzerland), and carboxymethyl cellulose (CMC, Blanose™ 7HOF, MW 725000; Ashland Specialties, Alizé, France), and Pluronic F-127 (Pluronic® F127, MW 12600; Sigma-Aldrich Chemie GmbH, Steinheim, Germany).

[0156] Preparation of TCP-25 hydrogels. To prepare the gel formulations, HPC, HEC, CMC, or Pluronic F-127 was added to 10 mM Tris pH 7.4 with 1.3% glycerol (for the HEC mixture, the buffer was preheated to 56°C). A magnetic stirrer was used to continuously stir the solution until a homogeneous gel was formed. To remove air bubbles, the gel formulation was centrifuged (3.5 × 1000 rpm) for 3 minutes. The desired amount of TCP-25 peptide was then dissolved in 10 mM Tris (pH 7.4) and 1.3% glycerol buffer and then added to the gel, and the stirring and centrifugation steps were repeated. For initial screening of TCP-25 formulations, we used either 40 μM TCP-25 supplemented with 1% formulation (HPC, CMC, or Pluronic in Figure 1A), different TCP-25 concentrations supplemented with 0.5% polymer or Pluronic (in Figure 1B), or 10 μM TCP-25 with 0.1% polymer or Pluronic (in Figure 1C or D). Unless otherwise explicitly stated, the term "TCP-25 Gel #1" refers to a gel containing 0.1% TCP-25 (0.3 mM), 10 mM Tris-HCl pH 7.4, 1.3% glycerol, and 1.5% HEC. TCP-25 Gel #1 was used for the in vitro and in vivo experiments described in this example. In the porcine wound model, the gels contained 0.1% or 1% TCP-25 (0.3 or 3 mM, respectively), 10 mM Tris-HCl at pH 7.4, 1.3% glycerol, and 2% HEC polymer. To the extent that the gel contained 0.1% TCP-25, it is referred to herein as "TCP-25 Gel #2," and the gel containing 1% TCP-25 is referred to as TCP-25 Gel #3. The gel formulations were stored at 4°C until further use. For fluorescent imaging of TCP-25, the gels were spiked with 2% fluorescently labeled TCP-25 (labeled with TAMRA, Cy3, or Cy5).

[0157] Bacterial isolates. The bacterial strains used in this study were Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (PAO1 and ATCC 27853), Staphylococcus aureus (ATCC 29213), Staphylococcus epidermidis (ATCC 14990), and Enterococcus faecalis (ATCC 29212). The bioluminescent bacteria used in this study were Pseudomonas aeruginosa and Staphylococcus aureus. We also used clinical isolates of Staphylococcus aureus (1779, 1781, 2278, 2279, 2404, 2405, 2528, 2788, 2789), Pseudomonas aeruginosa (10.5, 13.2, 23.1, 27.1, 51.1, 62.2, 15159, 18488), Staphylococcus epidermidis (2282), and Escherichia faecalis (2374), which were derived from either skin or wound infections. These strains were obtained from the Department of Bacteriology, University Hospital, Lund, Sweden.

[0158] Radial diffusion assay (RDA). Bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) were grown to mid-logarithmic phase in 10 mL of full-strength (3% w / v) tryptic soy broth (TSB; Becton, Dickinson and Company, Sparks, MD, USA), centrifuged (5600 rpm × 10 min), and then resuspended in 10 mM Tris buffer. 4 × 10 6CFU were added to 15 mL of underlay agarose gel consisting of 0.03% (w / v) TSB, 1% (w / v) low electroosmotic (EEO) agarose (Sigma-Aldrich, St. Louis, MA, USA), and 0.02% (v / v) Tween 20 (Sigma-Aldrich), which was then placed in a 144 mm Petri dish. Plates were then prepared by punching 4 mm wells into the agarose gel using a biopsy punch. TCP-25 (6 μL) was then added to the wells above the agarose and incubated for 3 hours at 37°C and 5% CO2 to allow the peptide to diffuse into the gel. The underlay gel was covered with 15 mL of molten overlay (6% TSB and 1% low EEO agarose in distilled H2O), and the plate was then incubated for 24 hours at 37°C. The antibacterial activity of the peptides was visualized as a clear zone around each well and expressed as the zone diameter excluding the punch diameter (4 mm). To assess the antibacterial properties of degraded TCP-25, RDA plates were prepared (4 × 10 in 15 mL of underlay agarose gel as described above). 6 CFU E. coli), samples were loaded as described in the above section. Samples were prepared by mixing digested peptide solutions with 10 mM Tris, pH 7.4, with or without 0.15 M NaCl.

