Biomarker for identifying hormone abnormality and method for measuring same

Abstract

The present disclosure provides a biomarker for identifying a hormone abnormality and a method for measuring the same. In one aspect, the present invention provides a method for measuring the substrate conversion capacity of 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) in a subject, the method comprising (i) a step for obtaining a test sample containing blood or one or more components thereof collected from the subject; (ii) a step for bringing the test sample into contact with the 11 beta HSD substrate; and (iii) a step for measuring the substrate conversion capacity by 11 beta HSD.

Description

Biomarkers for identifying hormonal abnormalities and methods for measuring them

[0001] The present invention relates to disease-related biomarkers and methods for measuring the same. The present invention particularly relates to biomarkers related to adrenal insufficiency and methods for measuring the same.

[0002] In living organisms, when subjected to physical or mental stress, the hypothalamus secretes corticotropin-releasing hormone (CRH), followed by adrenocorticotropic hormone (ACTH) from the pituitary gland and cortisol from the adrenal glands to cope with the stress. ACTH and cortisol levels also fluctuate throughout the day, peaking upon waking in the morning and peaking before bedtime. However, damage to the hypothalamus, pituitary gland, or adrenal glands leads to decreased cortisol secretion, resulting in adrenal insufficiency, a type of endocrine disorder.

[0003] In individuals suffering from adrenal insufficiency, cortisol deficiency disrupts homeostasis, leading to a variety of symptoms. For example, symptoms associated with adrenal insufficiency include central nervous system symptoms (loss of appetite, weight loss, nausea, pickiness, or dizziness), cardiovascular symptoms (hypotension or dehydration), hematological symptoms (anemia, lymphocytosis, or eosinophilia), electrolyte-related symptoms (hyponatremia, hyperkalemia, or hypercalcemia), neuropsychological symptoms (depression, fatigue, or decreased libido), gastrointestinal symptoms (diarrhea, vomiting, or abdominal pain), skin and hair symptoms (dry skin, primary or central hyperpigmentation, or loss of pubic or axillary hair), or musculoskeletal symptoms (muscle pain, joint pain, or weakness). However, these symptoms are all nonspecific and common to other diseases, making it difficult to distinguish adrenal insufficiency from other conditions. If adrenal insufficiency is left untreated and the disease progresses, it can lead to acute adrenal crisis (adrenal crisis), characterized by hypotension, hypoglycemia, or impaired consciousness, and in the worst case, death. The estimated annual incidence of adrenal crisis is approximately 5-9%, and the mortality rate from adrenal crisis is approximately 1% (Non-patent document 1: Stefanie, Hahner, et al. Nature Reviews: Disease Primers 7.1 (2021)).

[0004] Until now, diagnosing adrenal insufficiency required invasive tests such as rapid ACTH tests or CRH stimulation tests, and interpreting the test results required specialized expertise. Furthermore, the results did not always align with the patient's clinical symptoms. In addition, current diagnostic guidelines for adrenal insufficiency require frequent blood sampling or 24-hour urine collection, making it difficult to perform a simple diagnosis on an outpatient basis.

[0005] In recent years, FKBP5 mRNA, GDF15, or soluble CD16 and ADAM17 in the blood, as well as steroid metabolome profiles in urine, have been reported as candidate biomarker molecules for adrenal insufficiency. However, the analytical results of these biomarkers have not shown sufficient reproducibility with adrenal insufficiency, and none have reached practical application. Furthermore, among the previously reported biomarkers, FKBP5 mRNA exhibits mRNA instability, and urinary steroid profiles require 24-hour urine collection, making the testing of any of these biomarkers inconvenient for clinical use. Other molecules can also be altered by other pathological conditions and therefore cannot be considered specific markers for adrenal insufficiency.

[0006] Stefanie, Hahner, et al. Nature Reviews: Disease Primers 7.1 (2021).

[0007] The present invention relates to biomarkers associated with endocrine diseases, particularly adrenal insufficiency, and methods for measuring the same. In some aspects of the present invention, endocrine diseases, particularly adrenal insufficiency, are identified by evaluating the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11β-HSD) in mononuclear cells of a subject.

[0008] Cortisol is the major glucocorticoid in humans, synthesized in the adrenal cortex and released into the circulatory system. Cortisol is involved in gluconeogenesis in the liver, protein metabolism in muscles, lipolysis in adipose tissue, anti-inflammatory effects, and immunosuppression. Cortisone is the 11-keto form of cortisol and is inactive as a glucocorticoid. In humans, two isozymes with 11β-hydroxysteroid dehydrogenase activity (11βHSD) are involved in the interconversion of cortisone and cortisol. Type 1 11βHSD (11βHSD1) primarily functions as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductase, converting cortisone to cortisol. 11βHSD1 is mainly expressed in the liver, gonads, pituitary gland, cerebellum, or blood vessels. Type 2 11βHSD (11βHSD2) functions as a nicotinamide dinucleotide (NAD+)-dependent dehydrogenase, converting cortisol to cortisone. 11βHSD2 is primarily expressed in the kidneys, colon, lungs, and salivary glands. The action of both enzymes regulates cortisol levels in the body, maintaining homeostasis.

[0009] This invention is based on the inventors' discovery that the substrate conversion ability of 11βHSD in mononuclear cells derived from subjects with adrenal insufficiency differs from that of cells derived from healthy individuals. In some embodiments of this invention, the substrate conversion ability of 11βHSD1, a molecule involved in steroid metabolism, is measured.

