CN115089711A

CN115089711A - Use of HIF-1 alpha inhibitors for treatment of androgenetic alopecia

Info

Publication number: CN115089711A
Application number: CN202210465853.7A
Authority: CN
Inventors: 王大鹏
Original assignee: Suzhou Yipeng Pharmaceutical Technology Co ltd
Current assignee: Suzhou Yipeng Pharmaceutical Technology Co ltd
Priority date: 2022-04-29
Filing date: 2022-04-29
Publication date: 2022-09-23
Also published as: WO2023208013A1

Abstract

The invention discloses application of an HIF-1 alpha inhibitor in preparing a medicament for treating androgenetic alopecia. The HIF-1 alpha inhibitor can effectively inhibit the auxiliary effect of HIF-1 alpha on AR, and inhibit the expression of genes related to alopecia and inflammation (Nmur1, Pcolo, Fbln1, Prrx1, Vim, Tnfrsf1b and Fstl1) in an androgenetic alopecia mouse model, thereby promoting hair growth. The HIF-1 alpha inhibitor disclosed by the invention is selected from one or more of LW6,2-MeOE2, PX-4782HCI and BAY 87-2243. The HIF-1 alpha inhibitor has the advantages of low cost, small toxic and side effect and remarkable curative effect, and provides a new strategy for treating androgenetic alopecia.

Description

Use of HIF-1 alpha inhibitors for treatment of androgenetic alopecia

Technical Field

The invention belongs to the technical field of androgenetic alopecia treatment, and particularly relates to an application of an HIF-1 alpha inhibitor in treating androgenetic alopecia.

Background

Androgenetic alopecia (AGA) is one of the most common types of alopecia in clinical practice at present; the disease is clinically manifested as hair loss on forehead, crown of head (male type) or central region of scalp (female type), accompanied by fine and soft hair, reduced density or greasy scalp; differences in hair shaft diameter of >20% were seen under the skin mirror, vellus hair increased, number of hairs decreased in unit hair follicle, brown perifolliculus, yellow spots, etc.; the pathological feature is mainly the progressive reduction of hair follicle volume. The alopecia affects various aspects of work, life and the like of the patient, and easily destroys the self-confidence of the patient, so that the patient has behaviors of depressed mood, anxiety and the like.

The term "androgenetic alopecia" was first proposed in 1960 by 0rentreich, thereby revealing a close relationship between androgens and alopecia. AGA is currently considered to be an androgen-dependent multigenic genetic disease, which increases the sensitivity of hair follicles to androgen action, affects signal transduction between dermal papilla and hair follicle cells, and causes a series of changes such as progressive reduction of the forehead and/or top susceptible hair follicles, gradual transition of the hair follicles to vellus hair follicles, progressive reduction of the anagen phase, and concomitant reduction in the volume of hair matrix and hair papilla, and corresponding shortening and thinning of hair, resulting in the occurrence of baldness.

The androgen existing in the body is mainly testosterone (T), while Dihydrotestosterone (DHT) is a more active androgen produced by the conversion of T under the catalytic action of 5 α -reductase, the affinity of the Androgen Receptor (AR) is 5 times that of testosterone, and after testosterone or dihydrotestosterone is combined with AR, it enters into nucleus and is combined with DNA receptor, thus producing biological effect. Human 5 α -reductase is known to have two isozymes, type I which is mainly distributed in sebaceous glands, chest and back skin, liver, adrenal gland and kidney, and type II which is mainly distributed in scalp, in hair follicles (innermost layer of outer hair root sheath) and perifollicular tissue. McGinley et al found that patients with congenital type II 5 alpha-reductase deficiency had normal or slightly elevated serum testosterone, but reduced DHT synthesis, and homozygote patients exhibited pseudoamphoterism with reduced body hair, normal hair growth, and no AGA. In addition, the water levels of testosterone, dihydrotestosterone, AR and 5 α -reductase in the scalp of bald parts are higher on average than in non-bald parts (such as occipital scalp) or the same parts of non-balded persons, and thus it is possible to explain why AGA does not affect occipital hair even if it is severe again. In addition, there are reports that the serum T level of AGA patients is not obviously different from that of normal people, while the serum DHT level is higher than that of normal people, and the relevance of dihydrotestosterone to AGA pathogenesis is further proved.

