WO2023208013A1 - APPLICATION OF HIF-1α INHIBITOR IN TREATMENT OF ANDROGENIC ALOPECIA - Google Patents

APPLICATION OF HIF-1α INHIBITOR IN TREATMENT OF ANDROGENIC ALOPECIA Download PDF

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WO2023208013A1
WO2023208013A1 PCT/CN2023/090743 CN2023090743W WO2023208013A1 WO 2023208013 A1 WO2023208013 A1 WO 2023208013A1 CN 2023090743 W CN2023090743 W CN 2023090743W WO 2023208013 A1 WO2023208013 A1 WO 2023208013A1
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hif
inhibitor
hair
alopecia
androgenic alopecia
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王大鹏
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苏州翊鹏医药科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/235—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group
    • A61K31/24—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids having an aromatic ring attached to a carboxyl group having an amino or nitro group
    • A61K31/245—Amino benzoic acid types, e.g. procaine, novocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention belongs to the technical field of androgenetic alopecia treatment, and specifically relates to the application of HIF-1 ⁇ inhibitors in the treatment of androgenetic alopecia.
  • AGA Androgenic alopecia
  • the main clinical manifestations of this disease are hair loss on the forehead, top of the head (male type) or central area of the scalp (female type), which may be accompanied by There are conditions such as thin and soft hair, reduced density, or greasy scalp; under dermoscopy, the difference in hair shaft diameter is >20%, increased vellus hairs, reduced number of hairs per unit hair follicle, brown peritullary sign, yellow spot sign, etc.; the pathological characteristics are mainly hair follicles. Progressive reduction in size. Hair loss will affect many aspects of the patient's work and life, and can easily destroy the patient's self-confidence, leading to depression, anxiety and other behaviors.
  • Orentreich first proposed the term "androgenic alopecia", thus revealing the close connection between androgens and hair loss. It is currently believed that AGA is an androgen-dependent polygenic hereditary disease. This heritability increases the sensitivity of hair follicles to androgen, affects the signal transduction between the dermal papilla and hair follicle cells, leading to a series of changes such as the forehead. And/or the top susceptible hair follicles progressively shrink, the terminal hair follicles gradually transform into vellus hair follicles, the hair anagen phase progressively shortens, and the hair matrix and dermal papilla shrink at the same time, the hair also becomes shorter and thinner accordingly, leading to baldness. happened.
  • the main androgen present in the body is testosterone (testosterone, T), while dihydrotestosterone (DHT) is a more active androgen produced by the conversion of T under the catalysis of 5 ⁇ -reductase. It is similar to androgen.
  • the affinity of the androgen receptor (AR) is 5 times that of testosterone times, after testosterone or dihydrotestosterone combines with AR, it enters the nucleus and binds to the DNA receptor, thereby producing biological effects.
  • human 5 ⁇ -reductase has two isoenzymes, type I and type II. Type I is mainly distributed in sebaceous glands, chest and back skin, liver, adrenal glands and kidneys.
  • Type II is mainly distributed in the scalp and hair follicles (the outer hair root sheath is the most common). inner layer) and the tissue surrounding the hair follicle.
  • McGinley et al. found that patients with congenital type II 5 ⁇ -reductase deficiency had normal or slightly elevated serum testosterone, but reduced DHT synthesis. Homozygous patients showed pseudohermaphroditism, with reduced body hair, normal hair growth, and no AGA. occur.
  • the levels of testosterone, dihydrotestosterone, AR and 5 ⁇ -reductase in the scalp of bald areas are higher than those in non-bald areas (such as occipital scalp) or the same areas in non-bald people, thus explaining how severe AGA is.
  • minoxidil The therapeutic mechanism of minoxidil is not completely clear, and may include multiple pathways, such as opening ATP-dependent K + channels on the cell membrane, affecting the activity and stability of androgen receptor (AR), etc. Although both are currently widely used in clinical practice, neither can completely block the progression of the disease, and both have varying degrees of adverse reactions.
  • the screening method for cofactors includes the following steps: Step S1, use scalp biopsy technology to collect scalp tissue samples from normal volunteers and patients with androgenic alopecia; Step S2, detect different AR cofactors (c-Jun, NF- ⁇ B, Expression of SMAD3 and HIF-1 ⁇ ) in scalp tissue samples of normal volunteers and patients with androgenetic alopecia; Step S3, select the AR cofactors differentially expressed in step S2, and initially determine them as cofactors related to androgenetic alopecia.
  • the cofactor related to androgenic alopecia identified in the present invention is HIF-1 ⁇ .
  • the detection was performed using Western blotting.
  • a second aspect of the present invention provides the use of HIF-1 ⁇ inhibitors in the preparation of drugs for treating androgenic alopecia.
  • the HIF-1 ⁇ inhibitor can have a good effect on androgenic alopecia.
  • the HIF-1 ⁇ inhibitor can effectively inhibit the auxiliary effect of HIF-1 ⁇ on AR.
  • HIF-1 ⁇ HIF-1 ⁇ on AR is manifested by inhibiting the expression of one or more genes selected from Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, and Fstl1.
  • the HIF-1 ⁇ inhibitor is selected from one or more of LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243.
  • the third aspect of the present invention provides the use of a HIF-1 ⁇ detection reagent in preparing an androgenic alopecia risk prediction kit.
  • the HIF-1 ⁇ detection reagent includes antibodies for HIF-1 ⁇ Western blot detection, antibodies for immunohistochemical detection, or primers and probes for quantitative PCR.
  • the HIF-1 ⁇ detection object is the scalp tissue sample of the subject.
  • the HIF-1 ⁇ inhibitor is selected from one or more of LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243.
  • the fourth aspect of the present invention provides a method for constructing a mouse model of androgenetic alopecia, which is characterized in that The method includes the step of injecting testosterone propionate into male C57BL/6 mice.
  • the frequency of injection is daily.
  • the present invention performs scalp biopsy on normal people and patients with androgenetic alopecia to screen out the differentially expressed AR cofactor HIF-1 ⁇ .
  • the risk of androgenetic alopecia can be predicted through the detection of this differentially expressed factor.
  • HIF-1 ⁇ inhibitors prevent the transcription of related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) by effectively inhibiting HIF-1 ⁇ in the mouse model of androgenetic alopecia, causing related genes to The expression of the gene changes, and the decrease in the expression of related genes will promote hair growth in androgenetic alopecia model mice, and thus can be used as a therapeutic drug for androgenetic alopecia.
  • related genes Namur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1
  • the drug for androgenetic alopecia prepared by using HIF-1 ⁇ inhibitors provided by the present invention has the advantages of low cost, low toxic and side effects, and significant curative effect, and provides a new strategy for the treatment of androgenetic alopecia.
  • Figure 1 shows the expression of different AR cofactors in scalp samples from patients with androgenic alopecia and normal volunteers.
  • Figure 2A shows the therapeutic effects of various HIF-1 ⁇ inhibitor drugs in the constructed mouse model of androgenic alopecia.
  • Figure 2B is a statistical diagram of the weight of new shaved hair in the constructed mouse model of androgenic alopecia.
  • Figure 3 shows the GO enrichment analysis of differentially expressed genes between skin sample cells of androgenetic alopecia model mice after treatment with the inhibitor LW6.
  • Figures 4A-4G respectively show the effects of inhibitors LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243 on genes Nmur1 (Figure 4A), Pcolce ( Figure 4B), Fbln1 ( Figure 4C), Prrx1 ( Figure 4D), and Vim ( Figure 4D). 4E), Tnfrsf1b (Fig. 4F), and Fstl1 (Fig. 4G) expression levels.
  • AR androgen receptor
  • NTD N-terminal transcriptional activation domain
  • DBD DNA binding domain
  • LBD ligand binding domain
  • AR cofactor refers to a factor that modulates the transcriptional activity of the androgen receptor (AR) and thereby affects many functional properties of the AR, including ligand selectivity and/or DNA binding ability. Transcription factors.
