PHARMACEUTICAL COMPOSITION FOR TREATING, PREVENTING RELAPSE, OR MAINTAINING REMISSION OF INFLAMMATORY BOWEL DISEASE INCLUDING TRYPTOPHANYL tRNA SYNTHETASE-LIKE PROTEIN AS DYNAMIC NETWORK BIOMARKER AS ACTIVE COMPONENT

Abstract

To provide a pharmaceutical for treating, preventing relapse, or maintaining remission of inflammatory bowel disease.SOLUTION: The present invention provides a pharmaceutical for treating, preventing relapse, or maintaining remission of inflammatory bowel disease including tryptophanyl tRNA synthetase-like protein as an active component.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical agent for treating, preventing relapse, or maintaining remission of inflammatory bowel disease. [Background technology]

[0002] Inflammatory bowel disease (IBD), a debilitating intestinal disorder, is a term used to describe two conditions characterized by chronic intestinal inflammation: Crohn's disease (CD) and ulcerative colitis (UC). In many IBD cases, standard treatments, such as 5-aminosalicylic acid and immunosuppressants, are used to suppress the active inflammation associated with the onset and / or relapse of IBD. However, because IBD is a chronic inflammatory disease that undergoes repeated remissions and relapses, the current primary treatment goal has shifted from suppressing the active inflammation associated with onset and / or relapse to breaking the cycle of repeated relapses and achieving long-term remission. However, no IBD treatments have been developed specifically to prevent relapse or maintain remission, and no biomarkers have been established to predict relapse.

[0003] Although the exact cause of IBD is unknown, it has been suggested to be the result of an abnormally enhanced immune response, and analysis of the pathogenic mechanism is currently underway. It has been reported that the symptoms of colitis are alleviated by intraperitoneal injection of human umbilical cord blood-derived pluripotent stem cells (hUCB-MSCs) into mice (colitis model mice) in which experimental colitis was induced at 8 weeks of age by administration of the inflammation-inducing substance dextran sulfate sodium (DSS) solution. It has also been reported that serum tryptophanyl-tRNA synthetase levels in these model mice are reduced compared to control mice, but are restored by intraperitoneal injection of human umbilical cord blood-derived pluripotent stem cells. It has also been reported that in vitro treatment of hUCB-MSCs with IFN-γ increases tryptophanyl-tRNA synthetase secretion, and that tryptophanyl-tRNA synthetase treatment suppresses the proliferation of hUCB-MSCs or CD4+ T cells isolated from hUCB-MSCs (Non-Patent Document 1). However, it was not known that administration of tryptophanyl-tRNA synthetase could suppress the onset of experimental colitis.

[0004] The present inventors have proposed the Dynamic Network Biomarker (DNB) theory, which uses mathematical engineering techniques to scientifically and objectively predict "pre-illness," a concept previously only understood in traditional Chinese medicine. This theory is based on the hypothesis that because the human mind and body continuously fluctuates between health and illness, DNB analysis, a new mathematical analysis method, captures this fluctuation and maximizes it at the pre-symptomatic or pre-disease state, i.e., the pre-illness state. In other words, in a living organism, where various biological components interact to form complex networks, fluctuations are unlikely to occur in a healthy state where the biological system is stable. However, when virtual energy increases due to the complex intertwining of environmental factors (extrinsic factors) such as a deterioration in lifestyle habits in addition to genetic background (intrinsic factors), the endogenous factors of a specific subnetwork of the biological network fluctuate greatly with a strong correlation, and if virtual energy increases further, the state will irreversibly transition to another stable state with less "fluctuation," the onset / disease (abnormal) state. The pre-symptomatic (pre-disease) state, which is the critical point of this state transition, easily transitions to the onset / disease state, but there is a possibility of reversibly returning to a healthy state or a state of remission through appropriate medical intervention such as improving lifestyle habits or pharmaceutical intervention.

[0005] As a technology related to dynamic network biomarkers, for example, Patent Document 1 discloses a detection method using a detection device that detects candidate biomarkers that serve as indicators of symptoms of a living organism being measured, based on measurement data for multiple factor items obtained by measuring the organism. The detection device is characterized by performing a classification step of classifying multiple factor items into multiple clusters based on the correlation of time-series changes in the measurement data for each factor item, a selection step of selecting from each classified cluster a cluster that meets predetermined selection conditions based on the time-series changes in the measurement data for each factor item and the correlation of time-series changes in the measurement data between the factor items, and a detection step of detecting factor items included in the selected cluster as candidate biomarkers. Furthermore, Patent Documents 2 and 3 also disclose technologies related to dynamic network biomarkers. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2014 / 050160 [Patent Document 2] International Publication No. 2014 / 065155 [Patent Document 3] International Publication No. 2018 / 207925 [Non-patent literature]

[0007] [Non-Patent Document 1] BMB Rep.2019;52(5):318-323 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a pharmaceutical agent for treating inflammatory bowel disease, preventing relapse, or maintaining remission, and a companion diagnostic agent that enables the determination of patients to whom the pharmaceutical agent should be administered. Another object of the present invention is to provide a biomarker that can predict the relapse of inflammatory bowel disease, a companion diagnostic biomarker for the pharmaceutical agent, a method for assisting in the diagnosis of inflammatory bowel disease, a method for assisting in determining the possibility of relapse of inflammatory bowel disease, and a method for treating inflammatory bowel disease, preventing relapse, or maintaining remission. [Means for solving the problem]

[0009] The present inventors performed DNB analysis of gene expression levels in DSS-induced colitis model mice and identified the Wars gene (protein name: tryptophanyl-tRNA synthetase; WRS), which had not been identified in previous analyses, as a potential target for the treatment of inflammatory bowel disease. Furthermore, they investigated whether the Wars gene is a pathophysiologically significant gene in inflammatory bowel disease and found that fluctuations in Wars gene expression levels increased during the onset of colitis in colitis model mice. Furthermore, they found that administering WRS recombinant protein to colitis model mice suppressed the onset of colitis. The present invention is based on these novel findings.

[0010] That is, the present invention relates to, for example, the following inventions. [1] A pharmaceutical for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising a tryptophanyl-tRNA synthetase-like protein as an active ingredient. [2] The pharmaceutical according to [1], wherein the protein is of human origin. [3] The pharmaceutical agent according to [1] or [2], which is administered to a subject predicted to experience a relapse of inflammatory bowel disease. [4] The pharmaceutical according to [3], wherein the relapse of the inflammatory bowel disease is predicted by measuring the expression level of one or more proteins selected from tryptophanyl-tRNA synthetase, leucine-rich α2 glycoprotein, C-reactive protein, and calprotectin, or the amount of mRNA encoding the one or more proteins. [5] A diagnostic biomarker for inflammatory bowel disease, comprising human tryptophanyl-tRNA synthetase or mRNA encoding said enzyme. [6] measuring the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme contained in a sample derived from a test subject; and determining that the subject may be suffering from inflammatory bowel disease or is at high risk of suffering from inflammatory bowel disease if the amount of the enzyme is higher than the standard value for the enzyme amount or if the amount of the mRNA is higher than the standard value for the mRNA amount. [7] The method according to [6], wherein the sample is colon tissue or a sample prepared from the tissue. [8] The method according to [6] or [7], wherein the reference value of the enzyme is the amount of tryptophanyl-tRNA synthetase contained in a sample derived from a healthy control, and the reference value of the mRNA amount is the amount of mRNA encoding the enzyme contained in a sample derived from a healthy control. [9] The method according to any one of [6] to [8], further comprising a step of measuring the expression level of one or more proteins selected from leucine-rich alpha-2 glycoprotein, calprotectin, and C-reactive protein, or the amount of mRNA encoding the one or more proteins, contained in a sample derived from a test subject.

[10] Measuring the amount of a companion diagnostic biomarker for a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease contained in a sample from a test subject; A method for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising administering the pharmaceutical agent described in [1] or [2] to the subject when the amount of the companion diagnostic biomarker is higher than the reference value.

