ACTIVE AGENT FOR USE IN HAIR GROWTH REGULATION

Abstract

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 taste receptor TAS2R4 or interferes with the expression of the receptor. Furthermore, the present invention 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. An active agent for use in the treatment of hair growth, wherein the active agent activates, enhances, inactivates, blocks or dampens the cellular response of taste receptor TAS2R4 or interferes with the expression of the receptor.

2. The active agent of claim 1, wherein the active agent is an agonist of the taste receptor TAS2R4 for use in the treatment of unwanted hair growth.

3. The active agent of claim 1, wherein the active agent is an antagonist/inverse agonist of the taste receptor TAS2R4 for use in the treatment of unwanted hair loss.

4. The active agent of claim 1, wherein the active agent is a) any one of the TAS2R4 activating agonists amarogentin, arborescin, artemorin, azathioprine, chlorpheniramine, dapsone, D-camphor, denatonium benzoate, diphenidol, dulcoside A, (-)-epicatechin, leu-leu-leu, leu-trp, parthenolide, phe-trp, propylthiouracil, quassin, quinine, rebaudioside A, rebaudioside B, rebaudioside C, rubusoside, steviolbioside, stevioside, sucralose, taurocholic acid, trp-leu, trp-phe, trp-pro, trp-trp, trp-trp-trp, xanthoxin, yohimbine, or a combination thereof, or an aptamer binding to the TAS2R4 receptor and activating or enhancing the receptor to produce a cellular response: or b) any one of the TAS2R4 inactivating antagonists/inverse agonists carboxymethyllysine (CML), Nα,Nα-bis(carboxymethyl)-1-lysine (BCML), glyoxal-derived lysine dimer (GOLD) and 4-(2,2,3-trimethylcyclopentyl) butanoic acid (GIV3727) or a combination thereof, or an aptamer binding to the TAS2R4 receptor and inactivating, blocking or dampening the receptor to produce a cellular response.

5. The active agent of claim 1, wherein the active agent is miRNA, siRNA or a ribozyme targeted to TAS2R4.

6. An active agent according to claim 1 for use as a cosmetic in the treatment of hair growth.

7. An active agent according to claim 1 for use as a medicament in the treatment of hair growth disorder.

8. An active agent for use as a medicament according to claim 7, wherein the hair growth disorder to be treated is effluvium, nonscarring alopecia, scarring alopecia, hypertrichosis or hirsutism.

9. A composition for use as a cosmetic or medicament in the treatment of hair growth, the composition comprising at least one active agent according to claim 1; and at least one auxiliary agent selected from among the group consisting of carriers, recipients, adjuvants, diluents, or disintegrants.

10. The composition of claim 9, wherein the auxiliary agent is selected from among the group consisting of liposomes, nanoparticles, carboxymethyl cellulose, hydroxyethyl cellulose, mineral oil, petrolatum, glycerin, polysorbate 80, hydroxyethyl starch, dextran, or polyethylene glycol.

11. The composition of claim 9, further comprising at least one other active agent being effective in the treatment of hair growth.

12. The composition of claim 9, wherein the composition is formulated in the form of an ointment, a lotion, a cream, a shampoo, a gel, a solution, a spray, a plaster or a sustained release plaster.

13. A Non-therapeutic method of hair growth regulation, 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 the taste receptor TAS2R4 or interferes with the expression of the receptor.

14. The non-therapeutic method of claim 13, wherein the active agent is: a) any one of the TAS2R4 activating agonists, amarogentin, arborescin, artemorin, azathioprine, chlorpheniramine, dapsone, D-camphor, denatonium benzoate, diphenidol, dulcoside A, leu-leu-leu, leu-trp, parthenolide, phe-trp, propylthiouracil, quassin, quinine, Rebaudioside A, Rebaudioside B, Rebaudioside C, Rubusoside, steviolbioside, Stevioside, sucralose, taurocholic acid, trp-leu, trp-phe, tip-pro, trp-trp, trp-trp-trp, xanthoxin, yohimbine, and (-)epicatechin or a combination thereof, or an aptamer binding to the TAS2R4 receptor and activating or enhancing the receptor to produce a cellular response; or b) the TAS2R4 inactivating antagonists/inverse agonists carboxymethyllysine (CML), Nα,Nα-bis(carboxymethyl)-l-lysine (BCML), glyoxal-derived lysine dimer (GOLD) and 4-(2,2,3-trimethylcyclopentyl) butanoic acid (GIV3727), or an aptamer binding to the TAS2R4 receptor and inactivating, blocking or dampening the receptor to produce a cellular response.

