New technologies for the diagnosis, treatment, and screening of therapeutic agents for aortic aneurysms

Abstract

To provide a new technique for diagnosis of aortic aneurysm, a therapeutic agent, and screening of therapeutic agents.SOLUTION: A data acquisition method for diagnosing aortic aneurysm includes a step of detecting one or more selected from a group consisting of nucleoredoxin and Dishevelled in a sample collected from a subject. A diagnostic kit for aortic aneurysm includes a member for detecting one or more selected from a group consisting of nucleoredoxin and Dishevelled. A method of screening therapeutic agents for aortic aneurysm includes the steps of: bringing cultured cells capable of expressing one or more selected from a group consisting of nucleoredoxin and Dishevelled, into contact with a candidate substance of a therapeutic agent; and comparing one or more expressions selected from a group consisting of nucleoredoxin and Dishevelled in the cultured cells, before and after bringing the cultured cells into contact with the candidate substance.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a new technology for diagnosing aortic aneurysms, screening for therapeutic agents, and screening for therapeutic agents. More specifically, the present invention relates to a technology that utilizes nucleoredoxin and Dishevelled for diagnosing aortic aneurysms, screening for therapeutic agents, and screening for therapeutic agents for aortic aneurysms. [Background technology]

[0002] An aortic aneurysm can be defined as a circumferential or localized enlargement or protrusion of a portion of the aortic wall. Aortic aneurysms can be classified by their location of occurrence, for example, those occurring in the chest are called thoracic aortic aneurysms (TAA), those occurring in the chest and abdomen are called thoracoabdominal aortic aneurysms (TAAA), and those occurring in the abdomen are called abdominal aortic aneurysms (AAA).

[0003] Aortic aneurysms often progress asymptomatically and are only discovered when the blood vessels dissect and / or rupture. Therefore, there is growing interest in the development of diagnostic methods for aortic aneurysms, as well as therapeutic agents for aortic aneurysms.

[0004] The development of therapeutic agents for aortic aneurysms and diagnostic methods for aortic aneurysms requires a deeper understanding of the clinical risk factors for aortic aneurysms and the molecules involved in the formation of aortic aneurysms.

[0005] Clinical risk factors for aortic aneurysms include smoking, respiratory disorders, aging, diabetes, hyperlipidemia, and hypertension, while molecules involved in the formation of aortic aneurysms include osteoprotegerin (OPG) and matrix metalloproteinases (MMPs).

[0006] It is also known that sclerostin, which inhibits the Wnt signaling pathway, suppresses angiotensin II-induced aortic aneurysms and atherosclerosis (see, for example, Non-Patent Document 1). Furthermore, it is known that hypoxia is associated with atherosclerosis (see, for example, Non-Patent Document 2).

[0007] Based on these findings, the development of therapeutic agents for aortic aneurysms and diagnostic methods for aortic aneurysms is currently underway. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Smriti et al., Arteriosclerosis Thrombosis and Vascular Biology, Volume 37, Issue 3, March 2017, Pages 553-566, “Wnt Signaling Pathway Inhibitor Sclerostin Inhibits Angiotensin II-Induced Aortic Aneurysm and Atherosclerosis” [Non-patent document 2] Xingyu et al., Circulation Cardiovascular Imaging, Volume 13, Issue 1, e009791, January 8, 2020, “64Cu-ATSM Positron Emission Tomography / Magnetic Resonance Imaging of Hypoxia in Human Atherosclerosis” Summary of the Invention [Problem to be solved by the invention]

[0009] However, conventional therapeutic agents for aortic aneurysms and diagnostic methods for aortic aneurysms are not sufficient, and there is a demand for the development of new therapeutic agents and diagnostic methods.

[0010] Therefore, one aspect of the present invention aims to realize a new technique for diagnosing aortic aneurysms, developing a therapeutic agent, and screening for the therapeutic agent. [Means for solving the problem]

[0011] The present inventors have newly discovered that nucleoredoxin and Dishevelled are associated with aortic aneurysms (e.g., atherosclerotic plaques present in aortic aneurysms), and have completed the present invention. That is, one aspect of the present invention is as follows.

[0012] [1] A method for obtaining data for diagnosing aortic aneurysm, comprising the step of detecting one or more substances selected from the group consisting of nucleoredoxin and Dishevelled in a sample collected from a subject.

[0013] [2] A diagnostic kit for aortic aneurysm, comprising a component for detecting one or more selected from the group consisting of nucleoredoxin and Dishevelled.

