Endotoxin Neutralizer

Abstract

A peptide for use as an endotoxin neutralizing agent is provided. The peptide comprises an amino acid sequence having at least 80% sequence identity with the sequence RRPRPRPRP and at least four tryptophan residues provided at the C-terminus, N-terminus, or therebetween. A method for in vitro endotoxin neutralization is provided. The method includes contacting a patient sample with the peptide. An endotoxin-neutralizing amphiphilic hydrogel is also provided.

Description

[Technical Field]

[0001] The present disclosure relates to endotoxin neutralizing agents, and in particular to the use of peptides as endotoxin neutralizing agents. [Background technology]

[0002] Bacterial infections remain a threat to human health, due in part to increasing bacterial resistance, demographic changes with aging populations, and a lack of newly approved antibiotic treatments.

[0003] Severe bacterial infections can lead to sepsis, a severe inflammatory condition that affects the patient's entire body and is associated with high morbidity and mortality.

[0004] The problem in treating sepsis is that drugs must be able to kill bacteria without releasing pro-inflammatory toxins. In the case of Gram-negative bacteria, these toxins are lipopolysaccharides (LPS), and in the case of Gram-positive bacteria, they are lipoproteins (LP / LP).

[0005] Patent Document 1 (Amferia AB) describes antimicrobial hydrogels and antimicrobial peptides. Antimicrobial peptides have been shown to have significant antimicrobial efficacy. However, the antimicrobial efficacy of antimicrobial peptides does not necessarily correspond to their antiendotoxin efficacy. There is no simple model for either antimicrobial or antiendotoxin efficacy. There is also no clear correlation between folding structure, length, and sequence and antiendotoxin efficacy. (Non-Patent Document 1) Antimicrobial peptides that are suitable for killing bacteria and may therefore be considered potential treatments for sepsis along with numerous other symptoms resulting from bacterial infection, however, may actually be unsuitable due to the release of pro-inflammatory toxins when killing bacteria.

[0006] Because free antimicrobial peptides have been shown to degrade rapidly, for example, in blood, providing antimicrobial peptides covalently bound to a substrate is an ideal wound treatment device. However, due to structural changes, available binding sites, etc., it is believed that binding of antimicrobial peptides to a substrate may adversely affect the antiendotoxin efficacy of effective antimicrobial peptides. The efficacy of AMPs has been shown to decrease 500-1000-fold upon immobilization on a surface (Non-Patent Document 2), and therefore, endotoxin binding efficacy is also expected to decrease upon immobilization of AMPs on a surface. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2019 / 074422 [Non-patent literature]

[0008] [Non-Patent Document 1] Brandenburg,K et al.Peptides with dual mode of action:Killing bacteria and preventing endotoxin-induced sepsis,Biochimica et Biophysica Acta.2016,vol.1858,pp.971-979 [Non-patent document 2] The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopedic Medical Devices:A Review.Antibiotics(Basel).2023;12(2):211.19 Jan 2023 Summary of the Invention [Problem to be solved by the invention]

[0009] Improved anti-endotoxin therapies would be advantageous, particularly antimicrobial peptides that combine antibacterial and anti-endotoxin efficacy. [Means for solving the problem]

[0010] Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above identified shortcomings and disadvantages in the art, singly or in any combination, and solves at least the above problems by providing an amphipathic peptide for use as an endotoxin neutralizing agent, the amphipathic peptide comprising an amino acid sequence having at least 80% sequence identity with the sequence RRP9 (SEQ ID NO: 1) and at least four tryptophan residues provided at or between the C-terminus or N-terminus.

[0011] An amphiphilic hydrogel composition for use in endotoxin neutralization is provided.

[0012] Additionally, methods for in vitro endotoxin neutralization are provided.

[0013] Further advantageous embodiments are disclosed in the attached dependent claims. [Brief explanation of the drawings]

[0014] These and other aspects, features and advantages of the present invention will become apparent and elucidated from the following description of embodiments of the invention, which proceeds with reference to the accompanying drawings.

