JP2024078842A - Anti-inflammatory agent and pharmaceutical composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anti-inflammatory agent capable of simultaneously suppressing multiple signal transduction, and to provide a pharmaceutical composition for treating inflammatory reactions using the anti-inflammatory agent.

SOLUTION: Provided is an anti-inflammatory agent containing, as an active ingredient, a peptide having anti-inflammatory activity or a salt thereof selected from the group consisting of the following (a), (b) and (c): (a) a peptide comprising a portion of a full-length blood coagulation factor 9 excluding a trypsin domain portion and a light chain portion; (b) a peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in the portion of the full-length blood coagulation factor 9 excluding a trypsin domain portion and a light chain portion; and (c) a peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of the portion of the full-length blood coagulation factor 9 excluding a trypsin domain portion and a light chain portion.

SELECTED DRAWING: None

COPYRIGHT: (C)2024,JPO&INPIT

Description

The present invention relates to an anti-inflammatory agent and a pharmaceutical composition.

Infection with COVID-19 (coronavirus disease 2019), MERS, SARS, etc. can cause acute respiratory distress syndrome. One of the causes of this is said to be cytokine storm, an excessive biological defense response of the immune system.

During a cytokine storm, inflammatory cytokines such as interleukin-6 (IL-6) and various other inflammatory humoral factors are released in large quantities, causing neutrophil activation, activation of the blood coagulation mechanism, and vasodilation. This can result in pulmonary edema, thrombosis, etc.

It has been reported that administration of anti-IL-6 receptor monoclonal antibodies is somewhat effective against COVID-19 acute respiratory distress syndrome, but good results have not been obtained in terms of important indicators such as mortality rate.

On the other hand, the present inventors have reported that the activation peptide of blood coagulation factor IX inhibits the formation of lipid rafts (Patent Document 1).

JP 2015-214500 A

One of the reasons why acute respiratory distress syndrome is difficult to treat is thought to be that various humoral factors related to inflammatory responses form a network that enhances the inflammatory response. Therefore, even if some of the factors in this network are inhibited, the entire inflammatory response cannot be suppressed, and it is thought that acute respiratory distress syndrome cannot be treated.

The present invention therefore aims to provide an anti-inflammatory agent capable of simultaneously suppressing multiple inflammatory signal transduction pathways. It also aims to provide a pharmaceutical composition for treating inflammatory responses to which an anti-inflammatory agent is applied.

The present invention includes the following aspects.
[1] An anti-inflammatory agent comprising, as an active ingredient, a peptide or a salt thereof selected from the group consisting of the following (a), (b), and (c):
(a) a peptide comprising an amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding a trypsin domain portion and a light chain portion, and having anti-inflammatory activity;
(b) a peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in the amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and the light chain portion, and which has anti-inflammatory activity;
(c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity.
[2] The anti-inflammatory agent according to [1], which has activity of suppressing signal transduction induced by IL-6.
[3] The anti-inflammatory agent according to [1], which has activity of suppressing signal transduction induced by thrombin.
[4] A pharmaceutical composition for treating or preventing an inflammatory reaction, comprising an effective amount of the anti-inflammatory agent according to [1] and a pharma- ceutically acceptable carrier.
[5] The pharmaceutical composition described in [4], which is used for treating or preventing a cytokine storm.
[6] The pharmaceutical composition described in [5], wherein the cytokine storm is a cytokine storm caused by a viral infection.

The present invention provides an anti-inflammatory agent capable of simultaneously suppressing multiple inflammatory signal transduction pathways, and a pharmaceutical composition for treating inflammatory responses using the anti-inflammatory agent.

FIG. 1 shows the results of observing the intracellular localization of STAT3 after treating human umbilical vein endothelial cells with IL-6 in the absence or presence of F9-AP. FIG. 1 shows the results of observing intracellular actin fiber formation and STAT3 distribution after the action of thrombin on human umbilical vein endothelial cells in the absence or presence of F9-AP.

[Anti-inflammatory Agents]
In one embodiment, the present invention provides an anti-inflammatory agent comprising, as an active ingredient, a peptide selected from the group consisting of the following (a), (b), and (c) or a salt thereof:
(a) A peptide comprising the amino acid sequence of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity. (b) A peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in the amino acid sequence of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity. (c) A peptide comprising an amino acid sequence which has 90% or more sequence identity with the amino acid sequence of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity. In this specification, amino acid sequences are described using the commonly used single-letter code for amino acid residues.
The peptide selected from the group consisting of (a), (b) and (c) above has anti-inflammatory activity.