The etiology and pathogenesis of AGA are not clear, and it is believed that the expression of androgen and its receptor (AR) play a key role in the pathogenesis of the disease, while type II 5 α -reductase is an important factor in its pathogenesis. Currently, the national food and drug administration (SFDA) approved medicines for treating male AGA are mainly finasteride tablets and minoxidil solution. Finasteride is a type II 5 α -reductase inhibitor that blocks the conversion of Testosterone (Testosterone, T) to more active Dihydrotestosterone (DHT) and thereby reduces the inhibitory effect of androgens on hair follicles. The mechanism of minoxidil treatment is not completely defined and may involve multiple pathways, such as opening ATP-dependent K on the cell membrane ⁺ Channels, influence Androgen Receptor (AR) activity and stability, etc. At present, although the two drugs are widely applied to clinic, the two drugs can not completely block the progress of the disease course, and have adverse reactions of different degrees.

Therefore, it is highly desirable to find a new drug with better efficacy and less side effects for treating androgenetic alopecia.

Disclosure of Invention

In order to solve the above technical problems, a first aspect of the present invention provides a method for screening cofactors having inhibitory effects on AR activation, comprising the steps of: step S1, collecting scalp tissue samples of normal volunteers and androgenetic alopecia patients by using a scalp biopsy technology; step S2, detecting the expression of different AR accessory factors (c-Jun, NF-kB, SMAD3 and HIF-1 alpha) in scalp tissue samples of normal volunteers and androgenetic alopecia patients; in step S3, the AR cofactor differentially expressed in step S2 is selected and initially determined as a cofactor associated with androgenetic alopecia.

Furthermore, the accessory factor related to androgenetic alopecia determined by the invention is HIF-1 alpha.

Further, the detection is performed using western blotting (western blotting).

In a second aspect, the invention provides a use of an inhibitor of HIF-1 α in the manufacture of a medicament for treating androgenetic alopecia.

Further, the HIF-1 alpha inhibitor has good curative effect on androgenetic alopecia.

Further, the HIF-1 alpha inhibitor can effectively inhibit the auxiliary effect of the HIF-1 alpha on AR.

Further, the inhibition of HIF-1 α adjuvant effect on AR is manifested by inhibition of expression of one or more genes selected from Nmur1, Pcool, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl 1.

Further, the HIF-1 alpha inhibitor is selected from one or more of LW6,2-MeOE2, PX-4782HCI, BAY 87-2243.

In a third aspect, the invention provides use of HIF-1 α test agents in the preparation of a kit for predicting risk of androgenetic alopecia.

Further, the HIF-1 alpha detection reagent comprises an antibody for Western blot detection of HIF-1 alpha, an antibody for immunohistochemical detection or primers and probes for quantitative PCR.

Further, the HIF-1 alpha test subject is a scalp tissue sample of the subject.

The fourth aspect of the invention provides a method for constructing a mouse model of androgenetic alopecia, which is characterized by comprising the step of injecting testosterone propionate into male C57BL/6 mice.

Further, the frequency of the injections is daily.

Compared with the prior art, the invention has the following effects:

1) according to the invention, scalp biopsy is carried out on normal people and androgenetic alopecia patients, the AR accessory factor HIF-1 alpha which is differentially expressed is screened out, and the occurrence risk of androgenetic alopecia can be predicted through detection of the differential expression factor.

2) The HIF-1 alpha inhibitor is found to effectively inhibit HIF-1 alpha in an androgenetic alopecia mouse model, prevent the transcription of related genes (Nmur1, Pcole, Fbln1, Prrx1, Vim, Tnfrsf1b and Fstl1), change the expression level of the related genes, promote the hair growth of the androgenetic alopecia model mouse by reducing the expression of the related genes, and further can be used as a therapeutic drug for androgenetic alopecia.

3) The HIF-1 alpha inhibitor for preparing the medicament for treating the androgenetic alopecia has the advantages of low cost, small toxic and side effects and obvious curative effect, and provides a new strategy for treating the androgenetic alopecia.

Drawings

In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments will be briefly described below.

