Endotoxin neutralizing agent

Abstract

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

Description

Technical Field

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

[0003] Bacterial infections remain a threat to human health, driven in part by increasing antimicrobial resistance, demographic shifts brought on by an aging population, and a lack of newly approved antibiotic treatments.

[0004] Severe bacterial infections can lead to sepsis, a condition characterized by a systemic inflammatory state that carries significant morbidity and mortality.

[0005] One challenge in treating sepsis is that drugs must kill the bacteria without releasing toxins that induce inflammation. For Gram-negative bacteria, these toxins are lipopolysaccharides (LPS); for Gram-positive bacteria, these toxins are lipoproteins (LP / LPs).

[0006] WO 2019 / 074422 A1 (Amferia AB) describes an antimicrobial hydrogel and an antimicrobial peptide. The antimicrobial peptide has been shown to have significant antimicrobial efficacy. However, the antimicrobial efficacy of antimicrobial peptides does not necessarily correspond to their anti-endotoxin efficacy. There is currently no simple model for antimicrobial efficacy or anti-endotoxin efficacy. There is also no clear correlation between folded structure, length and sequence and anti-endotoxin efficacy. (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)

[0007] Antimicrobial peptides are suitable for killing bacteria and have been considered as potential treatments for sepsis and many other diseases caused by bacterial infections, but because killing bacteria also releases toxins that trigger inflammation, this may not be practical.

[0008] Since free antimicrobial peptides have been shown to degrade rapidly in environments such as blood, providing antimicrobial peptides covalently attached to a matrix is ​​an ideal wound treatment approach. However, attaching antimicrobial peptides to a matrix may negatively impact the anti-endotoxin activity of effective antimicrobial peptides due to changes in structure, available binding sites, and other factors. Studies have shown that AMP efficacy is reduced 500-1000-fold after immobilization on a surface (The Potential of Surface-Immobilized Antimicrobial Peptides for the Enhancement of Orthopaedic Medical Devices: A Review). Antibiotics (Basel). 2023; 12(2): 211. January 19, 2023). Therefore, the endotoxin binding efficacy of AMPs is expected to be reduced after immobilization on a surface.

[0009] Improved anti-endotoxin therapies would be advantageous. In particular, antimicrobial peptides that possess both antimicrobial and anti-endotoxin efficacy would be advantageous. Summary of the Invention

[0010] Therefore, the present invention preferably aims to alleviate, mitigate or eliminate one or more of the above-mentioned defects and disadvantages in the prior art (alone or in any combination) and solve at least the above-mentioned problems by providing an amphiphilic peptide for use as an endotoxin neutralizer, which peptide comprises an amino acid sequence having at least 80% sequence identity with the sequence RRP9 (SEQ ID: 1) and having at least four tryptophan residues at the C-terminus or N-terminus or in between.

[0011] The present invention provides an amphiphilic hydrogel composition for endotoxin neutralization.

[0012] In addition, an in vitro endotoxin neutralization method is also provided.

[0013] Further advantageous embodiments are disclosed in the additional dependent patent claims.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] These and other aspects, features and advantages of the present invention will become more apparent and clear through the following description of the embodiments of the present invention in conjunction with the accompanying drawings, in which:

[0016] Figure 1The results of the endotoxin neutralization test described in Experiment 1 are shown below. Endotoxin levels were measured after 3 hours and 24 hours, respectively. The left axis and left column in each pair of bar graphs represent endotoxin levels after 3 hours, and the right axis and right column in each pair of bar graphs represent endotoxin levels after 24 hours.

[0017] Figure 2 Results of the endotoxin neutralization test described in Experiment 2, detailed 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 bound to cross-linked hydrogel particles.

