ACTIVE AGENT MODULATING THE ACTIVITY OF AN ION CHANNEL FOR USE IN HAIR GROWTH REGULATION

Abstract

In order to provide a new agent for actively controlling hair growth in subjects, the present disclosure is directed to an active agent for use in hair growth regulation, particularly for use in the treatment of hair growth (stimulation or inhibition), wherein the active agent activates, enhances, inactivates, blocks or dampens the cellular response of the transient receptor potential ion channel TRPM5 or interferes with the expression of the ion channel. Furthermore, the present disclosure is directed to compositions for use as a cosmetic or medicament in the treatment of hair growth; the composition comprising at least one of the aforementioned active agents and at least one auxiliary agent. In addition, a non-therapeutic method of hair growth regulation is disclosed, wherein an effective amount of at least one of the aforementioned active agents is administered to a subject.

Claims

1.-15. (canceled)

16. A cosmetic or medicament for regulating hair growth comprising at least one active agent that activates, enhances, inactivates, blocks, or dampens the cellular response of transient receptor potential cation channel subfamily M member 5 (TRPM5), or interferes with the expression of TRPM5, and at least one auxiliary agent.

17. The cosmetic or medicament of claim 16, wherein the active agent that activates or enhances the cellular response of TRPM5 is an agonist of TRPM5.

18. The cosmetic or medicament of claim 17, wherein the agonist of TRPM5 is dimethylpyrazine, dimethylethylpyrazine, tetramethylpyrazine, 2-heptanone, eugenol, SID2848719 (CAS number 702636-90-6), rutamarin, bergapten, xanthotoxin, isopimpinellin, carbachol, 3-deoxyglucosone, glucagon-like peptide 1, (E)-N-(3,4dimethoxybenzylidene)-2-naphthalene-1-yl) acetohydrazide, or a combination thereof; or an aptamer that binds to TRPM5 and activates or enhances the TRPM5 ion channel.

19. The cosmetic or medicament of claim 16, wherein the active agent that inactivates, blocks, or dampens the cellular response of TRPM5 is an antagonist or inverse agonist of TRPM5.

20. The cosmetic or medicament of claim 19, wherein the antagonist or inverse agonist of TRPM5 is triphenylphosphine oxide, econazole, miconazole, chlorpromazine, or a combination thereof; or an aptamer that binds to TRPM5 and inactivates, blocks, or dampens the TRPM5 ion channel.

21. The cosmetic or medicament of claim 16, wherein the active agent that interferes with the expression of TRPM5 is a miRNA, siRNA, or a ribozyme targeted to the TRPM5 gene transcript.

22. The cosmetic or medicament of claim 16, wherein the auxiliary agent comprises a liposome, nanoparticle, carboxymethyl cellulose, hydroxyethyl cellulose, mineral oil, petrolatum, glycerin, polysorbate 80, hydroxyethyl starch, dextran, or polyethylene glycol.

23. The cosmetic or medicament of claim 16, further comprising at least one other active agent that regulates hair growth.

24. The cosmetic or medicament of claim 16, wherein the composition is formulated in the form of an ointment, a lotion, a cream, a shampoo, a hair conditioner, a gel, a solution, a spray, a plaster, or a sustained release plaster.

25. A method for regulating hair growth comprising administering to a subject an effective amount of at least one active agent that activates, enhances, inactivates, blocks, or dampens the cellular response of transient receptor potential cation channel subfamily M member 5 (TRPM5), or interferes with the expression of TRPM5 thereby regulating hair growth in the subject.

26. The method of claim 25, wherein the subject has unwanted hair loss and the step of administering to the subject an effective amount of at least one active agent that activates or enhances the cellular response of TRPM5 comprises administering to the subject an effective amount of an agonist of TRPM5.

