Method of detecting androgenetic alopecia

Abstract

To provide a method of detecting androgenetic alopecia or the risk thereof.SOLUTION: A method of detecting androgenetic alopecia or the risk thereof is provided, comprising measuring a ratio (DHT / T) of the dihydrotestosterone (DHT) level to the testosterone (T) level in skin surface lipids collected from the head of a subject.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for detecting androgenetic alopecia or the risk thereof, and a method for predicting one or more of the average hair diameter (μm), vellus hair rate (%), and short hair rate (%) of scalp hair. [Background technology]

[0002] Although hair loss is a physiological phenomenon, alopecia in which a large amount of hair falls out has a large impact on the appearance impression and therefore has a large impact on QOL (Quality of life). Androgenetic alopecia (AGA), a type of alopecia, is a type of alopecia that begins after puberty and progresses gradually. The pathology of AGA is that the growth phase shortens during the repeated hair cycle and the number of hair follicles that remain in the resting phase increases, and clinically, the hair on the frontal and vertex of the head becomes soft, thin and short, and finally the hair does not appear on the skin surface (Non-Patent Document 1).

[0003] It is believed that genetic background and male hormones are involved in AGA. In the hair follicle hair papilla cells that are distributed mainly on the frontal and vertex areas, the male hormone (androgen) testosterone (T) is converted to the more active dihydrotestosterone (DHT) by the action of 5α-reductase and binds to the male hormone receptor. It has been reported that the male hormone receptor of the hair follicle to which dihydrotestosterone is bound induces TGF-β and DKK1, suppressing the proliferation of hair matrix cells and shortening the anagen phase (Non-Patent Document 1).

[0004] For the treatment of AGA, various quasi-drugs and symptomatic treatments are recommended, mainly oral medications (finasteride, dutasteride) and topical medications (minoxidil). The oral medication finasteride is an inhibitor of type II 5α-reductase, which converts testosterone into a more potent dihydrotestosterone, while dutasteride simultaneously inhibits type I and type II 5α-reductase. The topical medication minoxidil was discovered and developed by chance when hirsutism appeared as a side effect in clinical trials of antihypertensive drugs, and its hair growth effects have been reported to include promoting blood flow and promoting the production of cell growth factors (Non-Patent Document 2). Many quasi-drugs have been approved, including adenosine, which promotes the production of FGF-7, a hair growth promoting factor, and t-flavanone, which is known to inhibit the activity of TGF-β, a hair matrix cell proliferation inhibitor, and to promote the production of DSG, a cell adhesion molecule (Non-Patent Document 3).

[0005] AGA is diagnosed mainly by interview and visual examination due to its visibility, but in treatment, androgen measurements in blood and hair are often performed. It has been reported that the amount of dihydrotestosterone in plasma and hair and the ratio of dihydrotestosterone to testosterone are higher than in healthy people, and that the ratio decreases after administration of finasteride (Non-Patent Documents 4, 5). Dihydrotestosterone levels are used to confirm AGA, confirm the risk of AGA progression, and confirm the effectiveness of treatment. However, blood sampling and hair plucking are painful, and each measurement is mentally painful. It has also been reported that the ratio of dihydrotestosterone to testosterone in hair on the top of the head decreases after administration of finasteride, while the ratio in hair on the back of the head does not decrease significantly (Non-Patent Document 5), and the relationship between the ratio and AGA pathology is unclear. Recently, a method has been developed to analyze the amount of dihydrotestosterone contained in sebum on the forehead to evaluate AGA risk, etc. (e.g., Non-Patent Document 6), but a method that can detect AGA with higher accuracy, non-invasiveness, ease, and painlessness is desired.

[0006] On the other hand, Patent Document 1 has discovered that it is possible to extract nucleic acids such as RNA from skin surface lipids (SSL), and has proposed the application of RNA from SSL, which can be collected non-invasively and easily, to biomarker discovery and diagnostic techniques. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 008319 [Non-patent literature]

[0008] [Non-Patent Document 1] Committee for the Creation of Guidelines for the Treatment of Male and Female Pattern Hair Loss, Male Pattern Hair Loss Guidelines 2017 Edition, Journal of the Japanese Society of Dermatology, 127:2763-2777, 2017 [Non-Patent Document 2] Susumu Kotomo, On the hair growth effect of minoxidil, Japanese Pharmacology Journal, 119:167-174, 2002 [Non-Patent Document 3] Tokuro Iwabuchi, Development and Evaluation Methods of Hair Growth Drugs (Past and Future), Journal of the Japanese Society of Cosmetic Science, 42(2);98-103, 2018 [Non-Patent Document 4] Journal of Dermatological Science, 34(1), 11-16, 2004 [Non-Patent Document 5] Br. J. Dermatol. 154(4), 730-734, 2006 [Non-Patent Document 6] “AGA Test by Milion”, [online], Milion Co., Ltd., [searched on September 25, 2023], Internet<URL:https: / / www.agatestlab.com / agadhttest> Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention relates to providing a method for detecting male pattern baldness or the risk thereof. [Means for solving the problem]

[0010] The present inventors have found that low molecular weight components derived from skin surface lipids other than nucleic acids can be prepared from skin surface lipids collected from subjects, and have previously filed a patent application (Patent Application No. 2023-24556). Then, skin surface lipids were collected from the heads of AGA sufferers and healthy subjects, and androgens derived from the skin surface lipids were analyzed. As a result, it was found that the ratio of the amount of dihydrotestosterone (DHT) to the amount of testosterone (T) increased depending on the severity of AGA, and that AGA can be accurately detected by using this ratio as an indicator. In addition, it was found that the ratio of the amount of dihydrotestosterone (DHT) to the amount of testosterone (T) significantly correlates with the average hair diameter, vellus hair rate, and short hair rate of the hair, and that the average hair diameter, vellus hair rate, and short hair rate can be predicted from this ratio.