"Anti-inflammatory activity" means activity to suppress inflammatory response. More specifically, it means activity to suppress signal transduction by IL-6 and/or activity to suppress signal transduction by thrombin. The anti-inflammatory agent according to the present embodiment exhibits anti-inflammatory activity by suppressing these signal transduction. The peptide having "anti-inflammatory activity" can be confirmed, for example, by culturing cells in the presence of the peptide and IL-6 and observing nuclear translocation of STAT3. For example, when the amount of nuclear localization of STAT3 in cells cultured in the presence of the peptide and IL-6 is reduced compared to the amount of nuclear localization of STAT3 in cells cultured in the absence of the peptide and in the presence of IL-6, the peptide can be determined to have anti-inflammatory activity. Alternatively, the peptide having "anti-inflammatory activity" can be confirmed, for example, by culturing cells in the presence of the peptide and thrombin and observing actin fibrosis. For example, if actin fibrillation in cells cultured in the presence of the peptide and thrombin is suppressed compared to actin fibrillation in cells cultured in the absence of the peptide and in the presence of thrombin, the peptide can be determined to have anti-inflammatory activity.

IL-6 is one of the factors that promote inflammatory responses. IL-6 activates the IL-6 receptor and promotes the nuclear translocation of the transcription factor STAT3, thereby promoting inflammatory responses (e.g., S. Kang et al., Targeting Interleukin-6 Signaling in Clinic, DOI: 10.1016/j.immuni.2019.03.026).

Thrombin is one of the factors that control blood coagulation. Thrombin promotes the formation of actin fibers, such as actin stress fibers, in vascular endothelial cells. Actin fibers cause vascular endothelial cells to contract, increasing vascular permeability (e.g., Mayumi Hirano & Katsuya Hirano, Myosin di-phosphorylation and peripheral actin bundle formation as initial events during endothelial barrier disruption, DOI: 10.1038/srep20989). As a result, inflammatory responses are enhanced. In other words, thrombin has the activity of enhancing inflammatory responses.

The inflammation targeted by the anti-inflammatory agent according to the present embodiment is not particularly limited as long as the effect of the present invention is exhibited, and may be inflammation caused by the following causes: Examples of the causes of inflammation that can be targeted include physical stimuli such as burns and bruises, chemical stimuli such as acids, bases, and poisons, stimuli due to infection with pathogens such as viruses and microorganisms, immune responses such as antigen-antibody reactions, humoral immunity, and cellular immunity, and necrosis of cells and tissues.
Examples of inflammation that can be treated with the anti-inflammatory agent according to this embodiment include acute inflammation, chronic inflammation, and cytokine storm.

Examples of acute inflammation include serous inflammation, fibrinous inflammation, suppurative inflammation, gangrenous inflammation, and ulcers.
Serous inflammation is inflammation that is mainly composed of liquid components and is accompanied by exudate with a low content of proteins such as fibrinogen, and examples thereof include pleurisy, peritonitis, and pericarditis.
Fibrinous inflammation is inflammation accompanied by exudate containing a high content of proteins such as fibrinogen, and examples thereof include meningitis, pleurisy, peritonitis, and pericarditis.
Purulent inflammation is inflammation accompanied by exudate containing pus and involving purulent bacteria, and examples thereof include purulent meningitis, lung abscess, liver abscess, cellulitis, and the like.
Gangrenous inflammation is inflammation involving putrefactive bacteria, and examples thereof include gangrenous appendicitis and gangrenous cholecystitis.
An ulcer is an inflammation accompanied by a localized defect in an organ, and examples thereof include gastric ulcer, duodenal ulcer, and arteriosclerosis obliterans.

Chronic inflammation includes, for example, inflammation caused by persistent infection, immune-related inflammation, and the like.
Inflammation caused by persistent infections includes, for example, infections caused by tuberculosis, viruses, fungi, parasites, and the like.
Examples of immune-related inflammation include autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis, inflammatory bowel diseases such as ulcerative colitis substitute and Crohn's disease, and allergic diseases such as bronchial asthma.

Inflammatory responses activate various signal transduction pathways. Examples of signal transduction pathways activated by inflammatory responses include signal transduction pathways activated by cytokines, chemokines, tumor necrosis factor (TNF), etc. Examples of cytokines include interleukins and interferons.

In a cytokine storm, the release of cytokines, chemokines, TNF, and the like is enhanced. Examples of cytokines include interleukins such as IL-1β, IL-2, IL-5, IL-6, and IL-8, interferons such as IFNα and IFNγ, and CSFs such as G-CSF and GM-CSF. Examples of chemokines include CCL2, CCL4, and CXCL10. Examples of TNF include TNFα and TNFβ. The anti-inflammatory agent according to this embodiment can suppress a cytokine storm by suppressing the signal transduction of IL-6 and thrombin. The anti-inflammatory agent according to this embodiment may suppress the signal transduction of one or more of the above-mentioned cytokines in addition to the signal transduction of IL-6 and thrombin.

<Peptide>
The anti-inflammatory agent according to the present embodiment contains a peptide derived from blood coagulation factor 9. Blood coagulation factor 9 (hereinafter also referred to as "F9") is one of the essential blood coagulation factors and is conserved in vertebrates such as mammals. In the living body, F9 is expressed as a precursor F9 having a signal peptide and a propeptide. The signal peptide and the propeptide are then removed from the precursor F9 to become mature F9. In the present specification and claims, "full length of blood coagulation factor 9" or "full length of F9" means the full length of "mature F9".