  • AR cofactors include c-Jun, NF- ⁇ B, SMAD3 and HIF-1 ⁇ , for verification.
  • hypoxia-inducible factor is a transcription factor widely present in mammals and humans under hypoxic conditions and is a key factor in response to hypoxic stress.
  • HIF-1 ⁇ is a subunit of hypoxia inducible factor-1 (HIF-1), which is regulated by hypoxia and regulates the activity of HIF-1.
  • HIF-1 ⁇ is transferred to the nucleus and combines with HIF-1 ⁇ to form active HIF-1, which regulates the transcription of multiple genes by binding to the hypoxia response element on target genes.
  • HIF-1 ⁇ can form different signaling pathways with multiple upstream and downstream proteins to mediate hypoxic signals and regulate cells to produce a series of
  • the compensatory response to hypoxia plays an important role in the growth and development of the body as well as physiological and pathological processes, and is a focus of biomedical research.
  • HIF-1 ⁇ inhibitor refers to a compound or composition that can be used to inhibit the activity and/or expression of HIF-1 ⁇ . Including but not limited to, LW6 (CAS number: 934593-90-5), 2-MeOE2 (CAS number: 362-07-2), PX-4782HCI (CAS number: 685898-44-6), BAY87-2243 (CAS No.: 1227158-85-1) and so on.
  • AGA androgenetic alopecia
  • androgenetic alopecia is a common and frequently-occurring disease in dermatology. It is a characteristic alopecia with genetic factors involved and dependent on androgen action, which often affects The patient's body image and mental health are adversely affected.
  • the main reason for the production of AGA is that when there is too much testosterone in the skin, under the catalysis of 5-alpha reductase, testosterone can generate dihydrotestosterone. Dihydrotestosterone competes with testosterone for the androgen receptor in the hair follicle target cells.
  • dihydrotestosterone Since dihydrotestosterone is significantly more active than testosterone, dihydrotestosterone has a stronger ability to bind to androgen receptors than testosterone. Once dihydrotestosterone enters the cell nucleus, it can inhibit the growth of hair follicle cells, prompting the hair follicles to enter the resting phase in advance, leading to hair loss. Androgens can also act on sebaceous glands, activating the SREBP pathway in sebaceous gland cells, promoting the development and differentiation of immature cells in the outer layer of sebaceous gland cells into mature secretory cells, causing sebaceous gland gland hypertrophy, enhanced secretion function, and excessive sebum production. When sebaceous glands secrete excessively, excess sebum will clog or compress pores, hinder normal hair growth and even induce inflammation, causing hair to wither and fall off.
  • This example uses Western blotting to detect the expression levels of c-Jun, NF- ⁇ B, SMAD3 and HIF-1 ⁇ in scalp tissue samples from patients with androgenic alopecia and normal volunteers.
  • Scalp tissue samples were collected through scalp biopsy technology. A total of 30 scalp tissue samples from patients with androgenic alopecia and 30 normal volunteers were collected. Then use RIPA tissue/cell lysis buffer and PMSF reagent to extract the protein, and use the extracted protein for the next step of Western blotting experiment.
  • the specific steps of the Western blotting experiment are as follows: First, prepare 10% SDS separation gel and stacking gel according to the formula, mix the sample with the loading buffer, boil it in an ice bath at 100°C for 5 minutes, add equal amounts of each sample using a microsampler after ice bath and centrifugation. Lanes were electrophoretically separated and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Merck Millipore, MA, USA) and incubated in 5% BSA for 1 hour.
  • c-Jun primary antibody (abcam, #ab40766), NF- ⁇ B primary antibody (abcam, #ab32536), SMAD3 primary antibody (abcam, #ab40854), HIF-1 ⁇ diluted 1:400 at 4°C.
  • the primary antibody (abcam, #ab179483) and ⁇ -actin primary antibody (abcam, #ab115777) were incubated overnight, washed three times with TBST and then incubated with goat anti-rabbit secondary antibody (abcam, #ab6721) for 1 hour. Wash 3 times with PBS buffer at room temperature, 5 min each time. Immerse the membrane in the ECL reaction solution at room temperature for 1 min. After removing the liquid, cover the film with food wrap, expose it to a line film in a dark room, develop and fix it, and observe the results.
  • the cause of hair loss is mainly due to the difference in sensitivity of local hair follicles to AR.
  • the inventor speculates that inhibiting the differential expression of AR cofactor-HIF-1 ⁇ will lead to changes in AR sensitivity and then lead to hair loss.
  • the inventors constructed a mouse model of androgenetic alopecia to explore the therapeutic effect of HIF-1 ⁇ inhibitors on androgenetic alopecia.
  • mice Male C57BL/6 mice aged 6-8 weeks and weighing 18-22g were selected for model preparation. After adaptive feeding for 1 week, they were numbered according to body weight and randomly divided according to body weight using Excel. There are 6 groups, 5 mice in each group. First, use a razor to shave the mouse hair, and then use depilatory cream to remove about 2 ⁇ 3cm 2 on its back. After depilating for 5-10 minutes, wipe the depilatory cream with warm water. The spine of the mice was used as the dividing line, and a skin area of 2 ⁇ 3 cm 2 was taken from the symmetrical part of the back as the experimental area. The drug solvent in all administration groups in the experiment was non-toxic and harmless corn oil, and the experimental animals were randomly Divided into the following groups, see Table 1 below:
  • mice in the blank group received no treatment except shaving.
  • the mice in the remaining groups were lightly anesthetized with 5% chloral hydrate and injected subcutaneously with testosterone propionate at a dose of 5 mg/kg daily to establish a mouse model of androgenic alopecia.
  • 30 days After 30 minutes of modeling treatment, the modeling group and the drug administration group were administered medication according to the above protocol. Photographs were taken every day to record the new hair of the mice. After 30 consecutive days, the mice were sacrificed and the new hair of the mice was shaved with a razor. Calculate the weight of new hair in mice.
  • Figure 2A shows the therapeutic effects of various drugs in the constructed mouse model of androgenic alopecia.
  • Figure 2B is a statistical diagram of the average weight of new hair of five shaved mice in the constructed mouse androgenic alopecia model. From Figures 2A and 2B, it can be seen that the model group experienced 30 days of hair growth. The new hair was sparse and the hair loss area was large. There was still a large bare hairless area on the back of the mouse and the average weight of the new hair was light. However, the HIF-1 ⁇ inhibitor treatment group , after 30 days of hair growth in the blank group, the new hair was dense and the area of hair loss was small. The back of the mice was visible to the naked eye with a large area of new hair and almost no exposed hairless area. The average weight of new hair was far higher than that of the modeling group.
  • HIF-1 ⁇ participates in inflammatory reactions.
  • tissue metabolic activity is enhanced due to the infiltration and high metabolic rate of inflammatory cells, resulting in an increase in oxygen demand.
  • the action of inflammatory factors causes blood vessel damage. Contraction reduces the oxygen supply to the inflammatory area, forming a hypoxic microenvironment, resulting in a large amount of HIF-1 ⁇ expression.
  • Current research shows that ILGF-1, IL-1, TNF- ⁇ , pGE2 and LPS and other pro-inflammatory cytokines can activate the transcriptional activity of HIF-1, suggesting the close relationship between HIF-1 ⁇ and the inflammatory process.
  • Example 3 Single-cell transcriptome sequencing detects cellular level gene changes in skin tissue of androgenetic alopecia mouse model
  • mice in the HIF-1 ⁇ inhibitor-LW6 treatment group peeled off and separated the skin tissue of mice in the HIF-1 ⁇ inhibitor-LW6 treatment group and the mice in the modeling group and performed single-cell transcriptome sequencing.
  • inflammation-related genes are significantly upregulated in scalp biopsy samples from patients with androgenic alopecia.