Figure 1 shows the expression of different AR co-factors in scalp samples from androgenetic alopecia patients as well as normal volunteers.

FIG. 2A is a graph of therapeutic efficacy of various HIF-1. alpha. inhibitor drugs in a constructed mouse androgenetic alopecia model. Fig. 2B is a statistical graph of shaved new hair weight in the constructed mouse androgenetic alopecia model.

Figure 3 is a GO enrichment analysis of differentially expressed genes between cells of skin samples from androgenetic alopecia model mice after treatment with inhibitor LW 6.

FIGS. 4A-4G show the effect of inhibitors LW6,2-MeOE2, PX-4782HCI, BAY87-2243 on the expression levels of genes Nmur1 (FIG. 4A), Pcool (FIG. 4B), Fbln1 (FIG. 4C), Prrx1 (FIG. 4D), Vim (FIG. 4E), Tnfrsf1B (FIG. 4F), Fstl1 (FIG. 4G), respectively.

Detailed Description

The invention will be better understood from the following examples. However, it is easily understood by those skilled in the art that the descriptions of the embodiments are only for illustrating and explaining the present invention and are not for limiting the present invention described in detail in the claims. Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional methods, and test materials used therein are available from commercial companies, unless otherwise specified.

As used herein, the term "AR", androgen receptor, belongs to the steroid receptor in the nuclear receptor superfamily. AR generally consists of four domains: an N-terminal transcriptional activation region (NTD), a DNA binding region (DBD), a hinge region, and a ligand binding region (LBD).

As used herein, the term "AR cofactor" refers to a transcription factor capable of modulating the transcriptional activity of the Androgen Receptor (AR) and thereby affecting a number of functional properties of the androgen receptor, including ligand selectivity and/or DNA binding capacity, etc. The inventors herein selected the 4 most common AR cofactors, c-Jun, NF- κ B, SMAD3, and HIF-1 α, for validation.

As used herein, the term "HIF-1 α", hypoxia-inducible factor, a transcription factor widely present in mammals and humans under hypoxic conditions, is a key factor in response to hypoxic stress. HIF-1 α is a subunit of hypoxia inducible factor-1 (HIF-1), and is regulated by hypoxia and regulates HIF-1 activity. Under hypoxic conditions, HIF-1 α translocates into the nucleus to bind HIF-1 β to form active HIF-1, which regulates transcription of a variety of genes by binding to hypoxia-responsive elements on target genes. HIF-1 alpha can form different signal channels with a plurality of proteins at the upstream and the downstream, mediate hypoxic signals, regulate and control cells to generate a series of compensatory responses to hypoxia, play an important role in the growth and development of organisms and physiological and pathological processes, and is a focus of biomedical research.

As used herein, the term "HIF-1 α inhibitor" refers to a compound or composition that can be used to inhibit HIF-1 α activity and/or expression. 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 number: 1227158-85-1), and the like.

As used herein, the term "androgenetic alopecia (AGA)", androgenic alopecia, is a common and frequently occurring disease of the dermatology family, a characteristic baldness with genetic factors involved and androgen-dependent action, often adversely affecting the physical image and mental health of the patient. The reason for AGA is mainly that when the content of testosterone in skin is excessive, testosterone can generate dihydrotestosterone under the catalysis of 5-alpha reductase, and the dihydrotestosterone and the testosterone compete for androgen receptor in hair follicle target cells. Since dihydrotestosterone has a significantly higher activity than testosterone, dihydrotestosterone has a greater ability to bind to the androgen receptor than testosterone. Once the dihydrotestosterone enters into cell nucleus, the growth of hair follicle cells can be inhibited, and the hair follicle is promoted to enter a resting period in advance, so that the hair is shed. The androgen can also act on the sebaceous gland to activate an SREBP (sequence-specific binding protein) pathway in the sebaceous gland cell, promote the immature cells on the outer layer of the sebaceous gland cell to develop and differentiate into mature secretory cells, so that the sebaceous gland is enlarged, the secretory function is enhanced, and excessive sebum is produced. When the sebaceous gland secretion is hypersecretion, excessive sebum can block or press pores, prevent normal growth of hair and even induce inflammation, thus causing hair withering and falling off.

