Topical preparations for stimulating hair growth containing oxidized low molecular weight carboxylic acids

Abstract

Topical formulations containing peroxyacids prepared in situ from trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, or acetic acid and hydrogen peroxide are useful for promoting hair growth and preventing hair loss.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to topical formulations comprising peroxyacids, preferably trichloroacetic acid, dichloroacetic acid, monochloroacetic acid or peroxyacids prepared in situ from acetic acid and hydrogen peroxide, for promoting hair growth.

[0002] Background of the Invention "Hair loss" can refer to the absence of hair on the skin where hair would normally be present, or to hair loss from the scalp. Hair loss can be due to a variety of causes, including various diets, environmental influences, genetic factors, hormonal imbalances, psychological stress, air pollution, radiation therapy, and exposure to certain medications (Dermatol Clin 31 (2013) 67-73).

[0003] Hair loss can be divided into various classes, such as hereditary male pattern baldness (baldness), alopecia areata (AA), tinea capitis (due to fungal infection), telogen effluvium, trichotillomania, hair production disorders, etc. Lupus alopecia, folliculitis barbae, lichen planus pilaris, and hair loss due to burns and trauma may properly be defined as examples of cicatricial alopecia.

[0004] Male pattern baldness (AGA) is a genetically determined disorder resulting from androgen overreaction, affecting up to 50% of men and 20% of women. AGA is characterized by the progressive loss of scalp hairline hair after puberty. AGA is associated with the activity of 5α-reductase type II, which converts testosterone to dihydrotestosterone. The conversion of testosterone to its most active metabolite, dihydrotestosterone, causes hair bulb miniaturization, leading to definitive hair bulb death and irreversible hair loss.

[0005] The most important drug for treating AGA is finasteride, a selective inhibitor of type II 5α-reductase. However, this treatment must be continued daily for life, and if discontinued, hair loss will resume. Furthermore, finasteride increases the circulating level of testosterone, which is aromatized to estrogen in the periphery, which may cause male sexual dysfunction, decreased libido, and gynecomastia.

[0006] AA is a common autoimmune disorder that affects hair follicles during the active (anagen) phase of the hair growth cycle. The most common method of treating AA is the use of corticosteroids (Canadian Family Physician, Vol 66: July 2020; 499-501), which are typically administered by local injection, topical ointment application, or orally. While medications can support hair growth, they can have serious side effects and cannot be taken long-term. Furthermore, hair loss can recur if medication is discontinued.

[0007] Other topical medications include minoxidil, a drug used in AA treatment (Drug Design, Development and Therapy 2019:13 2777-2786), immunosuppressants such as tacrolimus (American Academy of Dermatology, Inc 2005. doi: 10.1016 / j.jaad.2004.05.019) and cyclosporine A (Skin Pharmacol. 1994;7(1-2):101-4), and immune modulators such as diphenylcyclopropenone (Postepy Dermatol Allergol. 2018 Dec;35(6):577-581). Photochemotherapy and systemic immunosuppressive therapy may also be used. Recently, a class of drugs called Janus kinase (JAK) inhibitors has been demonstrated to be effective in treating AA and other skin diseases (J Cutan Med Surg. 2019 May / Jun;23(3):289-297).

[0008] Another approach to stimulating hair growth is the topical application of stimulants such as squaric acid dibutyl ester (International Journal of Dermatology, Vol. 3,5, No. 1, January 1996), anthralin (Dermatol Ther. 2017 Jul;30(4). doi: 10.1111 / dth.12500. Epub 2017 Jun 9), and trichloroacetic acid (J. Cosmet Dermatol. 2020; 00:1-7; KR No. 2012136519). The resulting inflammation is thought to inhibit the immune system's attack on hair follicles. On the other hand, some substances, such as hydrogen peroxide, suppress hair growth due to their cytotoxic effects and ability to induce oxidative stress (J.-A. Seo, et al., J. Dermatol. Sci. 66 (1) (2012) 12-19) and downregulate the GSK-3b / β-catenin signaling pathway (J Dermatol Sci. 2018 Jan; 89(1):91-94). To date, there is no universally proven treatment that induces and maintains remission of AA in all patients. [Brief explanation of the drawings]