[0159] Viable count assay (VCA). Bacterial strains were grown to mid-logarithmic phase in Todd-Hewitt (TH) medium and then centrifuged (5600 rpm x 10 min). The bacterial pellet was then washed with 10 mM Tris, pH 7.4, recentrifuged for 10 min, and then resuspended in the same 10 mM Tris buffer. 1 x 10 E. coli, P. aeruginosa, and S. aureus were cultured in 50 μL of 10 mM Tris, pH 7.4. 7CFU were added to test tubes containing different TCP-25 gel formulations (0.5% HPC, CMC, or Pluronic F-127 mixed with either 0, 1, 2, 5, or 10 μM TCP-25). The tubes were then incubated for 2 hours at 37°C (5% CO). After incubation, 10-fold serial dilutions were performed, and 10 μL from each dilution was plated in six replicates onto TH broth agar plates and incubated overnight at 37°C (5% CO), after which CFU were determined.

[0160] Antibacterial effect of TCP-25 gel against bioluminescent bacteria. Bioluminescent Pseudomonas aeruginosa Xen41 and Staphylococcus aureus SAP229 were grown to mid-logarithmic phase in TH medium, after which they were washed in 10 mM Tris and 1.3% glycerol buffer at pH 7.4 (5600 rpm) for 20 min. Bacterial pellets were diluted in 10 mM Tris buffer, and 50 μL (2 × 10) of each strain was removed. 8 CFU / mL) were mixed with a gel formulation (1.5% HEC, 10 mM Tris pH 7.4, 1.3% glycerol) with or without 0.1% TCP-25. After 2 hours of incubation at 37°C, each sample was gently mixed with a pipette tip. Bioluminescence was measured using an in vivo bioimaging system (IVIS). □ (Perkin Elmer, USA) at 1, 5, 30 and 120 minutes.

[0161] Minimum inhibitory concentration assay. MIC analysis, which defines the lowest concentration of antimicrobial substance that prevents microbial growth, was performed using the microtiter broth dilution method (Wiegand et al., 2008). Briefly, fresh overnight colonies were suspended to a turbidity of 0.5 units and further diluted in Mueller-Hinton broth (Becton Dickinson). To determine the MIC, TCP-25 was dissolved from the stock solution at concentrations 10-fold higher than the required range via serial dilutions. Then, 10 μL of each concentration was added to the corresponding wells of a 96-well microtiter plate (polypropylene, Costar Corp.). Bacteria were rinsed with Tris (pH 7.4), diluted in MH medium, and 90 μL of the suspension (approximately 1 × 10 5 CFU (CFU) were added to each well. The plates were incubated overnight (16-18 hours) at 37°C. The MIC was determined as the concentration at which no viable bacterial growth was observed.

[0162] NF-kB / AP-1 Assay. NF-kB activation was assessed using THP1-XBlue™-CD14 reporter cells (referred to herein as THP-1 cells, InvivoGen, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, THP-1 cells were cultured in RPMI1640 cell culture medium with 10% heat-inactivated FBS, 1% antibiotic-antimycotic (Invitrogen, Carlsbad, CA, USA), 100 μg / mL G418 (InvivoGen, CA, USA), and 200 μg / mL Zeocin (InvivoGen, CA, USA). Cells were cultured at a concentration of 1.8 × 10 5Cells / well were added to a 96-well plate. Different TCP-25 gel formulations (20 μL) described previously (in HPC, CMC, or Pluronic F-127) were mixed with 20 μL of LPS (1 μg / mL, derived from Escherichia coli O111:B4, Sigma-Aldrich) and added to THP-1 cells incubated overnight at 37°C. A portion of the supernatant (20 μL) was mixed with 180 μL of QUANTI-Blue reagent (InvivoGen, CA, USA) and incubated for an additional hour (the remainder of the well was used for the MTT assay). The concentration of secreted fetal alkaline phosphatase (SEAP), an indicator of NF-kB activation, was quantitatively determined using a spectrophotometer at 600 nm.

[0163] MTT assay. The viability of THP-1 cells subjected to different formulations (with or without TCP-25) was measured using the MTT assay. Sterile-filtered MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-tetrazolium bromide; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) solution (5 mg / mL in PBS) was stored at -20°C protected from light until use. MTT solution (20 μL) was added to each well along with the remainder (180 μL) from the NF-kB / AP-1 assay described previously. The plate was incubated for 90 minutes at 5% CO2 and 37°C. After incubation, the plate was centrifuged at 300 g for 10 minutes, and the MTT-containing medium was removed by aspiration. The blue formazan product was dissolved by adding 100 μL of 100% DMSO (Duchefa Biochemie, Haarlem, The Netherlands) to each well. The plate was then gently swirled for 30 minutes at room temperature to dissolve the precipitate, and absorbance was read at 550 nm. Lysed cells served as a positive control for the assay. The value of viable, untreated cells was considered 100%, and values ​​for other treatments were expressed relative to viable, untreated cells.

[0164] Wound fluid from patients with non-healing venous ulcers. Wound fluid was collected from patients with chronic venous leg ulcers with ulcer duration greater than 3 months as previously described (Lundqvist et al., 2004). An Op-Site dressing was applied to the wound, and wound fluid was collected via gentle aspiration under the film after 2 hours. Sterile wound fluid was obtained from surgical drainage after mastectomy. Wound fluid was centrifuged at 10,000 rpm in an Eppendorf centrifuge, aliquoted, and stored at -20°C. Wound fluid from patients with positive Pseudomonas aeruginosa and Staphylococcus aureus cultures was used in this study.

