Methods of promoting hair growth

Abstract

The present disclosure provides a method for promoting hair growth by modulating hair follicle cycling in an individual in need thereof. The method comprises inhibiting casein kinase 1 in an area of skin of the individual. The present disclosure further provides a method for increasing hair pigmentation in an individual in need thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to methods of promoting hair growth by inhibiting casein kinase 1 (CK1). The present disclosure further provides a method of modulating hair cycle phase by inhibiting casein kinase 1 and a method for increasing hair pigmentation. BACKGROUND

[0002] Hair has important physiological functions. For example, fur keeps most mammals warm, dry, and protected from harmful elements. Throughout an animal's life cycle, hair regrows through cycles of different phases of the hair follicle, including telogen (resting or dormant phase), anagen (regenerative or active hair growth phase), and catagen (regression or transitional phase). At the end of the telogen phase, the hair falls out and new hair replaces the old hair, resulting in the start of a new growth cycle. Each follicle has its own life cycle, which is affected by age, disease, and various other factors.

[0003] However, hair loss or insufficient hair growth is a common problem encountered by many humans and many animals. The hair follicle cycle is regulated by intrinsic and extrinsic signals that control the quiescence and activation of hair follicle stem cells (HFSCs). Insufficient activation and proliferation of hair follicle stem cells is the basis for alopecia in many biological and pathological conditions, including aging. Molecules that promote hair follicle stem cell activation and anagen initiation have been extensively studied as they can contribute to the regulatory approach for hair re-growth and provide therapeutic and cosmetic healing.

[0004] While there are some methods to address this problem, including therapeutic methods such as ultraviolet radiation, massage, psychotherapy, and exercise therapy, none of these methods are generally accepted as effective. Even methods such as vascular reconstruction surgery and acupuncture have little to offer in terms of hope.

[0005] Recently, the most common method of treating hair loss is drug therapy. Many types of drugs from vitamins to hormones have been tried, but only a few are considered to have a successful therapeutic effect. For example, androgens are known to be involved in the progression of male-pattern alopecia; therefore, systemic or topical administration of anti-androgens would provide an inhibitory effect for preventing or treating alopecia. However, anti-androgens are not as effective as expected.

[0006] Therefore, there is still an unmet need for compositions and methods that provide effective and easy-to-use for promoting hair growth in individuals in need thereof. SUMMARY

[0007] The present disclosure provides a method for enhancing or stimulating hair growth on a skin region of an individual in need thereof, the method comprising inhibiting casein kinase 1 in the skin region. In at least one embodiment, the casein kinase 1 is inhibited during the resting phase of the hair follicle cycle. In some embodiments, the casein kinase 1 is inhibited during the anagen phase of the hair follicle cycle. In some embodiments, the casein kinase 1 is inhibited in the sebaceous gland. In some embodiments, the casein kinase 1 in the skin region is casein kinase 1 alpha. In at least one embodiment of the present disclosure, the individual has hair loss. In some embodiments, the hair loss is hair loss caused by nutritional deficiency, drug-induced hair loss, radiation-induced hair loss, stress-induced hair loss, genetic hair loss, aging hair loss, or disease-induced hair loss. In some embodiments, the drug-induced hair loss is hair loss caused by chemotherapy drugs, lithium, arsenic, bismuth, boric acid, thallium, colchicine, retinoids, heparin, warfarin, beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, hormones, valproic acid, carbamazepine, phenytoin, cimetidine, anti-thyroid drugs, cholesterol-lowering drugs, interferons, anti-infective agents, amphetamines, anti-depressants, anti-fungal agents, anti-epileptic agents, birth control pills, vitamin A-based drugs, Parkinson's drugs, stomach disease drugs, or non-steroidal anti-inflammatory drugs. In some embodiments, the disease-induced hair loss is hair loss caused by an autoimmune disease, thyroid disease, metabolic syndrome, infection, or cancer. In some embodiments, the autoimmune disease is alopecia areata, lupus erythematosus, Sjogren's syndrome, scleroderma, Crohn's disease, inflammatory bowel disease, or psoriasis.

[0008] In some embodiments, the individual has alopecia. In some embodiments, the alopecia is at least one selected from the group consisting of androgenic alopecia, alopecia areata, anagen effluvium, spontaneous hair loss, telogen effluvium, and cicatricial alopecia.

[0009] In at least one embodiment of the present disclosure, the inhibition of casein kinase 1 comprises inhibiting the gene expression of casein kinase 1 in the skin region. In some embodiments, the inhibition comprises topically applying a casein kinase 1 alpha inhibitor to the skin region. In some embodiments, the casein kinase 1 inhibitor is selected from one of the group consisting of D4476, IC261, CKI7, and a compound represented by the following formulas II to VII:

[0010]

[0011] In some embodiments, the hair loss is due to delayed or slow hair growth, or premature hair loss. In at least one embodiment, the present disclosure provides a method of treating hair loss at different stages of the hair follicle cycle. In at least one embodiment of the present application, a method for modulating the hair follicle cycle in a skin region of an individual in need thereof is provided, comprising inhibiting casein kinase 1 in the skin region. In some embodiments, the casein kinase 1 in the skin region is casein kinase 1 alpha. In at least one embodiment of the present disclosure, a method for modulating the hair follicle cycle in a skin region of an individual in need thereof comprises inhibiting casein kinase 1 alpha in the skin region. In at least one embodiment, the modulation of the hair follicle cycle induces the hair follicle cycle to enter anagen, wherein the hair follicle cycle was in telogen prior to inhibiting casein kinase 1 in the skin region. In some embodiments, the modulation of the hair follicle cycle prolongs the anagen phase of the hair follicle, wherein the hair follicle cycle was in anagen prior to inhibiting casein kinase 1 in the skin region.