[11] The method according to

[10] , wherein the companion diagnostic biomarker is one or more selected from tryptophanyl-tRNA synthetase, leucine-rich alpha-2 glycoprotein, calprotectin, and C-reactive protein, and mRNAs encoding these proteins. [1'] A method for treating inflammatory bowel disease, comprising administering to a subject a pharmaceutical for treating inflammatory bowel disease, which pharmaceutical comprises a tryptophanyl-tRNA synthetase-like protein as an active ingredient. [2'] The method according to [1'], wherein the protein is a human tryptophanyl-tRNA synthetase-like protein. [3'] A method for preventing relapse or maintaining remission of inflammatory bowel disease, comprising administering a pharmaceutical for preventing relapse or maintaining remission of inflammatory bowel disease, which pharmaceutical contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient, to a subject predicted to experience a relapse of inflammatory bowel disease. [4'] The method according to [3'], wherein the protein is a human tryptophanyl-tRNA synthetase-like protein. [5'] The method according to [3'] or [4'], wherein the subject predicted to have a relapse of inflammatory bowel disease is a subject whose measured value of the amount of a companion diagnostic biomarker contained in a sample derived from the subject is determined to be higher than a reference value. [6'] The method according to [5'], wherein the companion diagnostic biomarker is the expression level of one or more proteins selected from leucine-rich alpha-2 glycoprotein, calprotectin, and C-reactive protein, or the amount of mRNA encoding the one or more proteins. [7'] A medicine containing 5-acetylsalicylic acid as an active ingredient for preventing relapse or maintaining remission of inflammatory bowel disease. [8'] The pharmaceutical agent described in [7'], which is administered to a subject predicted to experience a relapse of inflammatory bowel disease. [9'] The pharmaceutical agent according to [7'] or [8'], wherein the relapse of the inflammatory bowel disease is predicted by measuring the expression level of tryptophanyl-tRNA synthetase protein or the amount of mRNA encoding the protein. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a pharmaceutical agent for treating inflammatory bowel disease, preventing relapse, or maintaining remission, and a companion diagnostic agent that enables the determination of patients to whom the pharmaceutical agent should be administered. The present invention also provides a biomarker that can predict the relapse of inflammatory bowel disease, a companion diagnostic biomarker for the pharmaceutical agent, a method for assisting in the diagnosis of inflammatory bowel disease, a method for assisting in determining the possibility of relapse of inflammatory bowel disease, and a method for treating inflammatory bowel disease, preventing relapse, or maintaining remission. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a graph showing the results of evaluating the disease activity index in colitis model mice. [Figure 2] Graph (A) shows the results of calculating the standard deviation of the expression level of each DNB gene measured at each time point and averaging them at each time point, and graph (B) shows the results of calculating the absolute value of the correlation coefficient of the expression level for all combinations of two different DNB genes at each time point and averaging them at each time point. [Figure 3] This is a heat map created by standardizing the expression level data of DNB genes for each gene. [Figure 4] This graph shows the results of gene expression analysis of biopsy samples of the large intestine (colon) mucosa, using microarray data published in the GEO repository. [Figure 5]This graph shows the results of gene expression analysis of biopsy samples of the large intestine (colon) mucosa, using microarray data published in the GEO repository. [Figure 6] This graph shows the results of gene expression analysis of biopsy samples of the large intestine (colon) mucosa, using microarray data published in the GEO repository. [Figure 7] 1 is a graph showing the results of analyzing the expression of the Wars gene in colitis model mice. [Figure 8] 1 shows images showing the results of histological analysis of Wars gene expression in the colonic mucosa of normal mice. [Figure 9] 1 shows graphs depicting the results of evaluating the effect of WRS recombinant protein on the development of colitis using a mouse model of DSS-induced colitis. [Figure 10] 1 shows graphs depicting the results of evaluating the effect of anti-WRS antibodies on the development of colitis using a mouse model of DSS-induced colitis. [Figure 11] The images show the results of histological evaluation using hematoxylin and eosin staining to determine the effect of WRS recombinant protein on the pathology of colitis. [Figure 12] 1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. [Figure 13] 1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. [Figure 14] 1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. [Figure 15] 1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. [Figure 16]1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. [Figure 17] 1 is a graph showing the results of a real-time PCR analysis of the effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression. DETAILED DESCRIPTION OF THE INVENTION

[0013] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0014] [Medicines for treating inflammatory bowel disease, preventing relapse, or maintaining remission] A pharmaceutical agent for treating inflammatory bowel disease, preventing relapse, or maintaining remission according to this embodiment (hereinafter also referred to as "a pharmaceutical agent according to this embodiment") contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient.

[0015] As used herein, "treatment" refers to the alleviation, improvement, or complete cure of symptoms by administering a compound to a subject already suffering from inflammatory bowel disease in vivo. Furthermore, as used herein, "prevention of relapse or maintenance of remission" refers to the prevention of relapse or maintenance of remission of inflammatory bowel disease by administering a compound to a subject in whom a relapse of inflammatory bowel disease symptoms is predicted. The definitions of "remission" and "relapse" used herein are in accordance with the standards adopted in each country or region in which the present invention is implemented. For example, in the United States and Europe, the standard used to select patients in remission from Crohn's disease is a CDAI (Crohn's disease activity index) value of less than 150 (Clinical Gastroenterology and Hepatology, 14, 348-354, 2016). In the United States, the criteria used to select patients in remission of inflammatory bowel disease follow the definition of remission and relapse (including mild, moderate, and severe) activity indicators proposed by the American College of Gastroenterology, which incorporates both patient-reported outcomes (abbreviated as PROs), clinical test, and endoscopic examination-based values (Am. J. Gastroenterol., 114:384-413, 2019). In Europe, the criteria used to select patients in remission of inflammatory bowel disease are fewer than four bowel movements per day without bleeding or urgency (Clinical Gastroenterology and Hepatology, 14,348-354, 2016). In Japan, "remission" refers to an IOIBD score of 0 or 1 and normal erythrocyte sedimentation rate and C-reactive protein (CRP). "Maintained remission" means maintaining a state without progressing to relapse. "Relapse" means an IOIBD score of 2 or higher and an abnormal ESR or CRP value. Remission and relapse can be diagnosed by the IOIBD score as well as the ESR and CRP value.

[0016] As used herein, the term "tryptophanyl-tRNA synthetase-like protein" refers to a protein having a function possessed by tryptophanyl-tRNA synthetase or a modified version thereof. Examples of functions possessed by tryptophanyl-tRNA synthetase or a modified version thereof include the canonical aminoacylation activity function and non-canonical function that tryptophanyl-tRNA synthetase or a modified version thereof can exert intracellularly, as well as the non-canonical function that tryptophanyl-tRNA synthetase or a modified version thereof can exert in vivo after being secreted extracellularly. As used herein, the term "non-canonical function" refers to a function other than the aminoacylation activity function, which is the canonical function of tryptophanyl-tRNA synthetase.

[0017] Tryptophanyl-tRNA synthetase is an enzyme also known as tryptophan-tRNA ligase, TrpRS, WRS, etc. Tryptophanyl-tRNA synthetase has aminoacylation activity that catalyzes, for example, the reaction of attaching tryptophan, a type of amino acid, to tRNA (aminoacylation). More specifically, the reaction is represented by the following formula 1. The aminoacylation activity is a suitable example of a non-limiting form of the function that tryptophanyl-tRNA synthetase can exert in cells. (Equation 1): Tryptophan + tRNA + ATP → Tryptophanyl-tRNA + AMP + PPi

[0018] A non-limiting example of the function possessed by tryptophanyl-tRNA synthetase or a modified form thereof is a non-canonical function that can be exerted in vivo by being secreted extracellularly. Suitable examples of non-canonical functions analyzed in multiple publications include TLR2 / TLR4 receptor binding activity and macrophage-stimulating activity (Parker G Jobin et al.; J Biol Chem, 2019; 294(35):12866-12879), the immunostimulatory activity exerted by the VSE domain present at the amino terminus of tryptophanyl-tRNA synthetase (Young Ha Ahn et al.; Nat Microbiol, 2016; 17(2):16191), the angiogenesis-inhibiting activity exerted by the below-described mini TrpRS, T1 TrpRS, or T2 TrpRS, which are variants of tryptophanyl-tRNA synthetase, and the innate immune-stimulating activity in response to viral infection (Hyun-Cheol Lee et al.; J Virol, 2019; 93(2):e01291-18). Methods described in the above publications can be used as appropriate to evaluate functions other than aminoacylation activity. As used herein, a "tryptophanyl-tRNA synthetase-like protein" may have either the aminoacylation activity function or the non-canonical function, or may have both the aminoacylation activity function and the non-canonical function. Furthermore, as used herein, a "tryptophanyl-tRNA synthetase-like protein" may have, in addition to the aminoacylation activity function, a combination of one or more functions selected from non-canonical functions including a TLR2 / TLR4 receptor binding activity function, a macrophage-stimulating activity function, an immunostimulating activity function, and an anti-angiogenic activity function. Furthermore, as used herein, a "tryptophanyl-tRNA synthetase-like protein" may have a combination of one or more functions selected from a TLR2 / TLR4 receptor binding activity function, a macrophage-stimulating activity function, an immunostimulating activity function, and an anti-angiogenic activity function. In addition to a combination of one or more functions selected from the functions included in the non-canonical functions, a function other than the functions may also be possessed.