[0011] That is, the present invention relates to the following 1) to 4). 1) A method for detecting male pattern baldness or the risk thereof, comprising measuring the ratio of the amount of dihydrotestosterone (DHT) to the amount of testosterone (T) (DHT / T ratio) in lipids on the surface of the skin sampled from the subject's head. 2) A method for predicting one or more of the following: average hair diameter (μm), vellus hair rate (%), and short hair rate (%), which includes measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) (DHT / T ratio) in skin surface lipids collected from the subject's head. 3) A method for evaluating the effectiveness of a preventive or therapeutic intervention for male pattern baldness, comprising measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) (DHT / T ratio) in surface lipids of the skin collected from the subject's head in the presence of the intervention. 4) A test kit for detecting male pattern baldness or the risk thereof used in the above-described method, containing tools and reagents necessary for collecting and storing lipids on the skin surface, and a reagent for measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) in lipids on the skin surface (DHT / T ratio), a kit for predicting one or more of the average hair diameter (μm), vellus hair rate (%), and short hair rate (%), or a kit for evaluating the effect of an intervention. Effect of the Invention

[0012] According to the present invention, it is possible to detect male pattern baldness or its risk with high accuracy, non-invasive and simple.In addition, it is possible to predict at least one of the average hair diameter (μm), soft hair rate (%) and short hair rate (%) of head hair.Therefore, it is possible to take appropriate preventive or therapeutic measures against the male pattern baldness. [Brief description of the drawings]

[0013] [Figure 1] The androgen abundance ratio in patients with AGA is shown. [Diagram 2] The amount of androgen per weight of sebum in patients with AGA is shown. [Diagram 3] The relationship between the occipital DHA / T ratio and the average hair diameter (μm) and vellus hair rate (%) is shown. [Figure 4] The relationship between the amount of DHA on the back of the head and the average hair diameter (μm) and the rate of vellus hair (%) is shown. [Diagram 5] The relationship between the vertex DHA / T ratio and the average hair diameter (μm) and vellus hair rate (%) is shown. [Figure 6] The relationship between the amount of DHA on the top of the head and the average hair diameter (μm) and the rate of vellus hair (%) is shown. [Figure 7] The relationship between the DHT / T ratio and the short hair rate (%) at the back and top of the head is shown. [Figure 8] The relationship between the amount of DHT on the back and top of the head and the rate of short hair (%) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] All patents, non-patent publications, and other publications cited herein are hereby incorporated by reference in their entirety.

[0015] In the present invention, "androgenetic alopecia (AGA)" refers to alopecia that begins after puberty and progresses gradually. The pathology of AGA is that the growth phase shortens during the hair cycle and the number of hair follicles that remain in the resting phase increases, and clinically, the hair on the frontal and vertex of the head becomes soft, thin, and short, and finally the hair disappears from the skin surface.

[0016] The severity of male pattern baldness is measured by, for example, the Ogata classification (Itami Satoshi: Survey on awareness of hair (male pattern baldness) among Japanese adult men. Japan Medical News: No. 4209, 27-29, 2004), the Hamilton-Norwood classification, and the improved Takashima classification (Takashima I, Iju M, Sudo M: Alopecia androgenetica. Its incidence in Japanese and associated condition. In: Orfa- nos CE, Montagna W, Stuttgen G, eds. Hair Research, Berlin: Springer Verlag, 1981; 287-293.), which are classifications of progression patterns of hair thinning. Depending on these progression patterns of hair thinning and a doctor's diagnosis, hair may simply be classified as "not alopecia," "moderate," or "severe." The severity can be measured by, for example, the hair loss pattern according to the above classification, the average hair diameter (μm), the vellus hair rate (%), and the hair density (hairs / cm 2 ) and short hair rate (%), and visual examination by a doctor. 2In the present invention, the term "severe" refers to a condition classified as type II or IV in the final stage of the Ogata classification, in which the average hair diameter (μm) at the top of the head is 26.7±7.1, the vellus hair rate (%) is 88.2±14.4, and the density (hairs / cm 2 "Moderate" refers to a condition in which the average hair diameter (μm) at the top of the head is 40.5±6.7, the vellus hair rate (%) is 53.2±20.6, and the density (hairs / cm 2 "No alopecia" refers to a state in which the average hair diameter (μm) on the top of the head is 65.5±10.4, the vellus hair rate (%) is 19.7±9.3, and the density (hairs / cm 2 ) is 237.2±46.4 and the short hair rate (%) is 32.7%±12.9. Average hair diameter (μm) and hair density (hairs / cm 2 ) can be measured by the phototrichogram method. The vellus hair rate (%) can be expressed as the percentage of hairs with a diameter of less than 40 μm among the number of hairs in a certain area. The short hair rate (%) can be expressed as the percentage of hairs with tips of 3 cm or less among the number of hairs in a certain area (7 x 7 mm). An increase in the short hair rate (%) indicates a shortening of the hair cycle, and the short hair rate is thought to reflect the state of the hair cycle. It is well known that the hair cycle shortens in male pattern baldness.

[0017] In the present invention, "detection of androgenetic alopecia" means clarifying the presence or absence of androgenetic alopecia. "Risk of androgenetic alopecia" includes risk of onset of androgenetic alopecia and risk of worsening of androgenetic alopecia, and "detection of risk of androgenetic alopecia" includes detection of the possibility of onset of androgenetic alopecia or worsening of androgenetic alopecia, or the degree of possibility. "Detection" can also be expressed in other terms such as examination, measurement, judgment, or evaluation. In the present invention, the terms "detection," "examination," "measurement," "judgment," or "evaluation" of androgenetic alopecia or the risk thereof do not include diagnosis by a doctor.

[0018] In the present invention, the term "head" refers to the area that is covered by the scalp and can have hair, consisting of the "top", "back" and "temporal" parts excluding the face. The face is located on the front of the head and includes the sensory organs such as the eyes, nose, mouth and ears. The "top" is located at the top of the head and includes the hairline, which affects the appearance and hairstyle and characterizes the shape of the head. The "back" is the part at the back of the head and protects the brain as part of the skull. It supports the stability and movement of the head and is also the connection point with the neck. The "temporal" is the part that extends to both sides of the head and forms the sides of the skull.