[0018] Figure 3 Results of the endotoxin neutralization test described in Experiment 2, detailed below, are shown. Results show endotoxin levels detected after 24 hours. CP represents cross-linked hydrogel particles without antimicrobial agent attached. PBS-b represents PBS without bacteria, and PBS+b represents PBS with bacteria. AMP represents the antimicrobial peptide RRP9W4N in solution. AMP-P represents the same antimicrobial peptide bound to the cross-linked hydrogel particles. DETAILED DESCRIPTION

[0019] The present disclosure relates to the use of antimicrobial peptides as anti-endotoxin agents. Anti-endotoxin agents can be referred to as endotoxin neutralizers. Furthermore, due to their ability to inhibit inflammation, anti-endotoxin agents can also be referred to as anti-inflammatory agents. The anti-endotoxin agents can be used to treat bacterial sepsis. The endotoxin neutralizers can be used in vivo.

[0020] The antimicrobial peptide can be an antimicrobial peptide derived from the proline arginine rich end leucine rich repeat protein (PRELP). The antimicrobial peptide can comprise a sequence with 80% (e.g., 90%, e.g., 95%) identity to RRPRPRPRP (RRP9 SEQ ID: 1). Such peptides have been shown to be similar to LL-37 derived from human cathelicidin in many aspects, such as net charge and antimicrobial efficacy (Malmsten, M et al., Highly Selective End-Tagged Antimicrobial Peptides Derived from PRELP (derived from the highly selective end-tagged antimicrobial peptide of PRELP), PLoS ONE, 2011, 6(1):e16400.doi:10.1371 / journal.pone.0016400).

[0021] The antimicrobial peptide is advantageously a short chain peptide consisting of 1 to 50 amino acids, such as less than 40 amino acids, such as less than 30 amino acids, preferably less than 20 amino acids. The molecular weight of the antimicrobial peptide may be between 1 and 50 kDa.

[0022] As described above, the antimicrobial peptide may comprise a sequence that is at least 90% identical to the sequence RRPRPRPRP (SEQ ID: 1, RRP9). Ideally, the antimicrobial peptide comprises a stretch of at least two consecutive tryptophan or phenylalanine residues attached to or between the C-terminus or N-terminus. The antimicrobial peptide may comprise an N-terminal amidation. Advantageously, the antimicrobial peptide is amphiphilic, as this property has previously been shown to have improved efficacy and fixation.

[0023] The antimicrobial peptide can be, for example, RRPRPRPRPWWWW-NH2 (SEQ ID: 2, RRP9W4N), RRPRPRPRP-NH2 (SEQ ID: 3, RRP9N), RRPRPRPWWWWRP-NH2 (SEQ ID: 4, RRP7W4RPN) or RRPRPWWRPWWRP-NH2 (SEQ ID: 5, RRP5W2RPW2RPN). The above peptide sequence can be described as a peptide comprising the sequence RRP9 (SEQ ID: 1) and at least four tryptophan residues, wherein these tryptophan residues are continuous, or at least two pairs of two continuous residues separated by an arginine-proline pair (i.e., WWRPWW (SEQ ID: 6)).

[0024] As shown in the experimental part, antimicrobial peptides comprising sequences corresponding to the sequences defined herein surprisingly showed endotoxin neutralization efficacy.Antimicrobial peptides corresponding to the above definition have both a cationic and a hydrophobic part, thereby forming amphipathic antimicrobial peptides.

[0025] The peptide can be used in a method for neutralizing endotoxins in vivo. A therapeutically effective amount of the peptide can be administered to a patient. The peptide can be used to treat an acute inflammatory state in a patient. The peptide can be used to treat an inflammatory state induced by endotoxins. The peptide can be used to treat LPS-induced inflammation. The inflammatory state can be sepsis. Sepsis is defined as the presence or presumed presence of an infection accompanied by evidence of a systemic response, known as systemic inflammatory response syndrome (SIRS). Sepsis is typically caused by bacterial infection, including Gram-negative or Gram-positive bacteria, but can also be caused by other pathogens, such as viruses, fungi, and protozoa. The damage and symptoms caused by sepsis are not only caused by bacteria, but also by a component of the bacterial cell wall, called endotoxins or LPS. LPS molecules are glycolipids commonly found in the outer membrane of Gram-negative bacteria. When the immune system destroys invading bacteria, LPS is released. Released LPS binds to monocytes, macrophages, and endothelial cells and triggers the production of various mediators, such as TNF-α and interleukins (IL-1, IL-6, and IL-8). Excessive production of TNF-a, IL-1, IL-6, and IL-8 is the main cause of severe sepsis.

