JP7742129B2 - New technologies for the diagnosis, treatment, and screening of therapeutic agents for aortic aneurysms - Google Patents

Description

The present invention relates to new technologies for diagnosing aortic aneurysms, therapeutic agents, and screening for therapeutic agents. More specifically, the present invention relates to technologies that utilize nucleoredoxin and Dishevelled for diagnosing aortic aneurysms, screening for therapeutic agents for aortic aneurysms, and screening for therapeutic agents for aortic aneurysms.

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 the location of their occurrence; for example, those occurring in the chest are called thoracic aortic aneurysms (TAA), those occurring in the chest and abdomen are called thoracic-abdominal aortic aneurysms (TAAA), and those occurring in the abdomen are called abdominal aortic aneurysms (AAA).

Aortic aneurysms often progress without symptoms and are only discovered when the blood vessels dissect and/or rupture. Therefore, attention is being focused on developing diagnostic methods for aortic aneurysms, as well as developing therapeutic agents for aortic aneurysms.

To develop therapeutic agents for aortic aneurysms and diagnostic methods for aortic aneurysms, it is necessary to gain a deeper understanding of the clinical risk factors for aortic aneurysms and the molecules involved in their formation.

Clinical risk factors for aortic aneurysms include smoking, respiratory disorders, aging, diabetes, hyperlipidemia, and hypertension. Meanwhile, molecules known to be involved in the formation of aortic aneurysms include osteoprotegerin (OPG) and matrix metalloproteinases (MMPs).

Sclerostin, which inhibits the Wnt signaling pathway, is known to suppress aortic aneurysms and atherosclerosis induced by angiotensin II (see, for example, Non-Patent Document 1). Furthermore, hypoxia is known to be associated with atherosclerosis (see, for example, Non-Patent Document 2).

Based on these findings, efforts are currently underway to develop treatments for aortic aneurysms and diagnostic methods for aortic aneurysms.

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” 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”

However, existing treatments and diagnostic methods for aortic aneurysms are insufficient, and there is a need for the development of new treatments and diagnostic methods.

Therefore, one aspect of the present invention aims to realize new technologies for the diagnosis, treatment, and screening of aortic aneurysms.

The present inventors have newly discovered that nucleoredoxin and Dishevelled are associated with aortic aneurysms (e.g., atherosclerotic plaques present in aortic aneurysms), leading to the completion of the present invention. Specifically, one aspect of the present invention is as follows:

[1] A method for obtaining data for diagnosing aortic aneurysms, 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.

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

[3] A method for screening for 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.

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

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

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

One aspect of the present invention makes it possible to realize new technologies for the diagnosis of aortic aneurysms, therapeutic agents, and screening of therapeutic agents.

FIG. 10 is a diagram showing test results of ⁶⁴ Cu-ATSM PET/MRI in an example of the present invention. FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. FIG. 1 shows the expression of various molecules in the vicinity of atherosclerotic plaques in an example of the present invention. FIG. 1 shows changes in the expression levels of various molecules due to oxidative stress in an example of the present invention. 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. 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. FIG. 1 shows the results of screening for nucleoredoxin expression inducers in an example of the present invention. FIG. 1 shows the therapeutic effect of a nucleoredoxin expression inducer on aortic aneurysm in an example of the present invention.

One embodiment of the present invention is described below, but the present invention is not limited to this. The present invention is not limited to the 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, the expression "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

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 compounds selected from the group consisting of nucleoredoxin and Dishevelled in a sample collected from a subject.

If a decrease in nucleoredoxin expression is detected in the above process, it can be determined (Determination 1) that an aortic aneurysm will form in the future, an aortic aneurysm is forming, an aortic aneurysm has already formed, there is a possibility that an aortic aneurysm will form in the future, there is a possibility that an aortic aneurysm is forming, or there is a possibility that an aortic aneurysm has already formed.