[0010] This invention allows for the diagnosis or monitoring of diseases based on the substrate conversion ability of 11βHSD rather than the amount of cortisol. Furthermore, this invention allows for the diagnosis or monitoring of diseases based on the substrate conversion ability of 11βHSD in mononuclear cells contained in blood collected from a subject. The present invention, which uses the substrate conversion ability of 11βHSD (particularly 11βHSD1) as a biomarker, may have advantages over conventional biomarkers and diagnostic methods in that it can stably capture differences even if the enzyme expression level is small. Conventional diagnostic methods that measure disease-related mRNA have problems with diagnostic accuracy due to the instability of mRNA, but the method of the present invention does not involve mRNA detection, so such problems do not exist. In addition, since the method of the present invention can diagnose a disease with only one blood sample, it is simpler and less burdensome for patients compared to disease diagnostic methods that analyze urinary steroid profiles, which require 24 hours of urine collection. In some embodiments, a cortisol kit commonly used in clinical practice (e.g., Elecsys® Cortisol II reagent) can be used to measure cortisol concentration for evaluating 11βHSD1 substrate conversion ability, making the measurement much simpler compared to conventional methods for measuring other candidate molecules. The method of the present invention can reduce the burden on patients compared to highly invasive hormone secretion stimulation tests used in the diagnosis of adrenal diseases.

[0011] Accordingly, according to the main aspects of the present invention, the following inventions are provided: (Item 1) A method for measuring the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11βHSD) in a subject, the method comprising: (i) obtaining a test sample containing blood or one or more components thereof taken from the subject; (ii) contacting the test sample with a substrate of the 11βHSD; and (iii) measuring the substrate conversion ability of the 11βHSD. (Item 2) The method according to Item 1, characterized in that the substrate of the 11βHSD is added in step (ii). (Item 3) The method according to Item 1, wherein the step of measuring the substrate conversion ability of the 11βHSD comprises the step of measuring the concentration of a product from which the substrate has been converted. (Item 4) The method according to any of the above items, wherein the substrate conversion ability of the 11βHSD is an indicator for the diagnosis or monitoring of a disease related to the production of cortisol or its analogues in the subject. (Item 5) The method according to any of the above items, characterized in that the concentration of the product is equal to or greater than a specified concentration, or that the concentration increases by a specified amount or more upon contact, serves as the indicator. (Item 6) The method according to any of the above items, wherein the disease is an endocrine disease. (Item 7) The method according to any of the above items, wherein the endocrine disease is an adrenal-related disease. (Item 8) The method according to any of the above items, wherein the adrenal-related disease is selected from the group including adrenal insufficiency, primary aldosteronism, Cushing's syndrome, pheochromocytoma, adrenal incidentaloma, adrenal insufficiency, and congenital adrenal hyperplasia. (Item 9) The method according to any of the above items, wherein the adrenal-related disease is adrenal insufficiency. (Item 10) The method according to any of the above items, wherein the 11βHSD includes type 1 11βHSD (11βHSD1). (Item 11) The method according to any of the above items, wherein the substrate and the product are glucocorticoids. (Item 12) The method according to any of the above items, wherein the substrate is cortisone or an analog thereof, and the product is cortisol or an analog thereof. (Item 13) The method according to any of the above items, wherein the test sample is peripheral blood mononuclear cells.(Item 14) A therapeutic agent for a disease related to the production of cortisol or its analogs in a subject, comprising a hormone regulator, wherein the therapeutic agent is administered according to the substrate conversion ability measured by the method of any of the above items. (Item 14A) A method for treating a disease related to the production of cortisol or its analogs in a subject, comprising administering a hormone regulator to the subject according to the substrate conversion ability measured by the method of any of the above items. (Item 14B) A hormone regulator for treating a disease related to the production of cortisol or its analogs in a subject, wherein the hormone regulator is administered according to the substrate conversion ability measured by the method of any of the above items. (Item 14C) Use of a hormone regulator in the manufacture of a medicament for treating a disease related to the production of cortisol or its analogs in a subject, wherein the hormone regulator is administered according to the substrate conversion ability measured by the method of any of the above items.

[0012] In this disclosure, the one or more of the above features are intended to be provided in combinations other than those explicitly stated. Further embodiments and advantages of this disclosure will be apparent to those skilled in the art, by reading and understanding the detailed description below as necessary.

[0013] The symptoms of adrenal insufficiency are nonspecific and can be indistinguishable from other diseases. Current diagnostic methods for adrenal insufficiency involve performing invasive tests on patients exhibiting symptoms, and if the results are normal, adrenal insufficiency is ruled out. In other words, because symptoms alone are insufficient for diagnosing adrenal insufficiency, highly invasive diagnostic methods had to be performed on patients suspected of having the condition. This invention aims to improve upon this current situation.

[0014] Figure 1 shows the blood collection schedule for whole blood RNA expression differential gene analysis in patients with pituitary adrenal insufficiency. Figure 2 shows the scheme for evaluating HSD11B1 expression levels in primary cultured human mononuclear cells using dexamethasone. Figure 3 shows the results of HSD11B1 analysis in human mononuclear cells treated with different dexamethasone (Dex) concentrations. All quantitative data are shown as mean ± SEM, and the results of one-way ANOVA and subsequent Bonferroni tests are displayed (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 4-1 shows the relationship between time and human mononuclear cell HSD11B1 expression levels after dexamethasone (Dex) treatment. All quantitative data are shown as mean ± SEM, and the results of one-way ANOVA and subsequent Bonferrone tests are displayed (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 4-1 shows the results of HSD11B1 expression levels 48 hours after dexamethasone (Dex) treatment. All quantitative data are shown as mean ± SEM, and the results of one-way ANOVA and subsequent Bonferrone tests are displayed (*p < 0.05, **p < 0.01, ***p < 0.001). Figure 5 shows the results of 11βHSD1 substrate conversion ability in primary cultured human mononuclear cells. Figure 6 shows the measurement protocol for 11βHSD1 substrate conversion ability. Figure 7 shows the concentrations of mononuclear cell culture supernatant cortisol in patients with newly diagnosed and treated pituitary adrenal insufficiency and in healthy individuals. For patients with adrenal insufficiency, arrows indicate changes in mononuclear cell culture supernatant cortisol concentration under glucocorticoid administration and deficiency.