[0009] [Figure 1] Time course of cell viability and ROS production in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 2] Time course of NFkB, TNFα, and IL-1β quantification in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 3] Time course of proliferation rate, CDK1 activity, and WNT / beta-actin activity in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 4] Time course of BAX and gaspase 3 activities in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 5] Time course of cell viability in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 6] Time course of ROS production and SOD activity in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 7] Time course of NFkB, TNFα, and IL-1β quantification in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 8] Time course of CDK1 activity and WNT / beta-actin activity in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523 [Figure 9] Time course of BAX activity, cytochrome activity, and ERK / MAPK activity in Protocol A vs. control and Protocol B vs. control at various concentrations of THPF250523

[0010] Description of the Invention The present invention relates to topical formulations containing peroxyacetic acid or peroxyhalogenated acetic acid or a combination of acetic acid or halogenated acid and hydrogen peroxide solution in admixture with a suitable carrier, vehicle, or supplemental beneficial ingredient, for use in preventing hair loss or improving hair growth.

[0011] The peroxyacetic acid or peroxyhaloacetic acid is selected from peroxytrichloroacetic acid, peroxydichloroacetic acid, peroxymonochloroacetic acid, and peracetic acid, preferably in the range of 2.0 to 0.8% by weight.

[0012] The preparation preferably contains trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, or acetic acid in the range of 33.0 to 0.08% by weight, and 28 to 30% hydrogen peroxide. The weight ratio of acetic acid or halogenated acetic acid to 28 to 30% hydrogen peroxide is 22.0 to 5.3.

[0013] The formulation may be in the form of a kit containing separate dosage forms for the hydrogen peroxide solution and the acetic acid or halogenated acetic acid.

[0014] The carrier, vehicle or supplementary useful ingredient is preferably selected from glycerol at a concentration of 2.0 to 10.0% by weight, water or 0.9% aqueous NaCl solution (added individually in an amount of 52 to 94% by weight), 30% aqueous ammonia at a concentration range of 12.0 to 0.03% by weight.

[0015] The pH of the formulation is typically 2-3.

[0016] The present invention also provides a cosmetic, cosmetic, or therapeutic method for preventing hair loss or improving hair growth, comprising topically applying to the skin of a subject in need thereof an effective amount of a composition of the present invention.

[0017] According to a first embodiment of the invention, the composition is administered topically at TO and administration continues every other day for a total of 12 days of treatment.

[0018] According to an alternative embodiment of the invention, the composition is administered topically at T0 and administration continues every other day for 8 days of treatment.

[0019] The composition can be used as is or, typically, diluted with demineralized water or physiological 0.9% NaCl / demineralized water before administration. The final concentration can be adjusted to 0.25-25% w / v, e.g., 25, 1.25, and 0.25% w / v. Surprisingly, compounds recently investigated for promoting hair growth, such as optionally halogenated peroxyacetic acid, particularly trichloroacetic acid and hydrogen peroxide (known to suppress hair growth), can stimulate hair growth to a higher level than the individual components used alone (i.e., trichloroacetic acid or hydrogen peroxide). The hair growth-stimulating effect was confirmed in formulations containing trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, or acetic acid and 30% hydrogen peroxide in a weight ratio of 22.0 to 5.3. Similar hair growth stimulating effects were also observed when formulations containing peroxytrichloroacetic acid, peroxydichloroacetic acid, peroxymonochloroacetic acid, or peracetic acid at effective concentrations of 0.8 to 2.0% by weight in the final formulation were used.