The degree of decrease in nucleoredoxin expression is not limited. Let A be the expression level of nucleoredoxin in a comparison sample (e.g., normal tissue in a subject), and B be the expression level of nucleoredoxin in a sample collected from the subject (e.g., tissue in a subject suspected of being abnormal). For example, if "A>B," "0.9 x A≧B," "0.8 x A≧B," "0.7 x A≧B," "0.6 x A≧B," "0.5 x A≧B," "0.4 x A≧B," "0.3 x A≧B," "0.2 x A≧B," "0.1 x A≧B," or "0.01 x A≧B," then a determination can be made as in Determination 1 above.

If an increase in the expression of Dishevelled is detected in the above step, it can be determined (Determination 2) that an aortic aneurysm will form in the future, an aortic aneurysm is forming, an aortic aneurysm has already formed, there is a possibility that an aortic aneurysm will form in the future, there is a possibility that an aortic aneurysm is forming, or there is a possibility that an aortic aneurysm has already formed.

The degree of increase in Dishevelled expression is not limited. Let C be the expression level of Dishevelled in a comparison sample (e.g., normal tissue in a subject), and D be the expression level of Dishevelled in a sample collected from the subject (e.g., tissue in a subject suspected of being abnormal). 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," then the determination can be made as in Determination 2 above.

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

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.

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

Methods for detecting nucleoredoxin and Dishevelled protein are not limited, and examples include immunohistochemistry, immunohistochemistry, Western blotting, and ELISA. These methods are well known. Therefore, when using these methods to detect Dishevelled protein, it is sufficient to follow well-known methods.

The types of aortic aneurysms mentioned above are not limited, and examples include thoracic aortic aneurysms, thoracoabdominal aortic aneurysms, and abdominal aortic aneurysms.

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

The sample collected from the subject is not particularly limited, and examples thereof include samples collected from the thoracic aorta, thoracoabdominal aorta, and abdominal aorta of the subject (e.g., tissue fragments from these aortas).

The method for collecting samples from the subject is not limited, and any known method can be used.

[2. Diagnostic kit for aortic aneurysms]
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.

The configuration described above in [1. Method for acquiring data for diagnosing aortic aneurysms] will not be explained here.

An aortic aneurysm diagnostic kit 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.

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

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.

The length of the above probes and primers is not limited, as long as they are long enough to specifically detect nucleoredoxin or Dishevelled mRNA. The base sequences of the above probes and primers are not limited, as long as they are capable of specifically hybridizing to any site in nucleoredoxin or Dishevelled mRNA.

A component for detecting nucleoredoxin or Dishevelled protein may be a component for detecting nucleoredoxin or Dishevelled protein according to, for example, immunohistochemical staining, Western blotting, or ELISA.

Examples of components for detecting nucleoredoxin or Dishevelled protein include antibodies that bind to nucleoredoxin or Dishevelled protein.

The above-mentioned antibody may be a polyclonal antibody or a monoclonal antibody.

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]).

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)).

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).

The configurations described above in [1. Method for acquiring data for diagnosing aortic aneurysms] and [2. Aortic aneurysm diagnostic kit] will not be described here.

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.

Cultured cells capable of expressing one or more selected from the group consisting of nucleoredoxin and Dishevelled are not limited, and examples include aortic smooth muscle cells.

Candidate substances for the above-mentioned therapeutic agents are not limited, and examples include low molecular weight compounds, high molecular weight compounds, proteins, nucleic acids, and sugars.

In the first step, for example, a candidate therapeutic substance may be added to the 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.

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.

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 that have been contacted with the candidate substance.

In the second step, for example, cultured cells I that have been contacted with the candidate substance and cultured cells II that have not been 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.

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 x A'," "B' ≥ 1.2 x A'," "B' ≥ 1.3 x A'," "B' ≥ 1.4 x A'," "B' ≥ 1.5 x A'," "B' ≥ 1.6 x A'," "B' ≥ 1.7 x A'," "B' ≥ 1.8 x A'," "B' ≥ 1.9 x A'," "B' ≥ 2.0 x A'," "B' ≥ 5.0 x A'," "B' ≥ 10 x A'," or "B' ≥ 100 x A'" holds, the candidate substance can be determined to have potential as a therapeutic agent for aortic aneurysms.