27. The method of claim 26, wherein administering to the subject an effective amount of an agonist of TRPM5 comprises administering to the subject an effective amount of an agonist of TRPM5 selected from the group consisting of dimethylpyrazine, dimethylethylpyrazine, tetramethylpyrazine, 2-heptanone, eugenol, SID2848719 (CAS number 702636-90-6), rutamarin, bergapten, xanthotoxin, isopimpinellin, carbachol, 3-deoxyglucosone, glucagon-like peptide 1, (E)-N-(3,4dimethoxybenzylidene)-2-naphthalene-1-yl) acetohydrazide, and a combination thereof; and an aptamer that binds to TRPM5 and activates or enhances the TRPM5 ion channel.

28. The method of claim 25, wherein the subject has unwanted hair growth and the step of administering to the subject an effective amount of at least one active agent that inactivates, blocks, or dampens the cellular response of TRPM5 comprises administering to the subject an effective amount of an antagonist or inverse agonist of TRPM5.

29. The method of claim 28, wherein administering to the subject an effective amount of an antagonist or inverse agonist of TRPM5 comprises administering to the subject an effective amount of an antagonist or inverse agonist of TRPM5 selected from the group consisting of triphenylphosphine oxide, econazole, miconazole, chlorpromazine, and a combination thereof; and an aptamer that binds to TRPM5 and inactivates, blocks, or dampens the TRPM5 ion channel.

30. The method of claim 25, wherein the subject has unwanted hair growth and the step of administering to the subject an effective amount of at least one active agent that interferes with the expression of TRPM5 comprises administering to the subject an effective amount of an miRNA, siRNA, or a ribozyme targeted to the TRPM5 gene transcript.

31. The method of claim 25, wherein the subject has nonscarring (non cicatricial) alopecia, scarring (cicatricial) alopecia, or an excessive hair growth disorder.

32. The method of claim 31, wherein the nonscarring (non cicatricial) alopecia is selected from the group consisting of alopecia areata, telogen effluvium, androgenetic alopecia, anagen effluvium, loose anagen syndrome, and female pattern hair loss.

33. The method of claim 31, wherein the scarring (cicatricial) alopecia is selected from the group consisting of cutaneous lichen planopilaris, frontal fibrosing alopecia, discoid lupus erythematosus, dissecting cellulitis, and folliculitis decalvans.

34. The method of claim 31, wherein the excessive hair growth disorder is hypertrichosis or hirsutism.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] In the description of the examples it is referred to the following figures, wherein:

[0050] FIG. 1 is a photograph showing the result of an immunofluorescence analysis of TRPM5 expression in human skin,

[0051] FIG. 2 is a photograph showing the result of an immunofluorescence analysis of TRPM5 expression in human skin,

[0052] FIG. 3 is a graph showing that TRPM5 protein expression is significantly reduced in TRPM5-depleted human hair follicles,

[0053] FIG. 4 is a graph showing decreased cell proliferation (Ki67) in siTRPM5-treated hair follicles,

[0054] FIG. 5 is a graph showing increased apoptosis (TUNEL) in siTRPM5-treated hair follicles,

[0055] FIG. 6 is a graph showing that TRPM5 siRNA reduces expression levels of LEF1 and IGF1 in human hair follicles,

[0056] FIG. 7 is a graph showing that TRPM5 siRNA increases expression of TGFB2 and SFRP1 in human hair follicles,

[0057] FIG. 8 is a graph showing that 2,5-dimethylpyrazine (DMP) prolongs anagen in human hair follicle organ culture,

[0058] FIG. 9 is a graph showing that 2-heptanone (2-Hep) prolongs anagen in human hair follicle organ culture,

[0059] FIG. 10 is a graph showing that expression of catagen-promoting gene TGFB2 was down-regulated in 2-Hep-treated hair follicles, while Wnt target gene AXIN2 up-regulated,

[0060] FIG. 11 is a graph showing that triphenyl phosphine oxide (TPPO) promotes catagen in human hair follicle organ culture, and

[0061] FIG. 12 is a graph showing that expression levels of IGF1 and FGF7 genes were significantly down-regulated in TPPO-treated hair follicles, while TGFB2 transcript was up-regulated.