Mature F9 consists of a light chain, a trypsin domain, and an intermediate portion (activation peptide) located between the light chain and the trypsin domain (hereinafter referred to as "F9-AP"). F9-AP is "the portion of blood coagulation factor 9 minus the trypsin domain and light chain portions."

It has been reported that there is high homology between the amino acid sequences of F9-AP between mammalian species (for example, International Publication No. WO 2016/060158). An example of the amino acid sequence of human F9-AP is the amino acid sequence set forth in SEQ ID NO: 5. An example of the amino acid sequence of mouse F9-AP is the amino acid sequence set forth in SEQ ID NO: 10. The amino acid sequence of F9-AP of other mammals may be obtained from a known sequence database such as GenBank ^TM , or a gene encoding F9-AP may be searched for in the genome of a target mammal based on the amino acid sequence of the known F9-AP. Alternatively, an amino acid sequence having high homology to the known F9-AP may be searched for from amino acid sequences registered in a sequence database using homology search software such as BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

In mature F9, the light chain is adjacent to the N-terminus of F9-AP. In mature F9, the trypsin domain is adjacent to the C-terminus of F9-AP. In mature F9, the light chain, trypsin domain, and each domain of F9-AP can be determined using known domain search software, etc.

The animal from which F9 is derived is not particularly limited as long as the effects of the present invention are achieved, and mammals are preferred. Examples of mammals include rodents such as rats, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, and chimpanzees. Among these, humans are preferred.

The amino acid sequence of human precursor F9 consists of the amino acid sequence set forth in SEQ ID NO: 1 (GenBank accession number: CAA01140; 461 residues). The amino acid sequences of the signal peptide and propeptide of human F9 consist of the amino acid sequence set forth in SEQ ID NO: 2, which is a sequence consisting of the 1st to 28th amino acids of human precursor F9. The amino acid sequence of human mature F9 consists of the 29th to 461st amino acids of human precursor F9.

The amino acid sequence of the light chain of human mature F9 consists of the amino acid sequence set forth in SEQ ID NO:3, which corresponds to the amino acid sequence from the 29th to the 190th amino acids in the amino acid sequence of the human precursor F9 peptide (SEQ ID NO:1).
The amino acid sequence of the trypsin domain of human mature F9 consists of the amino acid sequence set forth in SEQ ID NO:4, which corresponds to the amino acid sequence from the 227th to the 461st amino acids in the amino acid sequence of the human precursor F9 peptide (SEQ ID NO:1).
The amino acid sequence of human F9-AP consists of the amino acid sequence set forth in SEQ ID NO:5, which corresponds to the amino acid sequence from the 191st to the 226th amino acids in the amino acid sequence of the human precursor F9 peptide (SEQ ID NO:1).

The amino acid sequence of mouse-derived precursor F9 consists of the amino acid sequence set forth in SEQ ID NO: 6 (GenBank accession number: BAE28840; 471 residues). The amino acid sequences of the signal peptide and propeptide of mouse F9 consist of the amino acid sequence set forth in SEQ ID NO: 7, which is a sequence consisting of the 1st to 46th amino acids of precursor F9. The amino acid sequence of mature F9 consists of the 47th to 471st amino acids of precursor F9.

The amino acid sequence of the light chain of mouse mature F9 consists of the amino acid sequence set forth in SEQ ID NO:8, which corresponds to the amino acid sequence from the 47th to the 191st amino acids in the amino acid sequence of the mouse precursor F9 peptide (SEQ ID NO:6).
The amino acid sequence of the trypsin domain of mouse mature F9 consists of the amino acid sequence set forth in SEQ ID NO: 9, which corresponds to the amino acid sequence from the 237th to the 471st amino acids in the amino acid sequence of the mouse precursor F9 peptide (SEQ ID NO: 6).
The amino acid sequence of mouse F9-AP consists of the amino acid sequence set forth in SEQ ID NO: 10, which corresponds to the amino acid sequence from positions 192 to 236 in the amino acid sequence of the mouse precursor F9 peptide (SEQ ID NO: 6).

Peptide (a) contains "the portion of blood coagulation factor 9 minus the trypsin domain and light chain," i.e., F9-AP. Peptide (a) has anti-inflammatory activity.

As shown in the Examples below, F9-AP has activity to suppress signal transduction induced by IL-6 and signal transduction induced by thrombin, and has anti-inflammatory activity due to the suppression of these signal transduction pathways.
The peptide (a) above may contain an amino acid sequence other than the amino acid sequence of F9-AP. The peptide (a) above may be a peptide in which an amino acid sequence is added to the amino acid sequence of F9-AP.
When the above peptide (a) is a peptide in which an amino acid has been added to F9-AP, the amino acid may be added to either the N-terminus or the C-terminus of the amino acid sequence of F9-AP, or may be added to both the N-terminus and the C-terminus.
In the above (a), the amino acid sequence added to F9-AP may be an amino acid or an amino acid sequence adjacent to the amino acid sequence of F9-AP in the amino acid sequence of mature F9 having the amino acid sequence of F9-AP.