In the second step, the expression level of Dishevelled in the cultured cells before contacting the cultured cells with the candidate substance is defined as C', and the expression level of Dishevelled in the cultured cells after contacting the cultured cells with the candidate substance is defined as D'. For example, if "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'" holds, the candidate substance can be determined to have potential as a therapeutic agent for aortic aneurysms.

4. Therapeutic Agent for Aortic Aneurysm
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.

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

The nucleoredoxin expression inducer may be any agent capable of increasing the expression level of nucleoredoxin protein, and may, for example, (i) induce the transcription of nucleoredoxin DNA into mRNA, (ii) induce the translation of nucleoredoxin mRNA into protein, or (iii) inhibit the degradation of nucleoredoxin mRNA and/or protein.

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

The above-mentioned nucleoredoxin expression inducers and Dishevelled expression inhibitors are not limited and may be, for example, low molecular weight compounds, high molecular weight compounds, proteins, nucleic acids, or sugars.

The above-mentioned nucleoredoxin expression inducers include, but are not limited to, tert-butylhydroxyquinone (tBHQ), sulforaphane (SFN), and 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid (CDDO).

The above-mentioned Dishevelled expression inhibitor is not limited to, but may include, for example, siRNA.

The amount of active ingredient contained in the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not particularly limited. For example, when the therapeutic agent is taken as 100% by mass, the amount may be 0.00001% 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.

The therapeutic agent for aortic aneurysms according to one embodiment of the present invention may contain ingredients other than the active ingredients described above.

Ingredients other than the active ingredient are not particularly limited and may include, for example, buffers, pH adjusters, isotonicity agents, preservatives, antioxidants, high molecular weight polymers, excipients, solvents, antibacterial agents, etc.

Examples of the buffering agent include phosphoric acid or its salts, boric acid or its salts, citric acid or its salts, acetic acid or its salts, carbonic acid or its salts, tartaric acid or its 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.

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

Examples of the isotonicity adjusting agent include ionic isotonicity adjusting agents (e.g., sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonicity adjusting agents (e.g., glycerin, propylene glycol, sorbitol, mannitol).

Examples of the preservatives include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

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

Examples of the high molecular weight polymers 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.

Examples of the above-mentioned excipients include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

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

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

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% to 99.99999% by mass, 0% to 99.9999% by mass, 0% to 99.9999% by mass, 0% to 99.999% by mass, 0% to 99.999% by mass, 0% to 99.99% by mass, or 5% to 99.9% by mass, assuming the therapeutic agent to be 100% by mass. It may be 10% to 99.9% by mass, 20% to 99.9% by mass, 30% to 99.9% by mass, 40% to 99.9% by mass, 50% to 99.9% by mass, 60% to 99.9% by mass, 70% to 99.9% by mass, 80% to 99.9% by mass, or 90% to 99.9% by mass.

The dosage form of the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not limited, and examples include tablets, capsules, liquid preparations, topical preparations, suppositories, injections, and inhalants.

The route of administration of the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is not limited, and examples 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.

The administration interval of the aortic aneurysm treatment agent according to one embodiment of the present invention is not limited, and can be, for example, once every hour to once every 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.

The subjects to which the therapeutic agent for aortic aneurysms according to one embodiment of the present invention is administered are not limited, and examples 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.

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

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

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

<3> The method for treating aortic aneurysms described in <2>, wherein the nucleoredoxin expression inducer is tBHQ, SFN, or CDDO.

<4> The method for treating aortic aneurysm described in <2>, wherein the Dishevelled expression inhibitor is siRNA.

<1. ⁶⁴ Cu-ATSM PET/MRI>
Basic data of two patients (patients no. 1 and 2) scheduled for surgery for aortic aneurysms were obtained according to known methods.