DETAILED DESCRIPTION

Examples

[0062] 1. Immunofluorescence Analysis of TRPM5 Expression in Human Skin

[0063] Cryosection without fixation was dried and then fixed in acetone at −20° C. Slides were then washed in phosphate-buffered saline (PBS). Sample sections were pre-treated with goat serum. Anti-TRPM5 was added to sample sections and incubated. Slides were washed in PBS and goat anti-rabbit-A488 antibody was added to sample sections and incubated. Slides were washed in PBS and sample sections were incubated with DAPI. Slides were washed again in PBS and mounted with coverslip using Southernbiotech FLUOROMOUNT®.

[0064] FIGS. 1 and 2 show the result of the immunofluorescence analysis of TRPM5 expression in human skin according to the above protocol. From FIG. 1 it can be identified that TRPM5 protein expression is predominately detected in the basal layer of the epidermis (arrowheads). From FIG. 2 it can be identified that strong TRPM5 expression is detected in the outer root sheath (arrowheads), with weaker expression in the hair matrix (arrows), while no expression was detected in the hair follicle mesenchyme (asterisks).

[0065] 2. Inactivation of TRPM5 by RNAi Knockdown

[0066] For analyzing the effect of inactivating TRPM5 by RNAi knock-down, human hair follicles were transfected with 1 μM TRPM5-targeting Accell siRNA (siTRPM5) and scrambled siRNA (siScr). After 24 hours, transfected hair follicles were snap frozen in liquid nitrogen for immunofluorescence analysis or collected in RNA extraction buffer for RNA isolation. TRPM5 protein expression was detected as above. Ki67 protein expression was detected as for TRPM5 using mouse anti-human Ki67 primary antibody and goat anti-mouse-A568 secondary antibody. For TUNEL immunolabelling, hair follicle cryosections were fixed in formalin/ethanol/acetic acid and labeled with digoxigenin-deoxyUTP (APOPTAG® Fluorescein In Situ Apoptosis detection kit; Millipore) in the presence of terminal deoxynucleotidyl transferase according to the manufacturer's protocol. TUNEL+ cells were visualized with anti-digoxigenin FITC-conjugated antibody (APOPTAG® kit). Cells positive for Ki-67 or TUNEL were counted per hair matrix and ORS and were normalized to the total number of nuclei (DAPI+).

[0067] Total RNA was isolated using PICOPURE® RNA Isolation Kit (Applied BioSystems) according to the manufacturer's protocol. After isolation, 100 ng of total RNA was reverse-transcribed into cDNA using a TETRO® cDNA synthesis kit (Bioline). Quantitative-PCR was performed using the StepOne Plus real time PCR system (Applied Biosystems) using TAQMAN® fast advanced master mix and TAQMAN® probes for LEF1, IGF1, TGFB2 AND SFRP1 (Applied Biosystems). The amount of the aforementioned transcripts was normalized to the expression of reference gene (GAPDH) using the ΔΔCt method. Results were performed in triplicate for each experiment.

[0068] FIGS. 3-6 show the results of siRNA targeting TRPM5 as mentioned above (Mean+/−sd, Mann Whitney U test, *p<0.05, **p<0.001).

[0069] According to FIG. 3, TRPM5 protein expression is significantly reduced in TRPM5-depleted human hair follicles. FIGS. 4 and 5 show decreased cell proliferation (Ki67) and increased apoptosis (TUNEL) in siTRPM5-treated hair follicles. According to FIGS. 6 and 7, TRPM5 siRNA reduces expression levels of LEF1 and IGF1 (FIG. 6), while increasing expression of TGFB2 and SFRP1 (FIG. 7) in human hair follicles.