15. The non-therapeutic method of claim 13, wherein the active agent is miRNA, siRNA or a ribozyme targeted to the TAS2R4 gene or targeted to the mRNA corresponding to the TAS2R4 gene.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The following examples show some of the features of specific embodiments of the present disclosure. However, the skilled reader will understand that those embodiments are just exemplary but do not restrict the inventive idea to exactly the features or the combination of features of the embodiments of the examples.

[0043] In the description of the examples it is referred to the following figures, wherein

[0044] FIG. 1 shows the result of an immunofluorescence analysis of TAS2R4 expression in human anagen hair follicle (cf. Example 1).

[0045] FIG. 2 shows effect of TAS2R4 knockdown on hair follicle biology, siRNA-targeting TAS2R4 (cf. Example 2) (Mean+/−SEM, n=5 HFs/group, GraphPad Prism 6 (GraphPad, San Diego, Calif.), ***p<0.001, Student's unpaired t-test).

[0046] FIG. 3 shows the data of the treatment of human hair follicles with 120 μM Nα,Nα-bis(carboxymethyl)-1-lysine hydrate (BCML) (cf. Example 3) (Mean+/−SEM, n=4-7 HFs/group, GraphPad Prism 6).

[0047] FIG. 4 shows the data of the treatment of human hair follicles with 200 μM Rebaudioside A (cf. Example 4) (Mean+/−SEM, n=4-7 HFs/group from 2 donors, GraphPad Prism 6).

[0048] FIG. 5 shows the data of the treatment of human hair follicles with 50 μM Rubusoside (cf. Example 5) (Mean+/−SEM, n=6-7 HFs/group, GraphPad Prism 6).

[0049] FIG. 6 shows the data of the treatment of human hair follicles with 200 μM Stevioside (cf. Example 6) (Mean+/−SEM, n=5-8 HFs/group, GraphPad Prism 6).

DETAILED DESCRIPTION

Examples

[0050] 1. Immunofluorescence Analysis of TAS2R4 Expression in Human Hair Follicles

[0051] Cryosection without fixation stored at −80° C. was dried for 10 min at room temperature (RT) and then fixed in acetone for 20 min at −20° C. Slides were then washed in Tris-buffered saline (TBS) for 5 min at RT (repeat ×2). The sample sections were encircled with a wax marker pen and the slides placed in a humidified chamber. Sample sections were pre-treated with 10% normal goat serum diluted in TBS for 20 min at RT. 1:100 dilution of rabbit polyclonal anti-TAS2R4 (ThermoFisher Cat No.: OSR00153W) in green antibody diluent was added to sample sections and incubated overnight (ON) in a humidified chamber at 4° C. Slides were washed in TBS for 5 min at RT (repeat ×2). 1:1000 dilution of goat anti-rabbit-A488 Ab (ThermoFisher Cat No.: A11070) in green antibody diluent was added to sample sections and incubated at RT for 45 min. Slides were washed in TBS for 5 min at RT (repeat ×2). Sample sections were incubated with DAPI for 5 min at RT. Slides were washed in TBS for 5 min at RT (repeat ×2) and mounted with coverslip using Southernbiotech Fluoromount.

[0052] FIG. 1 shows the result of the immunofluorescence analysis of TAS2R4 expression in human anagen hair follicle according to the above protocol.

[0053] TAS2R4 expression was detected in the outer root sheath, hair matrix, dermal papilla, and connective tissue sheath.