[0165] Circular dichroism spectroscopy. We performed circular dichroism spectroscopy using a Jasco J-810 spectropolarimeter (Jasco, Easton, MD, USA) equipped with a Jasco CDF-426S Peltier temperature control unit (25 °C). Sample matrices were prepared using 20 μM TCP-25, diluted, and mixed with different formulation components (TCP-25 with HPC / HEC, CMC, and Pluronic F-127 in a 1:1 ratio, respectively), LPS (20 μg / mL), or 10 mM Tris pH 7.4 alone. All mixtures were incubated at room temperature for 30 min, after which the samples were placed in 1 mm quartz cuvettes. After external purging with nitrogen, the samples were scanned over a wavelength interval of 200–260 nm (scan rate: 20 nm / min). An average of five scans was recorded for each sample. The baseline (10 mM Tris buffer, formulation components, or LPS) was subtracted from the spectrum of each sample. We calculated the α-helical content of TCP-25 from molar ellipsometry at 222 nm in the presence of 10 mM Tris buffer, LPS, and formulation components (at a 1:5 ratio) as previously described.

[0166] TCP-25 Gel Diffusion. Diffusion assays were performed in 6-well plates with polyethylene terephthalate (PET) inserts (0.4 μm, VWR International, Radnor, PA, USA). For peptide release analysis, TCP-25 gels (0.1% TAMRA-TCP-25, 10 mM Tris, pH 7.4, 1.3% glycerol, and 1.5% HEC) were prepared. Tris-glycerol buffer (4 mL) was added to the basolateral compartment of each well. TCP-25 gel (1 mL) was added to the apical compartment. The plate was then incubated at 37°C. 25 μL samples were taken from the basolateral compartment at different time points (5 min, 20 min, 30 min, 1 h, 2 h, 6 h, 24 h, and 48 h), and fluorescence was measured at 570 and 583 nm using a spectrophotometer. A 0.1% TCP-25 solution in buffer was used as a control.

[0167] Stability of TCP-25. The stability of TCP-25 in either HEC gel or buffer (10 mM Tris, pH 7.4, 1.3% glycerol) was investigated using MALDI-TOF mass spectrometry. Samples were prepared (1.5% HEC or 0.1% TCP-25 in buffer) and then placed in storage for 0, 14, 60, or 180 days. Sample matrices were assigned so that samples from each storage time were also held at different temperatures: -80°C, 4°C, 20°C, and 37°C. After storage, samples were prepared for mass spectrometry.

[0168] Mass spectrometry of TCP-25 fragments. TCP-25 (2 μg) in 10 mM Tris was digested with HNE (0.1 μg) in a total volume of 20 μL at 37°C for 30 minutes and / or 3 hours. 20 mg of gel formulation (0.1% TCP-25, 10 mM Tris pH 7.4, 1.3% glycerol, 1.5% HEC) was also digested with 0.2 μg HNE under the same conditions as in solution. The digestion of TCP-25 in solution and HEC gel was determined using MALDI mass spectrometry and LC-MS / MS analysis.

[0169] MALDI mass spectrometry. TCP-25 samples from gel or solution were diluted with 2% ACN / 0.1% TFA and mixed with a 0.5 mg / mL solution of α-cyano-4-hydroxycinnamic acid (CHCA) in 50% ACN / 0.1% TFA directly on a stainless steel MALDI target plate. Typically, 0.5 μL of sample was mixed with 0.5 μL of CHCA solution. Subsequent MS analysis was performed on a MALDI LTQ Orbitrap XL mass spectrometer (ThermoScientific, Bremen, Germany). All mass spectra were acquired at 60,000 resolving power (m / z 400) using an FT spectrometer (Orbitrap). Mass spectra were recorded in positive mode over the 800–4000 Da mass range. The nitrogen laser was operated at 10 μJ with automatic gain control in off mode, using 10 laser shots per position. Spectral evaluation was performed using Xcalibur v 2.0.7. software (Thermo Fisher Scientific, San Jose, CA, USA).

[0170] LC-MS / MS. MS analysis was performed on an Orbitrap Fusion Tribrid MS system (ThermoScientific) equipped with a Proxeon Easy-nLC 1000 (Thermo Fisher). Database searches were performed with PEAKS 7.5 with the following settings: enzymes and methionine oxidation were not treated as dynamic modifications. The maximum number of post-translational modifications was one per peptide. An MS mass error range of 7 ppm and an MS / MS mass error range of 0.05 Da were used.