[0015] The present disclosure is described below in best form. Throughout this specification, singular and other expressions should be understood to include the concept of their plural forms unless otherwise specified. Accordingly, singular articles (for example, "a," "an," and "the" in English) should be understood to include the concept of their plural forms unless otherwise specified. Furthermore, terms used in this invention should be understood to have the meaning commonly used in the art unless otherwise specified. Accordingly, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0016] The following provides definitions of terms used specifically in this specification and / or basic technical concepts as appropriate.

[0017] In this specification, "approximately" means ±10% of the following number.

[0018] In this specification, "measuring the substrate conversion ability" of an enzyme means measuring the amount of substrate that the enzyme being measured can convert to produce the converted product, and includes measuring the amount of product converted by the enzyme and / or the amount of substrate that was not converted, as well as measuring the amount of the enzyme itself. Measuring substrate conversion ability includes measurements at one time point, and measurements at two or more time points.

[0019] In this specification, “mononuclear cells” refers to blood cells having a spherical nucleus, including lymphocytes (such as T cells, B cells, and natural killer (NK) cells), monocytes, and dendritic cells. In this specification, mononuclear cells may originate from bone marrow, umbilical cord blood, or peripheral blood, and mononuclear cells derived from peripheral blood are referred to as peripheral blood mononuclear cells.

[0020] In this specification, “isolation” means that a particular component (such as a nucleic acid molecule, protein, or cell) has been substantially separated or purified from cells or a group of cells, tissue, or other mixtures.

[0021] As used herein, an "analogue" is a compound having a structure similar to another but different in a specific part. For example, an analogue has one or more different atoms, functional groups, or substructures, which are replaced by other atoms, groups, or substructures. A "cortisone analogue" refers to a compound having a steroid skeleton with an oxo group at the 11β-position, but having one or more different atoms, functional groups, or substructures and being capable of serving as a substrate for 11β-HSD. A "cortisol analogue" refers to a compound having a pregnane-type steroid skeleton with a hydroxy group at the 11β-position, but having one or more different atoms, functional groups, or substructures and being capable of serving as a substrate for 11β-HSD.

[0022] As used herein, "glucocorticoid" is a general term for a group including 11β-hydroxysteroids and 11-oxosteroids. 11β-hydroxysteroids is a general term for a group including cortisol and corticosterone, and their analogues. 11-oxosteroids is a general term for a group including cortisone and 11-dehydrocorticosterone, and their analogues.

[0023] As used herein, "the enzyme 11β-HSD (11β-hydroxysteroid dehydrogenase)" refers to an enzyme that catalyzes the interconversion of 11β-hydroxysteroids and 11-oxosteroids. There are two isotypes of 11β-HSD, type 1 (11β-HSD1) and type 2 (11β-HSD2). 11β-HSD1 converts 11-oxosteroids to 11β-hydroxysteroids, and 11β-HSD2 converts 11β-hydroxysteroids to 11-oxosteroids.

[0024] As used herein, "a disease associated with the production of cortisol or its analogue" refers to a disease that results in a state where the production of cortisol or its analogue is insufficient or excessive.

[0025] As used herein, the term "endocrine disease" refers to a disease caused by abnormal hormone secretion (i.e., excessive or decreased (including deficiency) hormone secretion) or an abnormality in an organ or tissue on which a hormone acts. Examples of endocrine diseases include pituitary-related diseases (such as acromegaly, amenorrheic diabetes, hypertension, etc.), thyroid diseases (such as Graves' disease, Hashimoto's disease, thyroid tumors, etc.), parathyroid diseases (such as abnormal blood calcium), adrenal-related diseases, pancreas-related diseases (such as diabetes, hypoglycemia, etc.), ovary-related diseases (such as menstrual disorders), and testis-related diseases (such as hypogonadism), but are not limited thereto.

[0026] As used herein, the term "adrenal-related disease" refers to a disease caused by an abnormality of the adrenal gland. Examples of adrenal-related diseases include, but are not limited to, adrenal insufficiency, primary aldosteronism, Cushing's syndrome, pheochromocytoma, adrenal incidentaloma, adrenocortical hypofunction, congenital adrenal hyperplasia, etc.

[0027] As used herein, the term "adrenal insufficiency" refers to a state in which the adrenal gland cannot sufficiently produce a necessary amount of cortisol.

[0028] As used herein, the term "specified amount" or "specified concentration" refers to a reference amount or concentration of a specific substance determined based on the amount or concentration of the specific substance in a sample derived from a healthy individual for evaluating the amount or concentration of the specific substance measured in the method of the present disclosure.

[0029] As used herein, the term "hormone regulator" refers to a drug that regulates the amount of hormone or the amount of hormone production in a subject. Examples of "hormone regulators" include hormone replacement drugs, hormone synthesis inhibitors, etc.