[0054] 2. Inactivation of TAS2R4 by RNAi Knockdown

[0055] For analyzing the effect of inactivating TAS2R4 by RNAi knock-down, hair follicles were cultured in William's E Medium (WEM) supplemented with 2 mM L-glutamine, 10 ng/mL hydrocortisone, 10 μg/mL insulin and 1% penicillin/streptomycin mix (WCM medium), and treated either with self-delivering scrambled oligos (Accell Non-targeting Control siRNA; Horizon Discovery Ltd, cat. D-001910-10-05) or with self-delivering siTAS2R4 (Accel siTAS2R4; Horizon Discovery Ltd, cat. E-013102-00-0005) according to the following procedure:

[0056] Day 0: Isolation of anagen hair follicles

[0057] Day 1: Change WCM media with self-delivery siRNA corresponding to experimental groups (Control group: 1 μM scrambled oligos, experimental group: 1 μM siTAS2R4)

[0058] Day 2: Rest

[0059] Day 3: Rest

[0060] Day 4: Collect 3 hair follicles/groups for qPCR (72h after siRNA delivery)

[0061] Day 5: Collect 5 hair follicles/groups for immunofluorescence (96h after siRNA delivery)

[0062] FIG. 2 shows the results of siRNA targeting TAS2R4 as mentioned above.

[0063] Inactivation of TAS2R4 by RNAi knock-down resulted in the anagen phase prolongation in the hair follicles ex vivo as shown with hair cycle staging and hair cycle score (HCS) (FIGS. 2A and 2B). HCS involves assigning an arbitrary unit for each stage of the hair cycle (Anagen VI=100; Early catagen=200; Mid-catagen=300; and Late catagen=400). After having classified each HF according to its hair cycle stage, the mean HCS was calculated. The closer the mean is to 100, the higher is the number of anagen VI HFs in a given group. The HCS provides a global readout parameter that looks at all HFs in a given experimental group and synthesizes them into a single number, which reflects how close the majority of HFs is to either anagen VI or catagen and also permits statistical analysis that it is not possible with hair cycle staging. Therefore, hair cycle staging and the HCS are independent readout parameters that complement each other.

[0064] TAS2R4 inactivation by RNAi knock-down was also associated with the increased number of proliferative Ki-67+ cells and the increased melanin content (FIGS. 2C and 2D).

[0065] In addition, the expression of a pro-anagenic growth factor IGF 1 and pro-catagenic growth factor TGFβ2 were increased and decreased respectively in response to the TAS2R4 inactivation by RNAi knock-down (FIG. 2E).

[0066] 3. Inactivation of TAS2R4 with Nα,Nα-bis(carboxymethyl)-l-lysine (BCML)

[0067] For analyzing the effect of inactivating TAS2R4, by the inverse agonist Nα,Nα-bis(carboxymethyl)-l-lysine (BCML), hair follicles were cultured in WEM medium supplemented with 2 mM L-glutamine, 10 ng/mL hydrocortisone, 10 μg/mL insulin and 1% penicillin/streptomycin mix (WCM medium), and treated either with 120 μM BCML (Sigma-Aldrich, cat. 14580) diluted in WCM or vehicle (WCM medium) according to the following procedure:

[0068] Day 0: Isolation of anagen hair follicles

[0069] Day 1: Change media, application of the substance corresponding to experimental groups (control group: WCM, experimental group: 120 μM BCML)

[0070] Day 2: Rest

[0071] Day 3: Change media

[0072] Day 4: Rest

[0073] Day 5: Change media

[0074] Day 6: Rest

[0075] Day 7: Collection and freezing of hair follicles for analyses

[0076] FIG. 3 shows the results of human hair follicles treated with 120 μM BCML.

[0077] Treatment of anagen hair follicles with the BCML increases the proportion of hair follicles in anagen stage (FIGS. 3A and 3B).

[0078] Further, treatment of human hair follicles with BCML increases hair matrix keratinocyte proliferation while does not affect apoptosis (FIG. 3C).

[0079] Finally, treatment of hair follicles with BCML increases pigmentation (FIGS. 3D and 3E).