Specifically, data on the patient's age, sex, type of aortic aneurysm, Body Mass Index, Brinkman Index, alcohol use, hypertension, diabetes, dyslipidemia, and ^64Cu -ATSM PET accumulation were obtained according to well-known methods.

In particular, data on " ^64Cu -ATSM PET accumulation" were obtained using the 64Cu-ATSM PET/MRI technique (see, for example, 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").

The following table lists basic data for two patients. In the "Alcohol" section of the table, a "+" is entered if the patient has a drinking habit, and a "-" is entered if the patient does not have a drinking habit. In the "Hypertension" section of the table, a "+" is entered if the patient has hypertension, and a "-" is entered if the patient does not have hypertension. In the "Diabetes" section of the table, a "+" is entered if the patient has diabetes, and a "-" is entered if the patient does not have diabetes. In the "Dyslipidemia" section of the table, a "+" is entered if the patient has dyslipidemia, and a "-" is entered if the patient does not have dyslipidemia. In the "Accumulation of ⁶⁴ Cu-ATSM PET" section of the table, a "+" is entered if accumulation of ⁶⁴ Cu-ATSM PET is observed in the patient's body, and a "-" is entered if accumulation of ⁶⁴ Cu-ATSM PET is not observed in the patient's body.

⁶⁴ Cu-ATSM PET is known to accumulate in living tissues, particularly in tissues under oxidative stress. In one patient (patient number 1), a greater accumulation of ⁶⁴ Cu-ATSM PET was observed compared to the other patient (patient number 2).

Upon closer observation of the tissues in which ⁶⁴ Cu-ATSM PET was accumulated, it was found that 64 Cu-ATSM PET was significantly accumulated in the arterial wall of the thoracic aortic aneurysm (particularly in the atheromatous plaques and in the vicinity of the ^atheromatous plaques in the arterial wall of the thoracic aortic aneurysm) (see 101 in Figure 1).

Furthermore, a significantly greater number of atheromatous plaques were observed in the arterial walls of thoracic aortic aneurysms in which ⁶⁴ Cu-ATSM PET accumulation was observed compared with the arterial walls of thoracic aortic aneurysms in which ⁶⁴ Cu-ATSM PET accumulation was not observed (see 102 in Figure 1).

In Figure 1, 102, "Case 1" indicates patient number 1, and "Case 2" indicates patient number 2. Also, in Figure 1, 102, "n" indicates the number of arterial wall samples of observed thoracic aortic aneurysms. Also, in Figure 1, 102, "Atheromatous plaque area (%)" indicates the ratio of the area of atherosclerotic plaque to the area of the arterial wall of observed thoracic aortic aneurysms.

2. Expression of various molecules in the vicinity of atherosclerotic plaques
The arterial walls of thoracic aortic aneurysms in which no accumulation of ⁶⁴ Cu-ATSM PET was observed and those in which accumulation of ⁶⁴ 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 plaques were observed.

The molecules used were 4-hydroxyxynonenal (4-HNE), nucleoredoxin (NRX), beta-catenin, dishevelled (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 the body. In other words, 4-HNE is an indicator of oxidative stress.

A commercially available staining kit was used for the immunostaining and fluorescent staining. Specific staining methods followed the protocol provided with the staining kit. For the molecules listed above, except for 4-HNE, protein expression levels were detected by immunostaining and fluorescent staining.

Figures 2 to 6 show the immunostained and fluorescent stained images.

As shown in Figure 2, it was revealed that (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.

As shown in Figure 3, it was revealed that (i) NRX expression levels were low near atherosclerotic plaques, (ii) Dvl expression levels were high near atherosclerotic plaques, and (iii) β-catenin expression levels were high near atherosclerotic plaques.

As shown in Figure 4, it was revealed that areas near atherosclerotic plaques with low NRX expression and areas with high β-catenin expression were the same areas.