[0015] [Figure 1] The results of the endotoxin neutralization test described in Experiment 1 below are shown. Endotoxin levels were detected after 3 hours and 24 hours. The left axis and left column of each column pair represent the endotoxin levels after 3 hours, and the right axis and right column of each column pair represent the endotoxin levels after 24 hours. [Figure 2]Results of the endotoxin neutralization assay described in Experiment 2 below are shown. Endotoxin levels were measured after 1 and 3 hours. AMP is the antimicrobial peptide RRP9W4N in solution. AMP-P is the same antimicrobial peptide conjugated to crosslinked hydrogel particles. [Figure 3] The results of the endotoxin neutralization test described in Experiment 2, detailed below, are shown. Results shown are endotoxin levels detected after 24 hours. CP refers to cross-linked hydrogel particles without any antimicrobial agent attached. PBS-b refers to PBS without bacteria, and PBS+b refers to PBS with bacteria. AMP is the antimicrobial peptide RRP9W4N in solution. AMP-P is the same antimicrobial peptide attached to cross-linked hydrogel particles. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention The present disclosure relates to the use of antimicrobial peptides as anti-endotoxin agents. The anti-endotoxin agents may be referred to as endotoxin neutralizing agents. Furthermore, the anti-endotoxin agents may be referred to as anti-inflammatory agents due to their anti-inflammatory effects. The anti-endotoxin agents may be for use in the treatment of bacterial sepsis. The endotoxin neutralizing agents may be for use in vivo.

[0017] The antimicrobial peptide may be derived from the proline-arginine-rich terminal leucine-rich repeat protein, PRELP. The antimicrobial peptide may comprise a sequence having 80%, for example 90%, for example 95% identity with RRPRPRPRP (RRP9 SEQ ID NO: 1). Such peptides have been shown to be similar to the human cathelicidin-derived LL-37 in several aspects, such as net charge and antimicrobial efficacy (Malmsten, M et al., Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP, PLoS ONE, 2011, 6(1):e16400. doi:10.1371 / journal.pone.0016400).

[0018] The antimicrobial peptide is advantageously a short peptide consisting of 1 to 50 amino acids, for example less than 40 amino acids, for example less than 30 amino acids, preferably less than 20 amino acids. The antimicrobial peptide may have a molecular weight between 1 and 50 kDa.

[0019] As described above, the antimicrobial peptide may comprise a sequence having at least 90% identity with the sequence RRPRPRPRP (SEQ ID NO: 1, RRP9). Ideally, the antimicrobial peptide comprises a stretch of at least two consecutive tryptophan or phenylalanine residues, either at the C-terminus or N-terminus, or added between them. The antimicrobial peptide may comprise N-terminal amidation. The antimicrobial peptide is advantageously amphipathic, as amphipathicity has previously been shown to have improved efficacy and immobilization properties.

[0020] The antimicrobial peptide may be, for example, RRPRPRPRPWWWW-NH2 (SEQ ID NO:2, RRP9W4N), RRPRPRPRP-NH2 (SEQ ID NO:3, RRP9N), RRPRPRPWWWWRP-NH2 (SEQ ID NO:4, RRP7W4RPN), or RRPRPWWRPWWRP-NH2 (SEQ ID NO:5, RRP5W2RPW2RPN). The above peptide sequence may be described as a peptide comprising the sequence RRP9 (SEQ ID NO:1) and at least four tryptophan residues, either consecutive or at least two pairs of two consecutive residues separated by an arginine-proline pair, i.e., WWRPWW (SEQ ID NO:6).

[0021] As shown in the experimental section, antimicrobial peptides containing sequences corresponding to those defined herein have surprisingly been shown to have endotoxin-neutralizing effects. Antimicrobial peptides corresponding to the above definition have both cationic and hydrophobic moieties, resulting in amphipathic antimicrobial peptides.

[0022] The peptide may be for use in a method for in vivo endotoxin neutralization. A therapeutically effective amount of the peptide may be administered to a patient. The peptide may be for use in treating an acute inflammatory condition in a patient. The peptide may be for use in treating an endotoxin-induced inflammatory condition. The peptide may be for use in treating LPS-induced inflammation. The inflammatory condition may be sepsis. Sepsis is defined as the presence or presumed presence of an infection accompanied by evidence of a systemic response called the systemic inflammatory response syndrome (SIRS). Sepsis is usually caused by bacterial infection with either gram-negative or gram-positive bacteria, but can also be caused by other pathogens such as viruses, fungi, and protozoa. The damage and symptoms resulting from sepsis are caused not only by bacteria but also by components of the bacterial cell wall known as endotoxin or LPS. LPS molecules are glycolipids ubiquitously present in the outer membrane of gram-negative bacteria. LPS is released when the immune system destroys invading bacteria. The released LPS binds to monocytes, macrophages, and endothelial cells, triggering the production of various mediators, such as TNF-α and interleukins (IL-1, IL-6, and IL-8). Excessive production of TNF-α, IL-1, IL-6, and IL-8 is a major cause of severe sepsis.