The peptide (a) is not particularly limited as long as the anti-inflammatory activity of F9-AP is maintained, and may be, for example, 5000 residues or less, 3000 residues or less, 2000 residues or less, 1000 residues or less, 500 residues or less, 300 residues or less, 100 residues or less, 60 residues or less, or 50 residues or less.

When the peptide (a) is a peptide in which one or more amino acids have been added to the amino acid sequence of F9-AP, the number of amino acids added to F9-AP is not particularly limited as long as the anti-inflammatory activity of F9-AP is maintained, but may be 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 100 residues, or 1 to 40 residues.

The peptide (b) comprises an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in the amino acid sequence of F9-AP. Hereinafter, "one or more amino acids that have been deleted, inserted, substituted and/or added" will also be referred to as "mutated amino acids". The peptide (b) has anti-inflammatory activity.

The peptide (b) may be a peptide consisting of an amino acid sequence in which one or more amino acids have been mutated in the amino acid sequence of F9-AP and has anti-inflammatory activity (hereinafter also referred to as "mutated F9-AP"), or may be a peptide in which one or more amino acid residues have been added to either or both of the N-terminus and C-terminus of mutant F9-AP. The mutation may be any of deletion, insertion, substitution, and addition, or a combination of these. The mutation is not particularly limited as long as the peptide resulting from the mutation has anti-inflammatory activity.

In the peptide (b) above, the number of mutated amino acids is not particularly limited. The "multiple" in (b) is not particularly limited, and examples include 2 to 20, 2 to 15, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, or 2. The "multiple" in (b) may also be "several." In this specification and claims, "several" means 2 to 5, 2 to 4, 2 to 3, or 2.

The mutant F9-AP may have a mutation in the amino acid sequence of F9-AP that does not affect the anti-inflammatory activity of F9-AP.
Such mutations include, for example, substitutions called conservative substitutions. Conservative substitutions are substitutions between amino acids with functionally similar side chains. Classifications of functionally similar amino acid side chains include hydrophobic side chains (A, I, L, M, F, P, W, Y, V), hydrophilic side chains (R, D, N, C, E, Q, G, H, K, S, T), aliphatic side chains (G, A, V, L, I, P), hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains (C, M), carboxylic acid and amide-containing side chains (D, N, E, Q), base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W), etc.
In addition, the amino acids described in the following groups 1) to 8) are known to be conservatively substituted for each other within each group.
1) Alanine (A), Glycine (G)
2) Aspartic acid (D), glutamic acid (E)
3) Asparagine (N), Glutamine (Q)
4) Arginine (R), Lysine (K)
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V)
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)
7) Serine (S), Threonine (T)
8) Cysteine (C), Methionine (M)

When the above peptide (b) is a peptide in which an amino acid has been added to a mutant F9-AP, the amino acid may be added to either the N-terminus or the C-terminus of the amino acid sequence of the mutant F9-AP, or may be added to both the N-terminus and the C-terminus.
In the above (b), the amino acid sequence added to the mutant F9-AP may be an amino acid or an amino acid sequence adjacent to the amino acid sequence of F9-AP in the amino acid sequence of mature F9 having the amino acid sequence of F9-AP.

The peptide (b) is not particularly limited as long as the peptide has anti-inflammatory activity, and may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, 100 residues or less, 60 residues or less, or 50 residues or less.

When the peptide (b) is a peptide in which one or more amino acids have been added to mutant F9-AP, the number of amino acid sequences added to mutant F9-AP is not particularly limited as long as it has anti-inflammatory activity, but may be 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 100 residues, or 1 to 40 residues.

The peptide (c) contains an amino acid sequence having 90% or more sequence identity with the amino acid sequence of F9-AP (hereinafter, this sequence is referred to as "F9-AP homologous sequence").
In the amino acid sequence of the peptide (c), the F9-AP homologous sequence has an amino acid sequence identity to the amino acid sequence of F9-AP of 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 97% or more, and particularly preferably 98% or more.

Here, the sequence identity of an amino acid sequence is a value indicating the percentage of identity between a target amino acid sequence (target amino acid sequence) and a reference amino acid sequence (reference amino acid sequence). The reference amino acid sequence is the amino acid sequence of F9-AP. The target amino acid sequence is the F9-AP homologous sequence in peptide (c).
The sequence identity of a subject amino acid sequence to a reference amino acid sequence can be determined, for example, as follows: First, the reference amino acid sequence and the subject amino acid sequence are aligned. Here, each amino acid sequence may contain gaps so as to maximize sequence identity. Next, the number of matching amino acids in the reference amino acid sequence and the subject amino acid sequence is calculated, and the sequence identity can be determined according to the following formula (F1).
Sequence identity (%) = number of matching amino acids/total number of amino acids in the subject amino acid sequence × 100
…(F1)

When the above peptide (c) is a peptide in which one or more amino acids have been added to an F9-AP homologous sequence, the amino acids may be added to either the N-terminus or the C-terminus of the F9-AP homologous sequence, or to both the N-terminus and the C-terminus.
In the above (c), the amino acid sequence added to the F9-AP homologous sequence may be an amino acid or an amino acid sequence adjacent to the amino acid sequence of F9-AP in the amino acid sequence of mature F9 having the amino acid sequence of F9-AP.

