Improvement of down-regulation of histone cytotoxicity by a complex of a negatively charged polysaccharide and a protease

Abstract

The present disclosure describes a complex of a negatively charged polysaccharide and a protein having serine protease activity. The negatively charged polypeptide is, for example, heparin or non-anticoagulant heparin. The serine protease is, for example, elastase or activated protein C. The complex can be used for the treatment, prevention or amelioration of diseases or disorders associated with immunothrombosis.

Description

Technical Field

[0001] The present invention relates to a complex of a negatively charged polysaccharide and a protein having protease activity. The inventors have shown that such complexes can be used to degrade extracellular histones. Such complexes are particularly useful for the treatment, prevention or amelioration of diseases or disorders mediated by extracellular histones, or diseases or disorders in which immunothrombosis is a problem, such as diseases or disorders such as sepsis and Covid-19. Non-limiting exemplary complexes according to the present invention are complexes of heparin or non-anticoagulant heparin and elastase or activated protein C.

Background Art

[0002] Extracellular histones (eHs) may be found in the circulating blood of patients suffering from related diseases and disorders mediated by extracellular histones, such as acute inflammatory diseases such as sepsis and COVID-19, but have also been demonstrated to play a role in trauma, stroke, and pancreatitis. Circulating eHs are cleaved by proteolysis. Proteolytic cleavage of eHs destroys cytotoxic activity. Patients with uncleaved circulating eHs have a significantly higher risk of worsening morbidity and mortality.

[0003] Over the past few years, it has become clear that eHs plays a major role in the development of diseases such as systemic inflammatory response syndrome, sepsis, and COVID-19. Recently, it has been shown that eHs significantly promotes the progression of prostate cancer through the inflammatory response via the NF-κB pathway. Therefore, neutralizing eHs is a promising therapeutic approach for treating cancer patients, especially those who have received chemotherapy or radiation therapy that dramatically increases eHs. Also, in organ preservation and transplantation, eHs worsens the outcome of transplantation. On the other hand, in atherosclerosis, it has been found that the lysis of smooth muscle cells (SMCs) mediated by eHs causes damage and inflammation in arterial tissue. Therefore, pharmacologically neutralizing eHs has been evaluated as an important strategy for treating this type of disease. Heparin has been proposed as a therapeutic strategy for several indications involving eHs, but the dual actions of heparin, namely its anti-inflammatory and anticoagulant effects, may result in undesirable effects. Therefore, low-anticoagulant heparin has been proposed as an alternative. Low-anticoagulant heparin that neutralizes eHs by electrostatic interaction is currently being investigated as a therapeutic agent for patients with sepsis and COVID-19. However, low-anticoagulant heparin may result in undesirable effects for patients, such as an undesired increase in activated partial thromboplastin time (aPTT), and thus formulations with improved efficacy or fewer side effects are desired. The present invention described in the appended claims aims, inter alia, to overcome these problems.

[0004] EP 0326014 A2 describes the use of a composition containing heparin and APC as an improved anticoagulant. Pejler et al. (Biofactors Volume 35, Number 1, January / February 2009, Pages 61-68) are concerned with the role of serglycin in promoting the storage and regulating the activity of many proteases expressed in hematopoietic cell types, particularly various mast cell proteases. It is also described that an interaction between mast cell proteases and heparin is likely to occur in vivo. Kummarapurugu et al. (J. Biol. Chem. (2018) 293(32) 12480-12490) describe that neutrophil elastase is inhibited by heparin in patients with cystic fibrosis. Wildhagen et al. (BLOOD, 13 FEBRUARY 2014 - VOLUME 123, NUMBER 7 pages 1098-1101) teach the use of heparin in sepsis by reducing coagulation. Kowalska et al. (Arterioscler Thromb Vase Biol. 2014;34:120-126) teach that heparin modulates activated protein C levels that may be beneficial in sepsis. SUMMARY OF THE INVENTION

[0005] In a first aspect, the present invention relates to a complex of a negatively charged polysaccharide and a protein having protease activity.

[0006] In a second aspect, the present invention relates to a pharmaceutical composition comprising a complex according to the first aspect of the present invention and a pharmaceutically acceptable carrier.

[0007] In a third aspect, the present invention relates to a complex according to the first aspect of the present invention or a pharmaceutical composition according to the second aspect of the present invention for use as a medicament.

[0008] In a fourth aspect, the present invention relates to a complex according to a first aspect of the present invention or a pharmaceutical composition according to a second aspect of the present invention for use in the treatment, prevention or amelioration of a disease or disorder mediated by extracellular histones. BRIEF DESCRIPTION OF THE DRAWINGS

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

[0010] In the present invention, the following terms are defined.

[0011] As used herein, the singular terms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes combinations of two or more cells, etc.

[0012] As used herein, the term "and / or" refers to a situation in which one or more of the recited cases may occur alone, or in combination with at least one of the recited cases, up to and including all of the recited cases.

[0013] In this specification, the term "at least" for a particular value means that value or more. For example, "at least 2" is understood to be the same as "2 or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,.... In this specification, the term "at most" for a particular value means that value or less. For example, "at most 5" is understood to be the same as "5 or less", i.e., 5, 4, 3,.... -10, -11, etc.

[0014] As used herein, the term "comprise", "comprises", "comprising" or variations thereof means including the recited element, integer or step, or group of elements, integers or steps, but not excluding other elements, integers or steps, or group of elements, integers or steps. The verb "comprising" includes the verbs "essentially consisting of" and "consisting of".

[0015] As used herein, the term "prior art" refers to situations where the methods of practicing the prior art used in the methods of the present invention are apparent to those of ordinary skill in the art. The practice of the prior art in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing, and related fields is well known to those of ordinary skill in the art and is described, for example, in the following references: Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

[0016] As used herein, the term "identity" refers to a measure of identity of nucleotide or amino acid sequences. Generally, sequences are aligned so as to obtain the highest order of match. "Identity" itself has the meaning recognized in the art and can be calculated using published techniques. For example, (Computational Molecular Biology, Lesk, A. M., ED., Oxford University Press, New York, 1988; Biocomputing: Informatics And Genome Projects, Smith, D. W., ED., Academic Press, New York, 1993; Computer Analysis Of Sequence Data, Part I, Griffin, A. M., And Griffin, H. G., EDS, Humana Press, New Jersey, 1994; Sequence Analysis In Molecular Biology, Von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer; Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). There are numerous methods for measuring identity between two nucleotide or amino acid sequences, but the term "identity" is well known to those skilled in the art (Carillo, H., and Lipton, D., SIAM J. Applied Math (1988) 48:1073). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide To Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo, H., and Lipton, D., Siam J. Applied Math (1988) 48:1073. Methods for determining identity and similarity are embodied in computer programs.Preferred computer program methods for determining identity and similarity between two arrays include, but are not limited to, the GCS program package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S. F. et al., J. Molec. Biol. (1990) 215:403).

[0017] By way of example, it is intended that a polynucleotide sequence be identical to a reference sequence, except that the polynucleotide sequence can contain up to 5 point mutations per 100 nucleotides of the reference amino acid sequence, by a polynucleotide having a nucleotide sequence that has at least, for example, 95% "identity" to a reference nucleotide sequence encoding a polypeptide of an array. In other words, up to 5% of the nucleotides in the reference sequence can be deleted and / or replaced with another nucleotide, and / or up to 5% of the number of all nucleotides in the reference sequence can be inserted into the reference sequence, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence. These mutations in the reference sequence can occur individually between nucleotides in the reference sequence, or be scattered in any one of one or more contiguous groups within the reference sequence, at the 5' or 3' terminal positions of the reference nucleotide sequence, or at any position between those terminal positions. Similarly, it is intended by a polypeptide having an amino acid sequence that has at least, for example, 95% "identity" to a reference amino acid sequence of SEQ ID NO: X, that the amino acid sequence of the polypeptide be identical to the reference sequence, except that the amino acid sequence can contain up to 5 amino acid changes per 100 amino acids of the reference amino acids of SEQ ID NO: X. In other words, up to 5% of the amino acid residues in the reference sequence can be deleted or replaced with another amino acid, or up to 5% of the number of all amino acid residues in the reference sequence can be inserted into the reference sequence, in order to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence. These modifications of the reference sequence can occur individually between residues in the reference amino acid sequence, or be scattered in any one of one or more contiguous groups within the reference sequence, at the amino-terminal or carboxy-terminal positions of the reference amino acid sequence, or at any position between those terminal positions.