[0019] In the present invention, "skin surface lipids (SSL)" refers to the fat-soluble fraction present on the surface of the skin, and is sometimes called sebum. Generally, SSL mainly contains secretions secreted from exocrine glands such as sebaceous glands in the skin, and is present on the skin surface in the form of a thin layer covering the skin surface. In addition, unless otherwise specified, "skin" is a general term for an area including tissues such as the stratum corneum, epidermis, dermis, hair follicles, sweat glands, sebaceous glands, and other glands. The site of the skin is the head, preferably the top of the head or the back of the head, and more preferably the back of the head.

[0020] As shown in the examples below, androgens prepared from sebum (SSL) collected from the heads (vertex and occipital regions) of AGA sufferers (moderate and severe AGA) and healthy individuals were analyzed, and the results showed that the ratio of dihydrotestosterone (DHT) to testosterone (T) (DHT / T ratio) increased in both regions depending on the severity of AGA. It was also shown that a discriminant (AGA detection model) using the DHT / T ratio as a feature value can detect AGA with high accuracy. On the other hand, no increase in the amount of dihydrotestosterone (DHT) depending on the severity of AGA was observed, suggesting that the average accuracy of the amount of dihydrotestosterone (DHT) is low and AGA cannot be detected. Furthermore, the DHT / T ratio was significantly correlated with the average hair diameter (μm), the rate of vellus hair (%), and the rate of short hair (%).

[0021] From the above, it is possible to detect AGA or its risk by using the ratio of dihydrotestosterone (DHT) to testosterone (T) in SSL taken from the head (DHT / T ratio) as an index. In the present invention, the head is preferably the vertex or the back of the head, more preferably the back of the head. In addition, the detection of AGA or its risk is preferably the detection of moderate AGA or its risk. Furthermore, in the present invention, the average hair diameter (μm), the vellus hair rate (%) or the short hair rate (%) of scalp hair can be predicted by utilizing the correlation between the ratio of the amount of dihydrotestosterone (DHT) to the amount of testosterone (T) (DHT / T ratio) in SSL collected from the head and the average hair diameter (μm), the vellus hair rate (%) or the short hair rate (%).

[0022] Therefore, the present invention provides a method for detecting AGA or a risk thereof, comprising measuring the DHT / T ratio in SSL collected from the head of a subject. The method may further comprise detecting the severity of AGA. The present invention also provides a method for predicting one or more of the average hair diameter (μm), vellus hair rate (%), and short hair rate (%), comprising measuring the DHT / T ratio in SSL collected from the head of a subject.

[0023] In the present invention, in order to improve accuracy, in addition to DHT / T ratio, other androgen ratios can be measured.Here, examples of other androgens other than dihydrotestosterone (DHT) and testosterone (T) include androstenedione (A-dione) and dehydroepiandrosterone (DHEA).In addition, examples of other androgen ratios other than DHT / T ratio include A-dione / DHEA ratio, T / DHEA ratio, DHT / DHEA ratio, T / A-dione ratio, and DHT / A-dione ratio. In the present invention, from the viewpoint of improving accuracy, it is preferable to use, in addition to the DHT / T ratio, one or more, two or more, three or more, or five or more selected from the A-dione / DHEA ratio, T / DHEA ratio, DHT / DHEA ratio, T / A-dione ratio, and DHT / A-dione ratio, i.e., a combination of six ratios, namely the DHT / T ratio, the A-dione / DHEA ratio, the T / DHEA ratio, the DHT / DHEA ratio, the T / A-dione ratio, and the DHT / A-dione ratio. The A-dione / DHEA ratio and the T / DHEA ratio were low depending on the severity of AGA, whereas the DHT / A-dione ratio and the DHT / T ratio were high depending on the severity of AGA.

[0024] The method for detecting AGA or the risk thereof in the present invention, and the method for predicting one or more of the average hair diameter (μm), the vellus hair rate (%), and the short hair rate (%) (hereinafter collectively referred to as the methods of the present invention) may further include collecting SSL from the subject's head.

[0025] A "subject" may be any person having an SSL on the skin. An example of a subject is a man who desires or needs to detect AGA or its risk. For example, a man who has subjective symptoms of softening of the scalp hair, a man who is suspected of having AGA, etc.

[0026] The SSL can be harvested from the subject's head by any means commonly used to retrieve or remove SSL from the skin, preferably using an SSL absorbent material, an SSL adhesive material, or a device for scraping the SSL from the skin, as described below. The SSL absorbent material or SSL adhesive material is not particularly limited as long as it has an affinity for SSL, and examples thereof include polypropylene and pulp. More detailed examples of procedures for collecting SSL from the skin include a method of absorbing SSL into a sheet-like material such as oil blotting paper or oil blotting film, a method of adhering SSL to a glass plate or tape, and a method of scraping SSL off and collecting it with a spatula, scraper, etc. In order to improve the adsorption of SSL, an SSL absorbent material that has been previously impregnated with a highly lipid-soluble solvent may be used. On the other hand, the SSL absorbent material preferably contains a low content of highly water-soluble solvent or water, since the adsorption of SSL is inhibited if the SSL absorbent material contains a highly water-soluble solvent or water. It is preferable to use the SSL absorbent material in a dry state.

[0027] The SSL collected from the subject may be stored for a certain period of time. In order to minimize the degradation of the RNA contained in the SSL, it is preferable to store the collected SSL under low temperature conditions as soon as possible after collection. The temperature condition for storing the SSL in the present invention may be 0° C. or lower, preferably −20±20° C. to −80±20° C., more preferably −20±10° C. to −80±10° C., even more preferably −20±20° C. to −40±20° C., even more preferably −20±10° C. to −40±10° C., even more preferably −20±10° C., and even more preferably −20±5° C. The period for storing the SSL under the low temperature condition is not particularly limited, but is preferably 12 months or less, for example, 6 hours or more and 12 months or less, more preferably 6 months or less, for example, 1 day or more and 6 months or less, even more preferably 3 months or less, for example, 3 days or more and 3 months or less.