The peptide (c) is not particularly limited as long as the peptide has anti-inflammatory activity, and may be, for example, 5,000 residues or less, 3,000 residues or less, 2,000 residues or less, 1,000 residues or less, 500 residues or less, 300 residues or less, 100 residues or less, 60 residues or less, or 50 residues or less.

When the peptide (c) is a peptide in which one or more amino acids are added to an F9-AP homologous sequence, the number of amino acids added to the F9-AP homologous sequence is not particularly limited as long as it has anti-inflammatory activity, but may be 1 to 5000 residues, 1 to 3000 residues, 1 to 2000 residues, 1 to 1000 residues, 1 to 500 residues, 1 to 300 residues, 1 to 100 residues, or 1 to 40 residues.

The peptide selected from the group consisting of (a), (b) and (c) may be a fusion peptide in which another protein or peptide tag is fused. For example, another protein or peptide tag may be bound to either or both of the N-terminus and C-terminus of a peptide (e.g., the peptides exemplified above) containing the amino acid sequence of F9-AP, mutant F9-AP or an amino acid sequence homologous to F9-AP. Examples of other proteins or peptides include marker enzyme proteins such as alkaline phosphatase or partial peptides thereof; fluorescent proteins such as GFP or partial peptides thereof; tag peptides such as His tag and FLAG tag, etc.

It is more preferable that the peptide selected from the group consisting of (a), (b), and (c) is an anti-inflammatory agent containing, as an active ingredient, a peptide selected from the group consisting of the following (a1), (b1), and (c1) or a salt thereof.
(a1) a peptide comprising the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10;
(b1) a peptide comprising an amino acid sequence in which one or more amino acids are deleted, inserted, substituted and/or added in the amino acid sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 10, and having anti-inflammatory activity;
(c1) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:5 or SEQ ID NO:10 and having anti-inflammatory activity.

The peptide or salt thereof selected from the group consisting of (a), (b), and (c) (hereinafter collectively referred to as "peptide component") contained in the anti-inflammatory agent according to this embodiment may be one type or two or more types. When there are two or more types of peptide components, the peptides contained therein may be one type or two or more types.

The salt of the peptide that can be contained in the anti-inflammatory agent according to this embodiment is not particularly limited as long as it is a physiologically acceptable salt, and may be an acid addition salt or a basic salt.
Examples of the acid addition salts include salts with inorganic acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid; and salts with organic acids such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid.
Examples of basic salts include salts with inorganic bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, and magnesium hydroxide; and salts with organic bases such as caffeine, piperidine, trimethylamine, and pyridine.

The anti-inflammatory agent according to this embodiment has activity of suppressing IL-6 signaling, and therefore can also be said to be an inhibitor of IL-6 signaling.
Abnormal activation of IL-6 signaling has been cited as one of the main causes of cytokine storm. The anti-inflammatory agent according to the present embodiment is capable of suppressing inflammatory responses such as cytokine storm by suppressing IL-6 signaling.

The anti-inflammatory agent according to this embodiment has activity of suppressing thrombin signaling, and therefore, the anti-inflammatory agent according to this embodiment can also be said to be an inhibitor of thrombin signaling.
Thrombin is one of the major blood coagulation factors and also enhances vascular permeability to enhance inflammatory responses. The anti-inflammatory agent according to the present embodiment is capable of suppressing inflammatory responses by inhibiting thrombin signal transduction.

Hereinafter, the peptides that may be included in the anti-inflammatory agent according to this embodiment may be collectively referred to as "F9-AP-like peptide."

<Method of producing peptide>
The F9-AP-like peptide may be produced as a recombinant protein by applying various known gene recombination techniques to a nucleic acid encoding the peptide.
The amino acid sequences of proteins having F9-AP-like peptides from mice, humans, etc., and the base sequences of the nucleic acids encoding them are registered in databases such as GenBank. Based on the base sequence information, a nucleic acid encoding an F9-AP-like peptide can be chemically synthesized or cloned from an appropriate gene source within the scope of ordinary skill in the art.

Alternatively, the F9-AP-like peptide may be obtained by a cell-free protein synthesis system. There is no particular limitation on the cell-free protein synthesis system, and examples of the cell-free protein synthesis system include synthesis systems that utilize cell extracts obtained from wheat germ, yeast, insect cells, cultured mammalian cells, rabbit reticulocytes, Escherichia coli, etc.; synthesis systems that reconstitute factors necessary for translation, etc.

Alternatively, the F9-AP-like peptide may be produced by a method commonly used in the art, such as an organic chemical synthesis method, such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). F9-AP-like peptide may also be produced using a commercially available device generally referred to as a peptide synthesizer.