[0023] The amount of peptide effective in treating a particular disorder or condition depends on the nature of the disorder or condition and the specific peptide. Effective amounts can be determined by standard clinical techniques known to those skilled in the art. In addition, in vitro assays can be optionally used to help identify optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration and the nature of the disease or disorder, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be estimated from dose-response curves obtained from in vitro or in vivo animal model test bioassays or systems. Compositions containing peptides can, for example, be introduced into the systemic circulation, which distributes the peptide within the patient.

[0024] The peptides can be used in vitro for endotoxin neutralization. A patient sample, such as a patient's blood or plasma, can be contacted with the peptide, thereby neutralizing endotoxins in the patient's blood, etc. A method for in vitro endotoxin neutralization can include contacting a patient sample extracted from a patient with a peptide, such as RRP9W4N (SEQ ID NO: 2) or other sequences disclosed herein.

[0025] Antimicrobial peptides for use as endotoxin neutralizing agents can be covalently or otherwise attached to the hydrogel substrate. Crosslinked hydrogels form ideal substrates onto which AMPs can be immobilized, as previously described, if the peptides are to be used for endotoxin neutralization in vitro.

[0026] As described in WO2019 / 074422 A1 (Amferia AB), when the hydrogel substrate is an amphiphilic hydrogel containing alternating hydrophilic and hydrophobic regions, antibacterial efficacy can be improved. However, as previously mentioned, improved antibacterial efficacy does not correspond to increased or even maintained antiendotoxin efficacy. While antibacterial efficacy generally decreases upon immobilization of a peptide to a surface, and therefore endotoxin binding efficacy is also expected to decrease upon immobilization, the present inventors have identified that the endotoxin binding efficacy of peptides containing the sequence RRP9 (SEQ ID NO: 1), e.g., RRP9W4N (SEQ ID NO: 2), or other sequences defined herein, is substantially maintained even when immobilized on a hydrogel. Furthermore,

[0027] As shown in the Experimental Section, we surprisingly demonstrated that conjugation of peptides to the hydrogel matrix did not inhibit antiendotoxin efficacy. As shown in Experiment 2, cross-linked hydrogel granules with immobilized AMP had similar endotoxin-binding efficacy as the corresponding amount of free AMP in solution.

[0028] The hydrogel substrate can be in the form of a crosslinked solid hydrogel, such as that disclosed in WO 2019 / 074422 A1 (Amferia AB). The hydrogel to which the antimicrobial peptide is attached can be in the form of individual crosslinked amphiphilic hydrogel particles, layers, sheets, or other suitable forms. When the hydrogel is provided as individual particles, the individual crosslinked hydrogel particles can be obtained, for example, by grinding a solid crosslinked hydrogel sheet into particles as described in the experimental section, or by other suitable methods for providing individual hydrogel particles. Individual crosslinked hydrogel particles can be a particularly suitable carrier medium to which the endotoxin neutralizing agent can be attached. For example, the particles have a high specific surface area to make the endotoxin neutralizing agent available for attachment. However, the hydrogel can be in any suitable form to which the endotoxin neutralizing particles can be attached. The hydrogel can also be a solid sheet to which the AMP is attached. The AMP-conjugated hydrogel can be provided as a wound dressing, a stoma dressing, a stoma base plate, an incision film, a patch, a bandage, a plaster, an adhesive, a bandage, a catheter, or a combination thereof. The AMP-conjugated hydrogel can also be provided as a filter or as a component of a filter for a fluid, such as a patient sample. In particular, for in vitro neutralization of endotoxin, the AMP-conjugated hydrogel can be provided as a component of a filter for a fluid sample.