[0070] 3. Activation of TRPM5 with 2,5-Dimethylpyrazine (DMP)

[0071] Human anagen hair follicles were isolated from the skin of males undergoing hair transplantation with written informed consent. Microdissected hair follicles were amputated at the level of dermal-subcutaneous junction and equilibrated in Williams' E medium (WEM) (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 10 ng/ml hydrocortisone, 10 μg/ml insulin, and antibiotics (all from Sigma-Aldrich). After 24 hours, growing undamaged anagen hair follicles were selected for treatment with DMP Human hair follicles were cultured in presence of DMP (12.5 The culture medium was changed every two days with addition of DMP. Hair follicles were photographed and percentage of hair follicles at different stages of hair cycle were calculated at day 3 and day 6 of the treatment and compared to vehicle.

[0072] According to the graph shown in FIG. 8, the TRPM5 agonist 2,5-dimethylpyrazine (DMP) prolongs anagen in human hair follicle organ culture as compared to vehicle (Veh).

[0073] 4. Activation of TRPM5 with 2-Heptanone (2-Hep)

[0074] Human anagen hair follicles were isolated from the skin of males undergoing hair transplantation with written informed consent. Microdissected hair follicles were amputated at the level of dermal-subcutaneous junction and equilibrated in Williams' E medium (WEM) (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 10 ng/ml hydrocortisone, 10 μg/ml insulin, and antibiotics (all from Sigma-Aldrich). After 24 hours, growing undamaged anagen hair follicles were selected for treatment with 2-Hep (12.5 The culture medium was changed every two days with addition of 2-Hep. Hair follicles were photographed and percentage of hair follicles at different stages of hair cycle were calculated at day 3 and day 6 of the treatment and compared to vehicle.

[0075] According to the graph shown in FIG. 9, the TRPM5 agonist 2-heptanone (2-Hep) prolongs anagen in human hair follicle organ culture.

[0076] For gene expression analysis, human hair follicles were microdissected as above and treated with 2-Hep (12.5 uM) for 6 hours. Following the treatment, total RNA was isolated and processed for cDNA synthesis as above. Quantitate PCR was performed using TAQMAN® assay with probes for TGFB2 and AXIN2 (Applied Biosystems).

[0077] FIG. 10 is a graph showing that expression of catagen-promoting gene TGFB2 was down-regulated in 2-Hep-treated hair follicles as compared to vehicle (Veh), while Wnt target gene AXIN2 was up-regulated (Mean+/−sd, Mann Whitney U test, *p<0.05).

[0078] 5. Inactivation of TRPM5 with Triphenyl Phosphine Oxide (TPPO)

[0079] Human anagen hair follicles were isolated from the skin of males undergoing hair transplantation with written informed consent. Microdissected hair follicles were amputated at the level of dermal-subcutaneous junction and equilibrated in Williams' E medium (WEM) (Life Technologies) supplemented with 2 mM L-glutamine (Life Technologies), 10 ng/ml hydrocortisone, 10 μg/ml insulin, and antibiotics (all from Sigma-Aldrich). After 24 hours, growing undamaged anagen hair follicles were selected for treatment with human hair follicles were cultured in presence of TPPO (150 The culture medium was changed every two days with addition of TPPO. Hair follicles were photographed and percentage of hair follicles at different stages of hair cycle were calculated at day 3 and day 6 of the treatment and compared to vehicle.

[0080] According to the graph shown in FIG. 11, the TRPM5 antagonist triphenyl phosphine oxide (TPPO) promotes catagen in human hair follicle organ culture as compared to vehicle (Veh).

[0081] For gene expression analysis, human hair follicles were microdissected as above and treated with TPPO (150 uM) for 6 hours. Following the treatment, total RNA was isolated and processed for cDNA synthesis as above. Quantitate PCR was performed using TAQMAN® assay with probes for IGF1, FGF7 and TGFB2 (Applied Biosystems).

[0082] FIG. 12 is a graph showing that expression levels of IGF1 and FGF7 genes were significantly down-regulated in TPPO-treated hair follicles as compared to vehicle (Veh), while TGFB2 transcript was up-regulated (Mean+/−SEM, Mann Whitney U test, *p<0.05).