[0018] As used herein, the term "in vitro" refers to experiments or measurements performed using components of an organism that have been isolated from their natural state.

[0019] As used herein, the term "ex vivo" refers to experiments or measurements conducted within or on biological tissue in an external environment with minimal modification of the natural state.

[0020] As used herein, the terms "nucleic acid", "nucleic acid molecule", and "polynucleotide" are intended to include DNA molecules and RNA molecules, as well as locked nucleic acids (LNA), bridged nucleic acids (BNA), morpholinos, or peptide nucleic acids (PNA). The nucleic acid (molecule) can be any nucleic acid (molecule) and can be single-stranded or double-stranded.

[0021] As used herein, the term "sequence" when referring to a nucleotide, or "nucleic acid sequence", "nucleotide sequence", or "polynucleotide sequence" refers to the order of nucleotides of a nucleic acid and / or polynucleotide, or the order of nucleotides within a nucleic acid and / or polynucleotide. In the context of the present invention, a first nucleic acid sequence may be incorporated within a further nucleic acid sequence or may overlap with a further nucleic acid sequence.

[0022] As used herein, the terms "subject", "individual", "animal", "patient", or "mammal" are used interchangeably and refer to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans, domestic animals, agricultural animals, and zoo animals, sports animals, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc. As defined herein, a subject may be alive or dead. A sample can be taken postmortem, i.e., after death, from a subject, and / or a sample can be taken from a living subject.

[0023] As used herein, the terms "treatment," "therapy," "alleviation," "mitigation," or "improvement" are used interchangeably and refer to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit. A therapeutic benefit means eradication, amelioration, or reduction (or delay) in the progression of the underlying disease being treated. Further, a therapeutic benefit is achieved by eradication, amelioration, or reduction (or delay) in the progression of one or more physiological symptoms associated with the underlying disease such that an improvement or a deceleration or decrease in deterioration is observed in a patient, even though the patient may still be afflicted with the underlying disease.

[0024] (Detailed Description) The headings of the various sections used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0025] Portions of the present invention contain material that is subject to copyright protection (including, but not limited to, figures, device photographs, or other aspects of the present submission for which copyright protection is available or may be available in any jurisdiction). The copyright owner does not object to the facsimile reproduction of the patent documents or patent inventions as recorded in the Patent and Trademark Office patent file or records, but reserves all other copyrights.

[0026] Various terms relating to the methods, compositions, uses, and other aspects of the present invention are used throughout the specification and claims. Such terms are given their ordinary meaning in the art to which the present invention pertains, unless otherwise indicated. Other specifically defined terms are interpreted in a manner consistent with the definitions provided herein. Preferred materials and methods are described herein, but methods and materials similar or equivalent to those described herein can be used in the practice of the present invention.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0028] The present invention is based on the observation that neutrophil elastase (NE), which is released by activated neutrophils involved in the acute inflammatory response, can cleave eHs. The inventors further demonstrated that the cleavage of eHs by NE is dramatically enhanced by heparins containing low anticoagulant heparin such as M6229. NE is a serine protease. Furthermore, it was found that another serine protease, APC, also cleaves histones and that heparin promotes the catalytic cleavage of eHs by APC, demonstrating a general principle of heparin - promoted proteolysis of eHs by proteases.

[0029] Without being bound by theory, the mechanism of catalysis is thought to be based on the approximate principle that the electrostatic interaction between heparin and eH enhances the functional collision between the protease and eH. Therefore, the inventors theorized that this principle is applicable to negatively charged polysaccharides complexed with any protein having proteolytic activity.

[0030] Accordingly, the present invention describes a novel composition that describes a complex of a protein having protease activity and a negatively charged polysaccharide with improved eHs neutralizing activity. In a first embodiment, the present invention thus relates to a complex of a negatively charged polysaccharide and a protein having serine protease activity. These complexes are particularly useful for the treatment, prevention or amelioration of diseases or disorders mediated by extracellular histones.

[0031] The present invention improves the ability to neutralize eHs of negatively charged polysaccharides such as heparin by adding proteolytic components (proteases such as serine proteases). Thereby, the neutralizing power of the negatively charged polysaccharide is enhanced, the dosage of the negatively charged polysaccharide (such as heparin) required to treat a patient is reduced, and thus the risk that the treated patient develops an autocatalytic cascade of histone release leading to severe tissue damage is reduced. As far as the inventors know, such a complex has not been described in the literature, so the present invention is novel. The present invention can also be considered an improvement of low-anticoagulant heparin, in which case the currently described complex has increased uses and indications due to improved specific activity and reduced risk of developing an autocatalytic cascade of histone release leading to severe tissue damage.

[0032] According to the literature, heparin inactivates leukocyte elastase (1), a serine protease, and activated protein C (2). Therefore, it is surprising that the inventors describe herein that heparin stimulates the cleavage of histones by proteases (see Examples 1 and 2). Low-anticoagulant heparin that neutralizes eHs by electrostatic interaction is currently being studied as a therapeutic agent for sepsis and COVID-19. The present invention improves the ability of heparin to inactivate eHs by adding a proteolytic component. Thereby, the dosage required to treat a patient is reduced, and the risk that the treated patient develops an autocatalytic cascade of histone release leading to severe tissue damage is reduced.

[0033] Therefore, the present invention aims to improve the treatment with low-anticoagulant heparin by using the complex described herein. The improved effect is expected to exist from the following observations.

[0034] · Heparin, a low anticoagulant, neutralizes eHs by electrostatic interaction, whereas the complex described herein is expected to lead to a more effective treatment as it degrades eHs into non-toxic protein fragments.

[0035] · Furthermore, since the protease can cleave eH and then move on to the next eH after such an action, it is expected that the complex described herein will enable the use of much lower concentrations of negatively charged polysaccharides (e.g., low - anticoagulant heparin, etc.) at least in part.

[0036] In a particularly preferred embodiment, the present invention further refers to a complex of a negatively charged polysaccharide and a protein having protease activity, where the protein having protease activity is a serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, or asparagine peptide lyase, and where the negatively charged polysaccharide is a glycosaminoglycan (GAG). Here, the negatively charged polysaccharide and the protein having protease activity are covalently bound.

[0037] Such a complex is also useful for the treatment, prevention, or amelioration of extracellular histone - mediated diseases or disorders, and has the advantage that the protease and the polysaccharide do not dissociate when administered to a subject in need thereof, but instead remain as a complex. Such a complex is expected to have even better protective properties against the cytotoxic effects caused by eHs.

[0038] One skilled in the art knows how such covalently bound complexes can be generated. For example, the negatively charged polysaccharides described herein can be cross-linked to the proteins having protease activity described herein using commonly used methods. Non-limiting examples include the use of cross-linking agents as described by Ohnishi et al. (9) using N-bromoacetylsulfanilyl chloride or N-bromoacetamidobenzoyl chloride as heterobifunctional cross-linking agents. Alternatively, polysaccharides such as heparin may contain residual amino acids such as serine (10), and for example, the polysaccharide can be bound to a protein such as a protease using the amino bond between the residual serine and the protein. One skilled in the art recognizes that these are merely exemplary methods for making the covalently bound complexes described herein, and that other options are readily available.