[0171] Mass Spectrometric Analysis of TCP-25 in Wound Fluid and Plasma. MS analysis of minipig wound fluid and plasma was performed at Q&Q Labs (Mölndal, Sweden). Briefly, 50 μL of internal standard solution (ISTD) was added to 300 μL of plasma or wound fluid sample and vortexed for 5 seconds. Then, 0.16% NH4OH and 30% CAN were added and vortexed for 10 seconds. The entire sample was then loaded onto an Oasis WCX column (Waters, Milford, MA, USA) and allowed to flow dropwise through the column. After rinsing and elution, the sample was dried by evaporation with N2. The sample was then diluted with 50:50 CAN:MQ, vortexed, and injected into the LC-MS / MS. The assay range was 30–3000 nM, with a limit of quantitation (LOQ) of 100 nM.

[0172] Stability of TCP-25 in plasma. TCP-25 (10 μM) was incubated in plasma of different species and in PBS at 37°C. Aliquots were taken at 0, 1, 3, and 5 hours and prepared for LC-MS analysis by protein precipitation with ice-cold acetone, with an internal standard to compensate for possible differences in final sample volume. LC-MS analysis was performed in full scan mode, and the ratio of the peak areas of TCP-25 and the internal standard was calculated and plotted against time. The k(h) obtained for disappearance was -1 ) values ​​were used to calculate the half-life of TCP-25.

[0173] Rheological Analysis. Rheological measurements on 2% HEC gels with or without 0.1% or 1% TCP-25 were performed on a Kinexus Pro rheometer (Malvern Panalytical Ltd., Malvern, UK) equipped with a plate / plate configuration and a 1 mm gap. Shear strains of 0.001 to 10 were applied to determine the linear viscoelastic region (LVR) and the pour point (shear stress at G' and G" crossovers) at 1 Hz and 25 °C. The shear stress at the pour point (Pa) was determined directly by the rheometer. In addition, the pour point as the strain at G' and G" crossovers was also determined. Measurements were performed in triplicate.

[0174] Murine Inflammation Model. 10-12-week-old male BALB / c tg(NFk□-RE-Luc)-Xen reporter mice (Taconic Biosciences, Albany, NY, USA) were used to test the anti-inflammatory effects of TCP-25 Gel #1 after subcutaneous co-treatment with LPS (5 μg of E. coli). The backs of the mice were carefully shaved and cleaned. LPS was mixed with 100 μL of TCP-25 Gel #1 and immediately injected subcutaneously while anesthetized with isoflurane (Baxter, Deerfield, IL, USA). Mice were immediately transferred to individually ventilated cages. TCP-25 in the gel formulation was spiked with TCP-25 Cy5 for fluorescent imaging of the peptide. Bioimaging with IVIS Spectrum was used to longitudinally determine NF-kB activation. Fifteen minutes before IVIS imaging, mice were intraperitoneally injected with 100 μL of D-luciferin (PerkinElmer, 150 mg / kg body weight). Bioluminescence from the mice was detected and quantified using Living Image 4.0 Software (PerkinElmer).

[0175] Murine Surgical Implant Model. 10-12-week-old male BALB / c mice were used in the surgical implant model. The backs of the mice were shaved and cleaned with 70% alcohol. Under isoflurane anesthesia, an approximately 10 mm incision was made in the dorsal skin of the mouse, and the tip of a pair of scissors was used to create a small pocket. A 6 mm diameter disk of polyurethane (PU) foam (Mepilex® Transfer, Molnlycke Heath Care, Gothenburg, Sweden) was inserted under the subcutaneous fascia. 100 μL of TCP-25 Gel #1 or control gel and LPS (Escherichia coli, 5 μg) were immediately deposited around the PU disk in the subcutaneous pocket, and the wound was closed with sutures (VICRYL®, Johnson & Johnson, Belgium). The mice were sacrificed at 24 hours, and the PU disks were collected. Wound fluid was extracted from the PU disks for further cytokine analysis.

[0176] Mouse model of subcutaneous infection. 10-12 week-old male SKH-1 hairless mice were anesthetized with a mixture of 2% isoflurane and oxygen. TCP-25 gel #1 spiked with TCP-25 Cy5 was used for fluorescent imaging of the peptide. Overnight cultures of the bioluminescent bacteria Pseudomonas aeruginosa Xen41 or Staphylococcus aureus 229 were refreshed and grown to mid-logarithmic phase in TH medium. Bacteria were washed (5.6°C, 1000 rpm) for 15 min and diluted in 10 mM Tris buffer (pH 7.4). The formulation was then applied to 100% bacteria. 6 The mixture was mixed with CFU. A total of 100 μL of the contamination mixture (80 μL gel + 20 μL bacterial suspension) was injected subcutaneously into the backs of mice. In vivo bacterial infection and peptide localization were assessed longitudinally in anesthetized mice using IVIS imaging to measure bioluminescence (bacteria) and fluorescence (TCP-25 Cy5). Animals were imaged in either bioluminescence or fluorescence mode, and the resulting data were analyzed using Living Image 4.0 Software (PerkinElmer). At termination, tissue samples from the wounds were collected and CFU determined.