[0019] Whether or not the "tryptophanyl-tRNA synthetase-like protein" of the present invention has the aminoacylation activity of tryptophanyl-tRNA synthetase can be confirmed, for example, by the method described in Methods in Enzymology Vol. 59, 1979, Pages 234-257. Whether or not the "tryptophanyl-tRNA synthetase-like protein" of the present invention has TLR2 / TLR4 receptor binding activity, macrophage-stimulating activity, immunostimulating activity, and angiogenesis inhibitory activity can be confirmed by publicly known methods disclosed in the above-mentioned multiple documents (Parker G jobin et al.; J Biol Chem, 2019; 294(35):12866-12879, Young Ha Ahn et al.; Nat Microbiol, 2016; 17(2):16191, Hyun-Cheol Lee et al.; J Virol, 2019; 93(2):e01291-18).

[0020] Tryptophanyl-tRNA synthetase-like proteins include wild-type tryptophanyl-tRNA synthetases and variants thereof (e.g., naturally mutated or artificially mutated tryptophanyl-tRNA synthetases). As used herein, "wild-type tryptophanyl-tRNA synthetases" refer to tryptophanyl-tRNA synthetases consisting of the amino acid sequence most frequently found in the population of each species present in nature. Those skilled in the art can appropriately determine whether a protein is a wild-type tryptophanyl-tRNA synthetase by referring to protein databases or literature.

[0021] As used herein, "tryptophanyl-tRNA synthetase" refers to wild-type tryptophanyl-tRNA synthetase. Furthermore, as used herein, "variant of tryptophanyl-tRNA synthetase" refers to a protein that has the function of tryptophanyl-tRNA synthetase (e.g., a function that can be exerted intracellularly, or a function that can be exerted in vivo by secretion outside the cell), and in which at least one amino acid residue or a region of consecutive amino acid residues has been substituted, deleted (deleted), or added to the amino acid sequence of wild-type tryptophanyl-tRNA synthetase. Variants of tryptophanyl-tRNA synthetase also include, for example, naturally occurring mutant tryptophanyl-tRNA synthetases that can be isolated from nature, and artificial mutant tryptophanyl-tRNA synthetase proteins that do not exist in nature.

[0022] The tryptophanyl-tRNA synthetase-like protein is not particularly limited, and may be derived from, for example, an animal, a plant, a unicellular eukaryote, or a prokaryote. When derived from an animal, it may be derived from a mammal or an animal other than a mammal. The protein is preferably derived from an animal, more preferably from a mammal, even more preferably from a human, rat, mouse, pig, or monkey, and particularly preferably from a human. Herein, for example, a human-derived tryptophanyl-tRNA synthetase-like protein includes not only wild-type or naturally mutated tryptophanyl-tRNA synthetase that can be isolated from a human, but also proteins in which at least one amino acid residue or a region of consecutive amino acid residues is deleted (deleted) from the amino acid sequence of the tryptophanyl-tRNA synthetase. In other words, a "human-derived tryptophanyl-tRNA synthetase-like protein" refers to a tryptophanyl-tRNA synthetase-like protein that contains a part or all of the amino acid sequence of a tryptophanyl-tRNA synthetase that can be isolated from a human.

[0023] As described above, a non-limiting example of a tryptophanyl-tRNA synthetase-like protein is a wild-type tryptophanyl-tRNA synthetase. The biological species from which tryptophanyl-tRNA synthetase is derived is not particularly limited, and suitable examples include animals, plants, or unicellular eukaryotes or prokaryotes. A suitable non-limiting example of the amino acid sequence of a wild-type human tryptophanyl-tRNA synthetase that can be isolated from nature is GenBank Accession Number NP_004175.2 (SEQ ID NO: 1). For convenience, herein, a tryptophanyl-tRNA synthetase comprising the amino acid sequence represented by NP_004175.2 is defined as a human full-length tryptophanyl-tRNA synthetase (human full-length WRS). Suitable examples of animal-derived tryptophanyl-tRNA synthetases that can be appropriately used in the present invention include human-derived tryptophanyl-tRNA synthetases as well as mammalian tryptophanyl-tRNA synthetases such as rat-derived, mouse-derived, pig-derived, or monkey-derived tryptophanyl-tRNA synthetases. The amino acid sequences of wild-type tryptophanyl-tRNA synthetases derived from mammals, animals other than mammals, plants, unicellular eukaryotes, or prokaryotes that are disclosed in databases can also be used appropriately in the present invention.

[0024] Full-length tryptophanyl-tRNA synthetases from prokaryotes, including Geobacillus stearothermophilus, contain the Rossmann Fold (RF) domain and anticodon-binding domain required for the aminoacylation activity described above, whereas full-length tryptophanyl-tRNA synthetases from eukaryotes contain an additional eukaryotic-specific extension (ESE) domain at the amino terminus of the RF domain, and full-length tryptophanyl-tRNA synthetases from mammals contain an additional vertebrate-specific extension (VSE) domain (also known as the WHEP domain) at the amino terminus of the ESE domain. The ESE domain contained in eukaryotic tryptophanyl-tRNA synthetases and the VSE domain containing a helix-turn-helix motif contained in mammalian tryptophanyl-tRNA synthetases are known to confer various functions to eukaryotic or mammalian tryptophanyl-tRNA synthetases, such as interactions with other proteins, in addition to aminoacylation activity.

[0025] A variant of tryptophanyl-tRNA synthetase may have an amino acid sequence in which one or more, for example, 1 to 100, 1 to 95, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acid residues have been deleted, substituted, or added from the amino acid sequence of a wild-type tryptophanyl-tRNA synthetase, and the substitutions, deletions, and additions may occur simultaneously. Here, addition of an amino acid includes both insertion into the original amino acid sequence and addition to the end of the original amino acid sequence.

[0026] As used herein, a non-limiting example of a variant of tryptophanyl-tRNA synthetase may be one having an amino acid sequence in which one or more amino acid residues have been deleted from the amino acid sequence of wild-type tryptophanyl-tRNA synthetase, such as T1-TrpRS (SEQ ID NO: 2) or T2-TrpRS (SEQ ID NO: 3), in which 70 amino acids have been deleted from the amino terminus of full-length human tryptophanyl-tRNA synthetase as a result of post-translational modification such as protease digestion or polyglutamylation. Another non-limiting example of a variant of tryptophanyl-tRNA synthetase is mini-TrpRS (SEQ ID NO: 4), in which 47 amino acids have been deleted from the amino terminus of full-length human tryptophanyl-tRNA synthetase as a result of translation from a splice variant of the gene encoding tryptophanyl-tRNA synthetase. Non-limiting examples of tryptophanyl-tRNA synthetase-like proteins having aminoacylation activity and non-canonical function include human full-length tryptophanyl-tRNA synthetase (SEQ ID NO: 1), T1-TrpRS (SEQ ID NO: 2), and mini-TrpRS (SEQ ID NO: 4). Non-limiting examples of tryptophanyl-tRNA synthetase-like proteins having aminoacylation activity but not non-canonical function include tryptophanyl-tRNA synthetases derived from prokaryotes. Non-limiting examples of tryptophanyl-tRNA synthetase-like proteins having non-canonical function but not aminoacylation activity include T2-TrpRS (SEQ ID NO: 3).

[0027] As used herein, among the modified tryptophanyl-tRNA synthetases, a non-limiting example of a naturally mutated tryptophanyl-tRNA synthetase is a tryptophanyl-tRNA synthetase translated from a tryptophanyl-tRNA synthetase gene having SNPs (single nucleotide polymorphisms) or the like.