[0014] [3] A method for screening a therapeutic agent for aortic aneurysm, comprising the steps of: contacting cultured cells capable of expressing one or more selected from the group consisting of nucleoredoxin and Dishevelled with a candidate therapeutic agent; and comparing the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in the cultured cells before and after contacting the cultured cells with the candidate agent.

[0015] [4] A therapeutic agent for aortic aneurysm, comprising a nucleoredoxin expression inducer or a Dishevelled expression inhibitor as an active ingredient.

[0016] [5] The therapeutic agent for aortic aneurysm according to [4], wherein the nucleoredoxin expression inducer is tBHQ, SFN, or CDDO.

[0017] [6] The therapeutic agent for aortic aneurysm according to [4], wherein the inhibitor of Dishevelled expression is siRNA. [Effects of the Invention]

[0018] According to one aspect of the present invention, a new technique for diagnosing aortic aneurysms, treating a patient with a disease, and screening for a treatment for a disease can be realized. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 shows test results of 64Cu-ATSM PET / MRI in an example of the present invention. [Figure 2] FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. [Figure 3] FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. [Figure 4] FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. [Figure 5] FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. [Figure 6] FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. [Figure 7] FIG. 1 shows changes in the expression levels of various molecules due to oxidative stress in an example of the present invention. [Figure 8] FIG. 1 shows the effect of a decrease in the expression level of NRX on the expression levels of other molecules in an example of the present invention. [Figure 9] FIG. 1 shows the effect of a decrease in the expression level of NRX on the expression levels of other molecules in an example of the present invention. [Figure 10]FIG. 1 shows the results of screening for nucleoredoxin expression inducers in an example of the present invention. [Figure 11] FIG. 1 shows the therapeutic effect of a nucleoredoxin expression inducer on aortic aneurysm in an example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0020] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

[0021] [1. Method of acquiring data for diagnosing aortic aneurysms] A method for obtaining data for diagnosing an aortic aneurysm according to one embodiment of the present invention includes detecting one or more selected from the group consisting of nucleoredoxin and Dishevelled in a sample collected from a subject.

[0022] If a decrease in nucleoredoxin expression is detected in the above steps, it can be determined (Decision 1) that an aortic aneurysm will form in the future, an aortic aneurysm is forming, an aortic aneurysm has already formed, an aortic aneurysm may form in the future, an aortic aneurysm may be forming, or an aortic aneurysm may already have formed.

[0023] The degree of decrease in nucleoredoxin expression is not limited. The expression level of nucleoredoxin in a comparison sample (e.g., normal tissue in a subject) is represented by A, and the expression level of nucleoredoxin in a sample collected from the subject (e.g., tissue suspected of being abnormal in a subject) is represented by B. For example, when "A>B," "0.9×A≧B," "0.8×A≧B," "0.7×A≧B," "0.6×A≧B," "0.5×A≧B," "0.4×A≧B," "0.3×A≧B," "0.2×A≧B," "0.1×A≧B," or "0.01×A≧B," the above-mentioned determination 1 can be made.

[0024] If an increase in the expression of Dishevelled is detected in the above steps, it can be determined (Decision 2) that an aortic aneurysm will form in the future, an aortic aneurysm is forming, an aortic aneurysm has already formed, an aortic aneurysm may form in the future, an aortic aneurysm may be forming, or an aortic aneurysm may already have formed.

[0025] The degree of increase in Dishevelled expression is not limited. The expression level of Dishevelled in a comparison sample (e.g., normal tissue in a subject) is defined as C, and the expression level of Dishevelled in a sample collected from the subject (e.g., tissue suspected of being abnormal in a subject) is defined as D. For example, if "D>C," "D≧1.1×C," "D≧1.2×C," "D≧1.3×C," "D≧1.4×C," "D≧1.5×C," "D≧1.6×C," "D≧1.7×C," "D≧1.8×C," "D≧1.9×C," "D≧2.0×C," "D≧5.0×C," "D≧10×C," or "D≧100×C," a determination can be made as in Determination 2 above.

[0026] In the above steps, (i) the mRNA of nucleoredoxin and Dishevelled may be detected, (ii) the protein of nucleoredoxin and Dishevelled may be detected, or (iii) both the mRNA and protein of nucleoredoxin and Dishevelled may be detected.

[0027] The process of translation from mRNA to protein can be negatively and / or positively regulated. Therefore, in the above process, it is preferable to detect nucleoredoxin and Dishevelled proteins, which are thought to be the final products and functional entities. This configuration can provide better data for diagnosing aortic aneurysms.

[0028] Methods for detecting nucleoredoxin and Dishevelled mRNA include, but are not limited to, in situ hybridization, PCR, Northern blotting, and microarray analysis. These methods are well known. Therefore, when detecting Dishevelled mRNA using these methods, it is sufficient to follow well-known methods.