[0039] As used herein, a protein or protease having protease activity refers to an enzyme that catalyzes proteolysis, breaks down proteins into smaller polypeptides or single amino acids, and causes the formation of new protein products by cleaving peptide bonds within the protein by hydrolysis. Proteases are classified into exopeptidases that cleave terminal amino acids from proteins or peptides and endopeptidases that hydrolyze internal peptide bonds. In the present invention, the protease or protein having protease activity is preferably an endopeptidase. Proteases can be further subdivided based on catalytic residues into serine proteases (which use serine alcohol), cysteine proteases (which use cysteine thiol), threonine proteases (which use threonine secondary alcohol), aspartic acid proteases (which use the carboxylic acid of aspartic acid), glutamic acid proteases (which use the carboxylic acid of glutamic acid), metalloproteases (which use a metal, usually zinc), and asparagine peptide lyases (which perform an elimination reaction (requiring no water) using asparagine). Thus, in one embodiment, the protein having protease activity is a serine protease, a cysteine protease, a threonine protease, an aspartic acid protease, a glutamic acid protease, a metalloprotease, or an asparagine peptide lyase. In a preferred embodiment, the protein having protease activity is a serine protease.

[0040] As used herein, a protein having serine protease activity refers to an enzyme that can cleave peptide bonds in a protein and in which serine functions as a nucleophilic amino acid at the active site of the enzyme. Preferably, the protein having protease activity is a serine protease, also known as a serine endopeptidase, or a derivative thereof. Serine proteases are classified, based on their substrate specificity, into the following groups: trypsin-like, chymotrypsin-like, thrombin-like, elastase-like, and subtilisin-like. It is contemplated that any of these groups may be used in the present invention. Thus, in one embodiment, the protein having serine protease activity is a trypsin-like, chymotrypsin-like, thrombin-like, elastase-like, or subtilisin-like serine protease. Serine proteases are classified by the EC number EC3.4.21 determined by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. Thus, proteins having serine protease activity include EC3.4.21.1: chymotrypsin, EC3.4.21.2: chymotrypsin C, EC3.4.21.3: metridin, EC3.4.21.4: trypsin, EC3.4.21.5: thrombin, EC3.4.21.6: factor Xa, EC3.4.21.7: plasmin, EC3.4.21.9: enteropeptidase, EC3.4.21.10: acrosin, EC3.4.21.12: alpha-lytic endopeptidase, EC3.4.21.19: glutamyl endopeptidase, EC3.4.21.20: cathepsin G, EC3.4.21.21: factor VIIa, EC3.4.21.22: factor IXa, EC3.4.21.25: cucumisin, EC3.4.21.26: prolyl oligopeptidase, EC3.4.21.27: factor Xia, EC3.4.21.32: brachyurin, EC3.4.21.34: plasma kallikrein, EC3.4.21.35: tissue kallikrein, EC3.4.21.36: pancreatic elastase, EC3.4.21.37: leukocyte elastase, EC3.4.21.38: factor XIIa, EC3.4.21.39: kexin, EC3.4.21.41: complement subcomponent C1r, EC3.4.21.42: complement subcomponent C1s, EC3.4.21.43: Classical complement pathway C3 / C5 convertase, EC3.4.21.45: Complement factor I, EC3.4.21.46: Complement factor D, EC3.4.21.47: Alternative complement pathway C3 / C5 convertase, EC3.4.21.48: Cerebrisin, EC3.4.21.49: Hypodermin C, EC3.4.21.50: Lysyl endopeptidase, EC3.4.21.53: Edopeptidase La, EC3.4.21.54: γ-Renin, EC3.4.21.55: Venombin AB, EC3.4.21.57: Leucyl endopeptidase, EC3.4.21.59: Tryptase, EC3.4.21.60: Scutellarin, EC3.4.21.61: Kexin, EC3.4.21.62: Subtilisin, EC3.4.21.63: Origin, EC3.4.21.64: Endopeptidase K, EC3.4.21.65: Thermomycolin, EC3.4.21.66: Thermitase, EC3.4.21.67: Endopeptidase So, EC3.4.21.68: t-Plasminogen activator, EC3.4.21.69: Protein C (activated), EC3.4.21.70: Pancreatic endopeptidase E, EC3.4.21.71: Pancreatic elastase II, EC3.4.21.72: IgA-specific serine endopeptidase, EC3.4.21.73: u-Plasminogen activator, EC3.4.21.74: Venombin A, EC3.4.21.75: Furin, EC3.4.21.76: Myeloblastin, EC3.4.21.77: Semenogelinase, EC3.4.21.78: Granzyme A, EC3.4.21.79: Granzyme B, EC3.4.21.80: Streptoglysin A, EC3.4.21.81: Streptoglysin B, EC3.4.21.82: Glutamyl endopeptidase II, EC3.4.21.83: Oligopeptidase B, EC3.4.21.84: Limulus coagulation factor C, EC3.4.21.85: Limulus coagulation factor B, EC3.4.21.86: Limulus coagulase, EC3.4.21.88: Repressor LexA, EC3.4.21.89: Signal peptidase I, EC3.4.21.90: Togavirilin, EC3.4.21.91: Flavivirilin, EC3.4.21.92: Endopeptidase Clp, EC3.4.21.93: Proprotein convertase 1, EC3.4.It may be selected from Protein convertase 2, EC 3.4.21.95: Snake venom factor X activator, EC 3.4.21.96: Lactocepin, EC 3.4.21.97: Assemblin, EC 3.4.21.98: Hepasibirin, EC 3.4.21.99: Spermosin, EC 3.4.21.100: Cedricin, EC 3.4.21.101: Xanthomonaricin, EC 3.4.21.102: C-terminal processing peptidase, EC 3.4.21.103: Physarolysin, EC 3.4.21.104: Mannan-binding lectin-associated serine protease 2, EC 3.4.21.105: Rhomboid protease, EC 3.4.21.106: Hepsin, EC 3.4.21.107: Peptidase Do, EC 3.4.21.108: HtrA2 protease, EC 3.4.21.109: Matriptase, EC 3.4.21.110: C5a peptidase, EC 3.4.21.111: Acridin 1, EC 3.4.21.112: Site-1 protease, EC 3.4.21.113: Pestivirus NS3 polyprotein peptidase, EC 3.4.21.114: Equine arterivirus serine peptidase, EC 3.4.21.115: Infectious pancreatic necrosis virus Vp4 peptidase, EC 3.4.21.116: SpoIVB peptidase, EC 3.4.21.117: Stratum corneum chymotrypsin-like enzyme, EC 3.4.21.118: Kallikrein 8, EC 3.4.21.119: Kallikrein 13, EC 3.4.21.120: Obiductin, and EC 3.4.21.121: Lys-Lys / Arg-Xaa endopeptidase.

[0041] In this specification, the term protein having cysteine protease activity or cysteine protease (also known as thiol protease) refers to a hydrolase enzyme that degrades proteins. Such proteases share a common catalytic mechanism that includes a nucleophilic cysteine thiol in a catalytic triad or dyad. Such enzymes are classified as EC3.4.22 in the Enzyme Commission number classification. Thus, cysteine proteases include EC3.4.22.1: cathepsin B, EC3.4.22.2: papain, EC3.4.22.3: ficin, EC3.4.22.6: chymopapain, EC3.4.22.7: asclepain, EC3.4.22.8: clostripain, EC3.4.22.10: streptopain, EC3.4.22.14: actinidin, EC3.4.22.15: cathepsin L, EC3.4.22.16: cathepsin H, EC3.4.22.24: cathepsin T, EC3.4.22.25: glycyl endopeptidase, EC3.4.22.26: cancer procoagulant, EC3.4.22.27: cathepsin S, EC3.4.22.28: picornain 3C, EC3.4.22.29: picornain 2A, EC3.4.22.30: calpain, EC3.4.22.31: ananain, EC3.4.22.32: stem bromelain, EC3.4.22.33: fruit bromelain, EC3.4.22.34: legumain, EC3.4.22.35: histolysin, EC3.4.22.36: caspase-1, EC3.4.22.37: gingipain R, EC3.4.22.38: cathepsin K, EC3.4.22.39: adenain, EC3.4.22.40: bleomycin hydrolase, EC3.4.22.41: cathepsin F, EC3.4.22.42: cathepsin O, EC3.4.22.43: cathepsin V, EC3.4.22.44: nuclear envelope-a endopeptidase, EC3.4.22.45: helper component protease, EC3.4.22.46: L-peptidase, EC3.4.22.47: gingipain K, EC3.4.22.48: staphostatin, EC3.4.22.49: separase, EC3.4.22.50: V-cath endopeptidase, EC3.4.22.51: cruzain, EC3.4.22.52: Calpain-1, EC 3.4.22.53: Calpain-2, EC 3.4.22.54: Calpain-3, EC 3.4.22.55: Caspase-2, EC 3.4.22.56: Caspase-3, EC 3.4.22.57: Caspase-4, EC 3.4.22.58: Caspase-5, EC 3.4.22.59: Caspase-6, EC 3.4.22.60: Caspase-7, EC 3.4.22.61: Caspase-8, EC 3.4.22.62: Caspase-9, EC 3.4.22.63: Caspase-10, EC 3.4.22.64: Caspase-11, EC 3.4.22.65: Peptidase 1 (tick), EC 3.4.22.66: Calicivirine, EC 3.4.22.67: Zingipain, EC 3.4.22.68: Ulp1 peptidase, EC 3.4.22.69: SARS coronavirus main protease, EC 3.4.22.70: Sortase A and EC 3.4.22.71: Sortase B may be selected from.