[0030] Preferred embodiments of the present disclosure will be described below. It should be understood that the embodiments provided below are for better understanding of the present disclosure, and the scope of the present disclosure should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present disclosure by referring to the descriptions in this specification. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0031] In one embodiment, a method is provided for measuring the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11βHSD) in a subject, the method comprising: (i) obtaining a test sample containing blood or one or more components thereof taken from a subject; (ii) contacting the test sample with a substrate of 11βHSD; and (iii) measuring the substrate conversion ability by 11βHSD.

[0032] In one embodiment, the present invention provides a method for measuring the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11βHSD) in a subject, the method comprising the steps of: obtaining a test sample containing blood taken from a subject; and measuring the substrate conversion ability of the 11βHSD in the test sample. The method of the present invention may further include the step of culturing cells contained in the test sample.

[0033] In one embodiment, the present invention provides a method for measuring the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11βHSD) in a subject, the method comprising: (i) obtaining a test sample including blood taken from the subject, wherein the test sample includes mononuclear cells; (ii) culturing the mononuclear cells in a predetermined amount of culture medium; and (iii) measuring the substrate conversion ability of the 11βHSD per predetermined amount of culture medium after culturing the mononuclear cells.

[0034] Test Sample The present invention may use a test sample containing blood or one or more components thereof, collected from a subject by blood sampling. The test sample may be whole blood, plasma, serum, or one or more polymorphonuclear cells or mononuclear cells. Since 11βHSD is contained in whole blood, plasma, and serum, and is expressed in polymorphonuclear cells and mononuclear cells, any of these may be used in the method of the present invention. Since mononuclear cells express 11βHSD (particularly 11βHSD1) more highly, for improved diagnostic accuracy, the test sample may preferably contain mononuclear cells or whole blood containing mononuclear cells, and more preferably isolated mononuclear cells may be used as the test sample.

[0035] Examples of suitable blood sampling times include early morning, daytime, noon, evening, night, before bedtime, or late at night. The substrate conversion ability of 11βHSD in subjects may be affected by blood cortisol levels, which exhibit physiological diurnal variations with high levels in the early morning and low levels at night. To improve the accuracy of measuring the substrate conversion ability of 11βHSD in subjects, preferred blood sampling times are early morning or nighttime (night, before bedtime, or late at night).

[0036] In some embodiments, subjects may undergo two or more blood draws for the diagnosis or monitoring of a disease in the subject. Disease monitoring is performed to confirm the progression of the condition or the effectiveness of treatment. When subjects undergo two or more blood draws, it is desirable that the conditions for each draw (such as the time of day or before / after medication, but not limited to these) be consistent.

[0037] In some embodiments, mononuclear cells are derived from bone marrow, umbilical cord blood, or peripheral blood. Preferably, from the viewpoint of the invasiveness and ease of blood collection, the mononuclear cells are peripheral blood mononuclear cells.

[0038] In some embodiments, cells including polymorphonuclear cells and mononuclear cells may be isolated or separated from a test sample containing whole blood. Since 11βHSD is expressed more highly in mononuclear cells, it is preferable to isolate or separate mononuclear cells.

[0039] In some embodiments, methods for isolation or separation may include centrifugation, immunomagnetic cell separation, microfluidic separation, aptamer technology, or other cell separation methods. Preferably, centrifugation, which allows for isolation or separation in a simple manner, may be used. In some examples, the centrifugation method is density gradient centrifugation, for example, centrifugation using lymphocyte separation solution LSM (Lymphocyte Separation Medium). The rotation speed for centrifugation is not limited but may range from about 100 × g to about 1800 × g, for example, about 200 × g, about 300 × g, about 400 × g, about 500 × g, about 600 × g, about 700 × g, about 800 × g, about 900 × g, about 1000 × g, about 1200 × g, about 1400 × g, about 1600 × g, and about 1800 × g. When performing centrifugation, the centrifugation time is not limited, but can range from approximately 5 minutes to 60 minutes, for example, approximately 10 minutes, approximately 15 minutes, approximately 20 minutes, approximately 25 minutes, approximately 30 minutes, approximately 40 minutes, approximately 50 minutes, and approximately 60 minutes. When performing centrifugation, the temperature can be controlled from approximately 0°C to approximately 40°C, preferably from approximately 0°C to room temperature, and more preferably from approximately 4°C to room temperature. The conditions for centrifugation (rotation speed, centrifugation time, or temperature control) can be appropriately set by those skilled in the art according to the desired cell recovery efficiency, viability, and purity. It is also desirable to adjust the acceleration and deceleration modes of the centrifuge to minimize the load on the cells.

[0040] In some embodiments, viability, cell count, or purity can be evaluated for cells such as mononuclear or polymorphonuclear cells. Any evaluation method known to those skilled in the art can be used for these evaluations.

[0041] In some embodiments, isolated or separated cells can be recovered or diluted in a liquid. The liquid may be an aqueous liquid, such as saline, a buffer, or a culture medium. If the cells are subsequently cultured after recovery or dilution, the liquid is preferably a culture medium.

[0042] In some embodiments, isolated cells can be cryopreserved as needed. When cryopreserving, it is effective to add a predetermined cryoprotective agent (e.g., dimethyl sulfoxide (DMSO) or glycerol) and control the freezing rate to maintain cell quality. The storage temperature should preferably be selected to maintain cell activity, such as -80°C or in liquid nitrogen.