[0029] Methods for detecting nucleoredoxin and Dishevelled proteins are not limited, and include, for example, immunohistochemical staining, immunohistochemical staining, Western blotting, and ELISA. These methods are well known. Therefore, when detecting Dishevelled proteins using these methods, well-known methods should be used.

[0030] The types of aortic aneurysms are not limited, and examples thereof include thoracic aortic aneurysms, thoracoabdominal aortic aneurysms, and abdominal aortic aneurysms.

[0031] The subject may include, but is not limited to, humans and non-human animals (e.g., livestock, pets, and laboratory animals), such as monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0032] The sample collected from the subject is not particularly limited, and may be, for example, a sample from the thoracic aorta, thoracoabdominal aorta, or the like of the subject. pulse, and samples taken from the abdominal aorta (eg, tissue strips from these aortas).

[0033] The method for collecting the sample from the subject is not limited, and any known method can be used.

[0034] [2. Aortic aneurysm diagnostic kit] A diagnostic kit for aortic aneurysm according to one embodiment of the present invention includes a member for detecting one or more selected from the group consisting of nucleoredoxin and Dishevelled.

[0035] The configuration explained in the above section (1. Method for acquiring data for diagnosing aortic aneurysm) will not be explained again.

[0036] The diagnostic kit for aortic aneurysm according to one embodiment of the present invention may include a component for detecting nucleoredoxin or Dishevelled mRNA, or a component for detecting nucleoredoxin or Dishevelled protein.

[0037] The member for detecting nucleoredoxin or Dishevelled mRNA may be, for example, a member for detecting nucleoredoxin or Dishevelled mRNA according to in situ hybridization, PCR, Northern blotting, or microarray techniques.

[0038] Examples of components for detecting nucleoredoxin or Dishevelled mRNA include probes that hybridize to nucleoredoxin mRNA or Dishevelled mRNA (e.g., polynucleotides that hybridize to mRNA), and primers for detecting nucleoredoxin mRNA or Dishevelled mRNA by PCR.

[0039] The length of the probe and primer is not limited as long as it can specifically detect nucleoredoxin or Dishevelled mRNA. The base sequence of the probe and primer is not limited as long as it can specifically hybridize to any site of nucleoredoxin or Dishevelled mRNA.

[0040] The member for detecting nucleoredoxin or Dishevelled protein may be a member for detecting nucleoredoxin or Dishevelled protein according to, for example, immunostaining, immunohistochemical staining, Western blotting, or ELISA.

[0041] Examples of a member for detecting nucleoredoxin or Dishevelled protein include antibodies that bind to nucleoredoxin or Dishevelled protein.

[0042] The antibody may be a polyclonal antibody or a monoclonal antibody.

[0043] The above antibodies can be produced according to well-known methods (see, for example, [Harlow (Ed.), "Antibodies: a laboratory manual", New York: Cold Spring Harbor Laboratory, 1988] and [Iwasaki Tatsuo et al., "Monoclonal Antibodies: Hybridomas and ELISA", Kodansha, 1991]).

[0044] Monoclonal antibodies can be produced according to methods well known in the art. Examples of methods for producing monoclonal antibodies include: (1) the hybridoma method (see, for example, Koehler G & Milstein C (1975) "Continuous cultures of fused cells secreting antibody of predefined specificity", Nature, Vol. 256 (No. 5517), pp. 447-518); (2) the trioma method; (3) the human B cell hybridoma method (see, for example, Kozbor D & Roder JC (1983) "The production of monoclonal antibodies from human lymphocytes", Immunology Today, Vol. 4 (Issue 3), pp. 72-79); and (4) the EBV-hybridoma method (see, for example, Cole SPC et al., "The EBV-hybridoma technique and its application to human lung cancer", In: Reisfeld RA & Sell S (Eds.), "Monoclonal antibodies and cancer therapy", New York: Alan R. Liss, Inc., 1985, pp.77-96 (UCLA Symposia on Molecular and Cellular Biology, Vol.27)].

[0045] 3. Screening method for therapeutic agents for aortic aneurysms A method for screening therapeutic agents for aortic aneurysms according to one embodiment of the present invention comprises the steps of contacting cultured cells capable of expressing one or more selected from the group consisting of nucleoredoxin and Dishevelled with a candidate therapeutic agent (also referred to as the first step), and comparing the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in the cultured cells before and after contacting the cultured cells with the candidate agent (also referred to as the second step).