[0020] A representative example of a formulation of the present invention contains trichloroacetic acid and hydrogen peroxide in a molar ratio of 15 / 1. In a preferred formulation, trichloroacetic acid and 30% hydrogen peroxide comprise 33% and 1.5% by weight of the final formulation, respectively, and the final formulation further comprises glycerol (10.0% by weight), water (43.5% by weight), and 28-30% aqueous ammonia (12% by weight).

[0021] Alternatively, the formulations of the invention contain trichloroacetic acid and peroxytrichloroacetic acid or acetic acid and peroxychloroacetic acid in relative molar ratios ranging from 3.6 to 3.8, and preferably the sum of these acids represents 8.4 to 3.2% by weight of the formulation. The invention is illustrated in more detail in the following examples.

[0022] Example 1 [Table 1]

[0023] Comparative Example 2 [Table 2]

[0024] Comparative Example 3 [Table 3]

[0025] Example 4 [Table 4]

[0026] Example 5 [Table 5]

[0027] Example 6 [Table 6]

[0028] Example 7 [Table 7]

[0029] Example 8 [Table 8]

[0030] Example 9 [Table 9]

[0031] Test results for proliferation of dermal papilla cells Confirmation of the higher efficacy of the formulations of Examples 1, 4, 5, 6, 7, 8 and 9 in hair follicle proliferation compared to the formulations of Comparative Examples 2 and 3 Human hair follicle dermal papilla cells were cultured in a 96-well plate at 4 x 10 5The cells were seeded at a density of 1.0 μL / well and cultured for 16 hours. The culture medium was replaced with serum-free medium, and the tested formulations were added in volumes of 1.0 μL, 5.0 μL, and 50.0 μL, respectively. The cells were cultured for 1 day. 5 μM minoxidil solution was used as a positive control for hair loss treatment. Cells were harvested, and nuclei were isolated using a nuclear protein extraction kit (Merck, USA). Western blot analysis was then performed for β-catenin using a β-catenin antibody (Cell Signaling Technology, USA).

[0032] The formulations of Examples 1, 4, 5, 6, 7, 8, and 9 demonstrated a higher promoting effect on hair follicle cell proliferation than the formulations of Comparative Examples 2 and 3. Treatment with the formulation of the present invention demonstrated an increase in β-catenin translocation from the cytoplasm to the nucleus of approximately +15÷20% compared to the comparative formulations, confirming that the proliferation mechanism of dermal papilla cells was activated at a higher level than with Comparative Formulations 2 and 3.

[0033] In vitro evaluation of hair growth stimulation using the formulations of Examples 1, 4, 5, 6, 7, 8 and 9 The advantageous effects of the present invention have been confirmed by using an in vitro model for hair follicle investigation (Huelsken, J. et al., Cell 2001, 105, 533-545; Topouzi, Het al., Blackwell Publishing Ltd.: Hoboken, NJ, USA, 2017; Volume 26, pp. 491-496; Lee, LF; Chuong, CM Building Complex. Tissues: High. Throughput Screening for Molecules Required in Hair Engineering; Nature Publishing Group: Berlin, German, 2009; Volume 129, pp. 815-817; Biomedicines 2021, 9, 435). Treatment of human hair follicle dermal papilla cells with the formulations of Examples 1, 4, 5, 6, 7, 8 and 9 significantly increased (+15 / 20%) the translocation of β-catenin from the cytoplasm to the nucleus compared to the reference formulations of Examples 2 and 3, which contained only trichloroacetic acid or 30% hydrogen peroxide, respectively.

[0034] Therefore, it was confirmed that the proliferation mechanism of dermal papilla cells was activated at a higher level in comparison with Comparative Preparations 2 and 3 due to increased translocation of β-catenin into the nucleus.

[0035] Further in vitro and ex vivo evaluation of hair growth stimulation using Formulation 9 (THPF250523) diluted 1 / 4 with 0.9% w / v NaCl in water To evaluate the properties of the topical formulation of the present invention, formulation 9 (experimental code THPF250523) after a 1 / 4 dilution using 0.9% w / v NaCl in water was further investigated either as is or after serial dilutions.