[0177] In the prophylactic model, all procedures were similar to those described above, except that the gel (80 μL) was first injected subcutaneously into the backs of BALB / c mice. Thirty minutes later, bioluminescent Staphylococcus aureus and Pseudomonas aeruginosa (10 in 20 μL) were injected. 6 CFU) were injected into the gel deposition site. In vivo bacterial infection was assessed longitudinally by measuring bacterial bioluminescence using IVIS imaging.

[0178] TCP-25 release in mice. Ten- to twelve-week-old male SKH-1 hairless mice were anesthetized with a mixture of 2% isoflurane (Baxter) and oxygen. TCP-25 gel #1 spiked with TCP-25 Cy5 was used for fluorescent imaging of the peptide. In some mice, TCP-25 gel #1 was mixed with 20 μg of LPS. The gel (100 μL) was injected subcutaneously into mice anesthetized with isoflurane. Peptide release was monitored using IVIS imaging in fluorescence mode, and the resulting data were analyzed using Living Image 4.0 Software (PerkinElmer).

[0179] Partial-thickness wound minipig model. A partial-thickness wound minipig model was used to test S. aureus wound infection in vivo. Female Göttingen minipigs weighing 14–16 kg were used. All procedures were performed according to strict aseptic technique by a qualified veterinarian surgeon. Prior to wound creation, the minipigs were acclimated for 1 week and fasted the night before wound creation. Hair on the back was clipped 24 h before surgery. On the day of wound creation, the minipigs' backs were scrubbed with chlorhexidine (MEDI-SCRUB sponge; Rovers, The Netherlands) and lukewarm water. The backs were then shaved, disinfected with chlorhexidine solution (4%), and dried with sterile gauze. Subsequent procedures were performed under general anesthesia. General anesthesia was achieved with a mixture of tiletamine and zolazepam (Zoletil 50, Virbac, Sweden). An induction dose of Zoletil (1 mL / 10 kg body weight) was given intramuscularly, and anesthesia was maintained intravenously via a catheter in the auricular vein (0.5 mL / 10 kg body weight). During anesthesia, the minipig received supplemental oxygen via a face mask. The wound outline was marked with a sterile triangular scale and tissue pen. Using an electric dermatome (Zimmer), 12 partial-thickness (750 μm deep) wounds measuring 2.5 × 2.5 cm were created on the back of the minipig (six wounds on each side). In the initial experiment, the depth of the fresh wound was confirmed by frozen sectioning of the biopsy and staining with DAPI nuclear stain. A minimum distance of 4 cm was maintained between wounds. For homeostasis, the wounds were covered with sterile gauze. An overnight Staphylococcus aureus (ATCC 29213) culture was refreshed and grown to mid-logarithmic phase in TH medium. Bacteria were washed (5.6 × 1000 rpm) for 15 min and diluted to 2 × 10 in 10 mM Tris buffer (pH 7.4). 8 For infection, the bacteria were diluted to a concentration of HEC (10 7The gel (100 μL) was applied to the uninfected control wound. After 15 minutes, 500 μL of TCP-25 Gel #2 or TCP-25 Gel #3 alone was applied to the wound, which was then covered with a primary foam dressing (Mepilex® Transfer; Molnlycke Healthcare, Gothenburg, Sweden). The primary dressing was a transparent, breathable, fixative dressing (Mepore □ The wound was then covered with a film (Mölnlycke, Gothenburg, Sweden). The dressing was then secured in place with skin staples (smi, St. Vith, Belgium). For further protection and padding, the wound area was then covered with two layers of sterile cotton gauze and secured with adhesive tape. Finally, a single layer of flexible self-adhesive bandage (Vet Flex, Kruuse, Denmark) was used to support and protect the underlying dressing. After recovery from anesthesia, the animals were monitored for any discomfort and provided with water and food. Animals were housed individually and monitored daily. 24 hours after wound formation and infection, the dressing was removed under general anesthesia and observations were performed. The wound was documented by imaging, and clinical scoring was performed by a qualified veterinarian. Swab samples were taken from the wound surface and used for bacterial analysis. Wound fluid recovered from the primary dressing was collected at each dressing change and further analyzed. TCP-25 Gel #2, TCP-25 Gel #3, or gel alone (500 μL) was applied to each wound group, and a new dressing was applied. In the short-term treatment regimen, dressings were changed daily, and the minipigs were sacrificed 4 days after infection. In the long-term treatment regimen, dressings were changed on days 2, 3, 5, 7, and 9, and the minipigs were sacrificed on day 10. Tissue samples from the wounds were also collected on the day of termination.

[0180] In a benchmark comparison study, Prontosan (B Braun, Sempach, Switzerland) and Mepilex Ag (Mölnlycke Healthcare, Gothenburg, Sweden) treatment groups were also added. In addition to the 1% TCP-25-2% HEC gel, some wounds were treated with Prontosan (500 μL), and others with Mepilex Ag (4.5 × 4.5 cm). In a wound healing study in minipigs, partial-thickness wounds were created as previously described, but bacterial infection was not introduced. Wounds were treated with TCP-25 Gel #2, TCP-25 Gel #3, or gel alone in a chronic treatment regimen.