[0028] The extraction of androgens from SSL involves the following steps (1) and (2): (1) A process in which a strong base aqueous solution is added to the organic layer obtained by phenol-chloroform extraction of the surface skin lipids (SSL) collected from a subject, mixed, and then the separated aqueous layer is removed. (2) A step of adding water to the organic layer after removing the aqueous layer, mixing the organic layer, removing the separated aqueous layer, and recovering the organic layer. This can be done by:

[0029] In step (1), phenol-chloroform extraction can be performed according to a conventional method. For example, phenol solution and then chloroform are added to the nucleic acid extraction sample, mixed, and then centrifuged. Centrifugation separates the sample into an aqueous layer and an organic layer (phenol-chloroform layer). The aqueous layer contains RNA in the upper layer, the organic layer contains androgens and proteins in the lower layer, and an intermediate layer containing DNA is formed at the boundary between the aqueous and organic layers. In the extraction of RNA from SSL, the upper aqueous layer is collected and the lower organic layer is discarded as RNA extraction residue, but in the present invention, the androgens contained in the lower organic layer are extracted from the organic layer.

[0030] The strong base aqueous solution used in step (1) may be an aqueous solution of a strong base such as an alkali metal or alkaline earth metal hydride, hydroxide, carbonate, or alkoxide. Specific examples include aqueous solutions of sodium hydride, potassium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, and sodium methoxide. Among these, an aqueous solution of sodium hydroxide is preferred from the viewpoints of availability and versatility. The strong base may be used alone or in combination of two or more. The water for the strong base aqueous solution may be tap water, purified water, distilled water, ion-exchanged water, pure water, ultrapure water, or the like.

[0031] In step (1), the amount of strong base aqueous solution added can be appropriately selected as long as the phenol in the organic layer is transferred to the aqueous layer, but from the viewpoint of facilitating the transfer of phenol from the organic layer to the aqueous layer, it is preferable that the amount is such that the value obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution [X(N)×Y(L)] is three times or more the volume (L) of the organic layer. The relationship between the normality X(N) of the strong base, the volume Y(L) of the strong base aqueous solution, and the volume (L) of the organic layer is shown in the following formula (1). X × Y ≧ ​​volume of organic layer × 3 (1) From the same viewpoint, the amount of the strong base aqueous solution added is preferably an amount such that the value [X(N)×Y(L)] obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution is 3 times or more the volume (L) of the organic layer, and from the viewpoint of improving the extraction efficiency and recovery rate of androgens, the amount is preferably an amount such that the value [X(N)×Y(L)] obtained by multiplying the normality X(N) of the strong base by the volume Y(L) of the strong base aqueous solution is 3 to 9.2 times, further 3 to 6 times, further 3 to 5 times, further 3 to 4 times the volume (L) of the organic layer.

[0032] The normality (N) of the strong base can be appropriately adjusted according to the amount of the strong base aqueous solution added and the volume of the strong base aqueous solution. The normality (N) of the strong base is preferably 0.2N or more, more preferably 0.4N or more, more preferably 0.5N or more, more preferably 0.8N or more, more preferably 1.0N or more, more preferably 1.8N or more, more preferably 3.0N or more, even more preferably 4.0N or more, even more preferably 4.3N or more, and even more preferably 6.0N or more, from the viewpoint of separating the organic layer and the aqueous layer and improving the recovery rate of the androgen. In addition, the upper limit of the normality (N) of the strong base is preferably 12.0N or less, more preferably 8.0N or less, from the viewpoint of improving the extraction efficiency and recovery rate of the androgen. The normality (N) of the strong base is preferably 0.2 to 12.0N, more preferably 0.4 to 12.0N, more preferably 0.5 to 12.0N, more preferably 0.8 to 12.0N, more preferably 1.0 to 12.0N, more preferably 1.8 to 8.0N, even more preferably 3.0 to 8.0N, even more preferably 4.0 to 8.0N, even more preferably 4.3 to 8.0N, and still more preferably 6.0 to 8.0N.

[0033] The volume (L) of the strong base aqueous solution can be appropriately adjusted depending on the amount of the strong base aqueous solution added and the normality (N) of the strong base, but it is preferable that the volume (L) is 0.9 times or more the volume of the organic layer from the viewpoint of improving the recovery rate of the androgen. From the same viewpoint, the volume (L) of the strong base aqueous solution is preferably 0.92 times or more, more preferably 1.0 times or more, more preferably 2.0 times or more, more preferably 3.8 times or more, more preferably 5.5 times or more, and more preferably 7.0 times or more, relative to the volume (L) of the organic layer, and from the viewpoint of operability, is preferably 20 times or less, more preferably 10 times or less.

[0034] In step (1), it is preferable to further add an organic solvent to the organic layer obtained by phenol-chloroform extraction from the SSL from the viewpoint of improving operability. The organic solvent is not particularly limited, but examples thereof include alcohols such as methanol, ethanol, propanol, and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol, and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; linear and cyclic ethers such as tetrahydrofuran and diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform, and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane, and petroleum ether; aromatic hydrocarbons such as benzene and toluene; and pyridines. These can be used alone or in combination of two or more. Among them, alcohols, halogenated hydrocarbons, and hydrocarbons are preferred, and chloroform and a chloroform-methanol mixed solution are more preferred. The ratio (volume ratio) of chloroform in the chloroform-methanol mixed solution is preferably 53% or more, more preferably 55% or more, from the viewpoint of facilitating the transfer of phenol from the organic layer to the aqueous layer and improving the recovery rate, and is preferably 75% or more, more preferably 90% or more, and even more preferably 100%, from the viewpoint of the extraction efficiency of androgens. The ratio (volume ratio) of chloroform in the chloroform-methanol mixed solution is preferably 53 to 100%, more preferably 55 to 100%, even more preferably 75 to 100%, and even more preferably 90 to 100%.

[0035] The amount of the organic solvent added is preferably 1 or more times the volume (L) of the organic layer.