[0012] The present disclosure provides a method for increasing hair pigmentation in a skin region of an individual in need thereof, comprising inhibiting casein kinase 1 in the skin region. In at least one embodiment of the present disclosure, the casein kinase 1 in the skin region is casein kinase 1 alpha. In at least one embodiment of the present disclosure, the skin region comprises white hair. BRIEF DESCRIPTION OF DRAWINGS

[0013] The present disclosure can be more fully understood by reading the following description in conjunction with the drawing figures, in which:

[0014] Figures 1A-1D K14-Cre-ERT2-CK1α fl / fl Results of intraperitoneal injection (i.p.) of tamoxifen (TMX) in mice (CK1α KO) to induce CK1α ablation in their keratinocytes. Figure 1A Experimental design is shown. Figure 1B Phenotypes of mice are shown before and after shaving at week 7 (W7), week 8 (W8), week 9 (W9), week 10 (W10), week 11 (W11), and week 12 (W12). Figure 1C Hematoxylin and eosin (H&E) staining is shown. Figure 1D β-catenin staining of skin samples taken at the indicated weeks of age is shown.

[0015] Figures 2A-2E K14-Cre-ERT2-CK1α fl / flResults of intraperitoneal injection (ip) of tamoxifen (TMX) into mice (CK1αKO) to induce CK1α knockout in their keratinocytes. Figure 2A Show the experimental design. Figure 2B The phenotypes of mice are shown before and after shaving at week 9 (W9), and at week 10 (W10), week 12 (W12), week 13 (W13), and week 16 (W16). Figure 2C Hematoxylin and eosin (H&E) staining are shown. Figure 2D This shows β-catenin staining in collected skin samples. Arrows indicate hair germs, where β-catenin is increased in CK1αKO ​​mice. Figure 2E BrdU staining is shown.

[0016] Figures 3A-3D The application of 4-OH-TMX in K14-Cre-ERT2-CK1α showed that... fl / fl Results of local induction of CK1α knockout in keratinocytes of mice (CK1αKO). Figure 3A The experimental design involved topical application of 4-OH-TMX to the backs of mice after shaving at week 9. Phenotypic behavior was recorded, and samples were collected at weeks 9 (W9), 11 (W11), and 13 (W13). Figure 3B The phenotypes of mice at week 9 (W9), week 11 (W11), and week 13 (W13) are shown. Figure 3C Hematoxylin and eosin (H&E) staining are shown. Figure 3D Displays β-catenin staining on skin samples collected at a specified time; "D" indicates the number of days after induction.

[0017] Figures 4A-4F The CK1 inhibitor A51 was shown to induce hair growth and pigmentation in C57 / BL6 wild-type mice at 8 weeks of age. Figure 4A For the experimental design, A51 was applied topically to the skin on the back of mice 2 or 3 times during week 8 after the back was shaved. Arrows indicate the sampling times at weeks 9, 10, and 11. Figure 4B The phenotypes of mice at weeks 8 (W8), 9 (W9), 10 (W10), and 12 (W12) are shown. 0.1*2Q.OD: 0.1 mg, every two days, for a total of 0.2 mg; N represents the number of mice in each group. Figure 4C Hematoxylin and eosin (H&E) staining are shown. Figure 4D This shows β-catenin staining on collected skin samples. Figure 4E and Figure 4F Fontana-Masson staining of hair follicle pigmentation at high and low magnification, respectively.

[0018] Figures 5A-5E CK1 inhibitors D4476 and IC261 were shown to induce hair growth and pigmentation in C57 / BL6 wild-type mice at 8 weeks of age. Figure 5A The experimental design is shown, where D4476 or IC261 were applied topically to the back skin of mice every other day at 0.04 mg each time, for a total of 0.12 mg. The sampling times at weeks 9 and 10 are indicated by arrows. Figure 5B The phenotypes of the mice at week 8 (W8), week 9 (W9) and week 10 (W10) are shown. Figure 5C Hematoxylin and eosin (H&E) staining, Figure 5D β-catenin staining of the harvested skin samples is shown. Figure 5E Fontana-Masson staining of hair follicle pigmentation is shown.

[0019] Figures 6A-6C Inhibition of CK1 was shown to prolong the anagen phase in the hair cycle. Figure 6A The experimental design is shown, where 4-OH-TMX was applied topically to the back of the mice after shaving at week 4. Figure 6B The phenotypes of the mice at week 4 (W4), week 6 (W6), week 7 (W7) and week 8 (W8) are shown. Figure 6C The results of hematoxylin and eosin (H&E) and β-catenin staining at week 8 are shown.

[0020] Figures 7A-7I CK1a deletion in keratinocytes was shown to rescue the Mc1r 549del pigmented hair in mice. Figure 7A The cross design to obtain CK1a deletion in keratinocytes in Mc1r 549del mice. Yellow hair color in Mc1r 549del mice with K14-CreER-CK1a f / f Cross of mice to generate K14-CreER-CK1a f / f ; Mc1r 549del mice. Figure 7B Intraperitoneal injection of TMX to delete K14-CreER-CK1a f / f ; Mc1r 549del Design of CK1a deletion in keratinocytes in Mc1r Figure 7C The phenotype of the back hair color in Mc1r 549del mice and CK1a deletion in keratinocytes in Mc1r 549del mice. - / - ; Mc1r 549del ). Figure 7D Hair shaft pigmentation under the microscope is shown. Figure 7E andFigure 7F Show Fontana-Masson staining of hair follicle pigmentation and Fontana-Masson staining of cell number, respectively. Figure 7G and Figure 7H Show Western blot analysis of protein expression concentration in KitL / c-Kit pathway in mouse back skin and quantified expression concentration in histogram, respectively. Figure 7I Show Mc1r 549del Mice with CK1a - / - ; Mc1r 549del Mouse back skin melanin analysis.

[0021] Figures 8A-8D Show the effect of CK1 inhibition on sebaceous glands during promotion of hair growth. Figure 8A Show K14-CreER-CK1a; ROSA mT / mG Intravital multiphoton imaging on mouse ears. Figure 8B Show WST-1 assay of SZ95 cells (human sebocyte cell line) treated with IC261 for 24 hours. Figure 8C Show expression of PPARy (a biomarker of lipogenesis) and c-Myc (a biomarker of differentiation) in SZ95 cells treated with different concentrations of DMSO or IC261. Figure 8C Show that IC261 increases LC3B in treated SZ95 cells, showing that autophagy is induced, in a concentration dependent manner. DETAILED DESCRIPTION

[0022] Embodiments of the present disclosure are illustrated by way of example in the following embodiments. Based on the content disclosed in the specification, those skilled in the art can easily understand the advantages and effects of the present disclosure. The present disclosure can also be implemented or applied by other different embodiments. Each detail in the specification can also be modified and changed based on different viewpoints and applications without departing from the spirit disclosed in the present disclosure.