[0046] The configurations described above in [1. Method for acquiring data for diagnosing aortic aneurysm] and [2. Diagnostic kit for aortic aneurysm] will not be described again.

[0047] [3-1. First step] The first step is a step of contacting a candidate substance for a therapeutic agent with cultured cells capable of expressing one or more selected from the group consisting of nucleoredoxin and Dishevelled.

[0048] The cultured cells capable of expressing any one or more selected from the group consisting of nucleoredoxin and Dishevelled are not limited, and examples thereof include aortic smooth muscle cells.

[0049] The candidate substances for the therapeutic agent are not limited, and examples thereof include low molecular weight compounds, high molecular weight compounds, proteins, nucleic acids, and sugars.

[0050] In the first step, for example, a candidate substance for a therapeutic agent may be added to a culture medium to a desired concentration, and the cultured cells may be cultured in the culture medium for a desired period of time (e.g., 1 hour to 100 hours, 1 hour to 72 hours, or 1 hour to 24 hours) to bring the cultured cells into contact with the candidate substance.

[0051] [3-2. Second step] The second step is a step of comparing the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in the cultured cells before and after contacting the cultured cells with a candidate substance.

[0052] In the second step, for example, cultured cells I are prepared, a portion of the cultured cells I is collected before contact with the candidate substance, and the remaining cultured cells I are contacted with the candidate substance. Next, the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in the previously collected cultured cells I may be compared with the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in the cultured cells I contacted with the candidate substance.

[0053] In the second step, for example, cultured cells I that are contacted with the candidate substance and cultured cells II that are not contacted with the candidate substance may be separately prepared, and the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in cultured cells I may be compared with the expression of one or more selected from the group consisting of nucleoredoxin and Dishevelled in cultured cells II.

[0054] In the second step, the expression level of nucleoredoxin in the cultured cells before contacting the cultured cells with the candidate substance is designated as A', and the expression level of nucleoredoxin in the cultured cells after contacting the cultured cells with the candidate substance is designated as B'. For example, if "B'>A'," "B'≧1.1×A'," "B'≧1.2×A'," "B'≧1.3×A'," "B'≧1.4×A'," "B'≧1.5×A'," "B'≧1.6×A'," "B'≧1.7×A'," "B'≧1.8×A'," "B'≧1.9×A'," "B'≧2.0×A'," "B'≧5.0×A'," "B'≧10×A'," or "B'≧100×A'" holds, the candidate substance can be determined to be a therapeutic agent for aortic aneurysm.

[0055] In the second step, the expression level of Dishevelled in the cultured cells before contacting the cultured cells with the candidate substance is designated as C', and the expression level of Dishevelled in the cultured cells after contacting the cultured cells with the candidate substance is designated as D'. For example, if the relationship is "C'>D'," "0.9 x C'≧D'," "0.8 x C'≧D'," "0.7 x C'≧D'," "0.6 x C'≧D'," "0.5 x C'≧D'," "0.4 x C'≧D'," "0.3 x C'≧D'," "0.2 x C'≧D'," "0.1 x C'≧D'," or "0.01 x C'≧D'," the candidate substance can be determined to be a potential therapeutic agent for aortic aneurysms.

[0056] 4. Treatment for aortic aneurysms A therapeutic agent for aortic aneurysm according to one embodiment of the present invention contains a nucleoredoxin expression inducer or a Dishevelled expression inhibitor as an active ingredient. A therapeutic agent for aortic aneurysm according to one embodiment of the present invention may contain both a nucleoredoxin expression inducer and a Dishevelled expression inhibitor as active ingredients.

[0057] The configurations described above in [1. Method for acquiring data for diagnosing aortic aneurysms], [2. Diagnostic kit for aortic aneurysms], and [3. Method for screening therapeutic agents for aortic aneurysms] will not be described here.

[0058] The nucleoredoxin expression inducer may be any agent capable of increasing the expression level of nucleoredoxin protein, and may be, for example, (i) an agent that induces the transcription of nucleoredoxin from DNA to mRNA, (ii) an agent that induces the translation of nucleoredoxin from mRNA to protein, or (iii) an agent that inhibits the degradation of nucleoredoxin mRNA and / or protein.

[0059] The Dishevelled expression inhibitor may be any agent capable of reducing the expression level of Dishevelled protein, and may be, for example, (i) an agent that inhibits the transcription of Dishevelled from DNA to mRNA, (ii) an agent that inhibits the translation of Dishevelled from mRNA to protein, or (iii) an agent that induces the degradation of Dishevelled mRNA and / or protein.

[0060] The nucleoredoxin expression inducer and Dishevelled expression inhibitor are not limited, and may be, for example, a low molecular weight compound, a high molecular weight compound, a protein, a nucleic acid, or a sugar.