[0080] 4. Activation of TAS2R4 with Rebaudioside A

[0081] For analyzing the effect of activating TAS2R4 by the agonist Rebaudioside A, human anagen hair follicles from 2 donors were cultured in WEM medium supplemented with 2 mM L-glutamine, 10 ng/mL hydrocortisone, 10 μg/mL insulin and 1% penicillin/streptomycin mix (WCM medium), and treated either with 200 μM Rebaudioside A (Sigma-Aldrich, cat. 01432) diluted in DMSO or vehicle (0.5% DMSO in WCM medium) according to the following procedure:

[0082] Day 0: Isolation of anagen hair follicles

[0083] Day 1: Change WCM media, application of the substance corresponding to experimental groups (control group: 0.5% DMSO, experimental group: 200 μM Rebaudioside A)

[0084] Day 2: Rest

[0085] Day 3: Change media

[0086] Day 4: Rest

[0087] Day 5: Change media

[0088] Day 6: Collection and freezing of hair follicles for analyses

[0089] FIG. 4 shows the results of human hair follicles treated with 200 μM Rebaudioside A.

[0090] From the figures, it can be identified that activation of TAS2R4 by treatment of hair follicles with the Rebaudioside A increases the proportion of hair follicles in catagen stage (FIGS. 4A and 4B).

[0091] Further, treatment of human hair follicles with Rebaudioside A decreases hair matrix keratinocyte proliferation, and increases apoptosis (FIG. 4C).

[0092] Finally, Rebaudioside A treatment induces hypopigmentation, which is a sign of catagen induction (FIGS. 4D and 4E).

[0093] 5. Activation of TAS2R4 with Rubusoside

[0094] For analyzing the effect of activating TAS2R4 by the agonist Rubusoside, hair follicles were cultured in WEM medium supplemented with 2 mM L-glutamine, 10 ng/mL hydrocortisone, 10 μg/mL insulin and 1% penicillin/streptomycin mix (WCM medium), and treated either with 50 μM Rubusoside (Sigma-Aldrich, cat. 62933) diluted in DMSO or vehicle (0.05% DMSO in WCM medium) according to the following procedure:

[0095] Day 0: Isolation of anagen hair follicles

[0096] Day 1: Change media, application of the substance corresponding to experimental groups (control group: 0.05% DMSO, experimental group: 50 μM Rubusoside)

[0097] Day 2: Rest

[0098] Day 3: Change media

[0099] Day 4: Rest

[0100] Day 5: Change media

[0101] Day 6: Collection and freezing of hair follicles for analyses

[0102] FIG. 5 shows the results of human hair follicles treated with 50 μM Rubusoside.

[0103] From the figures, it can be identified that activation of TAS2R4 by treatment of hair follicles with the Rubusoside increases the proportion of human hair follicles in catagen stage (FIGS. 5A and 5B).

[0104] 6. Activation of TAS2R4 with Stevioside

[0105] To determine the effect of activating TAS2R4 by the agonist Stevioside, anagen hair follicles were cultured in WEM medium supplemented with 2 mM L-glutamine, 10 ng/mL hydrocortisone, 10 μg/mL insulin and 1% penicillin/streptomycin mix (WCM medium), and treated either with 200 μM Stevioside (Sigma-Aldrich, cat. 50956) diluted in DMSO or vehicle (0.2% DMSO in WCM medium) according to the following procedure:

[0106] Day 0: Isolation of anagen hair follicles

[0107] Day 1: Change media, application of the substance corresponding to experimental groups (control group: 0.2% DMSO, experimental group: 200 μM Stevioside)

[0108] Day 2: Rest

[0109] Day 3: Change media

[0110] Day 4: Rest

[0111] Day 5: Change media

[0112] Day 6: Rest

[0113] Day 7: Collection and freezing of hair follicles for analyses

[0114] FIG. 6 shows the results of human hair follicles treated with 200 μM Stevioside.

[0115] From the figures, it can be identified that activation of TAS2R4 by treatment of anagen hair follicles with the Stevioside increases the proportion of hair follicles in catagen stage (FIGS. 6A and 6B).

[0116] Further, treatment of human hair follicles with Stevioside decreases hair matrix keratinocyte proliferation but does not affect apoptosis (FIG. 6C).