[0029] When used as an endotoxin-neutralizing filter, the AMP-conjugated hydrogel may be exposed to a fluid. The fluid may contain known or unknown amounts of endotoxin. Endotoxin present in the fluid is neutralized by the AMP-conjugated hydrogel. The fluid may be a patient-derived fluid sample, a non-patient-derived fluid sample, or a combination of a patient-derived sample and a non-patient-derived reagent. In vitro endotoxin filtration / neutralization may be used for reagents in various processes where endotoxin should not be present. For example, the AMP-conjugated hydrogel may be used to filter reagents and / or cellular material prior to an in vitro fertilization procedure. After contact with the AMP-conjugated hydrogel, the fluid sample is substantially free of endotoxin.

[0030] A process for preparing individual hydrogel particles includes providing a hydrogel composition containing a first amphiphilic polymer. The first amphiphilic component of the composition can be a crosslinkable amphiphilic polymer. A typical and suitable amphiphilic material is a diacrylate-modified poloxamer, such as polyethylene oxide-polypropylene oxide-polyethylene oxide (DA-PEOx-PPOy-PEOx-DA, where x and y refer to the number of PEO and PPO groups present, respectively), as described in WO 2019 / 074422 A1. Specifically, the amphiphilic material can be an amphiphilic triblock copolymer, polyethylene oxide (100)-polypropylene oxide (70)-polyethylene oxide (100) (Pluronic® F127, BASF Corporation), or polyethylene oxide (30)-polypropylene oxide (70)-polyethylene oxide (30) (Pluronic® 123, BASF Corporation).

[0031] As mentioned above, the first amphiphilic component may be a diacrylate derivative of a triblock copolymer, thus chemically crosslinking the copolymer. A process for diacrylate modification is provided in Experiment 1 of WO2019 / 074422 A1 (Amferia AB). Modification can be achieved by reacting the triblock amphiphilic copolymer with acryloyl chloride to form a diacrylate derivative. Other methods for forming crosslinkable amphiphilic polymers may be possible, such as forming a methacrylate derivative or using carboxyamine crosslinking.

[0032] Crosslinkable amphiphilic polymers, in the presence of water, can self-assemble to form ordered nanostructures called lyotropic liquid crystals (LLCs). In their crosslinked form, i.e., after crosslinking, hydrogels can be considered to be chemically crosslinked lyotropic liquid crystals (LLCs). Crosslinking of amphiphilic polymers can be considered to form polymerized lyotropic liquid crystals (PLLCs) with well-defined structures.

[0033] As previously mentioned, crosslinked hydrogels have a repeating, ordered nanostructure. The repeating, ordered nanostructure of amphiphilic hydrogels contains repeating and alternating hydrophobic-hydrophilic domains. The hydrogel contains the repeating, ordered nanostructure throughout the hydrogel, i.e., not just on the surface of the hydrogel. Crosslinked hydrogels are solids. Intermolecular crosslinks irreversibly fix the ordered structure, resulting in hydrogels with high integrity and mechanical elasticity.

[0034] Covalent attachment of peptides can be achieved through covalent bonds between carboxyl groups in the hydrophilic domains of the hydrogel. In the case of the peptide RRP9W4, strong amide bonds are formed between the peptide and the repeating hydrophilic domains of the hydrogel. Peptides can be covalently attached to the hydrogel via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS) activation of the carboxyl groups present in the hydrophilic domains of the hydrogel.

[0035] The endotoxin-neutralizing crosslinked hydrogel can be provided in a dispersion. In particular, when the crosslinked hydrogel is provided in particulate form, the endotoxin-neutralizing crosslinked hydrogel can be provided in a dispersion. The dispersion contains hydrogel particles as a suspension of particles in a solution. A peptide can be mixed with a solution containing individual crosslinked hydrogel particles to covalently bond the peptide to the particles. The solution can be an aqueous solution. The solution can be saline. The solution can be a biocompatible buffer, i.e., a buffer that is non-toxic to cells, such as PBS, as shown in the experimental section. Because the crosslinked hydrogel particles are amphiphilic, the solution can be a non-aqueous solution, such as a non-polar solvent.