[0028] Herein, among the modified forms of tryptophanyl-tRNA synthetase, non-limiting examples of artificial mutant tryptophanyl-tRNA synthetase include tryptophanyl-tRNA synthetase that has been subjected to codon optimization for the purpose of optimizing the expression efficiency during recombinant expression in heterologous cells, addition of a tag sequence used for the purpose of purifying or detecting recombinant proteins, fusion with an antibody Fc sequence or an albumin sequence, or chemical modification with hyaluronic acid or polyethylene glycol (PEG) for the purpose of extending the plasma half-life when administered in vivo, to the extent that the function that tryptophanyl-tRNA synthetase can exert intracellularly or the non-canonical function that tryptophanyl-tRNA synthetase can exert in vivo after being secreted extracellularly is not impaired.

[0029] Non-limiting examples of tryptophanyl-tRNA synthetase-like proteins include those that share 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence of full-length human tryptophanyl-tRNA synthetase (SEQ ID NO: 1), and may have a function that tryptophanyl-tRNA synthetase can exert intracellularly, or a function that can be secreted extracellularly and exerted in vivo. The above-mentioned function may be a combination of one or more functions selected from the group consisting of aminoacylation activity, which catalyzes the reaction of binding (aminoacylation) of tryptophan to tRNA, TLR2 / TLR4 receptor binding activity, macrophage-stimulating activity, immunostimulating activity, and angiogenesis inhibitory activity. Note that "sequence identity" as used herein refers to the percentage of identical bases or amino acid residues when two base sequences or amino acid sequences are aligned (e.g., aligned using the BLAST algorithm).

[0030] Tryptophanyl-tRNA synthetase-like proteins can be prepared by methods known to those skilled in the art. For example, a nucleic acid encoding the tryptophanyl-tRNA synthetase-like protein is prepared, operably linked to an expression promoter, introduced into an appropriate expression vector, and expressed in a host cell. Alternatively, tryptophanyl-tRNA synthetase-like proteins can be prepared by artificial synthesis using known peptide synthesis methods (e.g., solid-phase synthesis).

[0031] The inflammatory bowel disease is not particularly limited, and may be, for example, ulcerative colitis or Crohn's disease, preferably ulcerative colitis.

[0032] The content of tryptophanyl-tRNA synthetase-like protein in the pharmaceutical composition according to this embodiment is not particularly limited and may be appropriately determined depending on the formulation, etc., and may be 0.001% to 100% by mass. The content of tryptophanyl-tRNA synthetase activity-like protein in the pharmaceutical composition according to this embodiment may be, for example, 0.001% by mass or more, 0.005% by mass or more, 0.01% by mass or more, 0.05% by mass or more, 0.1% by mass or more, 0.5% by mass or more, or 1% by mass or more. The content of tryptophanyl-tRNA synthetase activity-like protein in the pharmaceutical composition according to this embodiment may be, for example, 100% by mass or less, 90% by mass or less, 70% by mass or less, 50% by mass or less, 40% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 1% by mass or less, 0.5% by mass or less, or 0.1% by mass or less.

[0033] The pharmaceutical according to this embodiment may contain only the active ingredient, or may further contain a pharmaceutically acceptable carrier (excipient, binder, disintegrant, filler, emulsifier, flow additive, etc.) or additive (isotonicity agent, lubricant, flavoring agent, solubilizer, suspending agent, diluent, surfactant, stabilizer, absorption enhancer, bulking agent, pH adjuster, moisturizer, adsorbent, disintegration inhibitor, coating agent, colorant, preservative, antioxidant, fragrance, flavoring agent, sweetener, buffer, soothing agent, etc.).

[0034] The administration route of the medicament according to this embodiment may be either oral administration, parenteral administration, or enteral administration. The dosage form of the medicament according to this embodiment may be appropriately selected depending on the administration route. For oral administration, examples of dosage forms include tablets, pills, granules, powders, capsules, etc. Furthermore, for oral administration, an enteric formulation is preferable from the viewpoint of efficiently delivering the active ingredient to the large intestine, and for example, a formulation with an enteric coating may be used. For parenteral administration, examples of dosage forms include injections whose formulation is optimized depending on the administration route, such as subcutaneous administration, intravenous administration, or intramuscular administration. For enteral administration, examples of dosage forms include enteral preparations or enteral foams. When administered parenterally by injection or the like, the pharmaceutical of this embodiment is transported into the plasma and then circulates throughout the body. To extend the half-life in plasma, tryptophanyl-tRNA synthetase-like proteins prepared by fusion with the above-mentioned antibody Fc sequence or albumin sequence, or by chemical modification with hyaluronic acid or polyethylene glycol (PEG), as well as modified tryptophanyl-tRNA synthetase encapsulated in nanoparticles exemplified by PEG, polylactic acid-co-glycolic acid (PLGA), chitosan, and the like can also be suitably used.

[0035] The dosage of the pharmaceutical according to this embodiment, when administered to an adult human (body weight 60 kg), is usually 0.001 mg to 5000 mg / day / person, preferably 0.01 mg to 500 mg / day / person, in terms of the amount of active ingredient. It may be administered in multiple divided doses.

[0036] The subjects to which the pharmaceutical of this embodiment is administered may be, for example, mammals such as humans, mice, rats, rabbits, guinea pigs, horses, pigs, and sheep, preferably humans or mice, and more preferably humans.

[0037] The medicament according to this embodiment may be administered to a subject predicted to have a relapse of inflammatory bowel disease. The subject predicted to have a relapse of inflammatory bowel disease may be a subject predicted to have a relapse of inflammatory bowel disease by measuring the expression level of one or more proteins selected from tryptophanyl-tRNA synthetase, leucine-rich alpha 2 glycoprotein (LRG), C-reactive protein (CRP), and calprotectin (FC), or the amount of mRNA encoding the one or more proteins. The subject predicted to have a relapse of inflammatory bowel disease is as described below in "Method 1 for determining a subject to administer a medicament for treating, preventing relapse, or maintaining remission of inflammatory bowel disease."

[0038] The pharmaceutical of this embodiment contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient, thereby enabling treatment, prevention of relapse, or maintenance of remission of inflammatory bowel disease. Therefore, another embodiment of the present invention provides a pharmaceutical for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising a nucleic acid encoding a tryptophanyl-tRNA synthetase-like protein as an active ingredient. A non-limiting example of such a pharmaceutical for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising a nucleic acid encoding a tryptophanyl-tRNA synthetase-like protein as an active ingredient, is a nanoparticle formulation encapsulating mRNA encoding tryptophanyl-tRNA synthetase. Another non-limiting aspect of this embodiment is the encapsulation of mRNA encoding tryptophanyl-tRNA synthetase in lipid nanoparticles with excellent endosomal escape function (e.g., Biol. Pharm. Bull. 43, 1617-1625, 2020, etc.), in order to allow the tryptophanyl-tRNA synthetase-like protein to exert its functions, such as aminoacylation activity, in the cytoplasm.

[0039] An example of a nucleic acid encoding a tryptophanyl-tRNA synthetase-like protein is GenBank Accession No. NM_004184.3 (SEQ ID NO: 5).

[0040] [Diagnostic biomarkers for inflammatory bowel disease] The diagnostic biomarker for inflammatory bowel disease according to this embodiment comprises human tryptophanyl-tRNA synthetase or mRNA encoding the enzyme. Human tryptophanyl-tRNA synthetase is as described above. The mRNA encoding the enzyme may be any mRNA encoding human tryptophanyl-tRNA synthetase, such as that represented by GenBank Accession No. NM_004184.3 (SEQ ID NO: 5).

[0041] As shown in the Examples below, the expression level of tryptophanyl-tRNA synthetase is significantly increased in subjects with inflammatory bowel disease compared to controls without inflammatory bowel disease (normal controls not affected by inflammatory bowel disease and controls before the onset of inflammatory bowel disease). Furthermore, DNB analysis has shown that the expression level of tryptophanyl-tRNA synthetase fluctuates in the pre-inflammatory bowel disease, pre-disease, or pre-disease state.