[0036] The organic layer and the aqueous layer can be separated by means of static separation, centrifugation, or the like. The separation conditions can be appropriately adjusted, but in the case of centrifugation, the conditions are preferably adjusted under normal pressure, 5,000 to 20,000 r / min, and for 5 to 30 minutes. The temperature during separation is preferably 20°C or lower, more preferably 10°C or lower, for example, 4°C. In step (1), by adding a strong base aqueous solution to the organic layer and mixing, the phenol in the organic layer becomes a phenoxide and migrates to the upper aqueous layer, and the aqueous layer containing the phenoxide is separated and removed, so that the phenol can be removed from the organic layer. The organic layer containing the androgen after the aqueous layer is removed is provided to the next step.

[0037] In step (2), the operation of adding water to the organic layer, mixing, and then removing the separated aqueous layer may be performed once or multiple times, for example, may be repeated two or three times. From the viewpoint of efficiently removing the strong base remaining in the organic layer, it is preferable to repeat the operation multiple times. Examples of water include tap water, purified water, distilled water, ion-exchanged water, pure water, and ultrapure water.

[0038] The amount of water added to the organic layer is preferably at least 1 time the volume (L) of the organic layer. The amount of water added is the value per one addition.

[0039] The organic layer and the aqueous layer can be separated by means of static separation, centrifugation, or the like. The separation conditions can be appropriately adjusted, but in the case of centrifugation, the conditions are preferably adjusted under normal pressure, 5,000 to 20,000 r / min, and for 5 to 30 minutes. The temperature during separation is preferably 20°C or lower, more preferably 10°C or lower, for example, 4°C. By adding water to the organic layer and mixing, the strong base remaining in the organic layer migrates to the upper aqueous layer. The aqueous layer containing the strong base is then separated and removed, allowing the organic layer containing the androgen to be recovered. The recovered organic layer may be dried, if necessary, by, for example, vacuum drying, freeze drying, spray drying, heat drying, or the like.

[0040] In this manner, androgens can be prepared from the SSL collected from the subject. In the present invention, the DHT / T ratio in SSL can be measured by various instrumental analyses such as mass spectrometry (e.g., LC-MS / MS, MALDI-TOF / MS, CE-MS, CE-TOF / MS) and nuclear magnetic resonance (NMR), physical property measurements (e.g., methods for measuring redox potential, pH, ultraviolet-visible absorbance, etc.), immunological measurements, and biochemical measurements, which are common methods for quantifying androgens.

[0041] Thus, the DHT / T ratio in the SSL collected from the subject's head is measured, and AGA or its risk in the subject is detected based on the DHT / T ratio. Also, the average hair diameter (μm), vellus hair rate (%) or short hair rate (%) of the hair is predicted by utilizing the correlation between the DHT / T ratio and the average hair diameter (μm), vellus hair rate (%) or short hair rate (%) of the hair. In one example, the method of the present invention is carried out by comparing the measured DHT / T ratio with a cutoff value (reference value). More specifically, by comparing the DHT / T ratio in the SSL collected from the subject's head with a cutoff value (reference value), AGA or its risk can be detected for the subject. In addition, any one or more of the average hair diameter (μm), vellus hair rate (%) and short hair rate (%) of the head hair can be predicted. Furthermore, the severity of AGA can be detected.

[0042] Here, the "cut-off value" ("reference value") can be set arbitrarily depending on the purpose, etc. For example, a certain population is divided into an AGA-afflicted group and an AGA-non-afflicted group, and a value determined with reference to statistical values ​​such as the average value and standard deviation of the DHT / T ratio of the present invention in each group can be determined as a cut-off value (reference value) for determining whether or not a subject belongs to each group, or predicting the average hair diameter (μm) and / or vellus hair rate (%). Furthermore, when detecting the severity of AGA, the group affected by AGA can be divided into groups with different severity of AGA (e.g., moderate and severe groups), and a value determined based on statistical values ​​such as the average value and standard deviation of the DHT / T ratio of the present invention in each group can be determined as a cutoff value (reference value) for determining whether or not the subject belongs to each group.

[0043] The cutoff value (reference value) can be determined by various statistical analysis methods. For example, values ​​based on ROC curve (Receiver Operating Characteristic Curve) analysis are exemplified. The ROC curve can be created by determining the probability (%) of a positive result in a positive patient (True Positive Rate (TPF), sensitivity) and the probability (%) of a negative result in a negative patient (specificity) based on the DHT / T ratio in SSL measured from a subject population, and plotting the sensitivity against [100-specificity] (False Positive Rate (FPF)). The point on the ROC curve to be adopted as the cutoff value (reference value) can be determined based on various conditions. In general, to increase both sensitivity and specificity (approaching 100%), the cutoff value (reference value) is set to the expression level at the point closest to (0,100) on the ROC curve with the true positive rate (sensitivity) on the vertical axis (Y axis) and the false positive rate on the horizontal axis (X axis), or to the expression level at the point where [true positive (sensitivity) - false positive (100 - specificity)] is maximum (Youden index). In the method of the present invention, when ratios between other androgens are used in addition to the DHT / T ratio, it is preferable to determine cutoff values ​​(reference values) for the ratios between each androgen. Groups may be formed based on race and age.

[0044] For example, if the DHT / T ratio in the SSL taken from the subject's head is higher than the cutoff value (reference value), the subject can be determined to have AGA or have a high probability of having AGA, or to have a high risk of AGA; if not, the subject can be determined to not have AGA, have a low probability of having AGA, or have a low risk of AGA. Furthermore, when detecting the severity of AGA, if the DHT / T ratio in the SSL sampled from the subject's head is higher than a cutoff value (reference value) based on a certain AGA severity group (e.g., moderate), the subject can be determined to have or be likely to have AGA severity of that level or higher (e.g., moderate), and if not, the subject can be determined to have or be likely to have AGA severity of that level or lower (e.g., mild or lower).

[0045] In the method of the present invention, if the DHT / T ratio in SSL is preferably 110% or more, more preferably 150% or more, and even more preferably 200% or more, relative to the cutoff value (reference value), the DHT / T ratio can be judged to be higher than the cutoff value (reference value). Alternatively, the difference between the DHT / T ratio in SSL and the cutoff value (reference value) can be judged, for example, by whether or not the two are statistically significantly different.