[0036] The particles in the dispersion are swollen, i.e., they absorb solution. However, in contrast to other hydrogel dispersions, they are truly suspensions of individual hydrogel particles in solution, not simply liquid hydrogels. Because some hydrogels contain individual particles that swell to form hydrogels within them, the hydrogel itself may sometimes be described as a dispersion or suspension itself. In this case, the composition contains multiple crosslinked hydrogel particles that are separate from each other and from a continuous medium, such as an aqueous solution. [Example]

[0037] Experimental section Experiment 1: Endotoxin binding of AMP-functionalized cross-linked hydrogel discs

[0038] Preparation of hydrogel discs DA-F127 was synthesized according to Experiment 1 of WO 2019 / 074422 A1 for Pluronic® F127. DA-F127 with a purity of at least 95% was mixed with water to a composition of 30% polymer and 70% water by weight. After mixing, the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone was added to the formed gel in an amount equivalent to 0.5% by weight of the polymer in the gel. The gel was refrigerated for at least two days. After refrigeration, the gel was crosslinked at 302 nm (UVP crosslinker; CL-3000M) for 3 minutes (1.5 minutes per side, total dose 0.8-0.9 J / cm). 2 The hydrogel was punched into 12 mm diameter disks. The crosslinked hydrogel disks were then washed in water for at least 2 days.

[0039] AMP binding to disk For AMP functionalization, hydrogel discs were placed in the bottom of a 12-well plate, and 2 ml of activation solution was added to each well. The activation solution consisted of 1 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in 0.5 M MES buffer (pH approximately 5.5, adjusted by adding NaOH). The solution was left for 30 minutes and then washed three times with Milli-Q water. Meanwhile, AMP (RRPRPRPRPWWWW-NH2, purity ≥90%, SEQ ID NO: 2) was weighed into a Falcon tube and PBS was added to form a 200 μM solution. This was then added to the activated and washed hydrogel discs at 2 ml per hydrogel and allowed to react for 2 hours. The discs were then washed three times with Milli-Q water and were ready for use.

[0040] Endotoxin sample preparation Control hydrogel and AMP-hydrogel discs with a diameter of approximately 14 mm (fully swollen) were prepared as described above. Standard cotton gauze was also cut into 14 mm squares to roughly match the same area and used as a reference material. Different samples were placed in the bottom of individual 20 ml glass vials that had been pre-autoclaved. Pseudomonas aeruginosa (CCUG 56489) was inoculated into TSB and incubated until an OD of 0.55–0.7, corresponding to approximately 10 CFU / ml. Next, TSB was added to a concentration of 5%, and the bacteria were diluted to 10 CFU / ml in PBS. 50 μl of this solution was then gently placed on top of the different samples and into the glass vials (hereafter referred to as PBS). A 13 mm diameter glass coverslip was then placed very gently on top of the droplets without applying pressure to ensure good and uniform contact with the sample. The caps of the glass vials were gently screwed on, and the vials were left on the bench at room temperature to ensure no liquid spilled. After 3 or 24 hours, the glass vials were opened and 4950 μl of PBS was added. Each vial was then vortexed at maximum speed for 10 seconds to ensure the glass cover slid away from the sample. 1 ml of each solution was drawn up into a syringe and passed through a 0.2 μm syringe filter (polyethersulfone, Whatman Puradisc 25) into an Eppendorf tube. Tubes from the 3-hour samples were frozen until analysis.

[0041] Endotoxin detection assay After collecting extracts from the 24-hour samples, the solutions were analyzed for endotoxin levels using the GenScript ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit. Following the manufacturer's protocol, the 3-hour samples were diluted 100-fold and the 24-hour samples were diluted 1000-fold before analysis. Data were statistically analyzed using the Mann-Whitney U test.

[0042] Endotoxin detection assay results Results from endotoxin binding after 3 and 24 hours of incubation with P. aeruginosa can be seen in Figure 1. PBS, cotton gauze, and the control hydrogel all exhibited similar endotoxin levels of approximately 450 EU / ml at the 3-hour incubation period, while the AMP-hydrogel sample exhibited significantly lower endotoxin levels of 55 EU / ml. The same general trend was observed for the 24-hour incubation, but at higher levels. PBS and cotton gauze recorded endotoxin levels of approximately 20,000 EU / ml, while the control hydrogel recorded a slightly lower level of 13,500 EU / ml. The endotoxin level observed for the AMP-hydrogel was again significantly lower at 2,300 EU / ml.