[0026] The amount of peptide that is effective for treating a particular disease or condition depends on the nature of the disease or condition and the specific peptide. The effective amount can be determined by standard clinical techniques known to those skilled in the art. In addition, in vitro assays can optionally be used to help determine the optimal dosage range. The precise dose to be used in the formulation will also depend on the route of administration, and the nature of the disease or condition, and should be determined based on the doctor's judgment and the circumstances of each patient. The effective dose can be derived from a dose-response curve obtained by an in vitro or in vivo animal model test bioassay or system. For example, a composition containing the peptide can be introduced into the systemic circulation so that the peptide is distributed in the patient's body.

[0027] The peptide can be used for in vitro endotoxin neutralization. Patient samples (e.g., blood, plasma, etc.) can be contacted with the peptide to neutralize endotoxins in the patient's blood, etc. In vitro endotoxin neutralization methods may include contacting a patient sample extracted from the patient with a peptide (e.g., RRP9W4N (SEQ ID: 2) or other sequences disclosed herein).

[0028] Antimicrobial peptides used as endotoxin neutralizers can be attached to the hydrogel matrix, for example covalently attached to the hydrogel matrix. As described above, when the peptides are used for in vitro endotoxin neutralization, the cross-linked hydrogel forms an ideal matrix to which AMPs can be immobilized.

[0029] As described in WO 2019 / 074422 A1 (Amferia AB), if the hydrogel matrix is ​​an amphiphilic hydrogel comprising repeating hydrophilic and hydrophobic regions, the antimicrobial efficacy may be improved. However, as mentioned above, the improvement in antimicrobial efficacy does not correspond to an improvement or even maintenance of anti-endotoxin efficacy. After the peptide is immobilized on the surface, the antimicrobial efficacy is generally reduced, and therefore the endotoxin binding efficacy after immobilization is also expected to be reduced. However, the inventors have found that even if fixed on a hydrogel, the endotoxin binding efficacy of peptides comprising the sequence RRP9 (SEQ ID: 1) (e.g., RRP9W4N (SEQ ID: 2)) or other sequences defined herein is substantially maintained. In addition,

[0030] As shown in the experimental section, the inventors unexpectedly demonstrated that attachment of peptides to the hydrogel matrix does not inhibit anti-endotoxin efficacy. As shown in Experiment 2, cross-linked hydrogel particles immobilized with AMP have similar endotoxin binding efficacy to the corresponding amount of free AMP in solution.

[0031] The hydrogel matrix can be in the form of a cross-linked solid hydrogel, such as that disclosed in WO 2019 / 074422 A1 (Amferia AB). The hydrogel to which the antimicrobial peptide is attached can be discrete cross-linked amphiphilic hydrogel particles, layers, sheets, or other suitable forms. If the hydrogel is provided in the form of discrete particles, the discrete cross-linked hydrogel particles can be obtained by, for example, grinding a solid cross-linked hydrogel sheet into particles (as described in the experimental section) or other suitable methods for providing discrete hydrogel particles. Discrete cross-linked hydrogel particles may be particularly suitable carrier media to which the endotoxin neutralizer can be attached. For example, the particles have a high relative surface area, allowing the endotoxin neutralizer to bind. However, the hydrogel can be in any suitable form to which the endotoxin neutralizing particles can be attached. The hydrogel can be a solid sheet to which the AMP is attached. The hydrogel to which the AMP is attached can be provided as a wound dressing, ostomy dressing, ostomy baseplate, incision membrane, patch, bandage, plaster, adhesive, fish gelatin plaster (court plaster), catheter, or a combination thereof. The hydrogel with AMP attached can be provided as a filter or used as a component of a fluid (eg, patient sample) filter. Specifically, for the in vitro neutralization of endotoxins, the hydrogel with AMP attached can be used as a component of a fluid sample filter.