Example 1 determination of AR cofactors associated with androgenetic alopecia

It has been experimentally confirmed that androgen levels in blood of most AGA patients are not elevated. Meanwhile, hair follicle transplantation tests show that the occipital hair follicle transplanted to the top of the head still has the characteristic of AGA resistance, and hair on the forehead is transplanted to the forearm and then still has hair loss with the forehead hair follicle synchronously. The main reason for this tendency to maintain the characteristics of the primary site of growth is due to differences in local AR sensitivity of the follicles. In 2002, it has been detected by foreign scholars that the activity of androgen receptor in alopecia area of AGA patients is increased, and the sensitivity to androgen is increased even though the androgen level in the patients is normal, thereby influencing the growth metabolism of hair follicles to cause alopecia, considering that AR only has increased activity but not increased expression in alopecia area of patients. The inventor speculates that the AR activity is increased probably due to the differential expression of the AR accessory factor, so the inventor researches the literature, inquires several previously verified AR accessory factors c-Jun, NF-kB, SMAD3 and HIF-1 alpha, detects the expression quantity of the AR accessory factors c-Jun, NF-kB, SMAD3 and HIF-1 alpha in androgenetic alopecia patients and normal volunteers scalp tissue samples, and verifies whether the AR accessory factors are differentially expressed in AGA patients.

In this example, the expression levels of c-Jun, NF- κ B, SMAD3 and HIF-1 α in scalp tissue samples from androgenetic alopecia patients and normal volunteers were determined by Western blotting.

Scalp tissue samples were collected by scalp biopsy techniques, for a total of 30 androgenic alopecia patients and 30 normal volunteers. Then, protein extraction was performed using RIPA tissue/cell lysate and PMSF reagent, and the extracted protein was subjected to the next western blotting experiment.

The western blot experiment comprises the following specific steps: first, 10% SDS separation gel and concentrated gel were prepared according to the formulation, the sample was mixed with a sample addition buffer, boiled in ice bath at 100 ℃ for 5min, electrophoretically separated by adding a microsyringe in equal amounts to each lane after ice bath and centrifugation, and the protein was separated by SDS-PAGE, transferred to PVDF membrane (Merck Millipore, MA, USA) and incubated in 5% BSA for 1 hour. The membrane was dried at 4 ℃ using a 1: 400 dilutions of c-Jun primary antibody (abcam, # ab40766), NF-. kappa.B primary antibody (abcam, # ab32536), SMAD3 primary antibody (abcam, # ab40854), HIF-1. alpha. primary antibody (abcam, # ab179483) and β -actin primary antibody (abcam, # ab115777) were incubated overnight, washed 3 times with TBST and incubated with goat anti-rabbit secondary antibody (abcam, # ab6721) for 1 hour. The cells were washed 3 times with PBS buffer at room temperature for 5min each time. The membrane was immersed in the ECL reaction solution at room temperature for 1 min. After removing the liquid, the film was covered with a food preservative film, exposed to light through a line film in a dark room, developed, and observed after fixation.

The analysis result is shown in figure 1, only HIF-1 alpha in four AR accessory factors of c-Jun, NF-kappa B, SMAD3 and HIF-1 alpha has differential expression in a sample of a patient suffering from androgenetic alopecia, the expression level of the patient suffering from androgenetic alopecia is obviously higher than that of a normal control, and no other AR accessory factors are differentially expressed.

Example 2 Effect of HIF-1 α inhibitors on the phenotype of mouse model of androgenetic alopecia

The reason for hair loss is mainly due to the difference in sensitivity of local hair follicles to AR, and based on the findings of example 1, the inventors speculate that inhibition of differential expression of HIF-1 α, which is an AR cofactor, causes a change in sensitivity of AR, which in turn causes hair loss, and based on this, the inventors constructed a mouse model of androgenetic alopecia and studied the therapeutic effect of HIF-1 α inhibitors on androgenetic alopecia.