[0043] Contact with Substrate The present invention includes the step of contacting blood collected from a subject with a substrate of 11βHSD (preferably 11βHSD1) in order to measure the substrate conversion ability of 11βHSD (preferably 11βHSD1). This substrate may be present in the blood collected from the subject or may be added separately. In some embodiments, the method of the present invention includes the step of culturing cells such as mononuclear cells or polymorphonuclear cells in contact with a substrate of 11βHSD (preferably 11βHSD1). This culturing can be carried out under general cell culture conditions. For example, at a temperature of about 37°C and CO2. ２ The concentration should be approximately 5%, and the pH should be maintained in the range of approximately 3.0 to approximately 10.0, preferably approximately 5.0 to approximately 9.0, more preferably approximately 7.0 to approximately 8.0, and even more preferably approximately 7.2 to approximately 7.4. When adding the culture medium to a blood sample, or when culturing cells isolated from a blood sample together with the culture medium, the culture medium is not particularly limited as long as it contains amino acids (such as glutamic acid), vitamins (such as B vitamins), inorganic salts (such as NaCl and KCl), or carbohydrates (such as glucose). In one example, a commercial medium such as RPMI-1640 medium or DMEM can be used as the culture medium. In some embodiments, growth factors, such as fetal bovine serum, may be added to the culture medium as needed.

[0044] In some embodiments, the initial cell seeding density is, for example, about 1 × 10⁶, depending on the size of the culture vessel or culture plate. ４ pieces / mL ~ approx. 1 x 10 ７ pieces / mL, preferably about 1 × 10 ５ pieces / mL ~ approx. 1 x 10 ７ pieces / mL, more preferably about 5 × 10５ cells / mL to about 5×10 ６ cells / mL, and can be, for example, about 5×10 ４ cells / mL, about 1×10 ５ cells / mL, about 2×10 ５ cells / mL, about 3×10 ５ cells / mL, about 4×10 ５ cells / mL, about 5×10 ５ cells / mL, about 6×10 ５ cells / mL, about 7×10 ５ cells / mL, about 7.5×10 ５ cells / mL, about 8×10 ５ cells / mL, about 9×10 ５ cells / mL, about 1×10 ６ cells / mL, about 2×10 ６ cells / mL, about 4×10 ６ cells / mL, about 6×10 ６ cells / mL, about 8×10 ６ cells / mL, about 1×10 ７ cells / mL, etc. In some embodiments, when the cells are cultured in a 24-well plate, the initial seeding density of the cells is preferably about 1×10 ５ cells / mL to about 4×10 ６ cells / mL, for example, about 2×10 ５ cells / mL, about 3×10 ５ cells / mL, about 4×10 ５ cells / mL, about 5×10 ５ cells / mL, about 6×10 ５ 個 / mL、約7×10 ５ 個 / mL、約7.5×10 ５ 個 / mL、約8×10 ５ 個 / mL、約9×10 ５ 個 / mL、約1×10 ６ 個 / mL、約2×10 ６ 個 / mL、約4×10 ６ cells / mL. It is desirable that the initial seeding density of the cells derived from the subject is equivalent to the initial seeding density of the cells derived from a healthy control subject.

[0045] In some embodiments, the contact time of the test sample with the substrate may be about 10 minutes to until the cells reach confluence, preferably about 1 hour to about 2 weeks, more preferably about 6 hours to about 1 week, and even more preferably about 24 hours to about 3 days, and may be about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 6 hours, about 12 hours, about 24 hours, about 2 days, about 3 days, about 5 days, about 1 week, about 2 weeks, or until the cells reach confluence. The culture time of cells derived from the test subject is preferably equivalent to the culture time of cells derived from a healthy control individual.

[0046] In some embodiments, a substrate of 11βHSD (typically 11βHSD1) is added to a culture medium containing cells such as mononuclear or polymorphonuclear cells. The substrate of 11βHSD and the products converted by 11βHSD may be glucocorticoids. The substrate may be cortisone or an analog thereof, and the product may be cortisol or an analog thereof. In certain embodiments, cortisone or a cortisone analog (e.g., 21-cortisone acetate) may be added to the cell culture medium as a substrate of 11βHSD1. Cortisone or a cortisone analog may be added as needed, adjusted in the test sample to a final concentration of about 5 nM to about 5 μM, preferably about 20 nM to about 1 μM, more preferably about 100 nM to about 800 nM, more preferably about 300 nM to about 700 nM, and even more preferably about 400 nM to about 600 nM, for example, to a final concentration selected from the group including about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 50 nM, about 100 nM, about 200 nM, about 300 nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about 800 nM, about 900 nM, about 1 μM, about 2 μM, about 3 μM, about 4 μM, or about 5 μM, and may be added as needed.

[0047] In the process of measuring the substrate conversion ability of 11βHSD, the substrate of 11βHSD in the test sample is consumed by an enzymatic reaction and converted to the corresponding product. In some embodiments, the concentration of the product converted by 11βHSD (preferably 11βHSD1) in the culture supernatant is measured after cell culture. In certain embodiments, the concentration of cortisol or cortisol analog converted from cortisone or a cortisone analog by 11βHSD1 in the culture supernatant may be measured after cell culture. In some embodiments, the decrease in the concentration of the added substrate in the culture supernatant is measured after culturing mononuclear cells.