[0023] In general, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, biochemistry, biology, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. However, where an otherwise-accepted definition in the art is inconsistent with the intended meaning of the term as used herein, the term as used herein prevails. In addition, some terms can be optionally selected by the applicant, in which case the meaning of the selected term will be described in detail in the description of the present disclosure. Therefore, the terms used herein must be defined based on the meaning of the terms and the description throughout the entire specification.

[0024] It is further noted that, as used in this disclosure, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "or" and "and / or" are used herein interchangeably and have the same meaning as the term "and."

[0025] In addition, the use of the term "including" or "comprising" in the specification of a component or step is not meant to be limiting, such that other components or steps are excluded. Rather, the term "including" or "comprising" is intended to be synonymous with the term "including, but not limited to."

[0026] The terms "individual," "patient," and "subject" are used interchangeably herein and refer to a warm-blooded animal, including, but not limited to, primates (e.g., humans), cows, pigs, sheep, goats, horses, dogs, cats, rabbits, rats, or mice. The terms "subject" and "patient" are used interchangeably herein, for example, to refer to a mammalian subject, such as a human subject. In some embodiments, the subject is a human.

[0027] The term "therapeutically effective amount" or "effective amount" is intended to include an amount of a compound that, when administered, is sufficient to prevent, alleviate, or ameliorate one or more symptoms of a disorder, disease, or condition under treatment to some extent. The term "therapeutically effective amount" or "effective amount" also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by the researcher, veterinarian, medical doctor, or clinician.

[0028] The terms "pharmaceutically acceptable carrier," "pharmaceutically acceptable excipient," "physiologically acceptable carrier," or "physiologically acceptable excipient" refer to a cosmetically or pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, solvent, or encapsulation material. In some embodiments, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of a cosmetically or pharmaceutical formulation, and suitable for use in contact with the tissue or organ of an individual (e.g., a human or animal) without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. See Remington: The Science and Practice of Pharmacy, 22ndEd.; Allen, Ed.; Philadelphia, PA, 2012; Handbook of Pharmaceutical Excipients, 7thEd.; Rowe et al., Eds.; Pharmaceutical Press and American Pharmaceutical Association: 2012; Handbook of Pharmaceutical Additives, 3rdEd.; Ash and Ash, Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2ndEd.; Gibson, Ed.; CRC Press LLC: Boca Raton, FL, 2009.

[0029] The terms "about" or "approximately" refer to an acceptable error for the particular value as determined by one of ordinary skill in the art, which is dependent upon how the value is measured or determined. In some embodiments, the terms "about" or "approximately" refer to within 1, 2, 3, or 4 standard deviations. In some embodiments, the terms "about" or "approximately" refer to within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value.

[0030] The terms "drug," "cosmetic agent," and "therapeutic agent" refer to a compound or a cosmetic or pharmaceutical composition thereof that is administered to an individual to prevent, ameliorate, or treat one or more symptoms of a disorder, disease, or condition.

[0031] In some embodiments of the present disclosure, the methods provided herein include treating an individual regardless of the age of the patient, although some diseases or disorders are more common in certain age groups.

[0032] The CK1 inhibitors of the present disclosure have at least twice, at least 5 times, or at least 10 times greater inhibitory activity against CK1 than other kinases, such as cyclin-dependent kinases (CDKs) that regulate the cell cycle (e.g., Cdk2, Cdk4, and Cdk6). In addition, the CK1 inhibitors have at least twice, at least 5 times, or at least 10 times greater inhibitory activity against CK1 than protein kinase C (PKC), protein kinase A (PKA), human epidermal growth factor receptor 2 (HER2), rapid accelerated fibrosarcoma 1 (RAF-1), mitogen-activated protein kinase 1 (MEK1), mitogen-activated protein kinase (MAP kinase), epidermal growth factor receptor (EGF receptor), platelet-derived growth factor receptor (PDGF receptor), insulin-like growth factor receptor (IGF receptor), phosphoinositide 3-kinase (PI3 kinase), Weel kinase, Src, and / or AbI.

[0033] In some embodiments of the present disclosure, the CKI is selective for CK1 alpha (CSNK1A; GenBank Accession Nos. NP_001020276, NM_001025105, and NM_001020276 at the genomic, mRNA, or protein level). In some embodiments of the present disclosure, the CKI also has inhibitory activity against other CK isozymes, such as CK1 delta, CK1 epsilon, and the like. In at least one embodiment of the present disclosure, the CKI has inhibitory activity against CK1 alpha, CK1 delta, CK1 epsilon, and any combination thereof.

[0034] In at least one embodiment of the present disclosure, the casein kinase 1 inhibitor is represented by the following Formula I, including any stereoisomer or salt thereof:

[0035]

[0036] wherein:

[0037] R1and R2are each independently selected from the group consisting of H, straight or branched C1-C8alkyl, straight or branched C1-C5alkoxy, straight or branched C1-C5acyl, C5-C15aryl, and C3-C7heteroaryl, each optionally substituted with at least one of halide, hydroxyl, ester, ether, C5-C15aryl, C3-C7heteroaryl, and amide; or R1and R2together with the nitrogen atom to which they are attached form a 4- to 7-membered saturated, unsaturated, or aromatic ring optionally including at least one of N, O, NH, C=N, C=O, and SO2, and optionally substituted with at least one of straight or branched C1-C5alkyl, C5-C15aryl, C3-C7heteroaryl, hydroxyl, halide, and cyano;

[0038] R3and R4are each independently selected from the group consisting of H, straight or branched C1-C8alkyl optionally substituted with at least one of halide, hydroxyl, alkoxy, C5-C15aryl, C3-C7heteroaryl, ester, and amide; or

[0039] R1or R2and R3together with the carbon and nitrogen atoms to which each is attached form a 4- to 7-membered saturated, unsaturated, or aromatic ring optionally including at least one of N, NH, O, C=N, C=O, and SO2, and can be optionally substituted with at least one of straight or branched C1-C5alkyl, C5-C15aryl, C3-C7heteroaryl, hydroxyl, carbonyl, and halide;