[0061] The nucleoredoxin expression inducer is not limited to, but includes, for example, tBHQ (tert-butylhydroxyquinone), SFN (sulforaphane), and CDDO (2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid).

[0062] The Dishevelled expression inhibitor is not limited to, but may be, for example, siRNA.

[0063] The amount of the active ingredient contained in the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not particularly limited, and may be, for example, 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.001% by mass to 100% by mass, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, 0.1% by mass to 95% by mass, 0.1% by mass to 90% by mass, 0.1% by mass to 80% by mass, 0.1% by mass to 70% by mass, 0.1% by mass to 60% by mass, 0.1% by mass to 50% by mass, 0.1% by mass to 40% by mass, 0.1% by mass to 30% by mass, 0.1% by mass to 20% by mass, or 0.1% by mass to 10% by mass, assuming the therapeutic agent to be 100% by mass.

[0064] The therapeutic agent for aortic aneurysm according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0065] The ingredients other than the active ingredient are not particularly limited and may be, for example, a buffering agent, a pH adjusting agent, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, an antibacterial agent, or the like.

[0066] Examples of the buffering agent include phosphoric acid or phosphate salts, boric acid or borates, citric acid or citrate salts, acetic acid or acetate salts, carbonic acid or carbonate salts, tartaric acid or tartrate salts, ε-aminocaproic acid, and trometamol. Examples of the phosphate salts include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borates include borax, sodium borate, and potassium borate. Examples of the citrate salts include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate salts include sodium acetate and potassium acetate. Examples of the carbonate salts include sodium carbonate and sodium bicarbonate. Examples of the tartrate salts include sodium tartrate and potassium tartrate.

[0067] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0068] Examples of the isotonic agent include ionic isotonic agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonic agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0069] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0070] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0071] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0072] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0073] Examples of the solvent include water, physiological saline, and alcohol.

[0074] Examples of the antibacterial agents include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and pyridonecarboxylic acid (quinolone), new quinolone, oxazolidinone, and sulfonamide synthetic antibacterial agents.

[0075] The amount of ingredients other than the active ingredient contained in the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not particularly limited, and may be, for example, 0% by mass to 99.99999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 0% by mass to 99.99% by mass, or 5% by mass to 99.9% by mass. The content may be 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0076] The dosage form of the therapeutic agent for aortic aneurysm according to one embodiment of the present invention is not limited, and examples thereof include tablets, capsules, liquid contents, external preparations, suppositories, injections, and inhalants.

[0077] The route of administration of the therapeutic agent for aortic aneurysm according to one embodiment of the present invention is not limited, and examples thereof include parenteral administration, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intranasal administration, epidural administration, oral administration, sublingual administration, intranasal administration, transdermal administration, rectal administration, inhalation, and topical administration.

[0078] The administration interval of the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not limited, and can be, for example, once every hour to six months. More specifically, examples include once every hour, once every two hours, once every three hours, once every six hours, once every 12 hours, once every day, once every two days, once every three days, once every four days, once every five days, once every six days, once every week, once every two weeks, once every three weeks, once every month, once every two months, once every three months, once every four months, once every five months, and once every six months.

[0079] The subjects to which the therapeutic agent for aortic aneurysm according to one embodiment of the present invention is administered are not limited, and examples thereof include humans and non-human animals (e.g., livestock, pets, and laboratory animals). Examples of non-human animals include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0080] [5. Other] The present invention can also be configured as follows.

[0081] <1> A method for diagnosing aortic aneurysm, comprising the step of detecting one or more selected from the group consisting of nucleoredoxin and Dishevelled in a sample collected from a subject.

[0082] <2> A method for treating aortic aneurysm, comprising the step of administering to a subject an agent for treating aortic aneurysm, the agent comprising, as an active ingredient, a nucleoredoxin expression inducer or a Dishevelled expression inhibitor.

[0083] <3> The nucleoredoxin expression inducer is tBHQ, SFN, or CDDO. <2> The method for treating an aortic aneurysm described above.

[0084] <4> The Dishevelled expression inhibitor is an siRNA. <2> The method for treating an aortic aneurysm described above. [Example]

[0085] <1. 64 Cu-ATSM PET / MRI Basic data of two patients (patients 1 and 2) scheduled for surgery for aortic aneurysms were obtained according to well-known methods.

[0086] Specifically, the patient's "age," "gender," "type of aortic aneurysm," "Body Mass Index," "Brinkman Index," "alcohol," "hypertension," "diabetes," "dyslipidemia," and 64Cu-ATSM PET accumulation data were obtained according to known methods.