[0048] In some embodiments, any known method can be used to measure the concentration of the product converted by 11βHSD or the substrate of 11βHSD after culturing cells such as mononuclear cells or polymorphonuclear cells, including spectrophotometrics, fluorescence measurement, electrochemical measurement, electrochemiluminescence immunoassay (ECLIA), high-performance liquid chromatography (HPLC), ultra-high-performance liquid chromatography (UPLC), liquid chromatography-mass spectrometry (LC-MS), or liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0049] Setting the Standard Concentration In some embodiments, the concentration of the substance converted by 11βHSD or the substrate of 11βHSD after culturing cells such as mononuclear or polymorphonuclear cells derived from a subject under culture conditions containing the substrate of 11βHSD can be compared to a standard concentration. By confirming whether the concentration is higher or lower than the standard concentration, or whether the concentration has increased or decreased by more than a specified amount, it is possible to diagnose, monitor, or determine a disease, or to adjust or determine the dosage of a drug to treat the disease. The standard concentration is set based on the concentration of the corresponding product after culturing cells such as mononuclear or polymorphonuclear cells derived from a healthy human. The standard concentration is set appropriately depending on the concentration of the added 11βHSD substrate, the initial cell seeding density, the cell culture time, or the object being measured (the substrate of 11βHSD or the product to which the substrate has been converted by 11βHSD). In some embodiments, the degree of deviation of the concentration from the standard concentration can be used to evaluate the severity of the disease suffered by the subject. In a specific embodiment, mononuclear cells (initial seeding density approximately 1.5 × 10⁶) were grown under conditions including cortisone (final concentration approximately 500 nM). ６ The specified concentration of cortisol after culturing cells / well for approximately 48 hours, when measured by the ECLIA method, can be selected from the group of approximately 0.5 to approximately 6.0 μg / dL, approximately 1.0 to approximately 5.0 μg / dL, approximately 1.0 to approximately 4.0 μg / dL, approximately 1.5 to approximately 3.0 μg / dL, and approximately 1.5 to approximately 2.5 μg / dL, preferably set to any value within the range of approximately 1.5 μg / dL to approximately 3.0 μg / dL, more preferably approximately 1.5 μg / dL to approximately 2.5 μg / dL, and even more preferably approximately 2.2 to approximately 2.5 μg / dL.

[0050] Types of Diseases In some embodiments, the substrate conversion ability of 11βHSD described herein can be used as an indicator for the diagnosis or monitoring of diseases related to the production of cortisol or its analogues in a subject. Diseases related to the production of cortisol or its analogues may be conditions in which the production of cortisol or its analogues is insufficient or excessive. In some embodiments, such diseases are endocrine diseases, and examples of endocrine diseases include pituitary-related diseases (e.g., acromegaly, amenorrhea, hypertension, etc.), thyroid diseases (e.g., Graves' disease, Hashimoto's disease, thyroid tumors, etc.), parathyroid diseases (e.g., abnormal blood calcium levels), adrenal-related diseases, pancreatic-related diseases (e.g., diabetes mellitus, hypoglycemia, etc.), ovarian-related diseases (e.g., menstrual disorders), and testicular-related diseases (e.g., hypogonadism). Examples of adrenal-related diseases include adrenal insufficiency, primary aldosteronism, Cushing's syndrome, pheochromocytoma, adrenal incidentaloma, adrenal insufficiency, and congenital adrenal hyperplasia.

[0051] The blood cortisol / cortisone ratio of a subject reflects the changes in the substrate conversion ability of 11βHSD in all tissues of the body, making it impossible to determine which tissue's changes in 11βHSD substrate conversion ability are responsible for the result. On the other hand, the inventors unexpectedly found that the measurement results obtained by the method described herein can reflect the disease state. For example, the inventors found that although there was no difference in the blood cortisol / cortisone ratio between subjects with adrenal insufficiency and healthy individuals, the method described herein can be used to distinguish between adrenal insufficiency and healthy individuals.

[0052] In some embodiments, the substrate conversion ability of 11βHSD can be used as an indicator for diagnosing, monitoring, or determining diseases related to the production of cortisol or its analogues in a subject (e.g., adrenal insufficiency). In this case, cells such as mononuclear cells or polymorphonuclear cells can be cultured under conditions containing cortisone, and the cortisol concentration in the culture supernatant after culturing may be above a specified concentration or increased by a specified amount, which can serve as an indicator.

[0053] In some embodiments, the dosage of a drug can be adjusted or determined to treat a disease related to the production of cortisol or its analogues in a subject (e.g., adrenal insufficiency). Such drugs may be hormone regulators, such as hormone replacement drugs and hormone synthesis inhibitors. Hormone replacement drugs are selected from the group consisting of dexamethasone, hydrocortisone, levothyroxine, desmopressin, prednisolone, methylprednisolone, and mineralocorticoids, and their pharmacologically acceptable salts, preferably selected from dexamethasone, hydrocortisone, prednisolone, and methylprednisolone and their pharmacologically acceptable salts. Hormone synthesis inhibitors are selected from the group consisting of mitotane, trilostane, metyrapone, ocilodrostat, and levocetoconazole and their pharmacologically acceptable salts.

[0054] In some embodiments, the method of the present invention can distinguish between a cortisol-deficient and a cortisol-sufficient state in a subject at the time of blood collection. By culturing cells such as mononuclear cells or polymorphonuclear cells under conditions containing cortisone, the method of the present invention can distinguish that (i) when the cortisol concentration in the culture supernatant after culturing is higher than the specified concentration or has increased by more than the specified amount, the subject is in a cortisol-deficient state at the time of blood collection, and (ii) when it is equal to or lower than the specified concentration, the subject is in a cortisol-sufficient state at the time of blood collection. In some embodiments, blood can be collected from the same subject two or more times, and the cortisol-deficient and cortisol-sufficient state of the subject at each blood collection can be distinguished.