[0042] Therefore, human tryptophanyl-tRNA synthetase can be used as a diagnostic biomarker for inflammatory bowel disease. The diagnostic biomarker according to this embodiment enables early determination of the presence or absence of inflammatory bowel disease and determination of the therapeutic effect of inflammatory bowel disease. Therefore, the diagnostic biomarker for inflammatory bowel disease according to this embodiment can also be used as a companion diagnostic biomarker for a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease. As used herein, "companion diagnosis" refers to predicting the effect of a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease without actual administration, and "companion diagnostic biomarker" refers to a biomarker for companion diagnosis. The drug according to this embodiment is also included in the drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease.

[0043] The diagnostic biomarker for inflammatory bowel disease according to this embodiment can be used, for example, in a method for assisting in the diagnosis of inflammatory bowel disease, which will be described later, etc. The companion diagnostic biomarker for a medicine for treating, preventing relapse, or maintaining remission of inflammatory bowel disease according to this embodiment can be used, for example, in methods 1 and 2 for determining a subject to which a medicine for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which will be described later, etc.

[0044] Method for assisting in the diagnosis of inflammatory bowel disease The method for assisting in the diagnosis of inflammatory bowel disease according to this embodiment includes a step of measuring the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme contained in a sample derived from a subject (measurement step A-1), and a step of determining that the subject may be suffering from inflammatory bowel disease or has a high risk of suffering from inflammatory bowel disease if the amount of the enzyme is higher than the reference value for the enzyme amount or if the amount of the mRNA is higher than the reference value for the mRNA amount (determination step A-2).

[0045] In the method for assisting in the diagnosis of inflammatory bowel disease according to this embodiment, the "subject" is not particularly limited as long as it is a human subject in need of a diagnosis of inflammatory bowel disease. Examples of such subjects include subjects with symptoms such as persistent or recurrent mucous and bloody stools or bloody diarrhea. Suitable examples of severely ill subjects include subjects with a bowel movement frequency of six or more times per day, bloody stools, a fever of 37.5°C or higher, tachycardia of 90 beats per minute or higher, anemia with a Hb concentration of 10 g / dL or lower, an ESR of 30 mm / h or higher, or a CRP level of 3.0 mg / dL or higher, or a combination of two or more of these.

[0046] The sample from the subject used in the measurement step A-1 may be any sample that allows measurement of the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme. Such a sample may be colon tissue obtained by biopsy during ileocolonoscopy, and is preferably a sample appropriately prepared from the tissue in accordance with its use in the measurement step. Examples of samples prepared from colon tissue include sections of colon tissue.

[0047] The amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme can be measured by known methods for measuring protein or RNA levels. Examples of methods for measuring protein levels include Western blotting, ELISA, and immunostaining. Examples of methods for measuring mRNA levels include quantitative reverse transcription (RT)-PCR, quantitative real-time reverse transcription (RT)-PCR, and quantitative northern blotting.

[0048] In the determination step A-2, the reference value refers to a value that can be compared with the measured amount of the enzyme or the amount of the mRNA to determine whether or not a test subject may or is likely to have inflammatory bowel disease, and can also be referred to as a threshold value. The reference value is set in advance. The reference value may be a cutoff value (Acta Paediatr. 96(5), 644-647, 2007) calculated using an ROC curve based on the amount of the enzyme contained in samples from multiple subjects (e.g., healthy control group) who do not have inflammatory bowel disease and the amount of the enzyme contained in samples from multiple controls (e.g., patient control group) who have been diagnosed with inflammatory bowel disease, or the amount of mRNA encoding the enzyme in both groups. In this case, the reliability of the reference value is considered to be higher with a larger number of healthy control subjects, and may be, for example, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. Furthermore, the reference value for a given subject may be a cutoff value determined using an ROC curve from the amount of the above enzyme contained in a sample collected during the remission period of the subject recorded in the subject's past history, and the amount of the above enzyme contained in a sample collected at a time when the subject was determined to be suffering from inflammatory bowel disease, or from the amount of mRNA encoding the above enzyme at both times.

[0049] As shown in the Examples, it is believed that patients suffering from inflammatory bowel disease have a higher expression level of tryptophanyl-tRNA synthetase compared to subjects without the disease. Therefore, in the determination step A-2, if the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme contained in a sample derived from the subject is higher than the reference value, or if the amount of mRNA is higher than the reference value for the mRNA amount, it can be determined that the subject may have inflammatory bowel disease or has a high risk of developing it. Conversely, in the determination step A-2, if the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding the enzyme contained in a sample derived from the subject is lower than the reference value, or if the amount of mRNA is lower than the reference value for the mRNA amount, it can be determined that the subject may not have inflammatory bowel disease or has a low risk of developing it. Here, "high risk of developing it" means that the subject is more likely to develop it in the future than subjects with a lower amount of tryptophanyl-tRNA synthetase or mRNA encoding the enzyme than the reference value.

[0050] The method for assisting in the diagnosis of inflammatory bowel disease according to this embodiment can also be considered as a data collection method, comprising the measurement step A-1, for determining whether a subject is likely to suffer from inflammatory bowel disease or is at high risk of suffering from inflammatory bowel disease.

[0051] [Method for determining (companion diagnostic for) a drug to be administered for the treatment, prevention of relapse, or maintenance of remission of inflammatory bowel disease, comprising a tryptophanyl-tRNA synthetase-like protein as an active ingredient, and a companion diagnostic for inflammatory bowel disease]

[0023] As used herein, the term "companion diagnostic" refers to an in vitro diagnostic drug used to identify patients for whom a specific drug is expected to be effective and safe, based on the analysis results of a companion diagnostic biomarker. The method for determining a subject to whom a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising a tryptophanyl-tRNA synthetase-like protein as an active ingredient, according to this embodiment (hereinafter also referred to as "determination method 1 according to this embodiment") comprises the steps of: measuring the amount of a companion diagnostic biomarker of the drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, contained in a sample derived from the subject (measurement step B); and, if the amount of the biomarker is higher than a reference value, determining the subject as a subject to whom a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, comprising a tryptophanyl-tRNA synthetase-like protein as an active ingredient (determination step B).

[0052] As previously mentioned, inflammatory bowel disease (IBD) is a chronic inflammatory disease that undergoes repeated remissions and relapses. Therefore, standardization of various diagnostic methods that can be used to predict relapses has been recommended in treatment guidelines (see, for example, the "ECCO-ESGAR Guideline for Diagnostic Assessment in IBD," compiled by the European Society of Crohn's Disease and Colitis (ESGAR) and the European Society of Gastrointestinal and Abdominal Radiology). For the purpose of early detection of relapses, the monitoring interval for patients with IBD who have achieved clinical and biochemical remission is 3–6 months, depending on the duration of remission and current treatment. To diagnose UC and CD, ileocolonoscopy with biopsies from the inflamed area is performed. For example, longitudinal ulcers, cobblestone lesions, or non-caseating epithelioid cell granulomas are among the key findings for the diagnosis of CD. For example, focal or diffuse basal plasmacytosis is recognized as one of the early features in the diagnosis of UC.

[0053] In the determination method 1 of this embodiment, the "subject" is not particularly limited as long as it is a human subject for whom it is necessary to determine whether to administer a pharmaceutical agent containing a tryptophanyl-tRNA synthetase-like protein as an active ingredient for the treatment of inflammatory bowel disease, prevention of relapse, or maintenance of remission.

[0054] A non-limiting example of a companion diagnostic biomarker for a drug containing a tryptophanyl-tRNA synthetase-like protein as an active ingredient for treating inflammatory bowel disease, preventing relapse, or maintaining remission, which is measured in the measurement step B, is the expression of one or more proteins selected from tryptophanyl-tRNA synthetase, leucine-rich alpha 2 glycoprotein (LRG), C-reactive protein (CRP), and calprotectin (FC), or mRNA encoding the one or more proteins. Companion diagnostics for LRG, CRP, FC, etc. have been approved by the Ministry of Health, Labor and Welfare, as described below. In another non-limiting example, normal values previously determined in treatment guidelines or package inserts of approved companion diagnostics can also be used as reference values for the companion diagnostic biomarker for a drug containing a tryptophanyl-tRNA synthetase-like protein as an active ingredient for treating inflammatory bowel disease, preventing relapse, or maintaining remission.