  • HIF-1 ⁇ inhibitors In order to further explore the mechanism of action of HIF-1 ⁇ inhibitors in the treatment of androgenetic alopecia, the inventors detected the transcription of inflammation-related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) after treatment with HIF-1 ⁇ inhibitors. Condition.
  • qPCR real-time fluorescence quantitative PCR
  • the specific steps are as follows: Cut the tissue sample into 2-4 mm slices. Then transfer it to the C tube of gentleMACS, add an appropriate amount of TRIzol, tightly cover the C tube, place it upside down on the gentleMACS tissue dissociator, and continue to rotate and incubate the tissue sample for 30 minutes. After the reaction is completed, the tissue sample can be Transfer the liquid to a centrifuge tube and centrifuge at 12,000 rpm for 10 minutes. After centrifugation is completed, transfer all the aspirated supernatant to a new centrifuge tube, then add 200 mL of chloroform to it, use a vortex shaker to shake the centrifuge tube for about 15 seconds, and then place it still in the centrifuge tube.
  • HIF-1 ⁇ inhibitor LW6, 2-MeOE2, PX-4782HCI, BAY87-2243 purchased from Selleck, product numbers: #S8441, #S1233, #S7612, #S7309).
  • HIF-1 ⁇ inhibitors will lead to a decrease in the expression levels of related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) in mouse skin samples.
  • the inventor believes that these genes are regulatory Downregulation of key genes for hair loss ultimately improved hair loss in mouse models of androgenic alopecia.

Abstract

Disclosed in the present invention is an application of a HIF-1α inhibitor in the preparation of a drug for treating androgenic alopecia. The HIF-1α inhibitor can effectively inhibit the auxiliary effect of HIF-1α on AR, and inhibit the expression of alopecia and inflammation-related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) in an androgenic alopecia mouse model, thereby promoting hair growth. The HIF-1α inhibitor disclosed in the present invention is selected from one or more of LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243. The HIF-1α inhibitor has the advantages of low costs, low toxic and side effects and significant curative effects, and provides a new strategy for the treatment of androgenic alopecia.

Description

HIF-1α抑制剂在雄激素性脱发治疗中的应用Application of HIF-1α inhibitors in the treatment of androgenic alopecia 技术领域Technical field
本发明属于雄激素性脱发治疗技术领域，具体涉及HIF-1α抑制剂在治疗雄激素性脱发中的应用。The invention belongs to the technical field of androgenetic alopecia treatment, and specifically relates to the application of HIF-1α inhibitors in the treatment of androgenetic alopecia.
背景技术Background technique
雄激素性脱发(Androgenic alopecia简称AGA)是目前临床上最为常见的脱发类型之一；本病主要临床表现为前额、头顶部(男性型)或头皮中央区(女性型)的毛发脱落，可伴有毛发细软、密度减低或头皮油腻等情况；皮肤镜下可见毛干直径差异>20％、毳毛增多、单位毛囊内毛发数量减少、褐色毛周征、黄点征等；病理特点主要为毛囊体积进行性缩小。脱发会影响到患者的工作、生活等多方面,且易摧毁患者的自信心，以致其出现情绪低落、焦虑等行为。研究报道，30岁白人男性中AGA的发病率高达30％，50岁男性中AGA患病率高达50％，70岁以上男性和女性中AGA患病率分别为80％和53％。我国AGA的发病率男性为21.3％，女性为6.0％。Androgenic alopecia (AGA) is one of the most common types of hair loss in clinical practice. The main clinical manifestations of this disease are hair loss on the forehead, top of the head (male type) or central area of the scalp (female type), which may be accompanied by There are conditions such as thin and soft hair, reduced density, or greasy scalp; under dermoscopy, the difference in hair shaft diameter is >20%, increased vellus hairs, reduced number of hairs per unit hair follicle, brown peritullary sign, yellow spot sign, etc.; the pathological characteristics are mainly hair follicles. Progressive reduction in size. Hair loss will affect many aspects of the patient's work and life, and can easily destroy the patient's self-confidence, leading to depression, anxiety and other behaviors. Studies have reported that the incidence of AGA is as high as 30% in 30-year-old white men, 50% in 50-year-old men, and 80% and 53% in men and women over 70 years old, respectively. The incidence of AGA in my country is 21.3% in men and 6.0% in women.
1960年0rentreich首先提出了“雄激素性脱发”一词，从而揭示了雄激素与脱发之间的密切联系。目前认为AGA是一种雄激素依赖的多基因遗传性疾病，这种遗传性使毛囊对雄激素作用的敏感性增加，影响真皮乳头与毛囊细胞之间的信号转导，导致一系列变化如前额和/或顶部易感毛囊进行性缩小、终毛毛囊逐渐转变为毳毛毛囊，毛发生长期进行性缩短，同时毛母质和毛乳头体积缩小，毛发也相应地变短变细，从而导致秃发的发生。In 1960, Orentreich first proposed the term "androgenic alopecia", thus revealing the close connection between androgens and hair loss. It is currently believed that AGA is an androgen-dependent polygenic hereditary disease. This heritability increases the sensitivity of hair follicles to androgen, affects the signal transduction between the dermal papilla and hair follicle cells, leading to a series of changes such as the forehead. And/or the top susceptible hair follicles progressively shrink, the terminal hair follicles gradually transform into vellus hair follicles, the hair anagen phase progressively shortens, and the hair matrix and dermal papilla shrink at the same time, the hair also becomes shorter and thinner accordingly, leading to baldness. happened.
机体内存在的雄激素主要为睾酮(testosterone，T)，而双氢睾酮(dihydrotestosterone，DHT)是由T在5α-还原酶催化作用下转化而产生的活性更高的雄激素，其与雄激素受体(androgen receptor，AR)的亲和力为睾酮的5 倍，睾酮或双氢睾酮与AR结合后，进入细胞核内与DNA受体结合，从而产生生物学效应。已知人的5α-还原酶有Ⅰ型和Ⅱ型两种同功酶，I型主要分布于皮脂腺、胸背部皮肤、肝脏、肾上腺和肾脏，II型主要分布于头皮、毛囊内(外毛根鞘最内层)和毛囊周围组织。McGinley等研究发现，先天性II型5α-还原酶缺乏症患者的血清睾酮正常或轻度增高，但DHT合成减少，纯合子患者表现为假两性畸形，伴有体毛减少、头发生长正常，无AGA发生。此外，秃发部位头皮中睾酮、双氢睾酮、AR和5α-还原酶的水平均高于非秃发部位(如枕部头皮)或非秃发者的相同部位，因此可以解释AGA即使再严重也不会累及枕部毛发的原因。此外，有报道称AGA患者血清T水平与正常人无明显差别，而血清DHT水平则较正常人高，进一步证明了双氢睾酮与AGA发病的相关性。The main androgen present in the body is testosterone (testosterone, T), while dihydrotestosterone (DHT) is a more active androgen produced by the conversion of T under the catalysis of 5α-reductase. It is similar to androgen. The affinity of the androgen receptor (AR) is 5 times that of testosterone times, after testosterone or dihydrotestosterone combines with AR, it enters the nucleus and binds to the DNA receptor, thereby producing biological effects. It is known that human 5α-reductase has two isoenzymes, type I and type II. Type I is mainly distributed in sebaceous glands, chest and back skin, liver, adrenal glands and kidneys. Type II is mainly distributed in the scalp and hair follicles (the outer hair root sheath is the most common). inner layer) and the tissue surrounding the hair follicle. McGinley et al. found that patients with congenital type II 5α-reductase deficiency had normal or slightly elevated serum testosterone, but reduced DHT synthesis. Homozygous patients showed pseudohermaphroditism, with reduced body hair, normal hair growth, and no AGA. occur. In addition, the levels of testosterone, dihydrotestosterone, AR and 5α-reductase in the scalp of bald areas are higher than those in non-bald areas (such as occipital scalp) or the same areas in non-bald people, thus explaining how severe AGA is. It also does not affect the hair on the occipital area. In addition, there are reports that the serum T level of AGA patients is not significantly different from that of normal people, while the serum DHT level is higher than that of normal people, further proving the correlation between dihydrotestosterone and the onset of AGA.