[0032] When used as an endotoxin neutralization filter, the hydrogel with AMP attached can be subjected to fluid. The fluid may contain known or unknown amounts of endotoxin. The endotoxin present in the fluid will be neutralized by the hydrogel with AMP attached. The fluid can be a fluid sample derived from a patient, or a fluid sample derived from a non-patient source, or a combination of a sample derived from a patient and a reagent derived from a non-patient source. In vitro filtration / neutralization of endotoxins can be used for reagents in a variety of processes that should be endotoxin-free. For example, the hydrogel with AMP attached can be used to filter reagents and / or cellular substances before an in vitro fertilization procedure. After contact with the hydrogel with AMP attached, the fluid sample is substantially free of endotoxin.

[0033] The process for preparing discrete hydrogel particles comprises providing a hydrogel composition comprising a first amphiphilic polymer. The first amphiphilic component of the composition can be a cross-linkable amphiphilic polymer. A typical and suitable amphiphilic material is a diacrylate-modified poloxamer, such as the polyethylene oxide-polypropylene oxide-polyethylene oxide (DA-PEO) described in WO 2019 / 074422A1. x -PPO y -PEO x -DA, where x and y refer to the number of PEO and PPO groups present, respectively). Specifically, the amphiphilic material can be an amphiphilic triblock copolymer, polyethylene oxide (100)-polypropylene oxide (70)-polyethylene oxide (100) ( F127 – BASF Corporation), polyethylene oxide (30)-polypropylene oxide (70)-polyethylene oxide (30) (

[0034] P123 – BASF).

[0035] As mentioned above, the first amphiphilic component can be a diacrylate derivative of the triblock copolymer, thereby enabling chemical crosslinking of the copolymer. WO 2019 / 074422 A1 (Amferia AB) Experiment 1 provides a diacrylate modification process. This modification can be performed by reacting the triblock amphiphilic copolymer with acryloyl chloride to form a diacrylate derivative. Other methods for forming crosslinkable amphiphilic polymers are also possible, such as forming methacrylate derivatives or via carboxylic acid-amine bridges.

[0036] Crosslinkable amphiphilic polymers can self-assemble in the presence of water to form ordered nanostructures known as lyotropic liquid crystals (LLCs). Hydrogels in their crosslinked form, i.e., crosslinked hydrogels, can be considered chemically crosslinked lyotropic liquid crystals (LLCs). Crosslinking of amphiphilic polymers can be considered the formation of polymeric lyotropic liquid crystals (PLLCs) with well-defined structures.

[0037] As described above, cross-linked hydrogels possess a repetitive and ordered nanostructure. The repetitive, ordered nanostructure of amphiphilic hydrogels comprises repeating and alternating hydrophobic-philic domains. The hydrogels contain an ordered, repetitive nanostructure throughout the hydrogel (not just on the surface). Cross-linked hydrogels are in a solid state. The intermolecular crosslinks irreversibly lock the ordered structure, resulting in a hydrogel with high integrity and mechanical resilience.

[0038] Covalent attachment of peptides can be achieved through covalent bonds between carboxyl groups on the hydrophilic domains of the hydrogel. In the case of peptide RRP9W4, strong amide bonds are formed between the peptide and the hydrophilic domains of the hydrogel repeats. Peptides can be covalently attached to the hydrogel by activating the carboxyl groups present on the hydrophilic domains of the hydrogel using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-N-hydroxysuccinimide (NHS).