[0043] These results clearly show that the solution that had been in contact with the AMP-functionalized hydrogel had lower endotoxin levels compared to the other samples. It was also very clear that allowing the bacteria to survive and grow released more endotoxin into the surrounding environment over time, as the endotoxin levels in the PBS sample increased from approximately 450 EU / ml at 3 hours to over 20,000 EU / ml at 24 hours.

[0044] Experiment 2: Endotoxin binding of AMP-functionalized cross-linked hydrogel particle dispersions

[0045] Preparation of hydrogel granules After washing, the hydrogel was ground into a coarse paste using a kitchen blender. The paste was then added to water and dispersed using an Ultra-Turrax® disperser to obtain a finer particle size and distribution. The resulting solution was then stable for convenient experimentation.

[0046] AMP binding to granules A known weight of granules (swollen form) was obtained by suction filtration and then placing the granules in a 15 ml Falcon tube. This was typically approximately 2 grams. The resulting batch of granulated particles was divided into two batches. The first batch was a control batch of particles without AMP attached. The second batch was activated with the AMP, RRP9W4N. To activate the particles, 10 ml of freshly prepared EDC / NHS (2 mg / ml) in MES buffer was added to the Falcon tube. The tube was sonicated for several minutes and then placed on a shaker plate for 30 minutes. The solution was then suction filtered and washed with water to separate the particles and wash away excess EDC / NHS. The granules were then weighed to record the loss, after which 10 ml of 400 μM AMP (RRPRPRPRPWWWW-NH2, purity ≥ 90%, SEQ ID NO: 2) (dissolved in PBS) was added to the granules. This was also quickly sonicated and then placed on a shaker plate for approximately 2 hours.

[0047] The solution was again filtered under suction and washed with 30 ml of water, but this time the wash was collected to measure the amount of peptide still remaining after activation. The granules were now activated with AMP and could be weighed into solution to obtain a known concentration for further experiments.

[0048] Endotoxin sample preparation A colony of E. coli (CCUG 29300) was inoculated into tryptic soy broth (TSB). 9 The test substance was prepared and diluted to the correct concentration. Then, 2x the target concentration was prepared. The goal was 50x the MIC for very rapid activity. For this test, 2ml of each of the following (for 2x 50x the MIC) was prepared:

[0049] [Table 1]

[0050] The prepared material was added to Eppendorf tubes, and 500 μl of PBS was added to each. A solution of 10% TSB and 90% PBS was prepared. 500 μl of the bacterial solution was added to each tube except the -bac tube. 6 The concentration of CFU / ml was adjusted. 500 μl of PBS was added to the -bac tube.

[0051] The tubes were vortexed and incubated at 37° C. Samples were removed at desired time intervals (1 hour, 3 hours, and 24 hours).

[0052] Endotoxin detection assay The endotoxin detection protocol was based on the ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit L00350 (Genscript) and the manufacturer's recommended protocol, with modifications based only on the target concentrations recommended in the manufacturer's protocol, version 07082022. Absorbance was read at 545 nm on a plate reader using distilled water as a reference. Endotoxin concentrations were determined using a standard curve from endotoxin standards.

[0053] result The results of the endotoxin detection assay after 1 and 3 hours are shown in Figure 2. In the 1- and 3-hour experiments, no control hydrogel particles were present. The 24-hour results, including control hydrogel particles, are shown in Figure 3. The baseline for both the 3- and 24-hour values ​​was PBS without bacteria, and after 24 hours, the AMP-conjugated hydrogel particles gave negative values.

[0054] The results show that the antimicrobial peptide neutralized endotoxin at all incubation periods measured. The results further demonstrate that the antimicrobial peptide bound to the hydrogel substrate exhibited similar efficacy to the antimicrobial peptide alone. The above results support the use of peptides containing the sequences defined herein as endotoxin neutralizing agents. Peptides can be advantageously bound to hydrogel substrates for use as endotoxin neutralizing agents. The antibiotic ciprofloxacin induced significantly more endotoxin release than free AMP or AMP bound to the hydrogel.