[0055] LRG, CRP, and FC are known biomarkers measured for the purpose of predicting the relapse of inflammatory bowel disease. It is known that the measured values of LRG, CRP, and FC are higher in subjects with a relapse of inflammatory bowel disease compared with controls without inflammatory bowel disease (normal controls without inflammatory bowel disease, controls in remission from inflammatory bowel disease, or controls before the onset of inflammatory bowel disease). Furthermore, in subjects predicted to experience a relapse of inflammatory bowel disease, the changes in the measured values of tryptophanyl-tRNA synthetase protein or the measured values of mRNA encoding the tryptophanyl-tRNA synthetase protein are significantly greater than those in controls without inflammatory bowel disease (normal controls without inflammatory bowel disease, controls in remission from inflammatory bowel disease, or controls before the onset of inflammatory bowel disease). In other words, fluctuations occur in the expression levels of tryptophanyl-tRNA synthetase protein and mRNA encoding the tryptophanyl-tRNA synthetase protein. In subjects experiencing an inflammatory bowel disease flare-up, the measured levels of tryptophanyl-tRNA synthetase protein or the measured levels of mRNA encoding the tryptophanyl-tRNA synthetase protein are elevated compared to controls without inflammatory bowel disease.

[0056] The sample derived from the subject used in the measurement step B may be a sample in which the amount of the biomarkers can be measured. The sample derived from the subject used in the measurement step B may be a sample in which one of the biomarkers can be measured, or a sample in which multiple types can be measured. When the amounts of multiple types of biomarkers are measured in the measurement step B, one sample in which all of the biomarkers can be measured may be measured, or multiple samples in which each biomarker can be measured may be measured.

[0057] Non-limiting examples of samples for measuring LRG or CRP include body fluids such as blood, lymph, urine, and tears. Serum obtained by fractionating blood is appropriately used as a sample for measuring LRG or CRP. Non-limiting examples of samples for measuring FC include bodily excretions including feces, or feces. Samples for measuring tryptophanyl-tRNA synthetase or mRNA encoding tryptophanyl-tRNA synthetase include tissue or sections thereof obtained by biopsy during ileocolonoscopy, or samples appropriately prepared from the tissue or sections according to the type of measurement method.

[0058] Tryptophanyl-tRNA synthetase, LRG, CRP, and FC, or mRNA encoding these proteins, can be quantified by known methods for quantifying proteins or mRNA. Protein measurement methods include, for example, Western blotting, ELISA, and immunostaining. Furthermore, mRNA levels can be measured using, for example, quantitative RT-PCR, quantitative real-time RT-PCR, and quantitative Northern blotting. Test sample preparation methods suitable for each measurement method are known.

[0059] In the determination step B, the reference value refers to a value, or threshold, at which a test subject can be determined to be a candidate for administration of a pharmaceutical agent for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient. The reference value is predetermined. The reference value refers to a cutoff value calculated using an ROC curve from measurements in multiple controls not suffering from inflammatory bowel disease (e.g., a normal control group not suffering from inflammatory bowel disease, a control group in remission from inflammatory bowel disease, or a control group before the onset of inflammatory bowel disease) and measurements in multiple controls determined to have inflammatory bowel disease (e.g., a patient control group) (Acta Paediatr. 96(5), 644-647, 2007). In this case, the reliability of the reference value is considered to be higher with a larger number of healthy controls, and may be, for example, 3 or more, 5 or more, 10 or more, 15 or more, or 20 or more. Furthermore, the reference value for a given subject may be a cutoff value determined using an ROC curve from the amount of the above enzyme contained in a sample collected during the remission period of the subject recorded in the subject's past history, and the amount of the above enzyme contained in a sample collected at a time when the subject was determined to be suffering from inflammatory bowel disease, or from the amount of mRNA encoding the above enzyme at both times.

[0060] As described above, in subjects with a relapse of inflammatory bowel disease, the measured values of LRG, CRP, FC, tryptophanyl-tRNA synthetase, or mRNA encoding the above enzymes are higher than in controls without inflammatory bowel disease. Furthermore, in subjects predicted to have a relapse of inflammatory bowel disease, the measured values of tryptophanyl-tRNA synthetase protein or mRNA encoding the tryptophanyl-tRNA synthetase protein change significantly (fluctuations occur) compared to controls without inflammatory bowel disease. That is, if the measured values in the measurement step B are higher than the reference value, the subject may have developed inflammatory bowel disease before (before) the onset of inflammatory bowel disease. Therefore, in the determination step B, if the amount of the biomarker is higher than the reference value, the subject is determined to be a candidate for administration of a medicament containing a tryptophanyl-tRNA synthetase-like protein as an active ingredient for treating, preventing relapse, or maintaining remission of inflammatory bowel disease.

[0061] In other words, in the determination step B, the effect of a medicine for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient, is predicted without actual administration, and the subject is determined to be a recipient of the medicine. Therefore, the determination method 1 according to this embodiment can also be considered a method for companion diagnostics of a medicine for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient. Therefore, the diagnostic agent for measuring biomarkers used in the determination method 1 according to this embodiment can be considered a companion diagnostic agent for a medicine for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which contains a tryptophanyl-tRNA synthetase-like protein as an active ingredient.

[0062] [Method for determining a target of administration of a medicine containing 5-aminosalicylic acid (5-ASA) or the like as an active ingredient for treating, preventing relapse, or maintaining remission of inflammatory bowel disease (companion diagnosis of said medicine)] In another non-limiting embodiment of the present invention, there is also provided a method for determining a subject to whom a medicament for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which comprises as an active ingredient 5-ASA or the like, which is an active ingredient of an existing medicament for inflammatory bowel disease, is to be administered (hereinafter also referred to as "determination method 2 according to this embodiment"). In one non-limiting embodiment of determination method 2, at least one companion diagnostic marker selected from tryptophanyl-tRNA synthetase protein or mRNA encoding tryptophanyl-tRNA synthetase protein is measured to determine a subject to whom a medicament for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, which comprises as an active ingredient 5-ASA, is to be administered. Furthermore, another non-limiting embodiment of determination method 2 according to this embodiment is a method for determining a subject to be administered a medicine containing 5-ASA as an active ingredient for preventing relapse or maintaining remission of inflammatory bowel disease, and in order to determine a subject to be administered a medicine containing a known active ingredient such as 5-aminosalicylic acid (5-ASA) for preventing relapse or maintaining remission of inflammatory bowel disease, the method may include the steps of: measuring at least one companion diagnostic marker selected from tryptophanyl-tRNA synthetase protein or the amount of mRNA encoding tryptophanyl-tRNA synthetase protein in the subject (measurement step C); and, when the value of at least one companion diagnostic marker selected from the enzyme amount or the mRNA amount is higher than the respective reference value, determining the subject as a subject to be administered a medicine containing 5-ASA or the like as an active ingredient for preventing relapse or maintaining remission of inflammatory bowel disease (determination step C).

[0063] The specific embodiments described in the measurement step A-1 can be applied without limitation to specific embodiments of the measurement step C. The specific embodiments described in the measurement step A-2 can be applied to specific embodiments of the determination step C.

[0064] Furthermore, determination method 2 according to this embodiment can also be considered a companion diagnostic method for a medicine containing, as an active ingredient, 5-aminosalicylic acid (5-ASA) or the like for treating, preventing relapse, or maintaining remission of inflammatory bowel disease. Therefore, the diagnostic agent for measuring the companion diagnostic marker (tryptophanyl-tRNA synthetase protein or mRNA encoding tryptophanyl-tRNA synthetase protein) used in determination method 2 according to this embodiment can be considered a companion diagnostic agent for a medicine containing a known active ingredient such as 5-aminosalicylic acid (5-ASA) for treating, preventing relapse, or maintaining remission of inflammatory bowel disease.

[0065] [Method for treating, preventing relapse, or maintaining remission of inflammatory bowel disease] The method for treating inflammatory bowel disease according to this embodiment includes the steps of: measuring the amount of a companion diagnostic biomarker for a drug for treating, preventing relapse, or maintaining remission of inflammatory bowel disease, contained in a sample derived from a subject (measurement step D); If the amount of the companion diagnostic biomarker is higher than the reference value, the method further comprises a step of administering the pharmaceutical agent according to this embodiment to the subject (administration step D).

[0066] As a specific embodiment of the measurement step D, the specific embodiment of the measurement step B can be applied without any restrictions.