[0055] In some embodiments, the method of the present invention may be used to evaluate the effectiveness of treatment for diseases related to the production of cortisol or its analogues (e.g., adrenal insufficiency). Using the method of the present invention, cells such as mononuclear or polymorphonuclear cells are cultured in cortisone-containing conditions in a subject receiving treatment for a disease related to the production of cortisol or its analogues. The following can be identified: (i) if the cortisol concentration in the culture supernatant after culturing is higher than or equal to a specified concentration, the subject at the time of blood collection is under-treated for the disease related to the production of cortisol or its analogues; (ii) if the concentration is equal to the specified concentration, the subject at the time of blood collection is adequately treated for the disease related to the production of cortisol or its analogues; or (iii) if the concentration is lower than or equal to a specified concentration, the subject at the time of blood collection is excessively treated for the disease related to the production of cortisol or its analogues.

[0056] In one embodiment, the present invention provides a therapeutic agent for a disease related to the production of cortisol or its analogues in a subject, comprising a hormone regulator, wherein the therapeutic agent is administered according to the substrate conversion ability measured by a method comprising: (i) obtaining a test sample comprising blood taken from the subject, wherein the test sample comprising cells such as mononuclear cells or polymorphonuclear cells; (ii) culturing the cells in a predetermined amount of culture medium; and (iii) measuring the substrate conversion ability of the 11βHSD per predetermined amount of culture medium after culturing the cells. The hormone regulator, each step, and the criteria for administration are described herein.

[0057] In this specification, "or" is used when "at least one" of the items listed in the text can be adopted. The same applies to "or else".

[0058] References such as scientific literature, patents, and patent applications cited herein are incorporated herein by reference to the same extent as they are specifically described herein.

[0059] The above description has provided preferred embodiments to prepare the reader for understanding this disclosure. The following description of the disclosure is based on examples, but the above description and the following examples are provided for illustrative purposes only and not to establish the disclosure. Accordingly, the scope of this disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0060] The following embodiments are provided to illustrate certain features of a particular aspect of the present disclosure, but the claims should not be limited to these illustrated features.

[0061] (Example 1) In Example 1, with the aim of exploring indicators for the diagnosis or monitoring of patients with adrenal insufficiency, blood samples were collected from patients with pituitary adrenal insufficiency according to the blood sampling schedule shown in Figure 1 (1 day after the last oral administration of Cortril® (cortisol) (after oral administration of cortisol, i.e., in a state of cortisol sufficiency) and 3 days after oral administration of cortisol (before oral administration of cortisol, i.e., in a state of adrenal insufficiency)). Whole blood RNA expression differential gene analysis was performed on the obtained whole blood samples. Table 1. Whole blood RNA gene changes before and after oral administration of Cortril in patients with adrenal insufficiency

[0062] Table 1 shows the results of gene analysis of differential RNA expression in whole blood. Table 1 lists genes with large variations in RNA expression levels. Among these genes, HSD11B1, which encodes 11β-hydroxysteroid dehydrogenase (11βHSD1), is a gene involved in steroid metabolism, so in the following examples, the expression and activity of 11βHSD1 were tested.

[0063] (Example 2) In Example 2, further analysis of HSD11B1 was performed. Since whole blood contains various blood cells, in this example, the expression levels of HSD11B1 in plasma, polymorphonuclear cells, and mononuclear cells contained in whole blood were evaluated by quantitative PCR.

[0064] As a result, higher HSD11B1 expression was observed in mononuclear cells compared to plasma and polymorphonuclear cells (Ct > 33-34). Therefore, in the following examples, HSD11B1 in mononuclear cells, which are thought to have higher expression levels and thus higher evaluation accuracy, was tested.

[0065] (Example 3) In Example 3, the expression level of HSD11B1 in mononuclear cells was analyzed. The protocol for this example is shown in Figure 2. Specifically, whole blood collected from the veins of healthy individuals was dispensed into EDTA-containing blood collection tubes, and mononuclear cells were separated from 10 mL of whole blood using lymphocyte separation solution LSM (MP Biomedicals, CA, USA). Then, the mononuclear cells were placed in 24-well cell culture dishes in a 1.5 × 10⁶ layer. ６ Seeds were seeded at a concentration of cells / well and cultured for 48 hours in the presence of dexamethasone (final concentrations of 1 nM, 10 nM, or 100 nM) or, as a control, in the presence of dimethyl sulfoxide (DMSO). The amount of HSD11B1 mRNA after culture was evaluated by quantitative PCR.

[0066] Figure 3 shows the relative amounts of HSD11B1 mRNA after culturing. The amount of HSD11B1 mRNA after culturing was significantly lower in the presence of dexamethasone compared to the control in the presence of DMSO. Furthermore, the amount of HSD11B1 mRNA after culturing decreased as the treatment concentration of dexamethasone increased.

[0067] Next, we performed a further analysis of the expression level of HSD11B1 in mononuclear cells. The isolated mononuclear cells were placed in 24-well cell culture dishes in a 1.5 × 10⁶ format. ６Seeds were seeded at a concentration of cells / well and cultured in the presence of dexamethasone (final concentration 10 nM), dexamethasone (final concentration 10 nM) + mifepristone (glucocorticoid receptor antagonist, final concentration 1 μM), or DMSO as a control. The amount of HSD11B1 mRNA over time was evaluated by quantitative PCR. The results are shown in Figures 4A and 4B. Compared to the amount of control mRNA, the amount of HSD11B1 mRNA in the presence of dexamethasone was lower at all time points, and was significantly lower after 48 hours of culture. However, the amount of HSD11B1 mRNA in the presence of dexamethasone + mifepristone was significantly higher than under conditions containing only dexamethasone, and approached the amount of control mRNA (see Figure 4B). This indicates that the decrease in HSD11B1 mRNA levels in the presence of dexamethasone is induced via the action of dexamethasone on glucocorticoid receptors. The results of Example 3 suggest that the expression level of HSD11B1 in human mononuclear cells may change in a manner dependent on blood cortisol concentration.