AGA的病因及发病机制尚不明确，一般认为雄激素及其受体(AR)的表达在本病的发生中起关键作用，而II型5α-还原酶是其发病的重要因素。目前国内经国家食品药品监督管理局(SFDA)批准的用于治疗男性AGA的药物主要是非那雄胺片和米诺地尔溶液。非那雄胺是一种II型5α-还原酶抑制剂，能够阻断睾酮(Testosterone，T)转变为活性更强的双氢睾酮(DHT)从而减弱雄激素对毛囊的抑制作用。米诺地尔的治疗机制尚不完全明确，可能包含了多条途径，如开放细胞膜上ATP依赖的K+通道、影响雄激素受体(AR)活性及稳定性等。目前二者虽广泛应用于临床，但均不能完全阻断病程进展，且都存在不同程度的不良反应。The cause and pathogenesis of AGA are still unclear. It is generally believed that the expression of androgen and its receptor (AR) plays a key role in the occurrence of this disease, and type II 5α-reductase is an important factor in its pathogenesis. At present, the domestic drugs approved by the State Food and Drug Administration (SFDA) for the treatment of male AGA are mainly finasteride tablets and minoxidil solution. Finasteride is a type II 5α-reductase inhibitor that blocks the conversion of testosterone (Testosterone, T) into the more active dihydrotestosterone (DHT), thereby weakening the inhibitory effect of androgens on hair follicles. The therapeutic mechanism of minoxidil is not completely clear, and may include multiple pathways, such as opening ATP-dependent K + channels on the cell membrane, affecting the activity and stability of androgen receptor (AR), etc. Although both are currently widely used in clinical practice, neither can completely block the progression of the disease, and both have varying degrees of adverse reactions.
因此，探寻一种新的疗效更好、副作用更小的药物来治疗雄激素性脱发是迫切需要的。Therefore, there is an urgent need to explore a new drug with better efficacy and fewer side effects to treat androgenic alopecia.
发明内容Contents of the invention
为了解决上述技术问题，本发明第一方面提供了一种具有抑制AR活化的辅 助因子的筛选方法，包括以下步骤：步骤S1，利用头皮活检技术，搜集正常志愿者，雄激素性脱发患者的头皮组织样本；步骤S2，检测不同AR辅助因子(c-Jun,NF-κB，SMAD3以及HIF-1α)在正常志愿者以及雄激素性脱发患者头皮组织样本中的表达；步骤S3，选择步骤S2中差异表达的AR辅助因子，初步确定为雄激素性脱发相关的辅助因子。In order to solve the above technical problems, the first aspect of the present invention provides an auxiliary agent capable of inhibiting AR activation. The screening method for cofactors includes the following steps: Step S1, use scalp biopsy technology to collect scalp tissue samples from normal volunteers and patients with androgenic alopecia; Step S2, detect different AR cofactors (c-Jun, NF-κB, Expression of SMAD3 and HIF-1α) in scalp tissue samples of normal volunteers and patients with androgenetic alopecia; Step S3, select the AR cofactors differentially expressed in step S2, and initially determine them as cofactors related to androgenetic alopecia.
进一步地，本发明确定的雄激素性脱发相关辅助因子为HIF-1α。Furthermore, the cofactor related to androgenic alopecia identified in the present invention is HIF-1α.
进一步地，所述检测采用蛋白质印迹(Westernblotting)进行。Further, the detection was performed using Western blotting.
本发明第二方面提供了HIF-1α抑制剂在制备治疗雄激素性脱发药物中的应用。A second aspect of the present invention provides the use of HIF-1α inhibitors in the preparation of drugs for treating androgenic alopecia.
进一步地，所述HIF-1α抑制剂可对雄激素性脱发有良好疗效。Furthermore, the HIF-1α inhibitor can have a good effect on androgenic alopecia.
进一步地，所述HIF-1α抑制剂能够有效抑制HIF-1α对AR辅助作用。Furthermore, the HIF-1α inhibitor can effectively inhibit the auxiliary effect of HIF-1α on AR.
进一步地，所述抑制HIF-1α对AR辅助作用表现为抑制选自Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1中一种或多种基因的表达。Further, the inhibitory effect of HIF-1α on AR is manifested by inhibiting the expression of one or more genes selected from Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, and Fstl1.
进一步地，所述HIF-1α抑制剂选自LW6,2-MeOE2,PX-4782HCI,BAY87-2243中的一种或多种。Further, the HIF-1α inhibitor is selected from one or more of LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243.
本发明第三方面提供了HIF-1α检测试剂在制备雄激素性脱发风险预测试剂盒中的用途。The third aspect of the present invention provides the use of a HIF-1α detection reagent in preparing an androgenic alopecia risk prediction kit.
进一步地，所述HIF-1α检测试剂包括用于HIF-1α蛋白印迹检测的抗体、用于免疫组化检测的抗体或用于定量PCR的引物和探针。Further, the HIF-1α detection reagent includes antibodies for HIF-1α Western blot detection, antibodies for immunohistochemical detection, or primers and probes for quantitative PCR.
进一步地，所述HIF-1α检测对象为受试者的头皮组织样本。Further, the HIF-1α detection object is the scalp tissue sample of the subject.
进一步地，所述HIF-1α抑制剂选自LW6，2-MeOE2，PX-4782HCI，BAY87-2243中的一种或多种。Further, the HIF-1α inhibitor is selected from one or more of LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243.
本发明第四方面提供了一种雄激素性脱发小鼠模型的构建方法，其特征在 于，包括给雄性C57BL/6小鼠注射丙酸睾酮的步骤。The fourth aspect of the present invention provides a method for constructing a mouse model of androgenetic alopecia, which is characterized in that The method includes the step of injecting testosterone propionate into male C57BL/6 mice.
进一步的，所述注射的频率为每日。Further, the frequency of injection is daily.
本发明相对于现有技术具有的效果：The present invention has the following effects compared to the prior art:
1)本发明通过对正常人和雄激素性脱发患者进行头皮活检，筛选出差异表达的AR辅助因子HIF-1α，通过该差异表达因子的检测可以预测雄激素性脱发的发生风险。1) The present invention performs scalp biopsy on normal people and patients with androgenetic alopecia to screen out the differentially expressed AR cofactor HIF-1α. The risk of androgenetic alopecia can be predicted through the detection of this differentially expressed factor.
2)本发明发现HIF-1α抑制剂通过有效抑制HIF-1α在雄激素性脱发小鼠模型中，阻止相关基因(Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1)的转录，使得相关基因的表达量发生改变，相关基因表达的降低会促进雄激素性脱发模式小鼠的毛发生长，进而能够作为雄激素性脱发的一个治疗药物。2) The present invention found that HIF-1α inhibitors prevent the transcription of related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) by effectively inhibiting HIF-1α in the mouse model of androgenetic alopecia, causing related genes to The expression of the gene changes, and the decrease in the expression of related genes will promote hair growth in androgenetic alopecia model mice, and thus can be used as a therapeutic drug for androgenetic alopecia.
3)本发明提供的采用HIF-1α抑制剂制备雄激素性脱发的药物，具有成本低，毒副作用小，疗效显著的优点，为雄激素性脱发的治疗提供了新策略。3) The drug for androgenetic alopecia prepared by using HIF-1α inhibitors provided by the present invention has the advantages of low cost, low toxic and side effects, and significant curative effect, and provides a new strategy for the treatment of androgenetic alopecia.
附图说明Description of drawings
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式描述中所需要使用的附图作简单地介绍。In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the drawings needed to describe the specific embodiments will be briefly introduced below.