[0036] In vitro evaluation of the formulation of Example 9 (THPF250523) on artificial dermis (pseudoderm) using two different administration protocols NHEK (normal human epidermal keratinocytes), HDF (primary dermal fibroblasts), and ORSK (outer sheath keratinocytes) cells were used to recapitulate a 3D organ system, taking advantage of their physiological functions and ability to develop stratified epithelium. Therefore, NHEK cells were placed on a layer of type I collagen mixed with human dermal fibroblasts (HDF) to create an "artificial dermis" that mirrors human anatomy. The artificial dermis was then overlaid with various cell mixtures and matrices containing basement membrane and extracellular matrix components, outer reticular sheath keratinocytes (ORSK) and mesenchymal cells (HDF). The artificial dermis was treated with both the original product (100%) and reduced doses of the product to find the minimum effective dose that mimics topical use.

[0037] The two administration protocols were as follows: In protocol A, cells were treated at T0, stimulated every other day, and evaluated after 4, 8, and 12 days of treatment. In protocol B, cells were always treated at T0, stimulated every other day, and evaluated after 4 and 8 days, with treatment discontinued on day 8 and cells maintained until day 12.

[0038] Using both protocols, the main biological activity of Formulation 9 was investigated, based on its antioxidant and anti-inflammatory properties. Because key mechanisms underlying hair bulb death may be due to the presence of oxidative stress and inflammatory processes, the production of reactive oxygen species (ROS) and the activation of NFKb (nuclear factor kappa B), TNFa (tumor necrosis factor), and the main cytokines involved were analyzed. In addition, mitochondrial metabolism and cell proliferation were analyzed using a crystal violet assay, and the crucial role of the WNT / beta-catenin pathway in hair follicle generation was explored. Finally, the main markers involved in the cell death process, such as BAX (bcl-2-like protein 4) and caspases (cysteine ​​aspartase), which are key proteins involved in the induction and activation of apoptosis, and cyclin-dependent kinases, which are key proteins involved in cell cycle regulation, were investigated.

[0039] cell culture Normal human epidermal keratinocytes (NHEK) derived from neonatal foreskin were purchased from Lonza (Basel, Switzerland) and cultured in keratinocyte basal medium (KBM, Lonza, Basel) containing keratinocyte growth medium-2 (KGM-2, Lonza, Basel, Switzerland) in an incubator at 37°C, 5% CO2, and 95% humidity [doi:10.4062 / biomolther.2012.20.2.171.]. Experiments were performed for passages 3 to 6, with 1 × 10 6 3D models were constructed using 3.5 × 10 cells / mL. Primary dermal fibroblasts (HDFs) were purchased from American Type Cell Culture (ATCC, Cansass, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM, Merck Life Sciences, Italy) supplemented with 10% fetal bovine serum (FBS, Merck Life Sciences, Italy), 2 mM L-glutamine, and penicillin / streptomycin (100 IU / 100 μg / mL). They were maintained in an incubator at 37°C, 5% CO2, and 95% humidity. For use as feeder cells, confluent HDFs were treated with 4 μg / mL mitomycin C (Acros Organics, supplied by Thermo Fisher Scientific) for 24 hours and then cultured at 3.5 × 10 cells / mL in a final volume of 2 mL. 4At confluence of 100 cells / well, cells were subcultured in 35 mm Petri dishes [Zarghami, A et al., Fibers and Polymers. 2015;16, 1201-1212]. ORSK cultures were used as previously described [doi:10.1371 / journal.pone.0240454]. Briefly, 5–10 hair follicles with clearly visible outer sheaths were collected and incubated for 30 min at 37°C in 2 ml of outer sheath keratinocyte (ORSK) medium consisting of DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 IU / mL penicillin G, 1 mM ascorbyl-2-phosphate, 2 μg / mL adenine, 10 ng / mL epidermal growth factor, 0.4 μg / mL hydrocortisone, 2 nM triiodothyronine, 0.1 nM cholera toxin, and 25 μg / mL gentamicin. After 30 minutes, the hair follicles were rinsed twice with 1 mL of 1x phosphate-buffered saline (PBS) containing 100 μg / mL gentamicin and then incubated with 0.5 mL of 0.25% trypsin-EDTA for up to 40 minutes, with pipetting and shaking at 10-minute intervals until the sheath was disrupted. This process resulted in a cell suspension composed solely of ORSK cells. These cells were then seeded onto a feeder culture of HDF cells pre-seeded in a 35 mm Petri dish and cultured for 5–6 days, with medium changes every 2 days. For the construction of artificial dermis, cells were cultured at 1x10 in EpiLife medium supplemented with Human Keratinocyte Growth Supplement (HKGS, Thermo Fisher Scientific, Waltham, Massachusetts, USA). 4 pieces / cm 2 The seeds were sown at a density of .