[0039] The endotoxin-neutralizing cross-linked hydrogel can be provided in the form of a dispersion. Specifically, if the cross-linked hydrogel is provided in particulate form, the endotoxin-neutralizing cross-linked hydrogel can be provided in the form of a dispersion. The dispersion comprises the hydrogel particles as a suspension of the particles in a solution. The peptide can be mixed with a solution comprising the cross-linked discrete hydrogel particles to covalently attach the peptide to the particles. The solution can be an aqueous solution. The solution can be a saline solution. The solution can be a biocompatible buffer, i.e., a buffer that is non-toxic to cells, such as PBS as described in the experimental section. Since the cross-linked hydrogel particles are amphiphilic, the solution can be a non-aqueous solution, such as a non-polar solvent.

[0040] The particles in the dispersion swell, i.e., they absorb the solution. However, unlike other hydrogel dispersions, they are actually suspensions of dispersed hydrogel particles in a solution, rather than simply liquid hydrogels. Hydrogels themselves are sometimes described as dispersions or suspensions, as some hydrogels contain dispersed particles that swell and form a hydrogel therein. In this case, the composition comprises a plurality of cross-linked hydrogel particles that are separated from each other and from a continuous medium (e.g., an aqueous solution).

[0041] Experimental part

[0042] Experiment 1: Endotoxin Binding of AMP-Functionalized Cross-Linked Hydrogel Discs

[0043] Preparation of hydrogel discs

[0044] According to WO 2019 / 074422 A1 Experiment 1 of F127 DA-F127 was synthesized by mixing DA-F127 having a purity of at least 95% with water to a composition of 30 wt% polymer and 70 wt% water. After mixing, a photoinitiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone, was added to the resulting gel in an amount corresponding to 0.5 wt% of the polymer in the gel. The gel was refrigerated for at least two days. After refrigeration, the gel was crosslinked (UVP crosslinker; CL-3000M) at a wavelength of 302 nm for 3 minutes (1.5 minutes per side, total dose 0.8-0.9 J / cm 2 ). 12 mm diameter discs were punched into the hydrogel. The newly cross-linked hydrogel discs were washed with water for at least two days.

[0045] Binding of AMP to the disc

[0046] To perform AMP functionalization, place the hydrogel discs at the bottom of a 12-well plate and add 2 ml of activation solution, consisting of 1 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in MES buffer, 0.5 M, pH ~5.5 (adjustable by adding NaOH). Let the solution sit for 30 minutes and then rinse three times with milli-Q water. Meanwhile, weigh AMP (RRPRPRPRPWWWW-NH2, ≥90% purity, SEQ ID: 2) into a falcon tube and make a 200 μM solution in PBS. This solution is then added to the activated and washed hydrogel discs, 2 ml per hydrogel, and allowed to react for 2 hours. Finally, the discs are washed three times with milli-Q water before use.

[0047] Endotoxin sample preparation

[0048] Control hydrogels and AMP-hydrogel discs were prepared as previously described and were approximately 14 mm in diameter (fully swollen). Standard cotton gauze was cut into 14 mm square pieces, roughly matching the same area, and used as standard reference material. The different samples were placed at the bottom of a pre-autoclaved 20 ml glass bottle. Pseudomonas aeruginosa (P. aeruginosa) (CCUG56489) was inoculated in TSB and incubated until the OD value was 0.55-0.7, corresponding to approximately 109 CFU / ml. The bacteria were then diluted to 107 CFU / ml in PBS and TSB was added to a 5% concentration. 50 μl of this solution was then gently placed on top of the different samples and just into the glass vial (hereafter the sample type is referred to as PBS). A 13 mm A glass coverslip was placed very gently on top of the droplet without applying any pressure to ensure good and even contact with the sample. The cap of the glass vial was gently screwed on and the vial was left to stand on the laboratory bench at room temperature to ensure that no liquid escaped. After 3 or 24 hours, the glass vial was opened and 4950 μl of PBS was added to the vial. Each vial was then vortexed at maximum speed for 10 seconds to ensure that the glass coverslip was separated from the sample. 1 ml of each solution was drawn up with a syringe and filtered through a 0.2 μm syringe filter (polyethersulfone, Whatman's Puradisc 25) into an Eppendorf tube. The tubes of the 3 hour sample were frozen until analysis.