[0181] For superinfection studies, the short-term treatment regimen described above was followed with one modification on day 2. Following wound formation, S. aureus infection, and treatment on day 1, P. aeruginosa was added to the wound on day 2. Overnight P. aeruginosa cultures were refreshed, grown to mid-logarithmic phase in TH medium, washed, and diluted with 10 mM Tris buffer (pH 7.4). To determine superinfection, P. aeruginosa (10 5 CFU / 100 μL Tris buffer) was applied to the wound surface. After 15 minutes, each treatment and dressing was applied. The dressing was changed on the third day, and the minipigs were sacrificed on the fourth day.

[0182] For confirmed infection studies, the chronic treatment regimen described above was followed with one modification on day 1 (Figure 7E). Immediately after wounding on day 1, S. aureus (10 7 CFU / 100 μL HEC gel was added to the fresh wound surface, but no treatment was applied. Each treatment was started on day 2, followed by dressing changes on days 3, 5, 7, and 9, and the minipigs were sacrificed on day 10.

[0183] Identification of contaminating bacteria from superinfected minipig wounds and preparation of conditioned media Fresh swab samples from the wounds were sent to the Department of Clinical Microbiology, Division of Laboratory Medicine, Skane University Hospital, Lund, for identification using standard microbiological methods. To prepare conditioned medium, bacteria were grown overnight (16 h) at 37°C in 5 mL TH medium in a shaking incubator (180 rpm) until an optical density of 1.5 was reached. The bacteria were then centrifuged at 3000 × g for 10 min. The supernatant was removed and filter-sterilized using a 0.2 μm Filtropur S filter (Sarstedt, Numbrecht). 1% conditioned medium was used to stimulate THP1-XBlue cells.

[0184] In vivo TCP-25 uptake. TCP-25 Cy3-spiked TCP-25 Gel #2 and TCP-25 Gel #3 were used to test TCP-25 skin penetration and tissue uptake. TCP-25 Gel #2 or TCP-25 Gel #3 was applied to either a partial-thickness wound (2 hours) or intact skin (2 and 24 hours) on the back of a minipig. After gel application, the wound was dressed as previously described. The pigs were sacrificed, and biopsies were snap-frozen and mounted in OCT compound for cryosectioning. Cryosections were washed in PBS at room temperature (RT) (once for 5 minutes), and 4',6-diamidino-2-phenylindole (DAPI) solution (0.5 mM in PBS) was used as a nuclear counterstain (1 minute, RT). Slides were washed with PBS (1x 5 min, RT), dried, and mounted with antifade mounting medium (PermaFluor, ThermoFisher Scientific). Sections were then imaged using a fluorescence microscope (AxioScope.A1, Carl Zeiss, Germany).

[0185] Ex vivo TCP-25 uptake. To test TCP-25 uptake in an ex vivo pig skin model, TCP-25 Gel #4 spiked with TCP-25 Cy3 was used. TCP-25 Gel #4 contained 2% TCP-25, 2% HEC, 1.3% glycerol, and 10 mM Tris-HCl, pH 7.4. Frozen skin was thawed and washed with ethanol (70%) and sterile water. The skin was partially submerged in PBS on a Petri dish to maintain moisture. TCP-25 Gel #4 (50 μL) was applied to wounded or intact skin and incubated at 37°C for 2 and 24 hours. At the end of the incubation period, tissue samples were excised using a scalpel, frozen, and mounted in OCT compound for cryosectioning. Cryosections were processed for fluorescence imaging as described above for in vivo uptake.

[0186] Wound fluid extraction. Wound dressing (Mepilex; Molnlycke Health Care, Gothenburg, Sweden) from the wound was transferred to a 5 mL pre-chilled test tube and kept on ice. To extract wound fluid, the dressing was immersed in 500 μL of ice-cold 10 mM Tris buffer, pH 7.4, and centrifuged (2000 g, 4°C) for 5 minutes. The extracted wound fluid was aliquoted into pre-chilled Eppendorf tubes with or without protease inhibitors and stored at -80°C until further analysis.

[0187] Bacterial analysis of wounds. Swab samples collected from the wounds were placed in Eppendorf tubes containing 500 μL of PBS and vortexed for 30 seconds. Diluted samples (10-fold diluted with PBS) were plated on TH broth agar and incubated overnight at 37°C for CFU analysis.

[0188] ELISA. Wound fluid collected from the dressings was used to determine IL-6 and TNF-α concentrations. Porcine IL-6 and TNF-α DuoSet □ELISA kits (R&D Systems, Minneapolis, MN, USA) were used according to the manufacturer's recommendations. IL-6 and TNF-α were assessed in mouse plasma using a mouse inflammation kit (Becton Dickinson AB, Franklin Lakes, NJ, USA) according to the manufacturer's instructions.