[0042] As used herein, the term protein having threonine protease activity or threonine protease refers to a family of proteolytic enzymes having a threonine (Thr) residue in the active site. Such enzymes are classified as EC 3.4.25 using the Enzyme Commission number classification. Thus, the threonine protease may be selected from EC 3.4.25.1: Proteasome endopeptidase complex and EC 3.4.25.2: HslU-HslV peptidase.

[0043] In this specification, the term protein having aspartic protease activity or aspartic protease refers to a catalytic type protease enzyme that uses an activated water molecule bound to one or more aspartic acid residues for the catalytic reaction of a peptide substrate. They generally have two highly conserved aspartic acids in the active site and are optimally activated at acidic pH. Such enzymes are classified as EC 3.4.23 in the enzyme commission number classification.Thus, aspartic proteases can be selected from EC3.4.23.1: pepsin A, EC3.4.23.2: pepsin B, EC3.4.23.3: gastricsin, EC3.4.23.4: chymosin, EC3.4.23.5: cathepsin D, EC3.4.23.12: nepenthesin, EC3.4.23.15: renin, EC3.4.23.16: HIV-1 retropepsin, EC3.4.23.17: proopiomelanocortin converting enzyme, EC3.4.23.18: aspergillopepsin I, EC3.4.23.19: aspergillopepsin II, EC3.4.23.20: penicillopepsin, EC3.4.23.21: rhizopuspepsin, EC3.4.23.22: endothiapepsin, EC3.4.23.23: mucorpepsin, EC3.4.23.24: candidapepsin, EC3.4.23.25: saccharopepsin, EC3.4.23.26: rhodotorulapepsin, EC3.4.23.28: acrosinodotropin, EC3.4.23.29: polyporopepsin, EC3.4.23.30: picnoporopepsin, EC3.4.23.31: sitalidopepsin A, EC3.4.23.32: sitalidopepsin B, EC3.4.23.34: cathepsin E, EC3.4.23.35: valipepsin, EC3.4.23.36: signal peptidase II, EC3.4.23.38: plasminopepsin I, EC3.4.23.39: plasminopepsin II, EC3.4.23.40: fitepsin, EC3.4.23.41: yapsin 1, EC3.4.23.42: thermopsin, EC3.4.23.43: prepyloric peptidase, EC3.4.23.44: nodavirus endopeptidase, EC3.4.23.45: memapsin 1, EC3.4.23.46: memapsin 2, EC3.4.23.47: HIV-2 retropepsin, EC3.4.23.48: plasminogen activator Pla, EC3.4.23.49: omptin, EC3.4.23.50: human endogenous retrovirus K endopeptidase, EC3.4.23.51: HycI peptidase and EC3.4.23.52: prefragilin peptidase.

[0044] As used herein, the term protein having glutamic protease activity or glutamic protease refers to a group of proteolytic enzymes that contain a glutamic acid residue within the active site. Such enzymes are described as EC 3.4.23.32 citrulline peptidase B. Thus, in an embodiment, the protein having glutamic protease activity is EC 3.4.23.32 citrulline peptidase B.

[0045] As used herein, the term protein having protease activity of a metalloprotease or metalloprotease means a protease enzyme in which a metal is involved in the catalytic mechanism. Such enzymes are classified into EC3.4.24 using the Enzyme Commission number classification. Thus, metalloproteases include EC3.4.24.1: atrolysin A, EC3.4.24.3: microbial collagenase, EC3.4.24.4: current EC3.4.24.40 serralysin, EC3.4.24.6: leucolysin, EC3.4.24.7: interstitial collagenase, EC3.4.24.11: neprilysin, EC3.4.24.12: embelysin, EC3.4.24.13: IgA-specific metalloendopeptidase, EC3.4.24.14: procollagen N-endopeptidase, EC3.4.24.15: symmet oligopeptidase, EC3.4.24.16: neurolysin, EC3.4.24.17: stromelysin 1, EC3.4.24.18: meprin A, EC3.4.24.19: procollagen C-endopeptidase, EC3.4.24.20: peptidyl-Lys metalloendopeptidase, EC3.4.24.21: astacin, EC3.4.24.22: stromelysin 2, EC3.4.24.23: matrilysin, EC3.4.24.24: gelatinase a, EC3.4.24.25: vibriolysin, EC3.4.24.26: pseudolysin, EC3.4.24.27: thermolysin, EC3.4.24.28: bacillolysin, EC3.4.24.29: aureolysin, EC3.4.24.30: coccolysin, EC3.4.24.31: mycolysin, EC3.4.24.32: β-lytic metalloendopeptidase, EC3.4.24.33: peptidyl Asp metalloendopeptidase, EC3.4.24.34: neutrophil collagenase, EC3.4.24.35: gelatinase B, EC3.4.24.36: raisimanolysin, EC3.4.24.37: saccarolysin, EC3.4.24.38: gametolysin, EC3.4.24.39: deuterolysin, EC3.4.24.40: serralysin, EC3.4.24.41: atrolysin B, EC3.4.24.42: atrolysin C, EC3.4.24.43: atroxase, EC3.4.24.44: atrolysin E, EC3.4.24.It may be selected from atrolysin F, EC 3.4.24.46: adamalysin, EC 3.4.24.47: horilysin, EC 3.4.24.48: lavarylisin, EC 3.4.24.49: bosolopasin, EC 3.4.24.50: bosololysin, EC 3.4.24.51: ohitorisin, EC 3.4.24.52: trimeroysin I, EC 3.4.24.53: trimeroysin II, EC 3.4.24.54: mukrolysin, EC 3.4.24.55: pitrilysin, EC 3.4.24.56: insulin, EC 3.4.24.57: O-sialoglycoprotein endopeptidase, EC 3.4.24.58: russellysin, EC 3.4.24.59: mitochondrial matrix peptidase, EC 3.4.24.60: ductylisin, EC 3.4.24.61: narjilysin, EC 3.4.24.62: magnolysin, EC 3.4.24.63: meprin B, EC 3.4.24.64: mitochondrial processing peptidase, EC 3.4.24.65: macrophage elastase, EC 3.4.24.66: coriolysin L, EC 3.4.24.67: coriolysin H, EC 3.4.24.68: tentoxin lysin, EC 3.4.24.69: botulinum toxin lysin, EC 3.4.24.70: oligopeptidase A, EC 3.4.24.71: endothelin converting enzyme 1, EC 3.4.24.72: fibrolase, EC 3.4.24.73: jarahagin, EC 3.4.24.74: flagylisin, EC 3.4.24.75: lysostaphin, EC 3.4.24.76: fluvastatin, EC 3.4.24.77: snapalysin, EC 3.4.24.78: gpr endopeptidase, EC 3.4.24.79: papalysin-1, EC 3.4.24.80: membrane type matrix metalloproteinase-1, EC 3.4.24.81: ADAM10 endopeptidase, EC 3.4.24.82: ADAMTS-4 endopeptidase, EC 3.4.24.83: Bacillus anthracis lethal factor endopeptidase, EC 3.4.24.84: Ste24 endopeptidase, EC 3.4.24.85: S2P endopeptidase, EC 3.4.24.86: ADAM17 endopeptidase and EC 3.4.24.87: ADAMTS13 endopeptidase.