As shown in Figure 5, it was revealed that (i) OPG expression levels were high near atherosclerotic plaques, (ii) MMP-2 expression levels were high near atherosclerotic plaques, (iii) MMP-7 expression levels were high near atherosclerotic plaques, and (iv) MMP-9 expression levels were high near atherosclerotic plaques.

As shown in Figure 6, it was revealed that the areas near atherosclerotic plaques with high levels of MMP-2 expression and high levels of β-catenin expression were the same areas.

<3. Changes in the expression levels of various molecules due to oxidative stress>
_H2O2 was added to the culture medium for culturing human aortic smooth muscle cells to a predetermined concentration (0 _μM , 10 μM, or 50 μM), and the human aortic smooth muscle cells were then cultured for a predetermined time (3 hours or 8 hours). Note that _H2O2 has the effect of _inflicting oxidative stress on the cells.

After culturing, the human aortic smooth muscle cells were harvested, and the amounts of NRX, β-catenin, and β-actin proteins expressed by the human aortic smooth muscle cells were quantified using the well-known Western blotting method.

The test results are shown in Figure 7.

701 in Figure 7 shows the results of detecting NRX _, β-catenin, and β-actin proteins by Western blotting. 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.

The ratio of the expression level of NRX to the expression level of β-actin is shown in 702 of Fig. 7. It can also be seen from 702 of Fig. 7 that the expression level of NRX decreases as the concentration _{of H2O2} in the culture medium increases, and also decreases as the culture time of human aortic smooth muscle cells increases.

The ratio of the expression level of β-catenin to the expression level of β-actin is shown in 703 of Fig. 7. It can also be seen from 703 of Fig. 7 that the expression level of β-actin increases as the concentration of H ₂ O ₂ in the culture medium increases, and also as the culture time of human aortic smooth muscle cells increases.

<4. Effect of decreased NRX expression on the expression of other molecules>
In order 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 then the human aortic smooth muscle cells were cultured for a predetermined period of time.

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 blot method.

The test results are shown in Figures 8 and 9.

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.

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

803 in Figure 8 shows the ratio of the expression level of β-catenin to the expression level of β-actin. From 803 in Figure 8, it can be seen that the expression level of β-catenin increases as the expression level of NRX decreases.

804 in Figure 8 shows the ratio of OPG expression level to β-actin expression level. From 804 in Figure 8, it can be seen that the expression level of OPG increases as the expression level of NRX decreases.

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.

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

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

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

<5. Screening of therapeutic agents for aortic aneurysms>
tBHQ, SFN, or CDDO was added to a 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.

After culturing, the human aortic smooth muscle cells were harvested, and the amounts of NRX and β-actin protein expressed by the human aortic smooth muscle cells were quantified using the well-known Western blot method.

Figure 10 shows the test results. 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 amount of NRX expression increased. When the concentration of SFN in the culture medium was set to 1 μM and 10 μM, the amount of NRX expression increased. When the concentration of CDDO in the culture medium was set to 0.5 nM, 1 nM, and 1.5 nM, the amount of NRX expression increased.

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

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 may have a therapeutic effect on aortic aneurysms. Therefore, in the following tests, tBHQ was used as a representative NRX expression inducer to confirm 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.

tBHQ was added to the culture medium in which human aortic smooth muscle cells were cultured 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.

After culturing, the human aortic smooth muscle cells were harvested, and the amounts of NRX, MMP-2, and β-actin proteins expressed by the human aortic smooth muscle cells were quantified using the well-known Western blot method.

The test results are shown in Figure 11.

Figure 11, 1101, shows the results of detecting NRX, MMP-2, and β-actin proteins by Western blotting. 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.

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

1103 in Figure 10 shows the ratio of MMP-2 expression level to β-actin expression level. From 1103 in Figure 10, it can be seen that the expression level of MMP-2 decreases as the concentration of tBHQ in the culture medium increases.

The present invention can be used to diagnose aortic aneurysms, screen for therapeutic agents for aortic aneurysms, and treat aortic aneurysms.

Claims (2)

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 . 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 .