[0067] In the administration step D, the method of administering the pharmaceutical agent according to this embodiment to the subject, the amount of the pharmaceutical agent according to this embodiment to be administered to the subject, etc. are as described in "Pharmaceutical agent for treating, preventing relapse, or maintaining remission of inflammatory bowel disease." [Example]

[0068] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0069] [Test Example 1: Evaluation of colitis onset in colitis model mice] In this test example, male BALB / c mice (manufactured by Japan SLC) were used. The mice were kept in the Animal Experiment Facility of the Advanced Life Science Research Support Unit, University of Toyama, under a constant temperature and humidity environment (summer: 23±2°C, humidity 55±10%, winter: 22±2°C, humidity 55±10%). Water and solid food were available ad libitum, and all animal experiments were conducted in accordance with the "University of Toyama Animal Experiment Handling Regulations" (Animal Experiment Protocol Approval Numbers: A2018 INM-3, A2021 INM-2).

[0070] Dextran sulfate sodium (DSS)-induced colitis was induced in the mice by allowing them to drink 3% DSS (MW: 36-50 kDa) (MP Biomedicals) ad libitum for 7 days to induce colitis. Symptoms of colitis were evaluated by calculating the disease activity index (DAI) based on scores for weight loss, diarrhea, and melena (Table 1) on days 0, 1, 3, 5, and 7 after the start of DSS drinking.

[0071] [Table 1]

[0072] The results are shown in Figure 1. As shown in Figure 1, the disease activity index was 0 until day 3, but increased by day 5, and symptoms of colitis were observed in the colitis model mice on day 5.

[0073] [Test Example 2: DNB analysis in colitis model mice] Colitis was induced in male BALB / c mice by allowing them to freely drink 3% DSS (MW: 36-50 kDa) (MP Biomedicals) aqueous solution for 7 days, as in Test Example 1. Gene expression profiles of mouse colon tissues 0, 1, 3, 5, and 7 days after the start of DSS drinking were comprehensively analyzed by microarray analysis as described below, and these data were analyzed using dynamic network biomarker (DNB) theory (DNB analysis).

[0074] Microarray analysis was performed using the SurePrint G3 Mouse Gene Expression 8x60K Microarray Kit (Agilent Technologies). Colonic tissues were isolated from mice 0, 1, 3, 5, and 7 days after the start of DSS drinking (n = 5 for each time point), and total RNA extracted from the colonic tissue was used for microarray analysis. Microarray analysis was performed according to the kit's protocol.

[0075] In this test example, DNB analysis was performed by calculating the score I represented by the following formula 2 using gene expression data in mouse colon tissue obtained by the above-mentioned microarray analysis. In the following formula 2, SDd represents the average standard deviation of gene expression levels of DNB candidates, and PCCd represents the average correlation coefficient between DNB candidate genes. More specifically, first, genes with significantly increased fluctuations in expression levels were selected from all genes using gene expression data in mouse colon tissue. Next, the correlation coefficient between the expression levels of the selected genes was calculated. Next, genes with high correlations among the selected genes were clustered. Then, the score represented by the following formula 2 was calculated. As a result, 27 DNB genes were detected: Mcpt1, Mcpt4, Ifit2, Cd274, Trim30d, Tnfsf10, Ccn1, Wars, Tap2, Plppr3, Ube2l6, Bst2, Cxcl16, B2m, 4933412E12Rik, H2-T23, Znfx1, H2-M2, H2-Bl, H2-Q2, Gm11127, H2-K2, H2-Q8, H2-Q7, H2-Q6, H2-D1, and Fbxo6. (Equation 2): Score I = SDd × PCCd

[0076] In addition, we extracted only the expression data for the DNB gene from the expression data for all genes analyzed. For each time point, we calculated the standard deviation of the expression level of each DNB gene and averaged them for that time point. Furthermore, we extracted only the expression data for the DNB gene from the expression data for all genes analyzed, and calculated the absolute value of the correlation coefficient for all combinations of two different DNB genes for each time point. These were then averaged for each time point. For correction, we subtracted the expected absolute value of the correlation coefficient for two independent random variables that follow a standard normal distribution for each time point. These results are shown in Figure 2. Furthermore, we extracted only the expression data for the DNB gene from the expression data for all genes analyzed, and then normalized each gene to create a heat map. The results are shown in Figure 3.

[0077] As shown in Figure 2, three days after the start of DSS drinking, the standard deviation (fluctuation in expression levels) and the strength of the correlation coefficient increased sharply. Furthermore, as shown in Figure 3, three days after the start of DSS drinking, there were individuals with high and low expression levels for each of the 27 DNB genes. This indicates that three days after the start of DSS drinking, there were fluctuations in the expression levels of each of the 27 DNB genes. These results suggest that the time three days after the start of DSS drinking corresponds to a pre-symptomatic / pre-disease state, or a pre-disease state, as defined by DNB theory.

[0078] [Test Example 3: DNB gene expression analysis using human IBD dataset] Using microarray data published in the GEO repository (GEO dataset accession number GSE9452), we performed gene expression analysis on colonic mucosa biopsy samples from control subjects and human IBD patients. Samples with and without macroscopic signs of inflammation were collected from human IBD patients, and the above dataset contains both sets of data. From the expression level data for all genes in the microarray data, we extracted and analyzed expression level data for 11 DNB genes detected in Test Example 2 that are present in humans and were present in the IBD data used. The results are shown in Figures 4 to 6. In Figures 4 to 6, the results using the control subject sample are labeled "Control," the results using the human IBD patient sample with macroscopic signs of inflammation are labeled "Ulcerative colitis_Inflammation," and the results using the human IBD patient sample without macroscopic signs of inflammation are labeled "Ulcerative colitis_No Inflammation."

[0079] As shown in Figures 4 to 6, the expression levels of WARS, CD274, FBXO6, TAP2, UBE2L6, and ZNFX1 fluctuated in samples from human IBD patients without macroscopic signs of inflammation.

[0080] In addition, the 27 genes detected by the DNB analysis in Test Example 2 were scored using network control theory. Scoring was performed using the following procedure. From the expression level data of all genes analyzed, only the expression level data of the DNB gene on day 3 was extracted. The eigenvector for the maximum eigenvalue of the covariance matrix of that data was calculated. The absolute value of each element of the eigenvector was taken, and each absolute value was divided by the maximum value. This value was then multiplied by the proportion of variation related to the maximum eigenvalue that explains the variance of the entire data. The results are shown in Table 2.

[0081] [Table 2]

[0082] Of the genes shown in Table 2, Mcpt1 and Mcpt4 are not present in humans, Ifit2, Cd274, and Tnfsf10 have been detected in conventional analyses using the average gene expression level as an indicator, while Trim30d, Ccn1, Plppr3, 4933412E12Rik, H2-T23, H2-M2, H2-Bl, H2-Q2, Gm11127, H2-K2, H2-Q8, H2-Q7, H2-Q6, and H2-D1 are not present in the above IBD data. Therefore, the present inventors focused on Wars among the genes shown in Table 2 and investigated whether it is a pathophysiologically significant gene in IBD below. Wars is also present in humans, but was not detected in conventional analyses using the average gene expression level as an indicator. It was first detected by DNB analysis and was a highly ranked gene.

[0083] [Test Example 4: Evaluation of Wars gene expression in colitis model mice] The expression of the genes listed in Table 3 below, particularly the Wars gene, in colitis model mice was analyzed by real-time PCR using the following procedure.

[0084] A mouse model of DSS-induced colitis was prepared as in Test Example 1. Colonic tissues from the model mice were collected daily at 1, 3, 5, and 7 days after the start of DSS drinking. RNA was extracted from the colonic tissue using Sepasol RNA I Super G (Nacalai Tesque) according to the manufacturer's protocol, and the RNA concentration and quality were measured using Nano Drop (ND-1000; Thermo Fisher Scientific). Reverse transcription was performed to synthesize cDNA from the extracted RNA using PrimeScript RT reagent Kit (Takara Bio).

[0085] Real-time PCR was performed using the synthesized cDNA as a template and TB Green Premix Ex Tag (manufactured by Takara Bio Inc.), and the expression levels of the target genes were measured. The expression levels of each target gene were normalized with the expression level of the TBP (TATA binding protein) gene, which serves as an internal standard, and the expression levels were measured using 2 -ΔΔCtThe expression of the Wars gene in the colitis model mice was analyzed by real-time PCR. The primers used to amplify the target gene and TBP gene were those shown in Table 3. As a control, normal BALB / c mice were also analyzed for expression of the genes listed in Table 3 below by real-time PCR. The analysis results of the expression of the Wars gene in the colitis model mice are shown in Figure 7.