[0046] As used herein, the term "protein having asparagine peptidase activity" or "asparagine peptidase" refers to a protease, also called a proteolytic enzyme, peptidase, or proteinase, which is one of seven groups classified by its catalytic residue. The catalytic mechanism of asparagine peptidase is such that the asparagine residue acts as a nucleophile, undergoes a nucleophilic elimination reaction rather than hydrolysis, and catalyzes the cleavage of peptide bonds. Asparagine peptidase proteases include EC 3.4.23.44: nodavirus endopeptidase, EC 7.1.2.2: H + transport-2-sector ATPase, and may be selected from EC 2.7.7.7: DNA-directed DNA polymerase.

[0047] In a particularly preferred embodiment, the protein having protease activity is a protein having elastase activity. As used herein, the term "elastase" or "protein having elastase activity" refers to a serine protease that can degrade the extracellular protein elastin in humans encoded by the ELN gene (ENSG00000049540). It is understood that elastase or a protein having elastase activity can also degrade other proteins, such as histones.

[0048] The inventors of the present invention have demonstrated in this specification that the proteolytic cleavage of histones by elastase and the disruption of cytotoxic activity are promoted by heparin (see Example 1). Therefore, in one embodiment, the protein having serine protease activity is elastase. In one embodiment, the protein having serine protease activity is selected from neutrophil elastase, chymotrypsin-like elastase family member 1, chymotrypsin-like elastase family member 2A, chymotrypsin-like elastase family member 2B, chymotrypsin-like elastase family member 3A, chymotrypsin-like elastase family member 3B, and chymotrypsin C, and preferably, the protein having elastase activity is neutrophil elastase.

[0049] Neutrophil elastase (EC 3.4.21.37, leukocyte elastase, ELANE, ELA2, elastase 2, neutrophil, elastzyme, serine elastase, subtype human leukocyte elastase (HLE)) is a serine protease belonging to the same family as chymotrypsin and has a broad substrate specificity. This protein is encoded in humans by the ELANE gene annotated as ENSG00000277571 and ENSG00000197561. Therefore, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% identical to the protein sequence encoded by the ELANE gene annotated as ENSG00000277571 or ENSG00000197561, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% identical to the nucleic acid sequence of the ELANE gene annotated by ENSG00000277571 or ENSG00000197561.

[0050] Chymotrypsin-like elastase family member 1, also known as pancreatic elastase 1 or EC 3.4.21.36, is a type of elastase produced by acinar cells of the pancreas. This protein is encoded by the CELA1 gene, which is annotated as ENSG00000139610 in humans. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the CELA1 gene annotated as ENSG00000139610, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the CELA1 gene annotated by ENSG00000139610.

[0051] Chymotrypsin-like elastase family member 2A (EC 3.4.21.71) is a protein encoded in humans by the CELA2A gene, which is annotated as ENSG00000142615. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the CELA2A gene annotated as ENSG00000142615, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the CELA2A gene annotated by ENSG00000142615.

[0052] Chymotrypsin-like elastase family member 2B (EC 3.4.21.71) is a protein encoded in humans by the CELA2B gene annotated as ENSG00000215704. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the CELA2B gene annotated as ENSG00000215704, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the CELA2B gene annotated by ENSG00000215704.

[0053] Chymotrypsin-like elastase family member 3A (EC 3.4.21.70) is a protein encoded in humans by the CELA3A gene annotated as ENSG00000142789. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the CELA2A gene annotated as ENSG00000142789, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the CELA2A gene annotated by ENSG00000142789.

[0054] Chymotrypsin-like elastase family member 3B (EC 3.4.21.70) is a protein encoded in humans by the CELA3B gene, which is annotated as ENSG00000219073. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the CELA3B gene annotated as ENSG00000219073, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the CELA3B gene annotated by ENSG00000219073.

[0055] Chymotrypsin C (EC 3.4.21.2) is an enzyme that catalyzes the cleavage of peptides and proteins. This protein is also known as CLCR or ELA4 in humans and is encoded by the chymotrypsin C gene, which is annotated as ENSG00000162438. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the chymotrypsin C gene annotated by ENSG00000162438, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the chymotrypsin C gene annotated by ENSG00000162438.

[0056] In this specification, the inventors further demonstrate (see Example 1) that proteolytic cleavage of histones and disruption of cytotoxic activity by (activated) protein C are promoted by heparin. Thus, in one embodiment, the protein having protease activity is protein C, and in a preferred embodiment, the protein having protease activity is activated protein C (APC). Protein C (EC 3.4.21.69), also known as autoprothrombin IIA and coagulation factor XIX, is a zymogen, i.e., an inactive enzyme. The activated form plays an important role in controlling anticoagulation, inflammation, cell death, and maintaining the permeability of the vascular wall in humans and other animals. Activated protein C (APC) performs these functions mainly by proteolytically inactivating factors Va and VIIIa. Since APC contains a serine residue at the active site, it is classified as a serine protease. This protein is encoded in humans by the PROC gene annotated as ENSG00000115718. Thus, in one embodiment, the neutrophil elastase according to the present invention has a protein sequence that is 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the protein sequence encoded by the PROC gene annotated with ENSG00000115718, or the neutrophil elastase according to the present invention has a protein sequence encoded by a nucleic acid sequence that is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or 100% identical to the nucleic acid sequence of the PROC gene annotated with ENSG00000115718.

[0057] As used herein, the term "negatively charged polysaccharide" means a polymer of sugars that contains or consists of negatively charged sugars. Preferably, the negatively charged polysaccharide is or comprises a glycosaminoglycan, which means that the negatively charged polysaccharide contains repeats of disaccharide units. As used herein, glycosaminoglycans (GAGs), also known as mucopolysaccharides, are long linear polysaccharides consisting of repeating disaccharide units (i.e., disaccharide units). The repeating disaccharide units are composed of uronic sugars and amino sugars, with the exception of keratan, which has galactose instead of uronic sugar. Thus, the repeating disaccharide unit preferably consists of or comprises a uronic sugar and an amino sugar, or a galactose and an amino sugar, or a combination of one of these. Uronic acid sugars (also called uronic acids) are sugars in which the hydroxyl group farthest from the carbonyl group is oxidized to a carboxylic acid. Non-limiting examples of uronic acid sugars are glucuronic acid, gluconic acid, iduronic acid and isaccharinic acid. Amino sugars as used herein are sugar molecules in which a hydroxyl group is substituted with an amine group. Non-limiting examples include glucosamine, N-acetylglucosamine, galactosamine, L-daunosamine, sialic acid. Thus, in one embodiment, the negatively charged polysaccharide is or comprises a glycosaminoglycan (GAG).

[0058] The polysaccharide according to the present invention is negatively charged. Preferably, the polysaccharide is negatively charged because it contains variably sulfated repeating disaccharide units. For example, iduronic acid is sulfated at the 2-O position to give 2-O-sulfated iduronic acid, and glucosamine is sulfated at the 6-O position and / or the N position to give 6-O-sulfated, N-sulfated glucosamine. Other sulfated sugars are known to those skilled in the art and can be incorporated into the negatively charged polysaccharides of the present invention. Thus, in one embodiment, the negatively charged polysaccharide consists of or comprises variably sulfated repeating disaccharide units.

[0059] Accordingly, as used herein, a negatively charged polysaccharide has at least 10%, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or more, such as 98%, 99% or even 100% of disaccharides selected from GlcA-GlcNAc, GlcA-GlcNS, IdoA-GlcNS, IdoA(2S)-GlcNS, IdoA-GlcNS(6S), IdoA(2S)-GlcNS(6S). Here, GlcA = β-D-glucuronic acid, IdoA = α-L-iduronic acid, IdoA(2S) = 2-O-sulfo-α-L-iduronic acid, GlcNAc = 2-deoxy-2-acetamido-α-D-glucopyranosyl, GlcNS = 2-deoxy-2-sulfamido-α-D-glucopyranosyl, GlcNS(6S) = 2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate.