The F9-AP-like peptide may be produced in a form in which a tag peptide such as a Flag tag, a polyhistidine tag, a c-Myc tag, an HA tag, an AU1 tag, a GST tag, or an MBP tag is added to its N-terminus and/or C-terminus, or in the form of a fusion protein with other proteins such as a fluorescent protein, glutathione transferase, or alkaline phosphatase.
These tag peptides and other proteins are preferably removed before administration to a target animal. The F9-AP-like peptide may be labeled with a fluorescent substance, a luminescent substance, biotin, or other appropriate labeling agent.

<Target subjects>
The subjects of administration of the anti-inflammatory agent according to this embodiment include the animals from which F9 is derived, as described above. The subjects of administration are preferably mammals, and more preferably humans. The subjects of administration are preferably animals belonging to the same species as the animal from which the F9-AP-like peptide is derived.

Administration Method
The method of administration of the anti-inflammatory agent in this embodiment is not particularly limited as long as the anti-inflammatory agent exerts an anti-inflammatory effect in the animal to which it is administered, and may be oral administration or parenteral administration, although parenteral administration is preferred.
Examples of parenteral administration include systemic administration such as intravenous administration and intra-arterial administration, and local administration such as intramuscular administration, subcutaneous administration, peritoneal administration, intraventricular administration, intrathecal administration, transdermal administration, intraocular administration, and intranasal administration.

The administration route may be, for example, intraarterial injection, intravenous injection, subcutaneous injection, intranasal injection, transbronchial injection, intramuscular injection, transdermal injection, or oral injection, which are known to those skilled in the art, and intravenous injection is preferred.
Injections can also be prepared as non-aqueous diluents (e.g., propylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions, or emulsions. Sterilization of such injections can be achieved by filtration sterilization using a filter, blending of a bactericide, etc. Injections can be produced in the form of preparations to be used. That is, they can be made into a sterile solid composition by freeze-drying or the like, and dissolved in distilled water for injection or other solvents before use.

Dosage:
The dosage of the anti-inflammatory agent according to this embodiment is appropriately adjusted taking into consideration the age, sex, weight, symptoms, treatment method, administration method, treatment time, etc. of the subject animal (various mammals including humans or non-human animals, preferably humans).
For example, in the case of intravenous injection, the amount of peptide administered to a subject animal (preferably a human) in one administration is preferably 100 μg or more per kg body weight, more preferably 200 μg to 3 mg, and particularly preferably 400 μg to 1 mg.
The frequency of administration is preferably one to several times per day on average.

According to the anti-inflammatory agent of the present embodiment described above, it is possible to suppress an inflammatory response. In an inflammatory response, various signal transduction pathways are activated, and it is difficult to suppress an inflammatory response by only suppressing a small number of them. Therefore, even if a highly specific normal drug is used, it is difficult to treat an inflammatory response such as a cytokine storm. The anti-inflammatory agent of the present embodiment contains an F9-AP-like peptide, and thus can simultaneously suppress at least IL-6 signal transduction and thrombin signal transduction. Thrombin signal transduction is a pathway unrelated to the signal transduction system by IL-6. The anti-inflammatory agent of the present embodiment can effectively suppress an inflammatory response by suppressing signal transduction of two independent pathways.
In addition, by using an F9-AP-like peptide derived from an animal belonging to the same species as the animal to be administered, the risk of side effects such as immune reactions can be reduced. Furthermore, since the F9-AP-like peptide is derived from F9 present in the blood of a living body, the anti-inflammatory agent according to this embodiment is considered to have few side effects and to be highly safe for a living body.

Pharmaceutical Compositions
In one embodiment, the present invention provides a pharmaceutical composition for treating or preventing an inflammatory response comprising an effective amount of an anti-inflammatory agent according to the embodiments, and a pharma- ceutically acceptable carrier.

The pharmaceutical composition according to this embodiment may contain a pharma- ceutical acceptable carrier in addition to an effective amount of the anti-inflammatory agent according to the embodiment. The term "pharma-ceutical acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and does not show substantial toxicity to the subject to which it is administered. The term "not substantially toxic" refers to a component that does not show toxicity to the subject to which it is administered at a dose normally used. In the pharmaceutical composition according to this embodiment, the pharma-ceutical acceptable carrier may be a component that does not inhibit the anti-inflammatory activity of the anti-inflammatory agent according to the embodiment and does not show substantial toxicity to the subject to which it is administered.

Examples of pharma- ceutically acceptable carriers include excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, and calcium carbonate; binders such as cellulose, methylcellulose, hydroxypropylcellulose, gelatin, gum arabic, polyethylene glycol, sucrose, and starch; disintegrants such as starch, carboxymethylcellulose, hydroxypropyl starch, sodium-glycol-starch, sodium bicarbonate, calcium phosphate, and calcium citrate; lubricants such as magnesium stearate, aerosil, talc, and sodium lauryl sulfate; fragrances such as citric acid, menthol, glycyrrhizin ammonium salt, glycine, and orange powder; preservatives such as sodium benzoate, sodium bisulfite, methylparaben, and propylparaben; stabilizers such as citric acid, sodium citrate, and acetic acid; suspending agents such as methylcellulose, polyvinylpyrrolidone, and aluminum stearate; dispersing agents such as surfactants; diluents such as water, buffer solutions, and physiological saline; and base waxes such as cacao butter, polyethylene glycol, and white kerosene.