[0040] Experimental Protocol NHEK, HDF, and ORSK cells were used to recapitulate 3D organ systems, taking advantage of their physiological functions and ability to develop stratified epithelia. Therefore, NHEK cells were placed on a layer of type I collagen mixed with human dermal fibroblasts (HDF) to create an "artificial dermis" that mirrors human anatomy. The artificial dermis was then covered with various cell mixtures and matrices, including basement membrane and extracellular matrix components, outer reticular sheath keratinocytes (ORSK) and mesenchymal cells (HDF). The artificial dermis was treated with both the original product (100%) and reduced doses of the product to find the minimally effective dose that mimics topical use. More specifically, cells were treated at TO, stimulated every other day, and evaluated after 4, 8, and 12 days of treatment (Protocol A). In addition, a second treatment protocol was applied (Protocol B). In this study, cells were always treated at TO, stimulated every other day, and evaluated after 4 and 8 days. On day 8, treatment was discontinued and cells were maintained until day 12. Using both protocols, the biological activity of THPF250523 was studied. Therefore, because key mechanisms responsible for hair bulb death may be due to the presence of oxidative stress and inflammatory processes, we analyzed the production of reactive oxygen species (ROS) and the activation of NFKb, TNFa, and other key cytokines involved. In addition, mitochondrial metabolism and cell proliferation were analyzed using a crystal violet assay, and the crucial role of the WNT / beta-catenin pathway in hair follicle generation was analyzed. Finally, we investigated key markers involved in the cell death process, such as BAX and caspases, which are involved in the induction and activation of apoptotic processes, and cyclin-dependent kinases, which are involved in cell cycle regulation.

[0041] Construction of artificial dermis Artificial dermis was prepared using type I collagen (Thermo Fisher Scientific, Waltham, Massachusetts, USA) at a final concentration of 4 mg / mL according to the protocol [doi:10.1111 / j.1365-2133.2004.06184.x]. First, type I collagen (prepared in Hank's buffered saline), FBS, and HDF cells (passages 2–8) were mixed in an 8:1:1 ratio to prepare coated feeders. The final concentrations were 3.2 mg / mL collagen and 2.5 × 10 5 The cell concentration was 1 × 10 cells / mL. 1.5 mL of this mixture was poured into a 24-well plate (Corning Costar, Corning, NY, USA) and allowed to gel at 37°C. After polymerization, DMEM containing 10% FBS was added, and the gel was grown for 5 days under immersion conditions, with the medium changed every other day. After this period, the gel had completely shrunk and was used as the artificial dermis. The artificial dermis was transferred with forceps to a tissue culture well (0.4 μm pore size, polycarbonate, Corning Costar). 1 × 10 cells were then added. 4 cells / mL ORSK cells, 1×10 6 NHEK cells (cells / mL) were mixed with previously prepared Matrigel™ in 1x PBS (ratio 9:1) and seeded onto the artificial dermis. The cells were maintained in culture at 37°C in DMEM supplemented with 10% fetal horse serum (FCS, Merck Life Science, Milan, Italy) until complete polymerization.