[0060] Ideally, the negatively charged polysaccharide has a molecular weight of at least 2 kDa, preferably at least 3 kDa, more preferably at least 4 kDa. Accordingly, the negatively charged polysaccharide has a chain length of at least 10 sugar units, preferably at least 15 sugar units, more preferably at least 20 sugar units, or at least 5 disaccharide units, more preferably at least 7 disaccharide units, most preferably at least 10 disaccharide units. The negatively charged polysaccharide may be a mixture of different but related polymers, in which case the molecular weight or chain length means the average molecular weight or chain length. The average molecular weight is determined by summing the weights of all chains and dividing by the total number of chains. The average chain length is determined by summing the lengths of all chains and dividing by the total number of chains. In one embodiment, the negatively charged polysaccharide has a molecular weight of at most 50 kDa, preferably at most 40 kDa, more preferably at most 30 kDa. Accordingly, the negatively charged polysaccharide has a chain length of at most 250, preferably at most 200, more preferably at most 150 sugar units, or at most 125, more preferably at most 100, most preferably at most 75 disaccharide units.

[0061] In one embodiment, the negatively charged polysaccharide is heparin. As used herein, heparin generally refers to polymers having a molecular weight in the range of 3 to 30 kDa, which is a member of the carbohydrate glycosaminoglycan family and also includes the closely related molecule heparan sulfate. Heparin contains variously sulfated repeating disaccharide units, and the main disaccharide units present in heparin are GlcA-GlcNAc, GlcA-GlcNS, IdoA-GlcNS, IdoA(2S)-GlcNS, IdoA-GlcNS(6S), IdoA(2S)-GlcNS(6S). The most common disaccharide unit in heparin is IdoA(2S)-GlcNS(6S) consisting of 2-O-sulfated iduronic acid and 6-O-sulfated N-sulfated glucosamine. Thus, in one embodiment, the negatively charged polysaccharide is heparin.

[0062] Furthermore, the inventors have found that in particularly beneficial embodiments of the present invention, low anticoagulant or non-anticoagulant heparin can be used. Heparin has both anticoagulant and anti-inflammatory effects, and it has been found that most of the anti-inflammatory pharmacological effects of heparin are independent of its anticoagulant effect (3). Thus, in one embodiment, the negatively charged polysaccharide is low anticoagulant heparin or non-anticoagulant heparin. Low anticoagulant heparin or non-anticoagulant heparin is known to those skilled in the art and refers to specific heparin fractions or modified heparins with reduced or no anticoagulant effect. Thus, as used herein, the term heparin encompasses "conventional" heparin and low anticoagulant or non-anticoagulant heparin. Heparin means naturally occurring heparin or synthetic heparin having a distinct anticoagulant function. Conventional heparin means natural or synthetic heparin having both anticoagulant and anti-inflammatory effects. An example of conventional heparin is unfractionated heparin (UFH).

[0063] For example, pentasaccharide-depleted heparin can be used as a low anticoagulant or non-anticoagulant heparin and is described, for example, in European Patent Application Publication No. 2731616, the entire disclosure of which is incorporated herein by reference. The term pentasaccharide-depleted heparin in this context means a fraction of heparin in which the content of pentasaccharides is substantially reduced compared to commercially available heparin. A non-limiting example of heparin lacking pentasaccharides is M6229 (see, for example, https: / / clinicaltrials.gov / ct2 / show / NCT05208112).

[0064] Other non-limiting examples of low anticoagulant or non-anticoagulant heparins include Dociparstat, chemically O-desulfated heparin, site-selectively desulfated heparin, and "glycol-split" heparin. Dociparstat is a glycosaminoglycan derived from porcine heparin and is also known as DSTAT, CX-01, 2-O,3-O desulfated heparin or ODSH. Dociparstat is currently being tested in combination with standard chemotherapy as a treatment for acute myeloid leukemia (AML). https: / / clinicaltrials.gov / ct2 / show / NCT04571645. Site-selectively desulfated heparin is heparin with partially removed sulfate groups and is described by Takano et al. (4), which is hereby incorporated by reference in its entirety. Chemically O-desulfated heparins include, for example, partially or fully 6-O-desulfated heparin, as described by Kariya et al. (5). Glycol-split heparin refers to heparin and low molecular weight heparin (LMWH) such as cebparine that have undergone periodate oxidation followed by borohydride reduction. This process converts well-known antithrombotic drugs into reduced oxyheparin (RO) type glycol-split (gs) derivatives, some of which are currently being developed as anti-cancer and anti-inflammatory agents and are described by Alekseeva et al. (6). Thus, in one embodiment, the negatively charged polysaccharide is selected from pentasaccharide deletion heparin, Dociparstat, chemically O-desulfated heparin, cebparine, site-selectively desulfated heparin, and glycol-split heparin.

[0065] The present invention relates to a complex of a negatively charged polysaccharide and a protein having protease activity. As used herein, the term complex should be construed as an association of the individual components. Thus, the term can mean the binding of the individual components (negatively charged polysaccharide and protein having protease activity) by one or more selected from, for example, covalent bonds, van der Waals force bonds, electrostatic force bonds, hydrogen bridge bonds, or ionic force bonds. Thus, in embodiments, it binds by van der Waals forces, by electrostatic forces, by hydrogen bridges, or by ionic forces. Thus, in embodiments, the negatively charged polysaccharide and the protein having protease activity are bound by a covalent bond, a bond by van der Waals forces, a bond by electrostatic forces, a bond by hydrogen bridges, a bond by ionic forces, or a combination of two or more of these. One option for the covalent bond is provided by the terminal serine group of heparin, which can be used to make an amide bond with the proteolytic site.

[0066] This complex is formed by utilizing the electrostatic interaction between the negatively charged polysaccharide and the protease. For example, such interactions may be mediated by recognition motifs such as the Cardin-Weintraub consensus sequence (see, for example, (7) and (8)) also found in NE, but the interaction does not necessarily have to be mediated by such sequences.

[0067] Thus, in an embodiment, the complex can be obtained or can be obtained by combining a negatively charged polysaccharide and a protein having protease activity. For example, the negatively charged polysaccharide and the protein having protease activity can be combined in an approximately equimolar ratio, or a slight excess of the negatively charged polysaccharide can be used. Thus, the present invention further relates to a method for obtaining the complex described herein, which comprises binding a negatively charged polysaccharide and a protein having protease activity. In a preferred embodiment, the molar ratio of the negatively charged polysaccharide to the protein having protease activity is about 1:1, such as 1:2, 1:1 or 2:1, or a slight excess amount of the negatively charged polysaccharide, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or even 10:1 can be used. Preferably, the negatively charged polysaccharide and the protein having protease activity are bound in a suitable solvent such as an aqueous buffer or physiological saline. For example, the negatively charged polysaccharide and the protein having protease activity can be bound by adding a solution containing the negatively charged polysaccharide to a solution containing the protein having protease activity, or vice versa, by adding a solution containing the protein having protease activity to a solution containing the negatively charged polysaccharide. Alternatively, the negatively charged polysaccharide and the protein having protease activity can be bound by dissolving the negatively charged polysaccharide in a solution containing the protein having protease activity, or vice versa, by dissolving the protein having protease activity in a solution containing the negatively charged polysaccharide.

[0068] Furthermore, it is assumed to be advantageous that a complex of a negatively charged polysaccharide and a protein having protease activity, wherein the complex is formed by a covalent bond, can prevent the dissociation of the negatively charged polysaccharide and the protein having protease activity. Such a complex can remove the excess (unbound) negatively charged polysaccharide, so that the amount of the negatively charged polysaccharide required can be further reduced. Methods for binding a negatively charged polysaccharide and a protein having protease activity to each other are known to those skilled in the art. For example, they can be covalently bound using the free amine or carboxyl group of the terminal serine group in heparin.