When the pharmaceutical composition according to this embodiment is used for parenteral administration, the pharmaceutical composition may contain an antioxidant, a buffer solution, a bacteriostatic agent, an isotonic agent, a suspending agent, a solubilizing agent, a thickening agent, a stabilizer, a preservative, and the like.
When the pharmaceutical composition according to this embodiment is used for parenteral administration, the pharmaceutical composition may be sealed in a container such as an ampoule, a vial, or a syringe cartridge in a unit dose or in multiple doses.

When the pharmaceutical composition according to this embodiment is to be administered orally, the pharmaceutical composition formulation can be produced by a conventional method using additives commonly used for oral preparations, such as excipients, fillers, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizing agents, antiseptics, flavoring agents, soothing agents, and stabilizers.
Examples of additives that can be used include lactose, fructose, glucose, starch, gelatin, methylcellulose, gum arabic, polyethylene glycol, citric acid, sodium sulfite, sodium phosphate, β-cyclodextrin, and hydroxypropyl-β-cyclodextrin.
When the pharmaceutical composition according to the present embodiment is to be administered orally, the pharmaceutical composition may be prepared in a dosage form suitable for oral administration, such as capsules, tablets, granules, powders, pills, fine granules, lozenges, etc.

<Target diseases>
The diseases to which the pharmaceutical composition according to the present embodiment is applicable include the inflammation described above in the anti-inflammatory agent according to the embodiment, and immune-related inflammation or cytokine storm is preferable, and cytokine storm is more preferable. The pharmaceutical composition according to the present embodiment can be used, for example, to suppress inflammation caused by viral infection. The pharmaceutical composition according to the present embodiment may be used to suppress cytokine storm caused by viral infection. The virus can be, for example, SARS-CoV-2.
The pharmaceutical composition of the present embodiment may be for treating inflammation, may be for preventing inflammation, or may be for treating and preventing inflammation.

<Target subjects>
The subjects to which the pharmaceutical composition according to this embodiment is administered include the animals mentioned above as the animals from which F9 is derived in the anti-inflammatory agent according to the embodiment, and humans are preferred.

Administration Method
Methods for administering the pharmaceutical composition according to this embodiment include the administration methods described above for the anti-inflammatory agent according to the embodiment, and parenteral administration is preferred.

<Effective amount>
The effective amount of the anti-inflammatory agent contained in the pharmaceutical composition according to this embodiment is not particularly limited as long as the pharmaceutical composition exerts a therapeutic effect, and can be appropriately determined by a person skilled in the art. The "effective amount" means an amount of the anti-inflammatory agent that is effective for treating or preventing a target disease.

The F9-AP-like peptide contained in the pharmaceutical composition according to this embodiment is preferably a peptide derived from F9 of the target animal, and more preferably a partial peptide of F9 of the target animal. When a peptide derived from F9 of the target animal is administered to the target animal, it is considered that side effects in the target animal are low.

The pharmaceutical composition according to this embodiment contains the above-mentioned anti-inflammatory agent, and therefore can simultaneously suppress multiple signal transduction pathways involved in inflammatory responses. Therefore, by administering the pharmaceutical according to this embodiment, it is possible to treat inflammatory responses in the subject. The F9-AP-like peptide contained in the pharmaceutical composition according to this embodiment is a peptide derived from a blood coagulation factor, and is therefore presumed to have few side effects and to be highly safe.

[Other embodiments]
In one aspect, the invention provides a method for treating inflammation comprising administering to an animal in need of treatment an effective amount of an anti-inflammatory agent according to the embodiments.
The animals to be treated include humans and non-human mammals. The effective amount of the anti-inflammatory agent may be an effective amount as described above for the pharmaceutical composition according to the embodiment.
The method of administration of the anti-inflammatory agent may be the administration method described above for the pharmaceutical composition according to the embodiment.

In one aspect, the present invention provides use of a peptide selected from the group consisting of the following (a), (b) and (c) or a salt thereof for the manufacture of a pharmaceutical composition for treating or preventing inflammation:
(a) a peptide comprising a portion of the full-length blood coagulation factor 9 minus the trypsin domain and the light chain, and having anti-inflammatory activity;
(b) a peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and the light chain portion, and having anti-inflammatory activity;
(c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity.

In one aspect, the present invention provides use of a peptide selected from the group consisting of the following (a), (b) and (c), or a salt thereof, for treating or preventing inflammation:
(a) a peptide comprising a portion of the full-length blood coagulation factor 9 minus the trypsin domain and the light chain, and having anti-inflammatory activity;
(b) A peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity. (c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence in a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity.

The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

Materials and Methods
(Preparation of peptides)
F9-AP consisting of the amino acid sequence shown in SEQ ID NO:10 was prepared by chemical synthesis.