Construction of a mouse model for androgenetic alopecia: selecting male C57BL/6 mice 6-8 weeks old and 18-22g in weight, performing model preparation, adaptively feeding for 1 week, numbering according to body mass, randomly dividing into 6 groups with 5 mice per group according to body mass by Excel, shaving off hair of mice with a shaver, and removing hair on the back by depilatory cream to obtain hair removal cream of 2 × 3cm ² Wiping the depilatory cream with warm water after depilating for 5-10min, taking the symmetrical part of the back with area of 2 × 3cm ² The skin of the size, as the experimental area, the drug solvent of all administration groups in the experiment was non-toxic and harmless corn oil, and the experimental animals were randomly divided into the following groups, as shown in table 1 below:

TABLE 1 treatment regimen for androgenetic alopecia mouse model

Wherein, the blank group of mice are not treated except for shaving, and the other groups of mice are slightly anesthetized by 5 percent chloral hydrate and subcutaneously injected with testosterone propionate every day according to the dose of 5mg/kg to establish an androgenetic alopecia mouse model for 30 days continuously. And (3) after the model building treatment and the administration treatment are carried out for 30min, the administration treatment is carried out according to the scheme, the condition of the new hairs of the mice is recorded by shooting every day, after 30 days continuously, the mice are killed, the new hairs of the mice are shaved by a shaver, and the weight of the new hairs of the mice is counted. FIG. 2A is a diagram showing the therapeutic effects of various drugs in the constructed mouse androgenetic alopecia model. Fig. 2B is a graph of the mean weight of shaved new hair of 5 mice in the constructed mouse androgenetic alopecia model. From fig. 2A and 2B, it can be seen that the modeling group has hair growth for 30 days, the new hairs are sparse, the hair loss area is large, the back of the mouse still has large bare hairless area and the average weight of the new hairs is light, while the HIF-1 α inhibitor treatment group and the blank group have hair growth for 30 days, the new hairs are dense, the hair loss area is small, the back of the mouse can be seen by naked eyes to cover the large fresh hairs and the bare hairless area is almost absent, and the average weight of the new hairs is far beyond the modeling group.

In cells, HIF-1 alpha is involved in inflammatory reaction, and the inventor believes that when inflammatory reaction occurs, tissue metabolic activity is enhanced due to infiltration and high metabolic rate of inflammatory cells, so that oxygen demand is increased, and then blood vessels are contracted due to the action of inflammatory factors, so that oxygen supply in inflammatory areas is reduced, a hypoxic microenvironment is formed, and HIF-1 alpha can be expressed in large quantity. The research shows that ILGF-1, IL-1, TNF-alpha, pGE2, LPS and other inflammatory cell factors can activate HIF-1 transcription activity, suggesting the close relation between HIF-1 alpha and inflammation process.

Example 3 Single-cell transcriptome sequencing detection of Gene alterations at cell level in skin tissues of mouse model of androgenetic alopecia

To further understand the changes occurring in skin tissue cells of mice treated with the HIF-1 α inhibitor, the inventors performed single cell transcriptome sequencing after exfoliating and separating skin tissue of the HIF-1 α inhibitor, LW 6-treated mice and model-built mice, respectively.

Results as shown in fig. 3, differential gene GO enrichment analysis of LW 6-treated skin tissue samples compared to the modeling group detected their enrichment into hair cycle pathways (hair cycles). This result demonstrates that treatment with HIF-1 α inhibitors results in altered expression of a number of genes in mouse skin tissue cells, and that the altered genes are highly correlated with the hair growth pathway.

Example 4 Effect of HIF-1 α inhibitors on Gene expression in a mouse model of androgenetic alopecia

Previous studies have reported significant upregulation of inflammation-associated genes in scalp biopsy samples from patients with androgenetic alopecia. To further explore the mechanism of action of HIF-1 α inhibitors in the treatment of androgenetic alopecia, the inventors examined the transcriptional status of inflammation-associated genes (Nmur1, Pcool, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) following HIF-1 α inhibitor treatment.

After the mice with the constructed androgenetic alopecia model are killed, the skin samples of the mice are taken down and digested. Then extracting tissue RNA by using TRIZOL, carrying out reverse transcription on the extracted RNA into cDNA, and finally detecting the expression level of related genes by real-time fluorescent quantitative PCR (qPCR). Primer sequences for Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1qPCR assays are shown in table 2 below.