[0087] especially," 64 Data on "Cu-ATSM PET accumulation" are available from the 64Cu-ATSM PET / MRI technique (e.g., Xingyu et al., Circulation Cardiovascular Imaging, Volume 13, Issue 1, e009791, January 8, 2020, " 64 The images were acquired using Cu-ATSM Positron Emission Tomography / Magnetic Resonance Imaging of Hypoxia in Human Atherosclerosis (see “Cu-ATSM Positron Emission Tomography / Magnetic Resonance Imaging of Hypoxia in Human Atherosclerosis”).

[0088] The table below lists the basic data of two patients. In the "Alcohol" section of the table below, if the patient has a drinking habit, it is marked with a "+", and if the patient does not have a drinking habit, it is marked with a "-". In the "High Blood Pressure" section of the table below, if the patient has high blood pressure, it is marked with a "+", and if the patient does not have high blood pressure, it is marked with a "-". In the "Diabetes" section of the table below, if the patient has diabetes, it is marked with a "+", and if the patient does not have diabetes, it is marked with a "-". In the "Dyslipidemia" section of the table below, if the patient has dyslipidemia, it is marked with a "+", and if the patient does not have dyslipidemia, it is marked with a "-". In the " 64 Cu-ATSM PET accumulation in the patient's body 64 If Cu-ATSM PET accumulation was observed, it was recorded as "+" and the patient's body 64 Cases where no accumulation of Cu-ATSM PET was observed are recorded as "-".

[0089] [Table 1]

[0090] 64Cu-ATSM PET is known to accumulate in tissues in the body, particularly in tissues under oxidative stress. In one patient (patient number 1), the accumulation was higher than in the other patient (patient number 2). 64 A large amount of Cu-ATSM PET accumulation was observed.

[0091] 64 A more detailed observation of the tissues in which Cu-ATSM PET was accumulated revealed that it was present in the arterial wall of the thoracic aortic aneurysm (particularly in the atheromatous plaques and areas adjacent to the atheromatous plaques in the arterial wall of the thoracic aortic aneurysm). 64 The accumulation of Cu-ATSM PET was significant (see 101 in Figure 1).

[0092] Also, 64 Cu-ATSM PET accumulation was observed in the arterial wall of thoracic aortic aneurysms. 64 Compared with the arterial wall of a thoracic aortic aneurysm in which no accumulation of Cu-ATSM PET was observed, a significantly greater number of atheromas (atherosclerotic plaques) were observed (see 102 in Figure 1).

[0093] In 102 of FIG. 1, "Case 1" indicates patient number 1, and "Case 2" indicates patient number 2. In 102 of FIG. 1, "n" indicates the number of samples of arterial wall of observed thoracic aortic aneurysm. In 102 of FIG. 1, "Atheromatous plaque area (%)" indicates the ratio of the area of ​​atheromatous plaque to the area of ​​the arterial wall of observed thoracic aortic aneurysm.

[0094] 2. Expression of various molecules in the vicinity of atherosclerotic plaques 64 The arterial wall of a thoracic aortic aneurysm showed no accumulation of Cu-ATSM PET. 64 The arterial walls of thoracic aortic aneurysms, where accumulation of Cu-ATSM PET was observed, were immunostained and fluorescently stained using various molecules as indicators, and changes in the expression of these molecules in and around the atherosclerotic plaque were observed.

[0095] The molecules used were 4-hydroxyxynonenal (4-HNE), nucleoredoxin (NRX), β-catenin, disheveled (Dvl), osteoprotegerin (OPG), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-7 (MMP-7), and matrix metalloproteinase-9 (MMP-9). 4-HNE is an aldehyde produced from unsaturated fatty acids (e.g., arachidonic acid) due to oxidative stress in vivo. Therefore, 4-HNE is an indicator of oxidative stress.

[0096] The immunostaining and fluorescent staining were performed using commercially available staining kits. The specific staining method followed the protocol provided with the kit. For the molecules listed above, except for 4-HNE, the protein expression levels were detected by immunostaining and fluorescent staining.

[0097] 2 to 6 show stained images of immunostaining and fluorescent staining.

[0098] As shown in Figure 2, (i) 4-HNE production is high within and near atherosclerotic plaques (in other words, oxidative stress is high within and near atherosclerotic plaques), (ii) NRX expression is low near atherosclerotic plaques, and (iii) β-catenin expression is high near atherosclerotic plaques.