图1显示雄激素性脱发患者以及正常志愿者头皮样本中不同AR辅助因子的表达情况。Figure 1 shows the expression of different AR cofactors in scalp samples from patients with androgenic alopecia and normal volunteers.
图2A为构建的小鼠雄激素性脱发模型中，各种HIF-1α抑制剂药物的治疗效果。Figure 2A shows the therapeutic effects of various HIF-1α inhibitor drugs in the constructed mouse model of androgenic alopecia.
图2B为构建的小鼠雄激素性脱发模型中，剃下的新生毛发重量统计图。Figure 2B is a statistical diagram of the weight of new shaved hair in the constructed mouse model of androgenic alopecia.
图3为抑制剂LW6处理后，雄激素性脱发模型小鼠皮肤样本细胞之间的差异表达基因的GO富集分析。 Figure 3 shows the GO enrichment analysis of differentially expressed genes between skin sample cells of androgenetic alopecia model mice after treatment with the inhibitor LW6.
图4A-4G分别显示抑制剂LW6,2-MeOE2,PX-4782HCI,BAY87-2243对基因Nmur1(图4A)、Pcolce(图4B)、Fbln1(图4C)、Prrx1(图4D)、Vim(图4E)、Tnfrsf1b(图4F)、Fstl1(图4G)的表达水平的影响。Figures 4A-4G respectively show the effects of inhibitors LW6, 2-MeOE2, PX-4782HCI, and BAY87-2243 on genes Nmur1 (Figure 4A), Pcolce (Figure 4B), Fbln1 (Figure 4C), Prrx1 (Figure 4D), and Vim (Figure 4D). 4E), Tnfrsf1b (Fig. 4F), and Fstl1 (Fig. 4G) expression levels.
具体实施方式Detailed ways
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施案例所描述的内容仅用于说明和解释本发明，并不用于限制权利要求书中所详细描述的本发明。除非特别说明，本发明采用的试剂、方法和设备如无特别说明，均为常规方法，所使用的试验材料如无特别说明，均可从商业公司获取。The present invention can be better understood from the following examples. However, those skilled in the art can easily understand that the content described in the implementation examples is only used to illustrate and explain the present invention, and is not used to limit the present invention described in detail in the claims. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional methods, and the test materials used can be obtained from commercial companies unless otherwise specified.
如本文所用，术语“AR”，雄激素受体，属于核受体超家族中的类固醇受体。AR一般由四个结构域组成：N端转录激活区(NTD)、DNA结合区(DBD)、铰链区和配体结合区(LBD)。As used herein, the term "AR", androgen receptor, is a steroid receptor in the nuclear receptor superfamily. AR generally consists of four domains: N-terminal transcriptional activation domain (NTD), DNA binding domain (DBD), hinge domain and ligand binding domain (LBD).
如本文所用，术语“AR辅助因子”是指能够调节雄激素受体(AR)的转录活性进而影响雄激素受体的包括配体选择性和/或DNA结合能力等在内的许多功能特性的转录因子。本文中发明人选择了c-Jun,NF-κB，SMAD3以及HIF-1α这4种最常见的AR辅助因子进行验证。As used herein, the term "AR cofactor" refers to a factor that modulates the transcriptional activity of the androgen receptor (AR) and thereby affects many functional properties of the AR, including ligand selectivity and/or DNA binding ability. Transcription factors. In this article, the inventor selected the four most common AR cofactors, c-Jun, NF-κB, SMAD3 and HIF-1α, for verification.
如本文所用，术语“HIF-1α”，缺氧诱导因子是缺氧条件下广泛存在于哺乳动物和人体内的一种转录因子，是应答缺氧应激的关键因子。HIF-1α是缺氧诱导因子-1(hypoxia inducible factor-1，HIF-1)的一个亚单位，受缺氧调控并调节HIF-1的活性。在缺氧条件下，HIF-1α转移到细胞核内结合HIF-1β形成有活性的HIF-1，通过与靶基因上的缺氧反应元件结合调节多种基因的转录。HIF-1α可以与上下游多种蛋白组成不同的信号通路，介导低氧信号，调控细胞产生一系列 对缺氧的代偿反应，在机体的生长发育及生理和病理过程中发挥重要作用，是生物医学研究的一个焦点。As used herein, the term "HIF-1α", hypoxia-inducible factor is a transcription factor widely present in mammals and humans under hypoxic conditions and is a key factor in response to hypoxic stress. HIF-1α is a subunit of hypoxia inducible factor-1 (HIF-1), which is regulated by hypoxia and regulates the activity of HIF-1. Under hypoxic conditions, HIF-1α is transferred to the nucleus and combines with HIF-1β to form active HIF-1, which regulates the transcription of multiple genes by binding to the hypoxia response element on target genes. HIF-1α can form different signaling pathways with multiple upstream and downstream proteins to mediate hypoxic signals and regulate cells to produce a series of The compensatory response to hypoxia plays an important role in the growth and development of the body as well as physiological and pathological processes, and is a focus of biomedical research.
如本文所用，术语“HIF-1α抑制剂”，指能够用于抑制HIF-1α活性和/或表达的化合物或者组合物。包括但不限于，LW6(CAS号：934593-90-5)，2-MeOE2(CAS号：362-07-2)，PX-4782HCI(CAS号：685898-44-6)，BAY87-2243(CAS号：1227158-85-1)等等。As used herein, the term "HIF-1α inhibitor" refers to a compound or composition that can be used to inhibit the activity and/or expression of HIF-1α. Including but not limited to, LW6 (CAS number: 934593-90-5), 2-MeOE2 (CAS number: 362-07-2), PX-4782HCI (CAS number: 685898-44-6), BAY87-2243 (CAS No.: 1227158-85-1) and so on.
如本文所用，术语“雄激素性脱发(AGA)”，androgenetic alopecia，是皮肤科的常见病与多发病,是一种具有遗传因素参与的且依赖雄激素作用的特征性秃发,往往会对病人的身体形象及心理健康产生不利的影响。AGA产生的原因主要是由于皮肤内的睾酮含量过多时，在5-α还原酶的催化下，睾酮能够生成双氢睾酮，双氢睾酮与睾酮竞争毛囊靶标细胞内的雄激素受体。由于双氢睾酮的活性明显高于睾酮，因此，双氢睾酮结合雄激素受体的能力强于睾酮。一旦双氢睾酮进入细胞核内，就能抑制毛囊细胞的生长，促使毛囊提前进入休止期，导致毛发的脱落。雄激素还能作用于皮脂腺，激活皮脂腺细胞中的SREBP通路，促进皮脂腺细胞外层的幼稚细胞发育分化为成熟的分泌细胞，使皮脂腺腺体肥大，分泌功能增强，产生过多的皮脂。当皮脂腺分泌亢进时，过多的皮脂会堵塞或者压迫毛孔，阻碍毛发的正常生长甚至诱发炎症，导致毛发枯萎、脱落。As used in this article, the term "androgenetic alopecia (AGA)", androgenetic alopecia, is a common and frequently-occurring disease in dermatology. It is a characteristic alopecia with genetic factors involved and dependent on androgen action, which often affects The patient's body image and mental health are adversely affected. The main reason for the production of AGA is that when there is too much testosterone in the skin, under the catalysis of 5-alpha reductase, testosterone can generate dihydrotestosterone. Dihydrotestosterone competes with testosterone for the androgen receptor in the hair follicle target cells. Since dihydrotestosterone is significantly more active than testosterone, dihydrotestosterone has a stronger ability to bind to androgen receptors than testosterone. Once dihydrotestosterone enters the cell nucleus, it can inhibit the growth of hair follicle cells, prompting the hair follicles to enter the resting phase in advance, leading to hair loss. Androgens can also act on sebaceous glands, activating the SREBP pathway in sebaceous gland cells, promoting the development and differentiation of immature cells in the outer layer of sebaceous gland cells into mature secretory cells, causing sebaceous gland gland hypertrophy, enhanced secretion function, and excessive sebum production. When sebaceous glands secrete excessively, excess sebum will clog or compress pores, hinder normal hair growth and even induce inflammation, causing hair to wither and fall off.