[0040] R5and R8are each independently selected from the group consisting of H, halide, straight or branched C1-C8alkyl, straight or branched C2-C8alkenyl, and straight or branched C2-C8alkynyl optionally substituted with at least one halide;

[0041] R6is selected from the group consisting of straight or branched C1-C8alkyl, straight or branched C2-C8alkenyl, straight or branched C2-C8alkynyl, C5-C10cycloalkyl, and saturated or unsaturated 4- to 6-membered heterocycle optionally substituted with at least one of straight or branched C1-C8alkyl, C3-C7cycloalkyl, 4- to 6-membered heterocycle, C5-C15aryl, C3-C7heteroaryl, halide, hydroxyl, and C1-C5alkylhalide;

[0042] R7is selected from the group consisting of straight or branched C1-C8alkyl, straight or branched C2-C8alkenyl, and straight or branched C2-C8alkynyl optionally substituted with at least one of C3-C7cycloalkyl, 4- to 6-membered heterocycle, C5-C15aryl, C3-C7heteroaryl, halide, hydroxyl, and C1-C5alkylhalide.

[0043] Other casein kinase I inhibitors include those described in International Publication No. WO 2017 / 021969; the disclosure of which is incorporated herein by reference in its entirety.

[0044] The cosmetic or pharmaceutical compositions provided herein can be formulated into any dosage form suitable for topical administration to produce a local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, foams, films, aerosols, irrigants, sprays, suppositories, bandages, and skin patches. The topical formulations of the cosmetic or pharmaceutical compositions provided herein can also include liposomes, micelles, microspheres, nanosystems, and any mixture thereof.

[0045] Cosmetically or pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations provided herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against microbial growth, stabilizers, solubility enhancers, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotective agents, lyoprotective agents, thickening agents, and inert gases.

[0046] Cosmetic or pharmaceutical compositions can also be administered topically by electroporation, iontophoresis, sonophoresis, phonophoresis, microneedle or needle-free injection, such as PowderJect (Chiron Corp., Emeryville, CA) and Bioject (Bioject Medical Technologies Inc., Tualatin, OR).

[0047] Cosmetic or pharmaceutical compositions provided herein can be provided in the form of an ointment, cream, and gel. Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard oil, benzoinated lard oil, white petrolatum, olive oil, cottonseed oil, and other oils; emulsifiable or absorbable vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointments; water-soluble ointment vehicles, including polyethylene glycols of various molecular weights; emulsion vehicles, water-in-oil (W / O) emulsions or oil-in-water (O / W) emulsions, including cetyl alcohol, glycerol monostearate, lanolin, and stearic acid (see, e.g., Remington: The Science and Practice of Pharmacy). These vehicles can be emollients, but often require the addition of antioxidants and preservatives.

[0048] Suitable cream bases can be either oil-in-water or water-in-oil. Suitable cream vehicles can be water-washable and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase, also known as the "internal" phase, is usually composed of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume and usually contains a humectant. The emulsifier in a cream formulation can be a nonionic, anionic, cationic, or amphoteric surfactant.

[0049] Suitable gels can be semi-solid, suspension-type systems. Single-phase gels contain organic macromolecules substantially uniformly distributed throughout a liquid carrier. Suitable gelling agents include, but are not limited to, cross-linked acrylic acid polymers such as carbomer, carboxy polyalkylene, and Carbopol; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol; cellulose polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl methyl cellulose phthalate, and methyl cellulose; gums such as tragacanth and xanthan gum; sodium alginate; and gelatin. To prepare a homogeneous gel, a dispersing agent such as an alcohol or glycerin can be added, or the gelling agent can be dispersed by grinding, mechanical mixing, and / or agitation.

[0050] Examples

[0051] Exemplary embodiments of the present disclosure are further described in the following examples, which should not limit the scope of the present disclosure.

[0052] Generally, the nomenclature and laboratory procedures used in this disclosure include molecular, biochemical, and recombinant DNA technologies. These technologies are explained in detail in the literature. For example, see Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989); Ausubel, RM, “The Current Handbook of Molecular Biology,” Volumes I-II (1994); John Wiley & Sons, “A Practical Guide to Molecular Cloning,” New York (1988); Watson et al., “Recombinant DNA,” Scientific American Books, New York; Birren et al., “Cell Biology: A Laboratory Manual,” Volumes I-III, edited by Cellis, JE (1994); “Animal Cell Culture—A Basic Technique Manual,” Freshney, Wiley-Liss, NY (1994), third edition; Hames, BD and Higgins, SJ, “Transcription and Translation” (1984); Freshney, RI, “Animal Cell Culture” (1986); IRL, “Immobilized Cells and Enzymes” (1986); Perbal, B., “A Practical Guide to Molecular Cloning” (1984), and Academic... The following publications are cited in accordance with reference to other sources: "Enzymological Methods," Volumes 1-317, Academic Press; "PCR Handbook: Methods and Applications," San Diego, Calif. (1990); and "Protein Purification and Characterization Strategies—A Laboratory Course Manual," Marshak et al., CSHL (1996). All of these are incorporated herein by reference as if fully described herein. Other general references are provided throughout this disclosure. The procedures described herein are considered well-known in the art and are provided for the reader's convenience. All information contained herein is incorporated herein by reference.

[0053] Materials and methods

[0054] laboratory mice

[0055] K14-Cr-ERT2-CK1α was obtained in cooperation with the Tainan Laboratory Animal Center in Taiwan. fl / fl Mice, Mc1r 549del Mice (Mc1r) 549del ) and CK1α knockout Mc1r in keratinocytes 549del Mice (CK1α) - / - Mc1r 549del) and wild-type C57BL / 6J mice were also purchased from the Tainan Experimental Animal Center. Experiments and animal care were performed at the Experimental Animal Center of Tzu Chi University, Hualien, Taiwan, and were in accordance with regulations and ethical standards approved by the Animal Care Committee. Male and female mice were used. The hair cycle of wild C57BL / 6J mice is age-dependent; the 4thto 6thweek is the anagen phase, the 6thto 7thweek is the catagen phase, and the 7thto 12thweek is the telogen phase.