[0046] Furthermore, a discriminant (prediction model) for detecting AGA or the risk of AGA, and a prediction model for predicting one or more of the average hair diameter (μm), the soft hair rate (%), and the short hair rate (%) can be constructed, and the prediction model can be used to detect AGA or the risk of AGA, or to predict one or more of the average hair diameter (μm), the soft hair rate (%), and the short hair rate (%). For example, a discriminant (prediction model) for separating AGA-affected and non-affected groups can be constructed using the measured values ​​of DHT / T ratio from AGA-affected and non-affected individuals as teacher samples. Furthermore, a cutoff value (reference value) for detecting AGA or its risk can be determined based on the discriminant. In creating the discriminant, dimensionality can be reduced by principal component analysis (PCA), and the main components can be used as explanatory variables. The DHT / T ratio in the SSL taken from the subject's head is then measured in the same manner, and the measured value obtained is substituted into the discriminant formula. Based on the results obtained from the discriminant formula, it may be determined whether or not the subject is classified as suffering from AGA. By comparing the results with a cutoff value (reference value), AGA or the risk of AGA can be detected for the subject.

[0047] Furthermore, when detecting the severity of AGA, a discriminant (prediction model) for distinguishing between AGA of different severity can be constructed using the measured values ​​of the DHT / T ratio from AGA patients with different severity and the DHT / T ratio from AGA-free patients, and the discriminant can be used to detect AGA as well as the severity of AGA. In other words, using the DHT / T ratios from AGA patients with different severity levels and the measured values ​​of the DHT / T ratios from AGA-free patients as teacher samples, a discriminant (prediction model) is constructed to separate AGA-free groups from groups with different severity levels (e.g., mild, moderate, and severe groups). Furthermore, a cutoff value (reference value) for distinguishing each AGA-free group from the AGA-free group is determined based on the discriminant. The DHT / T ratio in SSL taken from the subject's head is then measured in the same manner, and the measured value obtained is substituted into the discriminant formula. The severity of AGA can be determined based on the results obtained from the discriminant formula, and by comparing with a cutoff value (reference value), AGA can be detected in the subject as well as the severity of AGA.

[0048] The variables used to construct the discriminant equation include explanatory variables and objective variables. For example, the DHT / T ratio (feature) can be used as an explanatory variable. For example, the degree of AGA susceptibility of the sample (AGA susceptible group or non-AGA susceptible group) can be used as an objective variable.

[0049] The feature value can be a statistically significant difference between the two groups to be discriminated. The feature value for constructing the prediction model used in the method of the present invention can be the DHT / T ratio or the ratio between the other androgens mentioned above.

[0050] The algorithm for constructing the discriminant may be a known algorithm such as an algorithm used in machine learning. Examples of machine learning algorithms include random forest, support vector machine with linear kernel (SVM linear), support vector machine with rbf kernel (SVM rbf), neural net, generalized linear model, regularized linear discriminant analysis, regularized logistic regression, etc. A predicted value is calculated by inputting data for verification into the constructed prediction model, and a model whose predicted value is most compatible with the actual measured value, for example, a model with the highest accuracy rate, can be selected as the optimal prediction model. In addition, the detection rate (Recall), precision, and the F value, which is the harmonic mean thereof, can be calculated from the predicted value and the actual measured value, and the model with the highest F value can be selected as the optimal prediction model.

[0051] When using a random forest algorithm to construct a discriminant equation, the OOB error rate can be calculated as an indicator of the accuracy of the predictive model (Breiman L. Machine Learning (2001) 45;5-32).

[0052] In random forest, according to a technique called bootstrap method, about two-thirds of the total number of samples are randomly extracted, allowing overlaps, from all samples, to create a classifier called a decision tree. At this time, samples that are not extracted are called out of bug (OOB). By predicting the OOB objective variable using one decision tree and comparing it with the correct label, the error rate can be calculated (OOB error rate in decision tree). The same process is repeated 500 times, and the average value of the OOB error rates in the 500 decision trees can be used as the OOB error rate of the random forest model.

[0053] The number of decision trees (n_estimators value) used to construct a random forest model is 100 by default, but can be changed to any number as necessary. Furthermore, the number of variables (max_features value) used to create a sample discriminant in one decision tree is the square root of the number of explanatory variables by default, but can be changed to any value between 1 and the total number of explanatory variables as necessary.

[0054] The "scikit-learn" library in Python can be used to determine the max_features value. Random forest can be specified as the machine learning algorithm of the "scikit-learn" library, eight different max_features values ​​can be tried, and the max_features value that maximizes Accuracy can be selected as the optimal max_features value. Note that the number of trials for max_features values ​​can be changed to any number of trials as necessary.

[0055] In addition, the GridSearchCV function in the “scikit-learn” library can be used to find optimal hyperparameter values ​​such as the n_estimators value, max_features value, and max_depth, which is a parameter that controls how deeply the decision tree can branch, by using cross validation from all combinations of the specified parameters.

[0056] When using the random forest algorithm to construct a discriminant equation, the importance of the explanatory variables used to construct the model can be expressed as a numerical value (variable importance). For example, the mean decrease in the Gini coefficient (Mean Decrease Gini) can be used as the value of the variable importance.

[0057] The above-mentioned method for detecting AGA or the risk thereof of the present invention can be carried out in the presence of a preventive or therapeutic intervention against AGA to evaluate the effectiveness of the intervention. That is, the method for evaluating the intervention effect of the present invention involves measuring the DHT / T ratio in SSL taken from the subject's head in the presence of a preventive or therapeutic intervention against AGA. Furthermore, in the method for evaluating the intervention effect of the present invention, in addition to the DHT / T ratio, the ratio between the other androgens described above can be measured. The SSL, DHT / T ratio, ratio between other androgens, etc. are the same as those in the method of the present invention described above.

[0058] Here, the preventive or therapeutic intervention for AGA is an intervention carried out in the hope of preventing or treating AGA, and includes chemical intervention, physical or mechanical intervention. Chemical intervention includes the administration of natural substances, synthetic substances, compositions, and other substances, while physical or mechanical intervention includes the application of electromagnetic waves, light, and other radiation, application of heat, massage, and other treatments. Other options include interventions in the living environment and scalp environment, such as getting enough sleep, refraining from drinking and smoking, and eating a balanced diet.