[0189] Single-dose toxicity in mice. Ten-week-old female BALB / c mice were given 5 mg of TCP-25 subcutaneously in 100 μL Tris buffer and sacrificed 24 hours later. Tissues (lung, kidney, liver, skin, and spleen) were collected for histological examination and stained with H&E.

[0190] Histological examination. For mouse tissues, skin samples (4 mm or 6 mm by using a biopsy punch) collected from the infected area were placed on filter paper to prevent curling and fixed overnight in 4% paraformaldehyde; they were then stored in 70% ethanol. For minipig wounds, tissue samples were collected using a scalpel, fixed overnight in neutral buffered formalin, and then stored in 70% ethanol. After serial dehydration, tissues were embedded in paraffin blocks, sectioned, and stained with hematoxylin and eosin (H&E). Samples were imaged using bright-field microscopy (Axioplan2, Zeiss, Germany). H&E-stained sections of minipig wound biopsies were examined and scored in a blinded manner by an experienced pathology veterinarian (MP). Histological scoring was based on epithelialization, granulation tissue, inflammatory cells, abscesses, and tissue formation on a scale of 0 to 5 (0 being the worse score and 5 being the best score). For each wound section, five areas with 90-100% wound coverage were examined under a 10x objective.

[0191] result Evaluating the efficacy and structure of peptides in the presence of different formulation components The actions of TCP-25 involve structural transitions, such as the formation of C-turns and helical structures upon LPS binding, and depend in part on its ability to interact with both bacterial membranes and CD14. The gel formulations of the present invention support these TCP-25 functions. Antibacterial activity was determined for TCP-25 alone (see Figure 1E) or in the presence of hydroxypropyl cellulose (HPC), carboxymethyl cellulose (CMC), or Pluronic F-127 (hereafter referred to as Pluronic). The radial diffusion assay (RDA) is an agar diffusion-based method that measures bacteriostatic / bactericidal effects. Using RDA against Gram-negative Escherichia coli and Pseudomonas aeruginosa, as well as Gram-positive Staphylococcus aureus, we were able to demonstrate that TCP-25 activity against the Gram-negative bacteria E. coli and P. aeruginosa was largely retained. However, the addition of CMC inhibited TCP-25 activity against the peptide against S. aureus (Figure 1A). Analysis using a viable counting assay (VCA), which measures bactericidal efficacy in solution, demonstrated that both the anionic CMC polymer and the micelle-forming Plurornic interfered with the antibacterial action of TCP-25, whereas the antibacterial activity of the peptide was retained in HPC (Figure 1B). Clinical and regulatory considerations also prompted comparison with a related neutral polymer, hydroxyethyl cellulose (HEC), and results were similar to those obtained with HPC (Figures 1E and 1F).

[0192] Because the endotoxin-blocking effect of TCP-25 involves specific interactions with both LPS and cells, it is possible that the structural prerequisites for these anti-inflammatory activities may be separate from those required for antibacterial activity in specific formulations. The antiendotoxin activity of TCP-25 in the presence of different formulation components was determined using LPS-stimulated THP1-XBlue™-CD14 cells in vitro. Cells were incubated with E. coli LPS (10 ng / mL) and TCP-25 in the presence or absence of HPC, CMC, and Pluronic. After 18 to 24 hours of incubation, activation of NF-kB and AP-1 was assessed. Results indicated that CMC, and to a lesser extent Pluronic, in particular, interfered with the antiendotoxin activity of TCP-25. However, HPC did not exert any inhibitory effect on TCP-25 (Figure 1C). As mentioned above, comparison of the related polymer with hydroxyethylcellulose (HEC) yielded results si...

Claims

1. Amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 3), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 4), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 5), GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 6), GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 7), or Amino acid sequence A sequence having at least 90% sequence identity with GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1) Compounds containing peptides comprising or consisting of A composition comprising: the peptide has a length of 18 to 100 amino acid residues; the peptide has antibacterial and / or anti-inflammatory activity, the concentration of the compound in the composition is at least 0.08 wt %; composition.

2. a) Amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 3), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 4), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 5), GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 6), GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 7), or Amino acid sequence A sequence having at least 90% sequence identity with GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1) A compound comprising a peptide comprising or consisting of the peptide has a length of 18 to 100 amino acid residues; a compound, wherein the peptide has antibacterial and / or anti-inflammatory activity; b) EDTA, c) an aqueous buffer; A composition comprising: i. the composition has a pH of at most 8, and / or ii. The concentration of the compound in the composition is at least 0.08 wt %; composition.

3. a) Amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 3), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 4), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 5), GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 6), GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 7), or Amino acid sequence A sequence having at least 90% sequence identity with GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1) A compound comprising a peptide comprising or consisting of the peptide has a length of 18 to 100 amino acid residues; a compound, wherein the peptide has antibacterial and / or anti-inflammatory activity; b) a non-ionic polymer capable of forming a hydrogel when mixed with an aqueous solution; c) an aqueous solution; A composition comprising: i. the concentration of the compound in the composition is at least 0.08 wt %; and / or ii. the nonionic polymer is present in the composition at a concentration of at least 0.05 wt %; composition.