[0056] Targeted gene activation (inducing CK1a deletion)

[0057] To delete the expression of CK1a in keratinocytes, K14-Cre-ERT2-CK1a fl / fl Mice were injected intraperitoneally twice with 100 mg / kg tamoxifen (TMX) dissolved in corn oil (C8267; Sigma) or with 4-hydroxytamoxifen (4-OH-TMX) (H6278; Sigma) dissolved in 99.9% alcohol (32205; Sigma). After shaving the back skin 5 times per week, 200 μL of 3 mg / mL concentration was applied to the dorsal skin.

[0058] Preparation of CK1 inhibitors (CKIs)

[0059] A51 was obtained from the laboratory of Dr. Yinon Ben-Nerian (published in Cell, vol. 175, issue 1, September 20, 2018, pages 171-185). For topical application to the skin of mice, 1 mg of CKI was dissolved in 6 μL of dimethyl sulfoxide (DMSO) and mixed with 54 μL of vehicle containing 60% white wax and 40% paraffin oil. The final working concentration was 0.1 mg / cm 2 .

[0060] D4476 was purchased from Sigma Aldrich (#D1944). For topical application to the skin of mice, 4 mg of CKI was dissolved in 1 mL of DMSO as a stock solution. 10 μL of stock solution was mixed with 40 μL of vehicle containing 60% white wax and 40% paraffin oil. The final working concentration was 0.04 mg / cm 2 .

[0061] IC261 was purchased from Sigma Aldrich (#40090). For topical application to the skin of mice, 4 mg of CKI was dissolved in 1 mL of DMSO as a stock solution. 10 μL of stock solution was mixed with 40 μL of vehicle containing 60% white wax and 40% paraffin oil. The final working concentration was 0.04 mg / cm 2 .

[0062] Topical drug treatment

[0063] Eight-week-old mice were shaved to remove the hair on the dorsal skin and cleaned with 75% ethanol. CKI was topically applied on the dorsal skin 3 times within a week and tissue was collected on day 7, 14 and partially on day 28 after CKI application. Tissues were analyzed by the following methods.

[0064] Histopathology, immunohistochemistry staining and Fontana-Masson staining

[0065] Skin samples were collected at the indicated times for histological analysis. Paraffin-embedded specimens were cut into 5-μm sections. After deparaffination (immersion of tissue sections in xylene for 3 times, 5 min each) and rehydration (incubation of sections in the following graded ethanol series: 100%, 100%, 95%, 90% and 70%, 5 min each), slides were rinsed in distilled water for 5 min.

[0066] Hematoxylin and eosin (H&E) staining and Fontana-Masson staining

[0067] Fontana-Masson staining and hematoxylin and eosin (H&E) staining were performed using ScyTek Laboratories, Inc. kits according to the manufacturer's instructions.

[0068] Briefly, for Fontana-Masson staining, freshly mixed silver-ammonia solution was placed in a water bath at 58-60 °C and allowed to equilibrate. Slides were incubated in the warm silver-ammonia solution for 30-60 min or until the tissue sections turned yellow / brown, then rinsed in distilled water for 3-5 s. Slides were incubated in gold chloride solution (0.2%) for 30 s, then rinsed in distilled water for 3-5 s. Slides were incubated in sodium thiosulfate solution (5%) for 1 min, then rinsed in distilled water for 3-5 s. Slides were incubated in nuclear fast red solution for 5 min, rinsed in running tap water for 1 min, and dehydrated in four different fresh anhydrous alcohols (95%, 95%, 100% and 100%) and xylene, and mounted with Histokitt (Assistent).

[0069] For histopathology by hematoxylin and eosin (H&E) staining, skin tissues were fixed in 10% neutral buffered formalin at 4 °C overnight, then transferred to 70% ethanol, processed and embedded in paraffin. Paraffin sections were then stained with hematoxylin and eosin (H&E).

[0070] Immunohistochemistry staining

[0071] Paraffin sections were incubated in a humidity chamber at 60°C for 15 minutes. Sections were deparaffinised in two times xylene for 5 minutes each, hydrated in 100% ethanol for two times 5 minutes, then in 95% and 80% ethanol for 5 minutes each, and finally rinsed in distilled water. Antigen retrieval was enhanced by microwaving the slides in citrate buffer (DAKO; pH 6.0) for 20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in methanol. After blocking, sections were incubated overnight at 4°C with β-catenin antibody (1 / 100; BD Bioscience). The secondary antibody used was horse radish peroxidase (HRP)-polymer anti-mouse antibody (Nichirei). 3-amino-9-ethylcarbazole (AEC) chromogen (ScyTek) was used for detection, and haematoxylin was used as counterstain.

[0072] 5-Bromo-2'-deoxyuridine (BrdU) staining

[0073] For labelling proliferating cells in mice, an intraperitoneal injection of 200 μL BrdU (Amersham; Cell Proliferation Labeling Reagent, RPN201, GE Healthcare) was performed. Six hours later, skin samples were collected for paraffin embedding. 4 μm paraffin-embedded sections were prepared as above, then incubated with Target Retrieval Solution (pH 6.0) (DAKO, S1699) in a water bath at 95°C to 120°C for 20 minutes. The staining dish was moved to room temperature, allowing the slides to cool to 30°C, and rinsed once with PBS. Then, the slides were incubated with primary antibody BrdU (Thermo Fisher Scientific, B23151, 1:100) overnight at 4°C. The slides were rinsed 3 times with PBS for 5 minutes each. The slides were incubated with EnVision / HRP, Rabbit / Mouse (ENV) (DAKO, k5007) for 30 minutes, then rinsed 3 times with PBS for 5 minutes each. Then, the slides were incubated with AEC+High Sensitivity Substrate Chromogen Ready-to-Use (DAKO, K3461) for 5 minutes, and rinsed with running water for 5 minutes. The slides were then counterstained with haematoxylin for 30 seconds to 1 minute, and rinsed with running water for 10 minutes. The slides were mounted with Aqueous-Mount (ScyTek, 51934).