[0059] The effect of the intervention can be evaluated based on the change in the DHT / T ratio in the SSL collected from the subject's head caused by the intervention. For example, the effect can be evaluated by comparing the DHT / T ratio in the presence of the intervention with that in the control (absence of the intervention) or by comparing the DHT / T ratio before and after the intervention. An intervention that reduces the DHT / T ratio can be evaluated as an effective intervention for preventing or treating AGA.

[0060] The kit for detecting AGA or the risk thereof, the kit for predicting one or more of the average hair diameter (μm), the soft hair rate (%), and the short hair rate (%), or the kit for evaluating the effect of an intervention are a kit for detecting AGA or the risk of AGA in a subject according to the method for detecting AGA or the risk of AGA according to the present invention, a kit for predicting one or more of the average hair diameter (μm), the soft hair rate (%), and the short hair rate (%) according to the prediction method for one or more of the average hair diameter (μm), the soft hair rate (%), and the short hair rate (%) according to the present invention, and a kit for evaluating the effect of an intervention according to the method for evaluating the effect of an intervention according to the present invention. The kit of the present invention contains tools and reagents necessary for collecting and storing SSL, and reagents for measuring the DHT / T ratio. The tools and reagents necessary for collecting and storing SSL include, for example, tools for collecting SSL (e.g., oil-removing film for collecting SSL), reagents for storing the collected SSL, and storage containers. Reagents for measuring the DHT / T ratio include, for example, reagents for extracting androgens from the collected SSL, pretreatment reagents, reagents for various instrumental analyses, physical property measurement methods, immunological measurement methods, or biochemical measurement methods, internal standards, instruments necessary for testing, as well as indicators or guidance for measuring the DHT / T ratio. EXAMPLES

[0061] Example 1. Analysis of androgens in scalp sebum 1. Subject profile and collection of sebum samples Male subjects in their 30s to 50s were classified by a doctor into a healthy group (control group), a moderate group, and a severe group with male pattern baldness (AGA). Basic data for each classification is shown in Table 1. Note that the short hair rate (%) was not calculated for three subjects in the severe AGA group due to reasons such as the lack of hair at the measurement site. Sebum used for androgen analysis was collected from each subject's entire face (Face), top of the head (Vertex), back of the head (Occiput), and the border between the bald and non-bald areas (Border) using oil blotting film (3M Japan).

[0062] [Table 1]

[0063] 2. Extraction of sebum androgens The following was carried out with reference to the method described in Japanese Patent Application No. 2023-24556. After cutting the oil blotting film from which the sebum was collected, 5 μl of the internal standard mixed methanol solution (containing DHT-d3: 400 pg, T-d3: 100 pg, A-dione-13C3: 100 pg, DHEA-d5: 200 pg) was dropped onto the film, and the solvent was air-dried. The oil blotting film from which the sebum was collected was cut into appropriate sizes, and RNA extraction was performed using QIAzol (Qiagen) reagent (containing 50 v / v% phenol) according to the attached protocol. That is, 1,450 μl of QIAzol was added to the cut film, SSL was extracted, and then 260 μl of chloroform was added to the extract and mixed, and the centrifugation operation (4 ° C, 15,000 rpm, 15 minutes) was performed. The two layers that result are the upper aqueous layer containing RNA, and the lower pale red RNA extraction residue containing androgens. This lower layer was used for androgen extraction. 600 μl of the extraction residue was transferred to a 5 ml tube, and 900 μl of chloroform was added. 3.0 ml of ultrapure water and 350 μl of 8N sodium hydroxide solution were then added. After thorough mixing with a vortex, the mixture was centrifuged at 3,000 rpm for 5 minutes. After carefully removing the upper layer, 600 μl of ultrapure water was added to the remaining lower layer, mixed with a vortex, and centrifuged at 15,000 rpm for 5 minutes. The resulting upper layer was carefully removed. This operation of adding pure water → mixing → centrifugation → removing the upper layer was repeated twice in total. All centrifugation operations were performed at 4°C. The remaining lower layer was finally concentrated by centrifugation under reduced pressure to dryness.

[0064] 3. Derivatization and Quantification by LC-MS / MS Analysis The dried sample was redissolved in 90 μl of 50 mM methoxylamine hydrochloride in methanol and reacted at 60°C for 30 minutes to derivatize androgens. After cooling on ice for about 5 minutes, 110 μl of 40% acetonitrile was added, mixed by vortexing, and filtered through a 0.45 μm syringe filter (DISMIC 13HP045AN, ADVANTEC). The filtrate was analyzed by LC-MS / MS, and the amount of androgen present in the film (pg / film) was quantified from a calibration curve created relative to each internal standard. The amount of androgen (pg / film) was also calculated by dividing the amount of androgen (pg / film) by the weight of sebum (mg) measured in advance. The lower limit of quantification (LLOQ) for DHEA, A-dione, T, and DHT in this analysis was 10, 10, 2, and 10 pg / film, respectively, but for DHT, about half of the samples were below the LLOQ. In order to ensure as many samples as possible, the analysis was performed on samples with values ​​of 1 / 2 × LLOQ or higher.

[0065] 4.Analysis of androgen ratio Six sebum androgen ratios, namely A-dione / DHEA, T / DHEA, T / A-dione, DHT / DHEA, DHT / A-dione and DHT / T ratios in samples from the control group and the AGA group (moderate and severe), were compared and analyzed by site (whole face (Face), vertex (Vertex), occiput (Occiput) and border (Border)). The analysis showed that there was no significant difference in the androgen ratios in the whole face depending on the presence or absence of AGA or the severity. On the other hand, the A-dione / DHEA ratio and the T / DHEA ratio were significantly lower in the severe AGA group than in the control group at the occiput and vertex, respectively. On the other hand, the DHT / A-dione ratio was significantly higher in the severe AGA group than in the control group at the occiput, tending to be dependent on the severity of AGA. The DHT / T ratio was also shown to be significantly higher in both the parietal and occipital regions depending on the severity of AGA (Figure 1). The DHT / T ratio in the border region showed a similar trend. The DHT / T ratio of AGA sufferers (moderate and severe AGA) was about 1-2 in both the parietal and occipital regions. This suggests that androgen ratios, including the DHT / T ratio in scalp sebum, may be useful for evaluating the severity of AGA and the risk of developing the condition. Statistical tests were performed to test for significant differences between groups using the Dewas-Steele-Critchlow-Fligner method or the Conover test.