TABLE 2 inflammation-related genes qPCR primers

The specific operation steps are as follows: the tissue samples were cut into 2-4 mm slices. Then transferring the sample into a C tube of GentlemACS, adding a proper amount of TRIzol, then tightly covering the cover of the C tube, inverting the C tube on a GentlemACS tissue dissociator, continuously rotating and incubating the tissue sample for 30 minutes, transferring the liquid in the C tube into a centrifuge tube after the reaction is finished, carrying out 12000rpm, and carrying out centrifugation for 10 minutes. After centrifugation was complete, all of the aspirated supernatant was then transferred to a new centrifuge tube, and 200mL of chloroform was added thereto, the tube was shaken using a vortex shaker for about 15 seconds, and then placed on a centrifuge rack for 5-8 minutes at rest, and centrifuged at 12000rpm for 10 minutes. After the above centrifugation was completed, the liquid in the centrifuge tube was observed to be divided into three layers, and RNA in the uppermost aqueous layer was extracted, 500. mu.L of isopropyl alcohol was added thereto, and after standing for 10 minutes, centrifugation was carried out at 12000rpm for 10 minutes. Discarding the supernatant, adding 1mL of the previously prepared 75% ethanol prepared with DEPC water into the centrifuge tube, tightly covering the tube cap of the centrifuge tube, mixing up and down uniformly, placing the centrifuge tube into a centrifuge, rotating at 8000rpm, and centrifuging for 5 minutes. Discarding the supernatant, observing that there is a little white precipitate at the bottom of the centrifuge tube, placing the centrifuge tube on filter paper, absorbing the water, adding 20-30 μ L DEPC water to dissolve the white precipitate. After the RNA is dissolved, the extracted RNA concentration can be detected by using a NANODROP 2000 instrument and labeled on the outer wall of the centrifugal tube. RNA was reverse transcribed into cDNA using a 5XAll-In-One RT MasterMix reverse transcription kit, and a real-time fluorescent quantitative PCR experiment was completed using a SYBR SuperMix Plus qPCR kit produced by the national inshore protein company.

As shown in fig. 4A-4G, in comparison to the building block, the expression levels of relevant genes Nmur1 (fig. 4A), pcole (fig. 4B), Fbln1 (fig. 4C), Prrx1 (fig. 4D), Vim (fig. 4E), Tnfrsf1B (fig. 4F), Fstl1 (fig. 4G) were reduced in skin samples of mice treated with HIF-1 α inhibitors (LW6, 2-MeOE2, PX-4782HCI, BAY87-2243), approaching the blank control block.

HIF-1. alpha. inhibitors LW6,2-MeOE2, PX-4782HCI, BAY87-2243 (available from Selleck under the accession numbers # S8441, # S1233, # S7612, # S7309).

As can be seen from example 4, the use of HIF-1 α inhibitors resulted in a decrease in the expression levels of the genes of interest (Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl1) in the skin samples of mice, which were considered by the inventors to be key genes for controlling hair loss, and their down-regulation of expression ultimately improved hair loss in the androgenetic alopecia mouse model.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalent structural changes made to the above embodiment according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.

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Claims

Use of an HIF-1 α inhibitor for the manufacture of a medicament for the treatment of androgenetic alopecia.
2. The use of claim 1, wherein the HIF-1 α inhibitor is effective to inhibit the accessory effect of HIF-1 α on AR.
3. The use of claim 2, wherein the inhibition of HIF-1 α potentiation of AR is manifested by inhibition of expression of one or more genes selected from Nmur1, Pcolce, Fbln1, Prrx1, Vim, Tnfrsf1b, Fstl 1.
4. The use of claim 1, wherein the HIF-1 α inhibitor is selected from the group consisting of LW6,2-MeOE2, PX-4782HCI, BAY 87-2243.
Use of a HIF-1 α detection reagent in the preparation of a kit for risk prediction of androgenetic alopecia.
6. The use of claim 5, wherein the HIF-1 α detection reagent comprises an antibody for western blot detection of HIF-1 α, an antibody for immunohistochemical detection, or primers and probes for quantitative PCR.
7. The use of claim 6, wherein the HIF-1 α is detected in scalp tissue of a subject.
8. The use according to any one of claims 5 to 7, wherein the HIF-1 α inhibitor is selected from one or more of LW6,2-MeOE2, PX-4782HCI, BAY 87-2243.