[0074] Western blotting

[0075] For experiments involving tissues, tissue homogenates were lysed in 1x tissue protein extraction reagent (Thermo Fisher) containing a protease inhibitor (Millipore) and stored on ice. For experiments involving cells, cell pellets were collected and lysed in 1x radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor (Millipore) and stored on ice. Total protein concentration was determined using a microplate reader and protein assay dye concentrate (Bio-Rad) according to the manufacturer's instructions. Samples were diluted in SDS-PAGE sample buffer (Bio-Rad) and heated at 95°C for 5 minutes. Protein samples were then eluted on SDS-PAGE gels (TGX FastCast acrylamide solution, Bio-Rad) and transferred to poly(vinylidene fluoride) (PVDF) membranes (Millipore). The membranes were incubated overnight at 4°C with 5% BSA and primary antibody. The membranes were then washed with Tris-buffered saline containing Tween 20 (TBST) and incubated with secondary antibody at room temperature for 1 hour. Signals were detected using the Thermo Fisher Scientific iBright FL1000 imaging system.

[0076] Antibody

[0077] The antibodies used in the immunohistochemistry (IHC) and Western ink dot (WB) methods disclosed herein are listed in Table 1 below, which provides the manufacturer, catalog number, and dilution ratio used.

[0078]

[0079] Eumelanin Analysis

[0080] Measure skin samples and use Ca-free 2+ and Mg 2+ Blood was removed by rinsing with PBS (pH 7.4). For melanin content determination, tissue was minced with scissors and homogenized in 10 volumes of PBS at 28°C. Skin samples were processed for chemical analysis of eumelanin to detect a specific degradation product, pyrrole-2,3,5-tricarboxylic acid (PTCA). 1 ng of PTCA corresponds to 50 ng of eumelanin. The statistical significance of differences in eumelanin content was determined by Student's t-test to compare groups of similar size.

[0081] Establish K14-CreER-CK1α;ROSAmT / mG mice

[0082] K14-CreER-CK1α;ROSAmT / mG mice were bred by crossing K14-CreER-CK1α and mT / mG mice at the Tainan Laboratory Animal Center, and maintained under specific pathogen-free conditions. For the CK1α knockout keratinocyte marker, we administered TMX dissolved in corn oil via intraperitoneal injection at a dose of 1 mg / day for 5 consecutive days. When CK1α knockout was induced by TMX, the red fluorescence (tdTomato;mT) on keratinocytes changed to green fluorescence (EGFP;mG).

[0083] In vivo multiphoton imaging of mouse ear skin

[0084] We performed in vivo multiphoton imaging of mouse ears in Dr. Pei-Ling Chen's laboratory (Applied Science Research Center, Taipei Research Institute, Taiwan). The methods and procedures were modified from doi: 10.1038 / nprot.2011.438. The peak emission wavelengths of EGFR (green fluorescence) and tdTomato (ROSA26) (red fluorescence) were 510 nm and 580 nm, respectively.

[0085] SZ95 cell culture and WST-1 assay

[0086] SZ95 cells (human sebaceous cell line) were maintained in Sebomed basal medium (Sigma) supplemented with 10% FBS and 5 ng / mL epidermal growth factor under standard cell culture conditions of 37°C and 5% CO2.

[0087] WST-1 assay. The inoculum density in the 96-well plate was 1.5 x 10⁻⁶. 4 Cells / well cultured overnight. Remove the culture medium and add 100 μL of fresh medium containing different amounts of IC261 (1, 50, 100, 200, 500, 1000 nM) to each well, and incubate for 24 hours. Add 10 μL / well of WST-1 and incubate for 4 hours. Measure the absorbance at OD 450 nm and OD 655 nm using a microplate reader.

[0088] Statistical analysis

[0089] The statistical analyses in this disclosure present the results as mean ± standard deviation. Student's t-test was used for inter-group comparisons. A p-value less than 0.05 was considered statistically significant.

[0090] Example 1: Inhibition of CK1α induces the anagen phase in the hair cycle

[0091] The inhibition of CK1α in mouse keratinocytes was achieved through various mechanisms and all showed induction of the anagen phase in the hair cycle, starting from the resting phase, early resting phase (7 weeks), and mid-resting phase (9 weeks).

[0092] In the first study, 7-week-old K14-Cre-ERT2-CK1α was used. fl / fl Adult mice were induced by intraperitoneal injection (ip) of tamoxifen (TMX) to knock out CK1α expression in keratinocytes. Figure 1A The study design showed that mice had their back fur shaved at week 7 and were injected intraperitoneally with TMX. The injection was administered daily at a dose of 100 mg / kg on days 1 and 2 after shaving. Phenotypic and histological data of mouse skin sections were recorded weekly from week 7 to week 12, as shown below. Figure 1B and Figure 1C As shown. The results indicated that in control mice (N=6), hair follicles on the back of the mice remained in the resting phase from week 7 to week 12. However, in CK1α knockout mice (CK1αKO, N=6), the early anagen phase of the hair follicles first appeared at week 8 or 9 and continued to develop into the complete anagen phase from week 10 to week 12. The Wnt / β-catenin pathway acts on hair follicle precursor cells as a proximal signal for the telogen-anagen transition. Figure 1D As shown, compared with control mice, β-catenin staining was increased in the skin of CK1α knockout mice at weeks 8 and 9.

[0093] In the second study, 9-week-old K14-Cre-ERT2-CK1α was used. fl / fl Adult mice were induced by intraperitoneal injection (ip) of tamoxifen (TMX) to knock out CK1α expression in keratinocytes. Figure 2A The study design involved shaving the back hair of mice at week 9, and administering intraperitoneal injections of TMX at a daily dose of 100 mg / kg on days 1 and 2 post-shaving. Phenotypic characteristics of the mice were recorded from week 9 to week 16, as shown below. Figure 2B As shown in the diagram. The results indicated that in control mice (N=3), hair follicles on the back of the mice remained in the resting phase from week 9 to week 12, and entered the anagen phase from week 13. Visible hair was observed on the backs of control mice at week 16. In contrast, in CK1α knockout mice (CK1αKO, N=3), the early anagen phase of hair follicles occurred between weeks 10 and 11, with visible hair growth starting from week 13. By week 16, the backs of CK1α knockout mice were completely covered with hair. Figure 2CHistological findings of mouse skin sections stained with hematoxylin and eosin (H&E) are shown. Results indicate that both normal and CK1αKO ​​mice exhibited resting hair follicles prior to intraperitoneal injection of TMX at week 9. In control mice, the resting phase persisted from week 9 to week 11 or 12, with the anagen phase beginning at week 13 and fully developing by week 16. In contrast, in CK1αKO ​​mice, the anagen phase began at week 10 and continued to grow hair from week 11 to week 16. This indicates that CK1α inhibition during the keratinocyte resting phase induced and initiated the anagen phase earlier than expected. Figure 2D The results show the immunohistochemical staining of β-catenin. Similar to the first study, β-catenin staining was increased in the skin of CK1α knockout mice at week 10 compared to control mice. Figure 2E Immunohistochemical staining results of 5-bromo-2'-deoxyuridine (BrdU) are shown, in which BrdU labels proliferating cells, and the staining of proliferating cells in the hair follicle matrix of CK1αKO ​​mice is increased compared with control mice.