[0086] [Table 3]

[0087] As shown in Figure 7, Wars gene expression in colitis model mice was significantly higher than that in normal mice 5 days after the start of DSS drinking. Furthermore, Wars gene expression in colitis model mice did not show a significant increase compared to normal mice 3 days after the start of DSS drinking, and variation in expression level was observed.

[0088] [Test Example 5: Analysis of Wars gene expression in the colonic mucosa of normal mice] Histological analysis of Wars gene expression in the colonic mucosa of normal mice was performed using RNAscope (registered trademark) according to the following procedure.

[0089] RNAscope® was performed using frozen sections of normal mouse colonic mucosa. Colonic tissue excised from mice was embedded in Tissue-Tec® OCT compound (Sakura Finetech). 10 μm-thick frozen sections were prepared at -20°C using a cryostat (Leica Biosystems) and attached to glass slides (Matsunami). In situ hybridization was then performed using the RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics) according to the manufacturer's protocol. Mouse Wars-C1 probe (Advanced Cell Diagnostics) was used. Samples were observed and images were captured using a confocal laser microscope (LSM900Airyscan2) (ZEISS). The results are shown in Figure 8.

[0090] As shown in FIG. 8, expression of the Wars gene was confirmed in the colonic mucosa of normal mice.

[0091] Test Example 6: Evaluation of the effects of WRS recombinant protein and anti-WRS antibody on colitis pathology To elucidate the role of Wars (protein name: WRS) in the pathogenesis of IBD, we used a DSS-induced colitis model mouse and evaluated the effects of WRS recombinant protein and anti-WRS antibody on the development of colitis using the following procedure.

[0092] WRS recombinant protein (rWRS; CLOUD-CLONE) was dissolved in saline and administered intraperitoneally at 20 μg per mouse once daily on days 0, 2, 4, and 6 after the initiation of DSS drinking. Vehicle (saline) was administered in the same manner as a control. Anti-WRS antibody (Anti-WRS Ab) was generated using Cosmo Bio's first antibody and administered intraperitoneally at 150 μg per mouse once daily on days 0 and 2 after the initiation of DSS drinking. A control IgG antibody (rabbit IgG control (R&D Systems; catalog number: AB-105-C)) was also administered as a control. DAI was calculated on days 0, 1, 2, 3, 4, 5, 6, and 7 after the initiation of DSS drinking, as in Test Example 1, and colitis symptoms were evaluated. These results are shown in Figures 9 and 10.

[0093] As shown in Figure 9, administration of WRS recombinant protein to colitis model mice significantly reduced the DAI on days 6 and 7 compared to controls. In other words, administration of WRS recombinant protein significantly suppressed the onset of colitis in colitis model mice. Furthermore, as shown in Figure 10, administration of anti-WRS antibody to colitis model mice increased the DAI from days 1 to 7 compared to controls, with the DAI being significantly higher on days 5, 6, and 7. In other words, administration of anti-WRS antibody exacerbated the onset of colitis in colitis model mice. These results suggest that WRS / WRS plays a protective role in IBD pathology.

[0094] Test Example 7: Histological evaluation of the effect of WRS recombinant protein on colitis pathology The effect of WRS recombinant protein on the pathology of colitis was evaluated histologically using hematoxylin and eosin staining according to the following procedure.

[0095] A mouse model of DSS-induced colitis was generated by administering WRS recombinant protein as described in Test Example 6. A control mouse model of DSS-induced colitis was also generated in the same manner, except that vehicle (physiological saline) was used instead of WRS recombinant protein. Colonic tissues were excised from mice 7 days after the start of DSS drinking, fixed in 4% PFA for 24 hours, washed with 0.01 M PBS (20 min each, three times), and stored at 4°C in PBS containing 30% sucrose and 0.1% sodium azide. The fixed colonic tissues were embedded in Tissue-Tek® OCT compound and cryostat-treated to prepare 10 μm-thick frozen sections at -20°C. The sections were then fixed on glass slides, dried for 1 hour, washed in running water for 5 minutes, and stained with hematoxylin solution (Merck) for 7 minutes. After washing with running water (1 hour), the specimens were stained with eosin solution (Wako) for 2 minutes. The stained specimens were dehydrated and mounted using Marinol (MUTO PURE CHEMICALS). The specimens were observed using an all-in-one fluorescence microscope (BZ-X700; KEYENCE) and images were captured. Similar procedures were performed on normal male BALB / c mice, and histological evaluation was performed using hematoxylin and eosin staining. The results are shown in Figure 11.

[0096] As shown in Figure 11, disruption of the colonic mucosal structure was observed in the colonic tissue of DSS-induced colitis model mice administered with saline compared to that of normal mice. On the other hand, disruption of the colonic mucosal structure was improved in the colonic tissue of DSS-induced colitis model mice administered with WRS recombinant protein compared to that of DSS-induced colitis model mice administered with saline. Histological analysis also suggested that WRS recombinant protein significantly suppressed the onset of colitis in the colitis model mice.

[0097] [Test Example 8: Analysis of the effect of WRS recombinant protein and anti-WRS antibody on DNB gene expression] The effects of WRS recombinant protein and anti-WRS antibody on DNB gene expression were analyzed by real-time PCR using the following procedure.

[0098] DSS-induced colitis model mice were generated by administering WRS recombinant protein or anti-WRS antibody as described in Test Example 6. Three days after the start of DSS drinking, real-time PCR was performed as described in Test Example 4 to analyze the expression levels of B2m, Bst2, Ccn1, Cd274, Cxcl16, Fbxo6, H2-D1, H2-k2, H2-m2, H2-q2, H2-q6, H2-q7, H2-t23, Ifit2, Mcpt1, Mcpt4, Tap2, Trim30d, Ube2l6, Wars, and Znfx1. Primer combinations for each target gene listed in Table 3 were used for real-time PCR. The results are shown in Figures 12 to 17.

[0099] As shown in Figures 12 to 17, administration of WRS recombinant protein to colitis model mice suppressed fluctuations in the expression of Wars, as well as B2m, Cxcl16, and Ube2l6 in the pre-disease state (3 days after the start of DSS drinking).

[0100] The results of Test Examples 1 to 8 above suggest that Wars / WRS itself or substances that promote the expression and production of Wars / WRS may be useful novel therapeutic agents for medical intervention at the very early stages of IBD onset and relapse. Furthermore, it was suggested that Wars / WRS may be a biomarker for predicting pre-onset and pre-relapse states.

Claims

1. A pharmaceutical for treating, preventing recurrence of, or maintaining remission of inflammatory bowel disease, comprising a tryptophanyl-tRNA synthetase-like protein as an active ingredient.

2. The pharmaceutical according to claim 1 , wherein the protein is of human origin.

3. The pharmaceutical composition according to claim 1 or 2, which is administered to a subject predicted to experience a relapse of inflammatory bowel disease.

4. The pharmaceutical composition of claim 3, wherein the exacerbation of inflammatory bowel disease is predicted by measuring the expression level of one or more proteins selected from tryptophanyl-tRNA synthetase, leucine-rich α2 glycoprotein, C-reactive protein, and calprotectin, or the amount of mRNA encoding said one or more proteins.

5. A diagnostic biomarker for inflammatory bowel disease, comprising human tryptophanyl-tRNA synthetase or mRNA encoding said enzyme.

6. measuring the amount of tryptophanyl-tRNA synthetase or the amount of mRNA encoding said enzyme contained in a sample derived from a test subject; and determining that the subject may be suffering from inflammatory bowel disease or is at high risk of suffering from inflammatory bowel disease if the amount of the enzyme is higher than the standard value of the enzyme amount or if the amount of the mRNA is higher than the standard value of the mRNA amount.

7. The method of claim 6, wherein the sample is colon tissue or a sample prepared from the tissue.

8. The method according to claim 6 or 7, wherein the reference value of the enzyme is the amount of tryptophanyl-tRNA synthetase contained in a sample derived from a healthy control, and the reference value of the mRNA amount is the amount of mRNA encoding the enzyme contained in a sample derived from a healthy control.

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