[0099] As shown in Figure 3, (i) the expression level of NRX was low near atherosclerotic plaques, (ii) the expression level of Dvl was high near atherosclerotic plaques, and (iii) the expression level of β-catenin was high near atherosclerotic plaques.

[0100] As shown in FIG. 4, it was revealed that the region with low NRX expression and the region with high β-catenin expression were the same region near the atherosclerotic plaque.

[0101] As shown in Figure 5, it was revealed that (i) the expression level of OPG was high near atherosclerotic plaques, (ii) the expression level of MMP-2 was high near atherosclerotic plaques, (iii) the expression level of MMP-7 was high near atherosclerotic plaques, and (iv) the expression level of MMP-9 was high near atherosclerotic plaques.

[0102] As shown in FIG. 6, it was revealed that the region in the vicinity of the atherosclerotic plaque where the expression level of MMP-2 was high and the region in which the expression level of β-catenin was high were the same region.

[0103] <3. Changes in the expression levels of various molecules due to oxidative stress> H2O2 was added to the culture medium in which human aortic smooth muscle cells were cultured to a predetermined concentration (0 μM, 10 μM, or 50 μM), and the human aortic smooth muscle cells were then cultured for a predetermined period (3 hours or 8 hours). Note that H2O2 exerts oxidative stress on the cells.

[0104] After the culture, the human aortic smooth muscle cells were collected, and the amounts of NRX, β-catenin, and β-actin proteins expressed by the human aortic smooth muscle cells were quantified by the well-known Western blotting method.

[0105] Figure 7 shows the test results.

[0106] Figure 7, 701, shows the results of Western blotting to detect NRX, β-catenin, and β-actin proteins. The expression level of NRX decreased as the concentration of H2O2 in the culture medium increased, and also decreased as the culture time of human aortic smooth muscle cells increased. On the other hand, the expression level of β-catenin increased as the concentration of H2O2 in the culture medium increased, and also increased as the culture time of human aortic smooth muscle cells increased. The expression level of β-actin did not change even when the culture conditions of human aortic smooth muscle cells were changed.

[0107] The ratio of the expression level of NRX to the expression level of β-actin is shown in 702 of Figure 7. It can also be seen from 702 of Figure 7 that the expression level of NRX decreases as the concentration of HO in the culture medium increases, and also as the culture time of human aortic smooth muscle cells increases.

[0108] The ratio of the expression level of β-catenin to the expression level of β-actin is shown in 703 of Figure 7. It can also be seen from 703 of Figure 7 that the expression level of β-actin increases as the concentration of HO in the culture medium increases, and also as the culture time of human aortic smooth muscle cells increases.

[0109] 4. Effect of decreased NRX expression on the expression of other molecules To reduce the expression level of NRX, NRX siRNA was added to the culture medium in which human aortic smooth muscle cells were cultured to a predetermined concentration, and the human aortic smooth muscle cells were then cultured for a predetermined period of time.

[0110] After culturing, the human aortic smooth muscle cells were collected, and the amounts of NRX, β-catenin, OPG, MMP-2, MMP-7, MMP-9, and β-actin proteins expressed by the human aortic smooth muscle cells were quantified using the well-known Western blotting method.

[0111] Figures 8 and 9 show the test results.

[0112] Figure 8, 801, shows the results of Western blotting to detect NRX, β-catenin, OPG, and β-actin proteins. The expression level of NRX decreased as the concentration of NRA siRNA in the culture medium increased. On the other hand, the expression levels of β-catenin and OPG increased as the concentration of NRA siRNA in the culture medium increased. The expression level of β-actin did not change even when the culture conditions of human aortic smooth muscle cells were changed.

[0113] The ratio of the expression level of NRX to the expression level of β-actin is shown in 802 of Figure 8. From 802 of Figure 8, it can be seen that the expression level of NRX decreases as the concentration of NRX siRNA in the culture medium increases.

[0114] The ratio of the expression level of β-catenin to the expression level of β-actin is shown in 803 of Figure 8. From 803 of Figure 8, it can be seen that the expression level of β-catenin increases as the expression level of NRX decreases.

[0115] The ratio of the expression level of OPG to the expression level of β-actin is shown in 804 of Figure 8. From 804 of Figure 8, it can be seen that the expression level of OPG increases as the expression level of NRX decreases.

[0116] Figure 9, 901, shows the results of Western blotting to detect NRX, MMP-2, MMP-7, MMP-9, and β-actin proteins. The expression level of NRX decreased as the concentration of NRA siRNA in the culture medium increased. On the other hand, the expression levels of MMP-2, MMP-7, and MMP-9 increased as the concentration of NRA siRNA in the culture medium increased. The expression level of β-actin did not change even when the culture conditions of human aortic smooth muscle cells were changed.