[0094] Next, 4-OH-TMX was used to locally induce K14-Cre-ERT2-CK1α. fl / fl CK1α knockout in keratinocytes of mouse dorsal skin. (Example: Knockout of CK1α) Figure 3A As shown, 9-week-old mice were used. Their back hair was removed at week 9, and then 4-OH-TMX was locally induced on the shaved back skin. Figure 3B and Figure 3C The phenotype and histology recorded from week 9 to week 13 are shown. In normal mice (N=5), a resting phase was observed from week 9 to week 11, and the growth phase was noted to begin at week 13. In CK1α knockout mice (N=11), the growth phase with elevated papules on the skin surface was observed histologically at week 11, and visible hair was observed at week 13, with the growth phase fully developed. Figure 3D Immunohistochemical staining results for β-catenin are shown. At weeks 10 (W10) and 11 (W11), i.e., days 7 (D7) and 14 (D14) after CK1α knockout induction, β-catenin expression in hair germ was increased in CK1αKO ​​mice compared to control mice. β-catenin nuclear staining was observed in the basal layer of the epidermis, secondary hair germ, and matrix, indicating that Wnt / β-catenin signaling is activated during the telogen-anagen transition.

[0095] In addition, topical application of CK1 inhibitors to wild-type mice suppressed the expression of CK1 in the skin on the backs of the mice. For example... Figure 4AAs shown, at 8 weeks of age, a local CK1 inhibitor, such as A51, was applied to the shaved dorsal skin of mice at a concentration of 0.1 mg / cm². 2 . Figure 4B The study showed that back hair began to grow as early as week 9 in mice treated topically with the CK1 inhibitor A51, and most mice had their backs covered with hair by week 12. Figure 4C Hematoxylin and eosin (H&E) staining of the back skin revealed that in the control group, hair follicles remained in the resting phase at weeks 9, 10, and 12. However, mice treated topically with 0.2 mg CKI at week 8 showed induced anagen phase at week 9, and mice treated topically with 0.3 mg CKI at week 9 showed an even later anagen phase, with abundant pigmented hair shafts observed at week 12. This indicates that topical application of a CK1 inhibitor can also induce and initiate the anagen phase of hair follicles from the resting phase. Figure 4D Immunohistochemical staining results for β-catenin are shown. Topical application of the CK1 inhibitor increased the expression of β-catenin in the pubescent embryo, outer root sheath, and epidermis compared to control mice. Figure 4E The results of Fontana-Massen staining show, in addition to hair growth, follicular pigmentation in CK1-treated skin. Therefore, topical application of CK1 inhibitors can induce hair follicle growth and the formation of colored hair.

[0096] Other CK1 inhibitors, including D4476 and IC261, were also tested. Figure 5A As shown, CK1 inhibitors D4476 and IC261 were applied topically to the shaved back skin of 8-week-old mice every other day, three times a week, with a total dose of approximately 1 cm. 2 The amount of the skin on the back was 0.12 mg. Figure 5B The study showed that topical application of D4476 or IC261 significantly induced hair growth on the back skin of mice (N=3), which is a result of the hair cycle transitioning from the resting phase to the anagen phase. Figure 5C Hematoxylin and eosin (H&E) staining of the back skin revealed that topical application of a CK1 inhibitor induced the anagen phase of the hair cycle, and hair follicle formation was observed, showing hair follicles with anagen phase characteristics. Figure 5D Immunohistochemical staining results for β-catenin are shown. Topical application of the CK1 inhibitors D4476 and IC261 also increased the expression of β-catenin in the hair follicle matrix. Figure 5E The results of Fontana-Massen staining showed that topical application of additional CK1 inhibitors, such as D4476 and IC261, could induce hair follicle growth and the formation of colored hair.

[0097] Example 2: Inhibition of CK1α prolongs the anagen phase in the hair cycle

[0098] CK1α knockout during the anagen phase of keratinocytes was found to increase the duration of the anagen phase in the hair cycle. 4-OH-TMX was used for local induction of K14-Cre-ERT2-CK1α in 4-week-old keratinocytes. fl / fl CK1α knockout in keratinocytes of mouse dorsal skin, such as Figure 6A As shown. Phenotypic records were taken from week 4 to week 8. (See attached image.) Figure 6B As shown, in control mice (N=6), the growth phase lasted from week 4 to week 5, followed by a regression phase in week 6, and a resting phase from week 7 to week 8. In CK1α knockout mice (N=6), hair appeared and began to cover the shaved dorsal skin from week 6 to week 8, indicating a prolonged growth phase. Figure 6C The image shows hematoxylin and eosin (H&E) staining and β-catenin staining in mice at week 8. Control mice were found to be in a resting phase at week 8, but CK1α knockout mice remained in the growth phase at week 8, and showed increased β-catenin staining in the hair follicle matrix.

[0099] Example 3: Inhibiting CK1α from increasing hair pigmentation

[0100] Relapse revealed that inhibition of CK1α in keratinocytes increases hair pigmentation. CK1 inhibitors, including A51, D4476, and IC261, were topically applied to the shaved dorsal skin of mice at week 8, when the hair cycle was in the resting phase. Fontana-Massen staining was then performed to assess hair pigmentation. Figure 4E As shown, dark pigmented hair follicles and shafts were found in mouse hair treated topically with A51 and in mouse hair treated topically with D4476 and IC261, as Figure 5E As shown.