[0042] MTT test After treatment, cell viability analysis was performed using a classical technique based on the MTT in vitro toxicity test kit (Merck Life Science, Rome, Italy) [Ruga, S et al., Int. J. Mol. Sci. 2022, 23, 3805] according to the manufacturer's instructions. At the end of treatment, cells were incubated with 1% MTT dye for 2 hours in an incubator at 37°C, 5% CO2, and 95% humidity. The purple formazan crystals were then dissolved in an equal volume of MTT solubilization solution. Absorbance was analyzed at 570 nm and corrected at 690 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were expressed relative to the control (0% line). The control represents untreated cells. Results are expressed as the % viable cells compared to the control. This allows for the evaluation of the safety of the stimulation.

[0043] Reactive oxygen species (ROS) production Quantification of superoxide anion release was performed according to a standard protocol based on the reduction of cytochrome c [Molinari et al., Foods 2021, 10, 1885]. The absorbance of the culture supernatant was measured at 550 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Specifically, 100 μL of cytochrome c (Merck, Milan, Italy) was added to all wells, while 100 μL of superoxide dismutase (Merck, Milan, Italy) and 100 μL of cytochrome c were added to empty wells. The plate was then incubated for 30 minutes. Afterwards, 100 μL was removed from each well, and the absorbance was measured at 550 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). O2 rates were expressed as mean ± SD (%) of nanomoles of reduced cytochrome c per microgram of protein compared to the control (0 line).

[0044] ELISA assay for NFKB To analyze the DNA-binding activity of NFkB, an enzyme-linked immunosorbent assay (ELISA) was performed according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI, USA). Briefly, nuclear extracts were prepared using a nuclear extraction protocol [Uberti, F et al., J. Ovarian Res. 2017, 10, 61], and NFkB in these extracts was detected by adding a specific primary antibody. An HRP-conjugated secondary antibody was added to provide a highly sensitive colorimetric assay, measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Concentrations were calculated by comparing the results with a standard curve (range 1.6–400 ng / mL). Results are expressed as the mean ± SD (%) relative to the control (0 line) of five independent experiments performed in triplicate.

[0045] ELISA assay for TNFα TNFα concentrations were measured using a TNFα ELISA kit (Merck Life Science, Rome, Italy) according to the experimental protocol. Colorimetric intensity was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Data were calculated by constructing a calibration curve (range 24.58 pg / ml to 6000 pg / ml) relating the optical density of the samples to the concentrations of the standards. Results are expressed as the mean ± SD (%) relative to the control (0 line) of five independent experiments performed in triplicate.

[0046] ELISA assay for interleukin 1β (IL-1β) IL-1β was detected using a human IL-1β (interleukin 1β) ELISA kit (FineTest, Wuhan, China) according to the manufacturer's instructions. Plates were immediately read at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). A calibration curve relating color intensity (OD) to the concentration of the standard (31.25–2000 pg / mL) was plotted, and the results are expressed as the mean ± SD (%) relative to the control (0 line) of five independent experiments performed in triplicate.

[0047] Crystal violet staining Treated cells were also analyzed by crystal violet staining to examine proliferation. At the end of each time point, cells were fixed with 1% glutaraldehyde for 15 minutes at room temperature, washed, and stained with 100 μl of 0.1% crystal violet for 20 minutes at room temperature. To obtain estimated cell numbers, 100 μl of 10% acetic acid was added and mixed, and then the absorbance at 595 nm was read using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Estimated cell numbers were calculated by comparing the results with control cells (control TO). Results are expressed as the mean ± SD (%) compared to the control (0 line) of five independent experiments performed in triplicate.