实施例1雄激素性脱发相关AR辅助因子的确定Example 1 Determination of AR cofactors related to androgenic alopecia
已有实验证实大部分AGA患者血中雄激素水平并未升高。同时，毛囊移植试验表明，将枕部毛囊移植到头顶部仍具有抗AGA的特点,而额顶部的头发移植到前臂后仍然与额顶部毛囊同步发生脱发。说明这种维持其原生长部位特征趋势的主要原因是毛囊局部AR敏感性的差异所致。2002年国外的学者就已检测出AGA患者脱发区雄激素受体活性增高，即使患者体内雄激素水平处于 正常，亦对雄激素敏感性增加，从而影响毛囊的生长代谢引起脱发，鉴于AR只在患者脱发区活性增加而非表达增加。发明人推测可能是由于AR辅助因子的差异表达导致了AR活性增加，因此发明人调研文献，查询了几个前人已验证过的AR辅助因子c-Jun，NF-κB，SMAD3以及HIF-1α，并在雄激素性脱发患者以及正常志愿者头皮组织样本检测它们的表达量，验证它们是否在AGA患者中差异表达。Experiments have confirmed that most AGA patients have no elevated blood androgen levels. At the same time, hair follicle transplantation experiments have shown that transplanting occipital hair follicles to the top of the head still has anti-AGA characteristics, while hair on the top of the forehead will still lose hair synchronously with the hair follicles on the top of the forehead after being transplanted to the forearm. This indicates that the main reason for this tendency to maintain the characteristics of its original growth site is the difference in local AR sensitivity of hair follicles. In 2002, foreign scholars have detected increased activity of androgen receptors in the hair loss areas of AGA patients, even if the androgen levels in the patients are at Normally, it also has increased sensitivity to androgens, which affects the growth and metabolism of hair follicles and causes hair loss. In view of the fact that AR activity only increases in the patient's hair loss area but not its expression. The inventor speculates that the differential expression of AR cofactors may lead to increased AR activity. Therefore, the inventors researched the literature and queried several AR cofactors c-Jun, NF-κB, SMAD3 and HIF-1α that have been verified by previous researchers. , and detect their expression in scalp tissue samples from patients with androgenic alopecia and normal volunteers to verify whether they are differentially expressed in AGA patients.
本实施例通过蛋白质印迹法检测雄激素性脱发患者以及正常志愿者头皮组织样本的c-Jun，NF-κB，SMAD3以及HIF-1α的表达量。This example uses Western blotting to detect the expression levels of c-Jun, NF-κB, SMAD3 and HIF-1α in scalp tissue samples from patients with androgenic alopecia and normal volunteers.
头皮组织样本是通过头皮活检技术搜集而来，一共搜集了30例雄激素性脱发患者以及30例正常志愿者头皮组织样本。随后使用RIPA组织/细胞裂解液与PMSF试剂进行蛋白质提取，将提取而来的蛋白进行下一步蛋白质印迹实验。Scalp tissue samples were collected through scalp biopsy technology. A total of 30 scalp tissue samples from patients with androgenic alopecia and 30 normal volunteers were collected. Then use RIPA tissue/cell lysis buffer and PMSF reagent to extract the protein, and use the extracted protein for the next step of Western blotting experiment.
蛋白质印迹实验具体步骤如下：首先，按照配方配制10％SDS分离胶与浓缩胶，样品与加样缓冲液混合，100℃冰浴煮沸5min，冰浴、离心后用微量加样器等量加入各泳道进行电泳分离，通过SDS-PAGE分离蛋白质，转移至PVDF膜(Merck Millipore，MA，USA)并在5％BSA中孵育1小时。将膜在4℃条件下用1：400稀释的c-Jun一抗(abcam，#ab40766),NF-κB一抗(abcam，#ab32536)，SMAD3一抗(abcam，#ab40854)，HIF-1α一抗(abcam，#ab179483)和β-actin一抗(abcam，#ab115777)温育过夜,用TBST洗涤3次后与山羊抗兔二抗(abcam，#ab6721)温育1小时。室温下PBS缓冲液洗涤3次，每次5min。将膜浸入ECL反应液中，室温1min。移除液体后，将膜用食品保鲜膜覆盖，于阴暗室中环境下通过线片曝光，显影、定影后观察结果。The specific steps of the Western blotting experiment are as follows: First, prepare 10% SDS separation gel and stacking gel according to the formula, mix the sample with the loading buffer, boil it in an ice bath at 100°C for 5 minutes, add equal amounts of each sample using a microsampler after ice bath and centrifugation. Lanes were electrophoretically separated and proteins were separated by SDS-PAGE, transferred to PVDF membranes (Merck Millipore, MA, USA) and incubated in 5% BSA for 1 hour. Use c-Jun primary antibody (abcam, #ab40766), NF-κB primary antibody (abcam, #ab32536), SMAD3 primary antibody (abcam, #ab40854), HIF-1α diluted 1:400 at 4°C. The primary antibody (abcam, #ab179483) and β-actin primary antibody (abcam, #ab115777) were incubated overnight, washed three times with TBST and then incubated with goat anti-rabbit secondary antibody (abcam, #ab6721) for 1 hour. Wash 3 times with PBS buffer at room temperature, 5 min each time. Immerse the membrane in the ECL reaction solution at room temperature for 1 min. After removing the liquid, cover the film with food wrap, expose it to a line film in a dark room, develop and fix it, and observe the results.
分析结果如图1显示，c-Jun，NF-κB，SMAD3以及HIF-1α四种AR辅助因子中只有HIF-1α在雄激素性脱发患者样本中存在差异表达，脱发患者中的表达 量显著高于正常对照，其他AR辅助因子均无差异表达。The analysis results show in Figure 1 that among the four AR cofactors, c-Jun, NF-κB, SMAD3 and HIF-1α, only HIF-1α is differentially expressed in samples from patients with androgenetic alopecia. The amount was significantly higher than that of the normal control, and there was no differential expression of other AR cofactors.
实施例2 HIF-1α抑制剂对雄激素性脱发小鼠模型表型的影响Example 2 Effect of HIF-1α inhibitor on the phenotype of androgenic alopecia mouse model
脱发的原因主要是由于局部毛囊对AR的敏感性差异所致，基于实施例1的发现，发明人推测抑制AR辅助因子——HIF-1α的差异表达会导致AR的敏感性改变进而导致脱发，基于此，发明人构建雄激素性脱发小鼠模型，探究HIF-1α抑制剂对雄激素性脱发的治疗效果。The cause of hair loss is mainly due to the difference in sensitivity of local hair follicles to AR. Based on the findings in Example 1, the inventor speculates that inhibiting the differential expression of AR cofactor-HIF-1α will lead to changes in AR sensitivity and then lead to hair loss. Based on this, the inventors constructed a mouse model of androgenetic alopecia to explore the therapeutic effect of HIF-1α inhibitors on androgenetic alopecia.