(Preparation of IL-6)
As IL-6, Recombinant Human IL-6 (PeproTech, catalog number 200-06, lot number 031916) was used.

(Preparation of Thrombin)
Human alpha thrombin (Haematologic Technologies, catalog number HCT-0020, lot number LL0217-1MG) was used as thrombin.

[Experimental Example 1]
(Suppression of IL-6 signaling)
As described below, IL-6 was applied to cultured cells in the presence or absence of F9-AP, and then the nuclear translocation of STAT3 was observed.

Human umbilical vein endothelial cells were cultured on a fibronectin-coated cover glass for 24 hours. The medium was then changed to serum-free medium (DMEM). F9-AP (10 pmol/ml) was then added, and IL-6 (1 μg/ml) was added 30 minutes later. The cells were then fixed with 4% paraformaldehyde 30 minutes later.
Then, after fixation, blocking was performed with 1% albumin. STAT3 was stained using anti-STAT3 antibody (STAT3) (abcam) as the primary antibody and goat anti-mouse IgG-AlexaFlour488 (Life Technologies) as the secondary antibody. In addition, actin was stained using Phalloidin568 as a counterstain, and nuclei were stained using DAPI.
The specimen was then mounted on a slide glass and observed under a microscope. The results are shown in FIG.

In Figure 1, "Control" (top row) shows the staining results when IL-6 was not applied in the absence of F9-AP, "IL-6" (middle row) shows the staining results when IL-6 was applied in the absence of F9-AP, and "F9-AP, IL-6" (bottom row) shows the staining results when IL-6 was applied in the presence of F9-AP.

Compared to "Control," "IL-6" showed that STAT3 accumulated more in the nucleus and on the cell membrane. Compared to "IL-6," "F9-AP, IL-6" suppressed the distribution of STAT3 in the nucleus and rafts, and it was distributed throughout the cell.

These results demonstrate that F9-AP can suppress IL-6 signaling. They also suggest that F9-AP can suppress inflammatory responses, such as cytokine storm, caused by IL-6 signaling.

[Experimental Example 2]
(Inhibition of thrombin signaling)
Thrombin was allowed to act on the cultured cells in the presence or absence of F9-AP, and the formation of stress fibers was then observed.

Human umbilical vein endothelial cells were cultured on a fibronectin-coated cover glass for 24 hours. The medium was then changed to serum-free medium (DMEM). F9-AP (10 pmol/ml) was then added, and thrombin (1 μg/ml) was added 30 minutes later. The cells were then fixed with 4% paraformaldehyde 30 minutes later.
Then, after fixation, blocking was performed with 1% albumin. Actin was stained using Phalloidin 568. STAT3 was stained using anti-STAT3 antibody (STAT3) (abcam) as the primary antibody and goat anti-mouse IgG-AlexaFlour 488 (Life Technologies) as the secondary antibody, and nuclei were stained using DAPI.
The specimen was then mounted on a slide glass and observed under a microscope. The results are shown in FIG.

In Figure 2, "Control" shows the staining results when thrombin was not applied in the absence of F9-AP, "F9-AP" shows the staining results when thrombin was not applied in the presence of F9-AP, "Thrombin" shows the staining results when thrombin was applied in the absence of F9-AP, and "F9-AP, Thrombin" shows the staining results when thrombin was applied in the presence of F9-AP.

Compared to "Control," "Thrombin" showed that STAT3 accumulated more in the nucleus and on the cell membrane. Compared to "IL-6," "Thrombin" showed the formation of stress fibers that ran linearly through the center of the cell and cell contraction. Compared to "Thrombin," "F9-AP, Thrombin" suppressed both the formation of stress fibers and cell contraction.

These results demonstrate that F9-AP can suppress thrombin signaling. These results suggest that F9-AP can suppress inflammatory responses induced by thrombin.

The anti-inflammatory agent and pharmaceutical composition of the present invention can be suitably used to treat inflammatory reactions such as cytokine storm.

Claims (6)

An anti-inflammatory agent comprising, as an active ingredient, a peptide or a salt thereof selected from the group consisting of the following (a), (b) and (c):
(a) a peptide comprising an amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding a trypsin domain portion and a light chain portion, and having anti-inflammatory activity;
(b) a peptide comprising an amino acid sequence in which one or more amino acids have been deleted, inserted, substituted and/or added in the amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and the light chain portion, and which has anti-inflammatory activity;
(c) A peptide comprising an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a portion of the full-length blood coagulation factor 9 excluding the trypsin domain and light chain portions, and having anti-inflammatory activity. The anti-inflammatory agent according to claim 1, which has activity in suppressing signal transduction induced by IL-6. The anti-inflammatory agent according to claim 1, which has activity of suppressing signal transduction by thrombin. A pharmaceutical composition for treating or preventing an inflammatory response, comprising an effective amount of the anti-inflammatory agent according to claim 1 and a pharma- ceutical acceptable carrier. The pharmaceutical composition according to claim 4, which is used to treat or prevent a cytokine storm. The pharmaceutical composition according to claim 5, wherein the cytokine storm is a cytokine storm caused by a viral infection.