[0117] 902 in Figure 9 shows the ratio of the expression level of MMP-2 to the expression level of β-actin. From 902 in Figure 9, it can be seen that the expression level of MMP-2 increases as the expression level of NRX decreases.

[0118] 903 in Figure 9 shows the ratio of the expression level of MMP-7 to the expression level of β-actin. From 903 in Figure 9, it can be seen that the expression level of MMP-7 increases as the expression level of NRX decreases.

[0119] 904 in Figure 9 shows the ratio of the expression level of MMP-9 to the expression level of β-actin. From 904 in Figure 9, it can be seen that the expression level of MMP-9 increases as the expression level of NRX decreases.

[0120] <5. Screening of therapeutic agents for aortic aneurysms> tBHQ, SFN, or CDDO was added to the culture medium for culturing human aortic smooth muscle cells to a predetermined concentration, and the human aortic smooth muscle cells were then cultured for a predetermined period of time. When tBHQ was used, the tBHQ concentration in the culture medium was set to 0 μM, 5 μM, 20 μM, or 100 μM. When SFN was used, the SFN concentration in the culture medium was set to 0 μM, 1 μM, 10 μM, or 50 μM. When CDDO was used, the CDDO concentration in the culture medium was set to 0 nM, 0.5 nM, 1 nM, or 1.5 nM.

[0121] After the culture, the human aortic smooth muscle cells were collected, and the amounts of NRX and β-actin proteins expressed by the human aortic smooth muscle cells were quantified by the well-known Western blotting method.

[0122] The test results are shown in Figure 10. Figure 10 shows the results of detecting NRX and β-actin proteins by Western blotting. When the concentration of tBHQ in the culture medium was set to 100 μM, the expression level of NRX increased. When the concentration of SFN in the culture medium was set to 1 μM and 10 μM, the expression level of NRX increased. When the concentration of CDDO in the culture medium was set to 0.5 nM, 1 nM, and 1.5 nM, the expression level of NRX increased.

[0123] Next, the therapeutic effect of the above-mentioned compound that increases the expression level of NRX (nucleoredoxin expression inducer) on aortic aneurysm was confirmed.

[0124] MMP-2 is known to be involved in the formation of aortic aneurysms. This suggests that compounds that can reduce the expression level of MMP-2 have a therapeutic effect on aortic aneurysms. Therefore, in the following test, we used tBHQ as a representative NRX expression inducer to determine whether tBHQ has the effect of reducing the expression level of MMP-2, in other words, whether tBHQ has a therapeutic effect on aortic aneurysms.

[0125] tBHQ was added to a culture medium for culturing human aortic smooth muscle cells to a predetermined concentration (5 μM, 20 μM, or 100 μM), and the human aortic smooth muscle cells were then cultured for a predetermined period of time.

[0126] After the culture, the human aortic smooth muscle cells were collected, and the amounts of NRX, MMP-2, and β-actin proteins expressed by the human aortic smooth muscle cells were quantified by the well-known Western blotting method.

[0127] Figure 11 shows the test results.

[0128] Figure 11, 1101, shows the results of Western blotting to detect NRX, MMP-2, and β-actin proteins. The expression level of NRX increased as the concentration of tBHQ in the culture medium increased. On the other hand, the expression level of MMP-2 decreased as the concentration of tBHQ in the culture medium increased. The expression level of β-actin did not change even when the culture conditions of human aortic smooth muscle cells were changed.

[0129] The ratio of the expression level of NRX to the expression level of β-actin is shown in 1102 of Figure 10. From 1102 of Figure 10, it can be seen that the expression level of NRX increases as the concentration of tBHQ in the culture medium increases.

[0130] The ratio of the expression level of MMP-2 to the expression level of β-actin is shown in 1103 of Figure 10. It can be seen from 1103 of Figure 10 that the expression level of MMP-2 decreases as the concentration of tBHQ in the culture medium increases. [Industrial Applicability]

[0131] The present invention can be used for diagnosing aortic aneurysms, screening for therapeutic agents for aortic aneurysms, and treating aortic aneurysms.

Claims

1. detecting nucleoredoxin in a sample collected from a subject; A method for obtaining data for diagnosing aortic aneurysm, wherein the sample collected from the subject is a sample collected from the thoracic aorta, thoracoabdominal aorta, or abdominal aorta of the subject.

2. A diagnostic kit for aortic aneurysm, for diagnosing aortic aneurysm from a sample taken from the thoracic aorta, the thoracoabdominal aorta, or the abdominal aorta, comprising a component for detecting nucleoredoxin.

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