雄激素性脱发小鼠模型的构建：选取6-8周龄、体重18-22g的雄性C57BL/6小鼠进行模型制备，适应性饲养1周后,按体质量编号并用Excel根据体质量随机分为6组，每组5只，先用剃毛刀剃下小鼠毛发，再使用脱毛膏在其背部脱除约2×3cm2，脱毛5-10min后用温水将脱毛膏擦净，每只小鼠均以脊柱为分界线，在其背部对称部位取面积2×3cm2大小的皮肤，作为实验区，实验中所有给药组的药物溶剂均为无毒无害的玉米油，实验动物随机分为以下组别，见下表1：Construction of mouse model of androgenic alopecia: Male C57BL/6 mice aged 6-8 weeks and weighing 18-22g were selected for model preparation. After adaptive feeding for 1 week, they were numbered according to body weight and randomly divided according to body weight using Excel. There are 6 groups, 5 mice in each group. First, use a razor to shave the mouse hair, and then use depilatory cream to remove about 2×3cm 2 on its back. After depilating for 5-10 minutes, wipe the depilatory cream with warm water. The spine of the mice was used as the dividing line, and a skin area of 2 × 3 cm 2 was taken from the symmetrical part of the back as the experimental area. The drug solvent in all administration groups in the experiment was non-toxic and harmless corn oil, and the experimental animals were randomly Divided into the following groups, see Table 1 below:
表1雄激素性脱发小鼠模型的处理方式

Table 1 Treatment methods of mouse model of androgenic alopecia

其中空白组小鼠除剃毛不做任何处理，其余组小鼠经5％水合氯醛轻度麻醉后，按照5mg/kg剂量每日皮下注射丙酸睾酮建立雄激素性脱发小鼠模型，连续30天。建模组以及给药组于造模处理30min后，按照上述方案给药处理，每日拍照记录小鼠新生毛发情况，连续30天后，处死小鼠，用剃毛刀剃下小鼠新生毛发，统计小鼠新生毛发重量。图2A为构建的小鼠雄激素性脱发模型中，各类药物的治疗效果图。图2B为构建的小鼠雄激素性脱发模型中，剃下的5只小鼠新生毛发平均重量统计图。由图2A，2B可以看建模组经历30天的毛发生长，新生毛发稀疏，脱发面积大，小鼠背部仍有大片裸露无毛区域且新生毛发平均重量轻，而HIF-1α抑制剂处理组、空白组经历30天的毛发生长后，新生毛发浓密，脱发面积小，小鼠背部肉眼可见覆盖大片新生毛发且几乎没有裸露无毛区域，新生毛发平均重量远超建模组。The mice in the blank group received no treatment except shaving. The mice in the remaining groups were lightly anesthetized with 5% chloral hydrate and injected subcutaneously with testosterone propionate at a dose of 5 mg/kg daily to establish a mouse model of androgenic alopecia. 30 days. After 30 minutes of modeling treatment, the modeling group and the drug administration group were administered medication according to the above protocol. Photographs were taken every day to record the new hair of the mice. After 30 consecutive days, the mice were sacrificed and the new hair of the mice was shaved with a razor. Calculate the weight of new hair in mice. Figure 2A shows the therapeutic effects of various drugs in the constructed mouse model of androgenic alopecia. Figure 2B is a statistical diagram of the average weight of new hair of five shaved mice in the constructed mouse androgenic alopecia model. From Figures 2A and 2B, it can be seen that the model group experienced 30 days of hair growth. The new hair was sparse and the hair loss area was large. There was still a large bare hairless area on the back of the mouse and the average weight of the new hair was light. However, the HIF-1α inhibitor treatment group , after 30 days of hair growth in the blank group, the new hair was dense and the area of hair loss was small. The back of the mice was visible to the naked eye with a large area of new hair and almost no exposed hairless area. The average weight of new hair was far higher than that of the modeling group.
在细胞中，HIF-1α参与炎症反应，发明人认为是炎症反应发生时，由于炎性细胞的渗透和高代谢率使组织代谢活性增强，导致氧需求提高，再加炎症因子的作用导致的血管收缩，使得炎症区域的供氧降低，形成了一个低氧的微环境，导致HIF-1α就能大量表达。目前研究表明ILGF-1与IL-1、TNF-α、pGE2及LPS等致炎性细胞因子能激活HIF-1的转录活性，提示HIF-1α与炎症过程的密切关系。In cells, HIF-1α participates in inflammatory reactions. The inventor believes that when an inflammatory reaction occurs, tissue metabolic activity is enhanced due to the infiltration and high metabolic rate of inflammatory cells, resulting in an increase in oxygen demand. In addition, the action of inflammatory factors causes blood vessel damage. Contraction reduces the oxygen supply to the inflammatory area, forming a hypoxic microenvironment, resulting in a large amount of HIF-1α expression. Current research shows that ILGF-1, IL-1, TNF-α, pGE2 and LPS and other pro-inflammatory cytokines can activate the transcriptional activity of HIF-1, suggesting the close relationship between HIF-1α and the inflammatory process.
实施例3单细胞转录组测序检测雄激素性脱发小鼠模型皮肤组织中细胞水平基因改变Example 3 Single-cell transcriptome sequencing detects cellular level gene changes in skin tissue of androgenetic alopecia mouse model
为了进一步了解使用HIF-1α抑制剂处理后的小鼠皮肤组织细胞内发生的改 变，发明人分别将HIF-1α抑制剂——LW6处理组小鼠以及建模组小鼠的皮肤组织剥落分离后进行单细胞转录组测序。To further understand the changes that occur in mouse skin tissue cells after treatment with HIF-1α inhibitors To change, the inventors peeled off and separated the skin tissue of mice in the HIF-1α inhibitor-LW6 treatment group and the mice in the modeling group and performed single-cell transcriptome sequencing.
结果如图3所示，经过LW6处理的小鼠皮肤组织样本相较建模组的差异基因GO富集分析检测到了它们富集到毛发周期通路(hair cycle)。这个结果说明经过HIF-1α抑制剂处理造成小鼠皮肤组织细胞内大量基因的表达改变，且改变的基因与毛发生长通路高度相关。The results are shown in Figure 3. The differential gene GO enrichment analysis of LW6-treated mouse skin tissue samples compared with the modeling group detected that they were enriched in the hair cycle pathway. This result shows that treatment with HIF-1α inhibitors causes changes in the expression of a large number of genes in mouse skin tissue cells, and the changed genes are highly related to the hair growth pathway.
实施例4 HIF-1α抑制剂对雄激素性脱发小鼠模型基因表达的影响Example 4 Effect of HIF-1α inhibitors on gene expression in androgenetic alopecia mouse model
之前就有研究报道雄激素性脱发患者的头皮活检样本中炎症相关基因显著上调。为进一步探究HIF-1α抑制剂在雄激素性脱发治疗中的作用机制，发明人检测了HIF-1α抑制剂处理后炎症相关基因(Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1)的转录情况。Previous studies have reported that inflammation-related genes are significantly upregulated in scalp biopsy samples from patients with androgenic alopecia. In order to further explore the mechanism of action of HIF-1α inhibitors in the treatment of androgenetic alopecia, the inventors detected the transcription of inflammation-related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) after treatment with HIF-1α inhibitors. Condition.
将上述构建雄激素性脱发模型小鼠处死后，取下小鼠皮肤样本消化。随后使用TRIZOL提取组织RNA，将提取下来的RNA进行反转录为cDNA，最后通过实时荧光定量PCR(qPCR)检测相关基因的表达水平。用于Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1qPCR检测的引物序列如下表2所示。After the above-constructed androgenic alopecia model mice were sacrificed, the mouse skin samples were removed and digested. TRIZOL was then used to extract tissue RNA, and the extracted RNA was reverse transcribed into cDNA. Finally, real-time fluorescence quantitative PCR (qPCR) was used to detect the expression levels of relevant genes. The primer sequences used for qPCR detection of Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, and Fstl1 are shown in Table 2 below.