4. The composition described in claim 3, wherein the composition further contains EDTA.

5. The composition according to any one of claims 1 to 4, wherein the composition is a hydrogel or a viscous solution.

6. The composition comprises a nonionic polymer, and the nonionic polymer comprises polyallyl alcohol, polyvinyl alcohol, polyacrylamide, polyethylene glycol (PEG), polyvinylpyrrolidone, starch such as corn starch and hydroxypropyl starch, methyl cellulose, ethyl cellulose, and n-propyl cellulose. １ ~C ６ alkylcelluloses, such as alkylcelluloses; hydroxyalkylcelluloses, preferably hydroxy-C, such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxybutylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose and mixtures of the foregoing; １ ~C ６ -Alkylcellulose and hydroxy-C １ ~C ６ -Alkyl-C １ ~C ６ 6. A composition according to any one of claims 1 to 5, wherein the non-ionic polymer is selected from the group consisting of hydroxyalkyl celluloses, preferably hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC), including alkyl celluloses and substituted alkyl celluloses, for example, the non-ionic polymer is selected from the group consisting of hydroxyalkyl celluloses, preferably hydroxyethyl cellulose (HEC) and hydroxypropyl cellulose (HPC).

7. 7. A composition according to any one of claims 1 to 6, wherein the composition comprises a non-ionic polymer, and wherein the non-ionic polymer is present in the composition at a concentration of at least 0.5 wt%, such as a concentration in the range of 1 to 3 wt%, and / or the concentration of the non-ionic polymer is sufficient to obtain a viscosity of the composition of at least 10 mPas, for example between 10 mPas and 100,000 mPas.

8. The composition of any one of claims 1 to 7, wherein the compound is present in the composition at a concentration ranging from 0.08 to 3 wt%.

9. The peptide has a length of 18 to 35 amino acids, preferably 18 to 25 amino acids, and has the amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), FYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 2), GKYGFYTHVFRLKKWIQKVI (SEQ ID NO: 3), HVFRLKKWIQKVIDQFGE (SEQ ID NO: 4), KYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 5), GKYGFYTHVFRLKKWIQKVIDQF (SEQ ID NO: 6), GKYGFYTHVFRLKKWIQKV (SEQ ID NO: 7) comprising or consisting of any of the following: The composition according to any one of claims 1 to 8.

10. The peptide has the amino acid sequence:

10. The composition of any one of claims 1 to 9, wherein the peptide has at least 90% sequence identity with the amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1), preferably wherein the peptide consists of the amino acid sequence: GKYGFYTHVFRLKKWIQKVIDQFGE (SEQ ID NO: 1).

11. 11. The composition of any one of claims 1 to 10, wherein EDTA is present in the composition at a concentration of at least 1 mM, such as in the range of 1 to 100 mM, preferably at least 1.5 mM, such as at least 2 mM, for example in the range of 2 to 100 mM, such as in the range of 2 to 50 mM.

12. The pH of the composition is a) at most 8, such as lower than 7, such as lower than 6, such as lower than 5.5, and / or said pH is higher than 3, such as at least 3.5, or b) between 7 and 8, for example approximately 7.4; The composition according to any one of claims 1 to 11.

13. The composition comprising: at least 90%, at least 95%, etc. of the original content of said compound containing said peptide after storage for 2 months at 37°C; and / or at least 75%, such as at least 80%, for example at least 90% of the original content of said compound comprising said peptide after storage at 37°C for 4 months; and / or at least 70%, such as at least 80%, for example at least 85%, of the original content of said compound comprising said peptide after storage at 37°C for 6 months; and / or at least 90%, such as at least 95% of the original content of said compound containing said peptide after 8 months of storage at room temperature, The composition of any one of claims 1 to 12, comprising:

14. A product comprising the composition of any one of claims 1 to 13.

15. A pharmaceutical composition for use in treating a disorder in an individual in need thereof, comprising a composition according to any one of claims 1 to 13 or a product according to claim 14.

16. Use of a composition according to any one of claims 1 to 13 in the manufacture of a medicament for the treatment of a disorder in an individual in need thereof.

17. 16. The pharmaceutical composition for use according to claim 15, wherein the disorder is a disorder of the skin, ear, eye or nose.

18. 18. The pharmaceutical composition for use according to any one of claims 15 and 17, wherein the disorder is a wound.

19. 19. The pharmaceutical composition for use according to claim 18, wherein the wound is selected from the group consisting of burns, non-healing ulcers and surgical wounds.

20. The disorder is - involving or associated with inflammation; and / or - including or associated with bacterial infections, A pharmaceutical composition for use according to any one of claims 15 and 17 to 19.

21. The pharmaceutical composition for use according to any one of claims 15 and 17 to 20, wherein the disorder comprises the formation of a biofilm.

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