[0055] The claimed subject matter is limited only by the appended claims. In the claims, the term "comprises" does not exclude the presence of other elements or steps. Furthermore, although individual features may be included in different claims, these may conceivably be advantageously combined, and the inclusion of features in different claims does not imply that a combination of features is not feasible and / or advantageous. In addition, singular referents do not exclude a plurality. The use of terms such as "a," "an," "first," "second," etc. does not exclude a plurality.

Claims

1. 1. An amphipathic peptide for use as an endotoxin neutralizing agent, comprising: an amino acid sequence having at least 80% sequence identity with the sequence RRP9 (SEQ ID NO: 1), - at least four tryptophan residues provided at the C-terminus or N-terminus or therebetween.

2. 2. An amphiphilic peptide for use according to claim 1, having less than 40 amino acids, such as less than 30 amino acids, preferably less than 20 amino acids.

3. 3. An amphiphilic peptide for use according to claim 1 or 2, wherein the tryptophan residues are provided consecutively or in consecutive pairs separated by at most one arginine-proline pair.

4. The amphiphilic peptide for use according to any one of claims 1 to 3, wherein said use is in vivo.

5. 5. The amphipathic peptide for use according to claim 4, which is for use in the treatment of endotoxin-induced inflammation.

6. 6. The amphiphilic peptide for use according to any one of claims 1 to 5, comprising an amino acid sequence having at least 90% sequence identity with a sequence selected from RRP9W4N (SEQ ID NO: 2), RRP7W4RPN (SEQ ID NO: 4) or RRP5W2RPW2RPN (SEQ ID NO: 5).

7. The amphiphilic peptide for use according to any one of claims 1 to 6, which is RRP9W4N (SEQ ID NO: 2).

8. The amphiphilic peptide for use according to any one of claims 1 to 7, which is bound to a hydrogel substrate.

9. The amphiphilic peptide for use according to claim 8, which is covalently attached to the hydrogel matrix.

10. 10. The amphiphilic peptide for use according to claim 8 or 9, wherein the hydrogel matrix is ​​a solid cross-linked hydrogel.

11. 11. The amphiphilic peptide for use according to any one of claims 1 to 10, wherein the peptide and / or hydrogel to which the peptide is attached is provided on a substrate such as a wound dressing, a stoma dressing, a stoma base plate, an incision film, a patch, a bandage, a plaster, an adhesive, a bandage, a catheter, a filter for a fluid sample, or any combination thereof.

12. 1. A method of in vitro endotoxin neutralization, said method comprising contacting a fluid sample with an amphipathic peptide, said amphipathic peptide comprising: an amino acid sequence having at least 80% sequence identity with the sequence RRP9 (SEQ ID NO: 1), - at least four tryptophan residues provided at or between the C-terminus or N-terminus.

13. The in vitro method for endotoxin neutralization according to claim 12, wherein the amphiphilic peptide is covalently attached to a hydrogel substrate.

14. Use of an amphiphilic hydrogel composition in endotoxin neutralization, wherein the amphiphilic hydrogel in its chemically crosslinked state is a lyotropic liquid crystal and has an ordered nanostructure of hydrophilic and hydrophobic domains, and an endotoxin-neutralizing amphiphilic peptide is covalently bound to the hydrophilic and / or hydrophobic domains.

15. The endotoxin-neutralizing amphipathic peptide is an amino acid sequence having at least 80% sequence identity with the sequence RRP9 (SEQ ID NO: 1), - at least four tryptophan residues provided at or between the C-terminus or N-terminus.

16. 16. Use of an amphiphilic hydrogel composition according to claim 15, wherein the amphiphilic peptide has less than 40 amino acids, such as less than 30 amino acids, preferably less than 20 amino acids.

17. 17. Use of an amphiphilic hydrogel composition according to claim 15 or 16, wherein the peptide comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from RRP9W4N (SEQ ID NO: 2), RRP7W4RPN (SEQ ID NO: 4) or RRP5W2RPW2RPN (SEQ ID NO: 5).

18. Use of an amphiphilic hydrogel composition according to any one of claims 15 to 17, wherein said peptide comprises an amino acid sequence having at least 90% sequence identity with said sequence RRP9W4N (SEQ ID NO: 2).

19. Use of an amphiphilic hydrogel composition according to any one of claims 14 to 18, wherein the hydrogel is provided as discrete particles in a dispersion.

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