[0101] In addition to wild-type mice (black C57BL / 6), Mc1r 549del Mice have also been used as animal models to demonstrate increased hair pigmentation through inhibition of CK1α. Robbins Mountjoy et al. discovered Mc1r in 1992. 549del A single nucleotide deletion at position 549 of the MC1R in mice results in a 12-amino acid out-of-frame mutation: C57BL / 6J-Mc1r em1 The single nucleotide deletion at position 549 of the MC1R gene in mice was previously generated using the CRISPR / Cas9 system. This deletion results in a frameshift mutation and loss of Mc1r protein function, leading to yellowing of the mouse coat. This is due to the exclusive systhesis of melanin and the inability of melanocytes to synthesize eumelanin.Figure 7A As shown. Then, MC1R mutant mice were compared with K14-CreER-CK1α. f / f Mouse hybridization to produce K14-CreER-CK1α f / f Mc1r 549del Mice can be induced to knock out CK1α in keratinocytes. For example... Figure 7B As shown, 7-week-old mice were induced six times by intraperitoneal injection of tamoxifen at 100 mg / kg on days 1, 2, 5, 6, 8, and 9. Skin samples were collected on days 14, 28, 42, and 56 for analysis. Figure 7C As shown, with Mc1r 549del Compared to mice, Mc1r CK1α knockout in keratinocytes 549del Mice showed increased hair pigmentation on day 28. (Compared to Mc1r) 549del Compared to mice, on days 14 and 28, Figure 7D The dissecting microscope results shown in the image provide a closer and clearer view of Mc1r. 549del Increased hair shaft pigmentation in mice after CK1α knockout from keratinocytes. Figure 7E Fontana-Massen staining of skin samples showed that Mc1r was eliminated by CK1α knockout in keratinocytes. 549del Time-series analysis in mice demonstrated increased eumelanin intensity in the stroma and inner root sheath, which was observed in Mc1r. 549del It was not observed in mice. Figure 7F show Figure 7E The quantitative number of cells obtained by Fontana-Massen staining. Figure 7G and Figure 7H The results of Western blot analysis and corresponding quantitative expression levels reveal the stability of β-catenin and p53, as well as the upregulation of KitL, c-Kit, MITF, and tyrosinase. The KitL / c-Kit pathway is upregulated due to hair pigmentation. Figure 7I Display Mc1r 549del Mice and CK1α f / f Mc1r 549del Eumelanin analysis of dorsal skin in mice on days 14 and 28 was compared, with plate a indicating the experimental design for eumelanin analysis. Intraperitoneal injection of tamoxifen was used to eliminate Mc1r. 549del CK1α in mice. Back skin was collected from the control group and the CK1α knockout group on days 14 and 28 post-induction. As shown in Plate B, the data showed that Mc1r... 549del Eumelanin production increased in mice because eumelanin in the skin on the back of mice is mainly found in hair follicles, suggesting that inhibition of CK1α in hairy skin can increase hair pigmentation.

[0102] Example 4: Effects of CK1 inhibition on sebaceous glands and sebaceous cells

[0103] like Figure 8A As shown, in vivo multiphoton imaging of the ear skin of K14-CreER-CK1α;ROSAmT / mG mice. Before induction, keratinocytes of the sebaceous gland (SG) and hair follicles (HF) showed red fluorescence; after induction, CK1α-knockout keratinocytes showed green fluorescence. On day 3, sebaceous gland K14... + The basal layer exhibits green fluorescence, and during induced HF elongation, it can proliferate and differentiate into green fluorescent SGs by day 17. During CK1α inhibition of both SGs and HFs, SG size decreases, while HF growth is enhanced, suggesting that CK1α inhibition regulates SG size, which may be involved in the fate of pluripotent stem cells in the follicle-sebaceous unit. Figure 8B As shown, SZ95 (human sebaceous cell line) was treated with IC261 for 24 hours, with a cell count of 1.5 × 10⁻⁶. 4 Cells / well, IC50 approximately 475.6 nM. For example... Figure 8C As shown, SZ95 cell culture revealed sebaceous cells at different differentiation stages; PPARγ is a representative biomarker of adipogenesis in sebaceous cells, while c-Myc is a biomarker of undifferentiated sebaceous cells. In summary, IC261 treatment induced a decrease in PPARγ and an increase in c-Myc, indicating its inhibitory effect on adipogenesis or sebaceous cell differentiation. Figure 8D As shown, LC3B is a marker of autophagy. Overall, IC261 induces an increase in LC3B in SZ95 cells in a dose-dependent manner, indicating that IC261 induces autophagy in sebaceous cells.

[0104] This disclosure has been described in conjunction with its embodiments, and it should be understood that various modifications may be made to the embodiments of this disclosure without departing from the scope of this disclosure. Therefore, the described embodiments are intended to cover modifications within the scope of this disclosure, and not to limit it. Accordingly, the scope of the claims should be interpreted in the broadest possible sense to cover all such modifications.

Claims

1. The use of a casein kinase 1 inhibitor, characterized in that: For the preparation of a composition that promotes hair growth, the casein kinase 1 inhibitor is selected from the group consisting of D4476, IC261, CKI7, and compounds represented by formulas II to VI: The composition is applied topically to the skin area of ​​an individual who requires it.

2. The use of a casein kinase 1 inhibitor, characterized in that: For the preparation of a composition that increases hair pigmentation, the casein kinase 1 inhibitor is selected from the group consisting of D4476, IC261, CKI7, and compounds represented by formulas II to VI: The composition is applied topically to the skin area of ​​an individual who requires it.

3. The use as described in claim 2, wherein, The skin area includes gray hair.

4. The use of a casein kinase 1 inhibitor, characterized in that: For the preparation of a composition that reduces the size of sebaceous glands at hair follicles, the casein kinase 1 inhibitor is selected from the group consisting of D4476, IC261, CKI7, and compounds represented by formulas II to VI: The composition is applied topically to the skin area of ​​an individual who requires it.

5. The use as described in any one of claims 1 to 4, wherein, The casein kinase 1 inhibitor suppresses the gene expression of casein kinase 1 in the skin region.

6. The use as described in any one of claims 1 to 4, wherein, The casein kinase 1 inhibitor is casein kinase 1α.

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