[0068] (Example 4) In Example 4, the substrate conversion ability of 11βHSD1 in mononuclear cells was evaluated. Whole blood collected from the veins of healthy individuals was dispensed into EDTA-containing blood collection tubes, and mononuclear cells were separated from 10 mL of whole blood using lymphocyte separation solution LSM. The separated mononuclear cells were then placed in a 24-well cell culture dish in a 1.5 × 10⁶ dish. ６Mononuclear cells were seeded at a concentration of cells / well and cultured for 48 hours in the presence of cortisone (final concentration 500 nM) or cortisone (final concentration 500 nM) + PF-915275 (selective 11βHSD1 inhibitor, final concentration 1 μM). The cortisol concentration in the culture supernatant after culturing was measured by ECLIA. As a control, the cortisol concentration in the culture medium containing DMSO or cortisone (final concentration 500 nM) without mononuclear cells was measured after 48 hours. The results are shown in Figure 5. The cortisol concentration in the culture supernatant of mononuclear cells cultured in the presence of cortisone (final concentration 500 nM) was significantly higher than that of mononuclear cells cultured in the presence of cortisone + PF-915275, and was also significantly higher than the cortisol concentration in the culture medium containing cortisone but without mononuclear cells. This result indicates that mononuclear cells have the ability to convert 11βHSD1 substrates.

[0069] (Example 5) In Example 5, the substrate conversion ability of 11βHSD1 in mononuclear cells derived from adrenal insufficiency patients and healthy individuals was evaluated. Whole blood collected from the veins of human patients with adrenal insufficiency was dispensed into EDTA-containing blood collection tubes and refrigerated at 4°C for 30 minutes to 3 hours. Peripheral blood mononuclear cells were separated from 10 mL of whole blood using lymphocyte separation solution LSM, and cortisone (final concentration 500 nM) was added. The cells after addition were placed in a 24-well cell culture dish in a 1.5 × 10⁶ dish. ６ Seeds were seeded at a concentration of cells / well and incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C and 5% CO2. ２ Cell culture was performed in an incubator. After 48 hours, the cell supernatant was collected, and the substrate conversion ability of 11βHSD1 was evaluated by measuring the cortisol concentration in the culture medium using the ECLIA method. The same procedure was performed on healthy individuals. The procedure is illustrated in Figure 6.

[0070] Figure 7 shows the conditioned cortisol concentrations (μg / dL) of mononuclear cells derived from adrenal insufficiency patients or healthy individuals. The conditioned cortisol concentrations of mononuclear cells from healthy individuals ranged from approximately 1.93 to 2.16 μg / dL. On the other hand, the conditioned cortisol concentrations of mononuclear cells from adrenal insufficiency patients with glucocorticoid deficiency were higher than those of mononuclear cells from healthy individuals (ranging from approximately 2.39 to 4.36 μg / dL). However, the conditioned cortisol concentrations of mononuclear cells from adrenal insufficiency patients receiving glucocorticoid treatment were equivalent to or close to those of mononuclear cells from healthy individuals. This indicates that post-culture cortisol concentration, as an indicator of 11βHSD1 substrate conversion ability, is an excellent biomarker reflecting the clinical symptoms of adrenal insufficiency patients. This application claims the benefit of priority of Japanese Patent Application No. 2025-18333, filed on 6 February 2025, the contents of which are incorporated herein by reference.

[0071] This invention can be used as a novel biomarker for diseases such as adrenal insufficiency, and as a method for measuring the same.

Claims

1. A method for measuring the substrate conversion ability of 11β-hydroxysteroid dehydrogenase (11βHSD) in a subject, the method comprising: (i) obtaining a test sample containing blood or one or more components thereof taken from the subject; (ii) contacting the test sample with a substrate of 11βHSD; and (iii) measuring the substrate conversion ability by 11βHSD.

2. The method according to claim 1, characterized in that the substrate of 11βHSD is added in step (ii).

3. The method according to claim 2, wherein the step of measuring the substrate conversion ability of 11βHSD includes the step of measuring the concentration of the product from which the substrate has been converted.

4. The method according to claim 3, wherein the substrate conversion ability of 11βHSD is an indicator for diagnosing or monitoring a disease related to the production of cortisol or its analogues in the subject.

5. The method according to claim 4, characterized in that the indicator is that the concentration of the product is equal to or greater than a specified concentration, or that the concentration increases by a specified amount or more due to the contact.

6. The method according to claim 4, wherein the disease is an endocrine disease.

7. The method according to claim 6, wherein the endocrine disorder is an adrenal gland-related disorder.

8. The method according to claim 7, wherein the adrenal gland-related disease is selected from the group including adrenal insufficiency, primary aldosteronism, Cushing's syndrome, pheochromocytoma, adrenal incidentaloma, adrenal insufficiency, and congenital adrenal hyperplasia.

9. The method according to claim 8, wherein the adrenal gland-related disease is adrenal insufficiency.

10. The method according to claim 1, wherein the 11βHSD includes type 1 11βHSD (11βHSD1).

11. The method according to claim 3, wherein the substrate and the product are glucocorticoids.

12. The method according to claim 11, wherein the substrate is cortisone or an analog thereof, and the product is cortisol or an analog thereof.

13. The method according to claim 1, wherein the test sample is peripheral blood mononuclear cells.

14. A therapeutic agent for a disease related to the production of cortisol or its analogues in a subject, comprising a hormone regulator, wherein the therapeutic agent is administered according to the substrate conversion ability measured by the method of claim 1.

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