[0066] 5. Analysis of androgen content per sebum weight (Comparative example) The amount of androgen per sebum weight (pg / mg-sebum) in samples from the control group and the AGA group (moderate and severe) was compared and analyzed by site (whole face (Face), top of the head (Vertex), back of the head (Occiput), and border area (Border)). Samples with negative values ​​for sebum weight due to measurement errors, etc. were excluded from the analysis. The analysis showed that the amount of T was significantly lower in the severe AGA group than in the control group at the top of the head. Meanwhile, the amount of DHT was significantly higher in the severe AGA group than in the control group at the back of the head, and significantly higher in the severe AGA group than in the moderate AGA group, but there was no significant difference between the control group and the moderate AGA group, and the control group had a higher value in the median comparison (Figure 2). Therefore, no increase in DHT amount was observed depending on the severity. As with the analysis of the androgen ratio, no significant differences were observed in the face depending on the presence or absence of AGA or the severity of the condition.

[0067] 6. Discriminant model between the control group and the moderate AGA group A machine learning model was constructed to distinguish between the control group and the moderate AGA group using sebum androgen data. The data was divided into training data: evaluation data = 7:3, a model was constructed using the training data, and accuracy was evaluated using the evaluation data. A random forest was used as the algorithm, and the hyperparameters n_estimators and max_depth were optimized by grid search from among [1, 5, 10, 50, 100]. The contents of the divided data were randomly changed, and a total of five models were constructed and the average accuracy was calculated. Three types of models were constructed and evaluated based on differences in the features used in the models (Table 2).

[0068] As a result, when sebum androgens were used (Model_1-3), and when the DHT / T ratio was used as a feature, the average accuracy was 76%, which was relatively high, and the impact of split data on accuracy was also small. When all six androgen ratios were used, the accuracy did not improve much compared to the DHT / T ratio alone, suggesting that the DHT / T ratio is particularly important for discrimination. However, when the amount of DHT was used, the average accuracy was below 50%, suggesting that discrimination was not possible.

[0069] [Table 2]

[0070] 7. Correlation between sebum androgen ratio and average hair diameter, vellus hair rate, and short hair rate on the scalp The data on hair diameter, vellus hair rate, and short hair rate obtained from each subject were used to analyze correlations with the data on sebum androgens at the "top" and "back of the head."

[0071] The results showed that the DHT / T ratio was significantly correlated with the average hair diameter (μm), vellus hair rate (%), and short hair rate (%) in the occipital region (Fig. 3 and Fig. 7, left). In addition, a simple regression analysis was performed on the same data to obtain the regression equation, and the following equations were obtained: for the average hair diameter, y = -15.090x + 60.505 (x is the DHT / T ratio in the occipital region, y is the average hair diameter), for the vellus hair rate, y = 29.596x + 22.571 (x is the DHT / T ratio in the occipital region, y is the vellus hair rate), and for the short hair rate, y = 27.607x + 36.370 (x is the DHT / T ratio in the occipital region, y is the short hair rate, but subjects with less than 10 hairs within a 7 x 7 mm area were excluded from the analysis). On the other hand, there was no significant correlation with the DHT amount (pg / mg-sebum) (Fig. 4, Fig. 8, left). The same tendency was observed in the top of the head, where the DHT / T ratio was significantly correlated with the average hair diameter (μm), vellus hair rate (%), and short hair rate (%) (Fig. 5, Fig. 7 right). On the other hand, there was no significant correlation with the DHT amount (pg / mg-sebum) (Fig. 6, Fig. 8 right).

Claims

1. A method for detecting male pattern baldness or the risk thereof, comprising measuring the ratio of the amount of dihydrotestosterone (DHT) to the amount of testosterone (T) (DHT / T ratio) in lipids on the surface of the skin collected from the head of a subject.

2. The detection method according to claim 1, further comprising extracting dihydrotestosterone and testosterone from the RNA extraction residue after extracting RNA from lipids on the skin surface.

3. The method according to claim 1 or 2, further comprising comparing the DHT / T ratio with a reference value to detect the severity of male pattern baldness.

4. The method according to any one of claims 1 to 3, further comprising measuring one or more selected from the ratio of the amount of androstenedione (A-dione) to the amount of dehydroepiandrosterone (DHEA) (A-dione / DHEA ratio), the ratio of the amount of testosterone (T) to the amount of dehydroepiandrosterone (DHEA) (T / DHEA ratio), the ratio of the amount of dihydrotestosterone (DHT) to the amount of dehydroepiandrosterone (DHEA) (DHT / DHEA ratio), the ratio of the amount of testosterone (T) to the amount of androstenedione (A-dione) (T / A-dione ratio), and the ratio of the amount of dihydrotestosterone (DHT) to the amount of androstenedione (A-dione) (DHT / A-dione ratio).

5. A method for predicting one or more of the average hair diameter (μm), vellus hair rate (%), and short hair rate (%) of scalp hair, comprising measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) (DHT / T ratio) in skin surface lipids collected from the subject's head.

6. A method for evaluating the effectiveness of a preventive or therapeutic intervention for male pattern baldness, comprising measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) (DHT / T ratio) in surface lipids of the skin collected from the subject's head.

7. A test kit for detecting androgenetic alopecia or the risk thereof, which is used in the method according to any one of claims 1 to 6, comprising tools and reagents necessary for collecting and storing lipids on the skin surface, and a reagent for measuring the ratio of dihydrotestosterone (DHT) to testosterone (T) in lipids on the skin surface (DHT / T ratio), a kit for predicting one or more of the average hair diameter (μm), vellus hair rate (%), and short hair rate (%), or a kit for evaluating the effect of an intervention.

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