[0049] Endotoxin detection test

[0050] After collecting extracts from 24-hour samples, the ToxinSensor TMEndotoxin levels in the solutions were analyzed using a chromogenic LAL endotoxin test kit. Samples taken at 3 hours were diluted 100-fold, and samples taken at 24 hours were diluted 1000-fold, according to the manufacturer's instructions. Data were statistically analyzed using Mann-Whitney U-tests.

[0051] Endotoxin detection test results

[0052] Figure 1 Endotoxin binding results after 3 and 24 hours of incubation with P. aeruginosa are shown. PBS, cotton gauze, and the control hydrogel all showed similar endotoxin levels of approximately 450 EU / ml during the 3-hour incubation period, while the AMP-hydrogel sample showed a significantly lower endotoxin level of 55 EU / ml. The same general trend was observed after the 24-hour incubation, but with higher endotoxin levels. PBS and cotton gauze showed endotoxin levels of approximately 20,000 EU / ml, while the control hydrogel showed a slightly lower endotoxin level of 13,500 EU / ml. Endotoxin levels were again significantly lower in the AMP-hydrogel, at 2,300 EU / ml.

[0053] These results clearly demonstrate that the AMP-functionalized hydrogels contained lower levels of endotoxin compared to the other samples. It is also clear that surviving and proliferating bacteria release increasing amounts of endotoxin into the surrounding environment over time, as endotoxin levels in the PBS sample rise from approximately 450 EU / ml within 3 hours to over 20,000 EU / ml within 24 hours.

[0054] Experiment 2: Endotoxin Binding of AMP-Functionalized Cross-Linked Hydrogel Particle Dispersions

[0055] Preparation of hydrogel particles

[0056] After washing, grind the hydrogel into a coarse paste using a kitchen blender. Then add the paste to water and use The disperser achieves finer particle size and distribution. The resulting solution is then stable and convenient for experiments.

[0057] Binding of AMP to particles

[0058] The particles are then filtered and placed in a 15ml Falcon tube to obtain particles of known weight (swollen form). Usually about 2 grams. The resulting granulation batch is divided into two batches. The first batch is a control particle batch, without AMP attached. The second batch is activated with AMP, RRP9W4N. To activate the particles, 10ml of freshly prepared EDC / NHS (2mg / ml) is dissolved in MES buffer and added to a falcon tube. The tube is ultrasonicated for a few minutes and then placed on a rocking plate for 30 minutes. The solution is then filtered and washed with water to separate the particles and wash away excess EDC / NHS. The particles are then weighed to record any losses, and then 10ml of 400μM AMP (RRPRPRPRPWWWW-NH2, purity ≥90%, SEQ ID: 2) (dissolved in PBS) is added to the particles. Similarly, rapid ultrasonication is performed and then placed on a rocking plate for about 2 hours.

[0059] The solution was filtered again and washed with 30 ml of water, but this time the wash was recovered to measure the amount of peptide remaining after activation. The particles were now activated with AMP and could be weighed and placed in solution to obtain a known concentration for further experiments.

[0060] Endotoxin sample preparation

[0061] E. coli colonies (CCUG 29300) were inoculated into tryptone soy broth (TSB). 9 CFU / ml). Prepare the test substance and dilute it to the correct concentration. Subsequently, prepare a 2x concentration of the substance of interest. The goal is to achieve 50x the MIC, which results in reasonably rapid activity. For this study, prepare 2 ml of each of the following solutions (for 2x50xMIC):

[0062]

[0063] Add the prepared material to Eppendorf tubes and add 500 μl of PBS to each tube. Prepare a solution of 10% TSB and 90% PBS. Add 500 μl of bacterial solution to each tube except the -bac tube to make a 10% solution. 6 CFU / ml concentration. Add 500 μl PBS to the -bac tube.

[0064] The tubes were vortexed and incubated at 37° C. Each sample was removed at the desired time intervals (1 hour, 3 hours, and 24 hours).