[0069] In a second aspect, the present invention relates to a pharmaceutical composition comprising the complex according to the first aspect of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are known to those skilled in the art and can be, for example, salt solutions such as physiological saline solutions or buffer solutions. However, those skilled in the art will recognize other suitable pharmaceutically acceptable carriers or will be able to determine a pharmaceutically acceptable carrier suitable for the intended application of the complex as described herein.

[0070] The complex as described herein is envisioned to be used in medicine. Thus, in a third aspect, the present invention relates to the complex according to the first aspect of the present invention or to the pharmaceutical composition according to the second aspect of the present invention for use as a medicament. Alternatively, the present invention relates to a method of treatment comprising administering the complex according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention to a subject in need thereof.

[0071] Extracellular histones Histones are nuclear proteins, and the core histones H2A, H2B, H3, and H4, together with the linker histone H1, organize DNA into chromatin and control gene expression. The pathological release of extracellular histones is known to have clinical implications because these proteins are cytotoxic to host tissues and function as damage-associated molecular patterns (DAMPs). Extracellular histones are associated not only with immunothrombotic diseases such as sepsis and renal ischemia-reperfusion injury (IRI), but also with trauma.

[0072] The inventors have already demonstrated, for example, the negative role of eHs in immunothrombosis. Thus, the results provided here in Examples 1 and 2 make it plausible that the complexes as described herein can be used for treating, preventing or ameliorating a disease or disorder mediated by extracellular histones. This is made plausible because while the complex can assist in the degradation of eHs (in the extracellular space), when using such a complex, the amount of heparin required is much less than when using heparin alone, thereby counteracting the anticoagulant effect of heparin and the potential problems associated with the development of the self-reinforcing cascade of histone release leading to significant tissue damage. Thus, in a fourth aspect, the invention relates to a complex according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for use in the treatment, prevention or amelioration of a disease or disorder mediated by extracellular histones. In an alternative embodiment, the invention relates to a method of treating, preventing or ameliorating a subject suffering from a disease or disorder mediated by extracellular histones, the method comprising administering to the subject a complex according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention. As used herein, the term extracellular histone-mediated disease or disorder refers to any disease, disorder or event in which histones are released extracellularly to exert a cytotoxic effect. Examples of diseases or disorders mediated by extracellular histones are immunothrombosis-related diseases or disorders (defined below herein), and trauma.

[0073] Immunothrombosis Immune thrombotic disease refers to the complex reaction of the human body to sterile and non-sterile inflammatory stimuli. This complex reaction is caused by the interaction of the activated hemostatic system, the activated immune system, and the activated complement system. The activated systems include platelet activation and coagulation (hemostatic system), neutrophil activation and neutrophil extracellular trap (NET) formation (immune system), C5a formation and C5b-9 complex formation (complement system). The interaction between these systems includes an amplification loop that promotes the immune thrombotic reaction and increases the risk of worsening morbidity and mortality. Therefore, in this specification, the term immune thrombotic disease or disorder should be interpreted as a disease or disorder in which one or more mechanisms of immune thrombotic disease occur. Preferably, the disease or disorder is characterized in that at least one of the symptoms of the disease or disorder results from immune thrombotic disease.

[0074] Therefore, in an embodiment, the present invention relates to a complex according to a first aspect of the present invention or a pharmaceutical composition according to a second aspect of the present invention for use in the treatment, prevention or amelioration of a disease or disorder associated with immune thrombotic disease. In an alternative embodiment, the present invention relates to a method of treating, preventing or ameliorating a subject suffering from an immune thrombotic disease or disorder, the method comprising administering to the subject a complex according to a first aspect of the present invention or a pharmaceutical composition according to a second aspect of the present invention.

[0075] Non-limiting examples of diseases or disorders associated with immune thrombotic disease include sepsis, inflammation, cancer, burns, severe trauma, stroke, ischemia-reperfusion, acute myocardial infarction, organ transplantation, etc., antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, thrombotic microangiopathy, acute respiratory distress syndrome, thrombotic thrombocytopenic purpura, endotoxemia, pancreatitis, peritonitis, and thromboembolic diseases. Therefore, in one embodiment, the immune thrombotic disease or disorder is selected from sepsis, inflammation, cancer, burns, severe trauma, stroke, ischemia-reperfusion, acute myocardial infarction, organ transplantation, antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, thrombotic microangiopathy, acute respiratory distress syndrome, thrombotic thrombocytopenic purpura, endotoxemia, pancreatitis, peritonitis, thromboembolic diseases.

[0076] References 1. Redini F, Tixier JM, Petitou M, Choay J, Robert L, Hornebeck W. Inhibition of leucocyte elastase by heparin and its derivatives. Biochem J 1988;252(02):515-519 2. Nicolaes GAF, Sorensen KW, Friedrich U, et al. Altered inactivation pathway of factor Va by activated protein C in the presence of heparin. Eur J Biochem 2004;271(13):2724-2736 3. Rao et al. Am J Physiol Cell Physiol. 2010 Jul;299(1):C97-110. 4. Takano et al., Japanese Journal of Thrombosis and Hemostasis. 10. 45-55. 5. Kariya et al. J Biol Chem. 2000 Aug 25;275(34):25949-58. 6. Alekseeva et al. Anal Bioanal Chem. 2014 Jan;406(1):249-65. 7. Gandhi et al. Chem Biol Drug Des 2008;72:455-482 8. Munoz et al. Arterioscler Thromb Vasc Biol. 2004 Sep; 24(9): 1549-1557. 9. Ohnishi et al. Chem. Pharm. Bull. 33(2) 674-678 (1985) 10. Chen et al. Carbohydr Polym. 2017 Feb 10;157:244-250.

Example

[0077] Example 1: Proteolytic cleavage of histones by elastase and disruption of cytotoxic activity are promoted by unfractionated heparin (UFH) and low-anticoagulant heparin (M6229). Materials and methods Histones H2B, H3, and H4 were purchased from Roche or NEB. Native human neutrophil elastase (NE) (ab91099) was purchased from Abcam.

[0078] Cleavage of histone isoforms To evaluate the possibility that NE cleaves histones, 5 nM of NE was added to 5 μg / ml of histones H2B, H3, and H4 in HN buffer (25 mM HEPES, 140 mM NaCl, 5 mM CaCl2, pH 7.4) and incubated at 37 °C. Samples were taken at several time points and the reaction was stopped by adding the reaction volume to Laemmli sample buffer. The samples were then incubated at 95 °C for 5 minutes. The samples were run on an SDS-PAGE gel and analyzed by Western blotting using antibodies against H2B, H3, and H4. To examine the effect of heparin, heparin was complexed with NE before adding it to the reaction mixture. The complex of NE and heparin decreased the conversion of a small chromogenic substrate induced by NE (not shown).

[0079] Histone Western blotting Fragmentation of histones was measured by a semi - quantitative method. Briefly, samples were subjected to SDS - PAGE gel electrophoresis and transferred to a PVDF membrane (Bio - Rad Laboratories) using semi - dry blotting. The membrane was blocked and incubated overnight at 4°C with primary specific histone antibodies: mouse monoclonal anti - H2B (SC - 515808, Bio - connect), rabbit polyclonal anti - histone H3 (ab94817, Abcam), mouse monoclonal anti - H4 (L64C1, Cell signaling). Subsequently, for histone H3, biotinylated donkey anti - rabbit IgG (ab97083, Abcam), and for histone H2B or H4, HRP - labeled goat anti - mouse (p0477, Dako) were incubated at RT for 1 hour each. For histone H3, it was incubated with streptavidin - biotin / HRP complex (Vectastain) at RT for 30 minutes. Histone bands were detected using a chemiluminescent ECL substrate (Advansta). The obtained band densities were quantified using ImageQuant TL software (GE Healtcare, Little Chalfont, UK).