表2炎症相关基因qPCR引物

Table 2 Inflammation-related gene qPCR primers

具体的操作步骤如下：将组织样本切成2-4毫米的薄片。然后将其转移到gentleMACS的C管当中，加入适量的TRIzol后盖紧C管的盖子，并将其倒置于gentleMACS组织离解器上，持续旋转孵育组织样本30分钟，反应结束后，可将其中的液体转移到离心管中，12000rpm，进行10分钟离心。离心完成后，然后将所有吸出的上清转移到新的离心管当中，然后向其中再加入200mL三氯甲烷，使用涡旋振荡器震动摇晃离心管15秒左右，然后将其静止放置在离心管架上5-8分钟后，12000rpm的10分钟离心。完成上步离心之后，可观察到此时离心管的液体分为三层，提取最上层的水相层的RNA，向其中加入500μL异丙醇，静止放置10分钟后12000rpm离心10分钟。弃上清，向离心管中加入之前预备好的使用DEPC水配制的75％乙醇1mL，盖紧离心管的管盖，上下混匀后将离心管放入离心机中，转8000rpm，5分钟离心。弃上清，此时可以观察到离心管底部有少许白色沉淀物，将离心管倒置于滤纸上，使其水分被吸尽，加入20-30μL DEPC水溶解其中的白色沉淀物。待溶解好了以后，可以使用NANODROP 2000仪器检测提取好的RNA浓度，并在离心管外壁进行标注。使用5X All-In-One RT MasterMix逆转录试剂盒将RNA逆转录为cDNA，使用中国近岸蛋白公司生产的SYBR SuperMix Plus qPCR试剂盒来完成实时荧光定量PCR实验。 The specific steps are as follows: Cut the tissue sample into 2-4 mm slices. Then transfer it to the C tube of gentleMACS, add an appropriate amount of TRIzol, tightly cover the C tube, place it upside down on the gentleMACS tissue dissociator, and continue to rotate and incubate the tissue sample for 30 minutes. After the reaction is completed, the tissue sample can be Transfer the liquid to a centrifuge tube and centrifuge at 12,000 rpm for 10 minutes. After centrifugation is completed, transfer all the aspirated supernatant to a new centrifuge tube, then add 200 mL of chloroform to it, use a vortex shaker to shake the centrifuge tube for about 15 seconds, and then place it still in the centrifuge tube. After racking for 5-8 minutes, centrifuge at 12,000 rpm for 10 minutes. After completing the previous centrifugation step, it can be observed that the liquid in the centrifuge tube is divided into three layers. Extract the RNA from the uppermost aqueous layer, add 500 μL isopropyl alcohol to it, let it stand for 10 minutes, and then centrifuge at 12,000 rpm for 10 minutes. Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water to the centrifuge tube, close the cap of the centrifuge tube tightly, mix up and down, put the centrifuge tube into the centrifuge, turn to 8000 rpm, and centrifuge for 5 minutes. . Discard the supernatant. At this time, you can observe a little white precipitate at the bottom of the centrifuge tube. Place the centrifuge tube upside down on the filter paper to absorb all the water. Add 20-30 μL DEPC water to dissolve the white precipitate. After it is dissolved, you can use the NANODROP 2000 instrument to detect the concentration of the extracted RNA and mark it on the outer wall of the centrifuge tube. The 5X All-In-One RT MasterMix reverse transcription kit was used to reverse transcribe RNA into cDNA, and the SYBR SuperMix Plus qPCR kit produced by China Nearshore Protein Company was used to complete the real-time fluorescence quantitative PCR experiment.
实验结果如图4A-4G所示，相较于建模组，使用HIF-1α抑制剂(LW6,2-MeOE2,PX-4782HCI,BAY87-2243)处理小鼠的皮肤样本中，相关基因Nmur1(图4A)、Pcolce(图4B)、Fbln1(图4C)、Prrx1(图4D)、Vim(图4E)、Tnfrsf1b(图4F)、Fstl1(图4G)的表达水平下降，接近于空白对照组。The experimental results are shown in Figures 4A-4G. Compared with the modeling group, in the skin samples of mice treated with HIF-1α inhibitors (LW6, 2-MeOE2, PX-4782HCI, BAY87-2243), the related gene Nmur1 ( Figure 4A), Pcolce (Figure 4B), Fbln1 (Figure 4C), Prrx1 (Figure 4D), Vim (Figure 4E), Tnfrsf1b (Figure 4F), and Fstl1 (Figure 4G) expression levels decreased and were close to those in the blank control group.
HIF-1α抑制剂LW6，2-MeOE2，PX-4782HCI，BAY87-2243(购自Selleck，货号：#S8441、#S1233、#S7612、#S7309)。HIF-1α inhibitor LW6, 2-MeOE2, PX-4782HCI, BAY87-2243 (purchased from Selleck, product numbers: #S8441, #S1233, #S7612, #S7309).
由实施例4可知，使用了HIF-1α抑制剂会导致小鼠皮肤样本中的相关基因(Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1)的表达水平下降，发明人认为这些基因是调控脱发的关键基因，它们的表达下调最终改善了雄激素性脱发小鼠模型的毛发脱落的情况。It can be seen from Example 4 that the use of HIF-1α inhibitors will lead to a decrease in the expression levels of related genes (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) in mouse skin samples. The inventor believes that these genes are regulatory Downregulation of key genes for hair loss ultimately improved hair loss in mouse models of androgenic alopecia.
以上所述，仅是本发明的较佳实施例，并非对本发明作任何限制，凡是根据本发明技术实质对以上实施例所作的任何简单修改、变更以及等效结构变化，均仍属于本发明技术方案的保护范围内。 The above are only preferred embodiments of the present invention and do not limit the present invention in any way. Any simple modifications, changes and equivalent structural changes made to the above embodiments based on the technical essence of the present invention still belong to the technology of the present invention. within the protection scope of the scheme.

Claims (8)

  1. HIF-1α抑制剂在制备治疗雄激素性脱发药物中的应用。Application of HIF-1α inhibitors in the preparation of drugs for the treatment of androgenic alopecia.
  2. 根据权利要求1所述的应用，其特征在于，所述HIF-1α抑制剂能够有效抑制HIF-1α对AR的辅助作用。The application according to claim 1, characterized in that the HIF-1α inhibitor can effectively inhibit the auxiliary effect of HIF-1α on AR.
  3. 根据权利要求2所述的应用，其特征在于，所述抑制HIF-1α对AR辅助作用表现为抑制选自Nmur1、Pcolce、Fbln1、Prrx1、Vim、Tnfrsf1b、Fstl1中一种或多种基因的表达。The application according to claim 2, characterized in that the inhibitory effect of HIF-1α on AR is expressed by inhibiting the expression of one or more genes selected from the group consisting of Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, and Fstl1. .
  4. 根据权利要求1所述的应用，其特征在于，所述HIF-1α抑制剂选自LW6，2-MeOE2，PX-478 2HCI，BAY87-2243中一种或多种。The application according to claim 1, characterized in that the HIF-1α inhibitor is selected from one or more of LW6, 2-MeOE2, PX-478 2HCI, and BAY87-2243.
  5. HIF-1α检测试剂在制备雄激素性脱发风险预测试剂盒中的用途。Use of HIF-1α detection reagent in preparing androgenetic alopecia risk prediction kit.
  6. 根据权利要求5所述的用途，其特征在于，所述HIF-1α检测试剂包括用于HIF-1α蛋白印迹检测的抗体、用于免疫组化检测的抗体或用于定量PCR的引物和探针。The use according to claim 5, wherein the HIF-1α detection reagent includes an antibody for HIF-1α Western blot detection, an antibody for immunohistochemical detection, or a primer and probe for quantitative PCR. .
  7. 根据权利要求6所述的用途，其特征在于，所述HIF-1α检测的对象为受试者头皮组织。The use according to claim 6, characterized in that the object of HIF-1α detection is the subject's scalp tissue.
  8. 根据权利要求5-7任一项所述的用途，其特征在于，所述HIF-1α抑制剂选自LW6，2-MeOE2，PX-478 2HCI，BAY87-2243中一种或多种。 The use according to any one of claims 5 to 7, characterized in that the HIF-1α inhibitor is selected from one or more of LW6, 2-MeOE2, PX-478 2HCI, and BAY87-2243.
PCT/CN2023/090743 2022-04-29 2023-04-26 APPLICATION OF HIF-1α INHIBITOR IN TREATMENT OF ANDROGENIC ALOPECIA WO2023208013A1 (en)

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CN102361853A (en) * 2008-12-30 2012-02-22 欧洲分子生物学实验室 Toluidine sulfonamides and their use
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