[0065] Endotoxin detection test

[0066] Endotoxin detection solution based on ToxinSensor TMThe chromogenic LAL endotoxin test kit L00350 (Genscript) and the manufacturer's recommended protocol were used, with modifications based on the concentration of interest suggested in the manufacturer's protocol version 07082022. The absorbance was read at 545 nm using a plate reader with distilled water as a reference. Endotoxin concentrations were measured using a standard curve prepared with endotoxin standards.

[0067] result

[0068] Figure 2 Endotoxin detection test results are shown after 1 hour and 3 hours. In the 1 hour and 3 hour experiments, no control hydrogel particles were present. Figure 3 Results for the 24-hour assay are shown for control hydrogel particles. The baseline for both the 3-hour and 24-hour values ​​was PBS without bacteria, resulting in negative values ​​for the AMP-attached hydrogel particles after 24 hours.

[0069] The results show that the antimicrobial peptides can neutralize endotoxins in all measured incubation durations. The results further show that the antimicrobial peptides attached to the hydrogel matrix have similar efficacy to the independent antimicrobial peptides. The above results support the use of peptides comprising the sequences defined herein as endotoxin neutralizers. The peptides can advantageously be attached to the hydrogel matrix to serve as endotoxin neutralizers. The endotoxin release induced by the antibiotic ciprofloxacin is significantly higher than that of free AMP or AMP attached to the hydrogel.

[0070] The claimed subject matter is limited only by the appended claims. In the claims, the term "comprises / comprising" does not exclude the presence of other elements or steps. Furthermore, although individual features may be included in different claims, these features may be advantageously combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. Furthermore, singular references do not exclude plural references. Terms such as "a", "an", "first", "second", etc. do not exclude plural references.

Claims

1. An amphiphilic peptide for use as an endotoxin neutralizer, the peptide comprising: - an amino acid sequence having at least 80% sequence identity to the sequence RRP9 (SEQ ID: 1), and - at least four tryptophan residues located at the C-terminus or N-terminus or in between.

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

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

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

5. The amphiphilic peptide for use according to claim 4, wherein the peptide is used to treat endotoxin-induced inflammation.

6. The amphiphilic peptide for use according to any one of claims 1 to 5, wherein the peptide comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from RRP9W4N (SEQ ID: 2), RRP7W4RPN (SEQ ID: 4) or RRP5W2RPW2RPN (SEQ ID: 5).

7. The amphiphilic peptide for use according to any one of claims 1 to 6, wherein the peptide is RRP9W4N (SEQ ID: 2).

8. An amphiphilic peptide for use according to any one of claims 1 to 7, wherein the peptide is attached to a hydrogel matrix.

9. An amphiphilic peptide for use according to claim 8, wherein the peptide is covalently attached to a hydrogel matrix.

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

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

12. An in vitro endotoxin neutralization method comprising contacting a liquid sample with an amphiphilic peptide comprising: - an amino acid sequence having at least 80% sequence identity to the sequence RRP9 (SEQ ID: 1), and - at least four tryptophan residues located at the C-terminus or N-terminus or in between.

13. The in vitro endotoxin neutralization method of claim 12, wherein the amphiphilic peptide is covalently attached to the hydrogel matrix.

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

15. The use of the amphiphilic hydrogel composition according to claim 14, wherein the endotoxin-neutralizing amphiphilic peptide comprises: - an amino acid sequence having at least 80% sequence identity to the sequence RRP9 (SEQ ID: 1), and - at least four tryptophan residues located at the C-terminus or N-terminus or in between.

16. Use of the 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. Use of the 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: 2), RRP7W4RPN (SEQ ID: 4) or RRP5W2RPW2RPN (SEQ ID: 5).

18. Use of the amphiphilic hydrogel composition according to any one of claims 15 to 17, wherein the peptide comprises an amino acid sequence having at least 90% sequence identity with the sequence RRP9W4N (SEQ ID: 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.

Images