[0080] Detection of histone cytotoxicity EA.hy926 cells were plated in 24-well plates and grown to 80% confluence in complete DMEM medium (Thermofisher Scientific) supplemented with HAT (hypoxanthine-aminopterin-thymidine, Gibco). After washing twice with phosphate-buffered saline (PBS, Gibco), the cells were incubated with 2.7 nM histone H3 and various amounts of UFH or M6229 in the presence or absence of 5 nM NE in DMEM without fetal bovine serum. After incubation at 37 °C for 1 h, the medium containing detached cells was collected, and the adherent cells were collected using 0.05% trypsin-EDTA (Gibco). The collected cells were pelleted and reconstituted in binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 3.3 mM CaCl2) containing 2.5 μg / ml propidium iodide (Sigma). After incubation in the dark for 10 min, the percentage of PI-positive cells was measured by flow cytometry using a BD Accuri C6 and analyzed using BD CFlow plus software.

[0081] Results NE cleaves histones NE was shown to cleave histones H2B, H3, and H4 in a time-dependent manner. Figure 1 shows the proteolytic cleavage of histones by NE. Histone H4 is cleaved into small fragments that are not visualized by Western blotting using the monoclonal antibodies used here.

[0082] NE-catalyzed histone cleavage is promoted by UFH The complex of NE and UFH promoted the degradation of histones H2B, H3, and H4 by NE catalysis. The time course of NE-induced histone degradation by free NE or the NE-UFH complex is shown in Figure 2.

[0083] NE enhances the cytoprotective effects of UFH and M6229 When endothelial cells (EA.hy926) are incubated with histone H3, the survival rate decreases due to the cytotoxic effect of H3. When H3 is incubated with heparins such as UFH or M6229, the cytotoxic effect of H3 is neutralized in a dose-dependent manner (Figure 3). The complexes of UFH or M6229 with NE enhance the cytoprotective effect of UFH or M6229 (Figure 3). NE itself did not affect the survival rate of EA.hy926 cells (not shown).

[0084] Example 2: Proteolytic cleavage of histone and disruption of cytotoxic activity by activated protein C are promoted by unfractionated heparin (UFH). Materials and Methods Histone H3 was purchased from Roche. Human activated protein C (APC) was generated by recombinant technology.

[0085] Cleavage of histone isoforms To evaluate the possibility that APC cleaves histones, 10 nM NE was added to 5 μg / ml histone H3 in HN buffer (25 mM HEPES, 140 mM NaCl, 5 mM CaCl2, pH 7.4) and incubated at 37°C. Samples were taken at several time points and the reaction was stopped by adding the reaction volume to Laemmli sample buffer. The samples were then incubated at 95°C for 5 minutes and analyzed by Western blotting using an antibody against H3 on an SDS-PAGE gel. To examine the effect of heparin, heparin was complexed with APC before adding to the reaction mixture.

[0086] Histone Western blotting Fragmentation of histone H3 was determined by a semi - quantitative method. Briefly, samples were subjected to SDS - PAGE gel electrophoresis and transferred to a PVDF membrane (Bio - Rad Laboratories) using semi - dry blotting. The membrane was blocked and incubated overnight at 4°C with a primary specific rabbit polyclonal anti - histone H3 (ab94817, Abcam). Subsequently, it was incubated at RT with a secondary biotin - labeled donkey anti - rabbit IgG (ab97083, Abcam) and streptavidin - biotin / HRP complex (Vectastain). Histone bands were detected using a chemiluminescent ECL substrate (Advansta). The obtained band density was quantified using ImageQuant TL software (GE Healtcare, Little Chalfont, UK).

[0087] Detection of histone cytotoxicity EA.hy926 cells were plated in 24 - well plates and grown to 80% confluence in complete DMEM medium (Thermofisher Scientific) supplemented with (hypoxanthine - aminopterin - thymidine, Gibco). After washing twice with phosphate - buffered saline (PBS, Gibco), the cells were incubated in serum - free DMEM in the presence or absence of 10 nM APC, with 2.7 nM H3 and various amounts of UFH for 1 hour at 37°C. After incubation, the medium containing detached cells was collected, and the attached cells were collected using 0.05% trypsin - EDTA (Gibco). The collected cells were pelleted and resuspended in binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 2 mM MgCl2, 3.3 mM CaCl2) containing 2.5 μg / ml propidium iodide (Sigma). After incubation in the dark for 10 minutes, the percentage of PI - positive cells was measured by flow cytometry using a BD Accuri C6 and analyzed using BD CFlow plus software.

[0088] Results APC cleaves histones, and heparin promotes the cleavage by APC catalysis. APC cleaves histone H3 in a time-dependent manner (Figure 4). When APC is complexed with UFH, as shown in Figure 5, APC-catalyzed degradation of histone H3 was promoted.

[0089] APC enhances the cytoprotective effect of UFH. When endothelial cells (EA.hy926) are incubated with histone H3, the viability decreases due to the cytotoxic effect of histone H3. When histone H3 is incubated with heparin, the cytotoxic effect of histone H3 is neutralized in a dose-dependent manner (Figure 5). The complex of UFH and APC enhances the cytoprotective effect of UFH (Figure 5). APC itself had no effect on the viability of EA.hy926 cells (not shown).

Claims

1. A complex of a negatively charged polysaccharide and a protein having protease activity for the treatment, prevention or improvement of a disease or disorder mediated by extracellular histones, wherein the protein having protease activity is a serine protease, a cysteine protease, a threonine protease, an aspartic acid protease, a glutamic acid protease, a metalloprotease, or an asparagine peptidase, and the negatively charged polysaccharide is a glycosaminoglycan (GAG).

2. The complex according to claim 1, wherein the protein having protease activity is elastase, preferably neutrophil elastase, chymotrypsin-like elastase family member 1, chymotrypsin-like elastase family member 2A, chymotrypsin-like elastase family member 2B, chymotrypsin-like elastase family member 3A, chymotrypsin-like elastase family member 3B, or chymotrypsin C, and more preferably, the protein having elastase activity is neutrophil elastase.

3. The complex according to claim 1, wherein the protein having protease activity is protein C, and preferably, the protein having protease activity is activated protein C (APC).

4. The complex according to any one of claims 1 to 3, wherein the negatively charged polysaccharide consists of or contains variably sulfated repeating disaccharide units.

5. The complex according to any one of claims 1 to 4, wherein the negatively charged polysaccharide is heparin.

6. The complex according to any one of claims 1 to 5, wherein the negatively charged polysaccharide is low anticoagulant heparin or non-anticoagulant heparin.

7. The complex according to any one of claims 1 to 6, wherein the negatively charged polysaccharide is selected from pentasaccharide-depleted heparin, dosiparstat, chemically O-desulfated heparin, sebrparin, site-selectively desulfated heparin, and "glycol-split" heparin.

8. The complex according to any one of claims 1 to 7, wherein the negatively charged polysaccharide and the protein having protease activity are bound by a covalent bond, a bond by van der Waals force, a bond by electrostatic force, a bond by hydrogen bridge, an ionic force, or a combination of two or more thereof.

9. The complex according to any one of claims 1 to 8, wherein the complex is contained in a pharmaceutical composition, preferably in a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

10. The complex according to any one of claims 1 to 9, wherein the disease or disorder mediated by the extracellular histone is a disease or disorder associated with immunothrombosis, or trauma.

11. The complex according to claim 10, wherein the disease or disorder associated with immunothrombosis is selected from sepsis, inflammation, cancer, burns, severe trauma, stroke, ischemia-reperfusion, acute myocardial infarction, organ transplantation, antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis, thrombotic microangiopathy, acute respiratory distress syndrome, thrombotic thrombocytopenic purpura, endotoxemia, pancreatitis, peritonitis, and thromboembolic diseases.

12. A complex of a negatively charged polysaccharide and a protein having protease activity, wherein the protein having protease activity is serine protease, cysteine protease, threonine protease, aspartic protease, glutamic protease, metalloprotease, or asparagine peptide lyase, the negatively charged polysaccharide is glycosaminoglycan (GAG), and the negatively charged polysaccharide and the protein having protease activity are covalently bonded.

Images