[0048] Assays for Wnt activity Wnt activity was measured using an ELISA kit (Human Wnt-3a ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instructions [doi:10.1016 / j.bbrc.2017.04.110.]. The absorbance of samples was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were compared with a standard curve (range 1.57–100 ng / mL) and expressed as mean ± SD (%) normalized to the control value (0 line).

[0049] Assay for BAX activity BAX activity was measured using an ELISA kit (Human Bax ELISA Kit, MyBiosource, San Diego, CA, USA) according to the manufacturer's instructions [doi:10.1016 / j.bioorg.2021.105035]. The absorbance of samples was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were compared with a standard curve (range 0–2000 pg / mL) and expressed as mean ± SD (%) normalized to the control value (0 line).

[0050] Caspase 3 assay Caspase 3 activity was measured in the lysates using an ELISA kit (Caspase 3 (Cleaved) Human ELISA Kit, Thermoscientific, Waltham, MA, USA) and by reading the absorbance of the samples at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland) according to the manufacturer's instructions [doi:10.1038 / s41598-020-61871-w.] Data were obtained by comparison with a standard curve (0.039–2.5 ng / mL), and results are expressed as the mean ± SD (%) compared to the control value (0 line) of five independent experiments performed in triplicate.

[0051] ELISA for CDK1 A CDK1 ELISA kit (MyBiosource, San Diego, CA, USA) was used according to the manufacturer's instructions [doi:10.1016 / j.stemcr.2015.01.019.]. HOSEpiC cells were lysed in chilled 1x phosphate-buffered saline (PBS, Merck Life Science, Rome, Italy) and centrifuged at 5,000 x g for 5 minutes. 100 μL of each sample was analyzed by reading the absorbance of the sample at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were obtained by comparing the data to a standard curve (range 0.156–10 ng / mL) and expressed as a percentage (%) of the control (0 line) from five independent experiments performed in triplicate.

[0052] statistical analysis Results are expressed as the mean ± SD of at least five biological replicates for each experimental protocol, with each replicate repeated three times for each experimental protocol. Statistical comparisons between groups were performed using one-way analysis of variance combined with Bonferroni's post-hoc test using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA). A p-value of p<0.05 was considered statistically significant. All densitometric analysis data were normalized to the control value (defined as 0). All other data from each experimental protocol were normalized to the % control value (defined as 0%).

[0053] result To investigate the effects of various concentrations of THPF250523 on cell viability and ROS production, artificial dermis was treated with two protocols (A and B) for 12 days. As can be seen in Figure 1A, after application of Protocol A, cell viability was statistically significantly increased throughout the entire stimulation period after stimulation with the undiluted test product (100%) compared to the other concentrations tested (p<0.05). These experiments were also performed after application of Protocol B (Figure 1C). Figure 1C shows that cell viability was statistically significantly improved between days 8 and 12 of treatment compared to the other concentrations tested (p<0.05). Furthermore, a better effect was exerted by Protocol B than by Protocol A (p<0.05). This was due to the high cell viability maintained from day 8 to day 12 of treatment (plateau phase). Both protocols were also evaluated in terms of oxidative stress by analyzing various THPF250523 concentrations after 4, 8, and 12 days of treatment. Indeed, as shown in Figures 1B and 1D, ROS production was reduced in a dose-dependent manner (p<0.05), consistent with cell viability for both treatment protocols. Therefore, the obtained data suggest that undiluted THPF250523 has a strong potential to maintain cellular homeostasis without activating oxidative processes.

[0054] Based on the results obtained, we then investigated the main biological markers involved in the induction of inflammatory processes to further confirm the positive effects promoted by THPF250523 on hair bulb health. The data shown in Figure 2 demonstrate that 100% THPF250523 promoted the maintenance of reduced levels of TNFα, NFkB, and IL-1β during the treatment period of Protocol A compared with the other formulations (p<0.05). In fact, the data demonstrate a favorable preventive and protective effect against inflammatory mechanisms, confirming the results regarding cell vitality and oxidative stress. Similarly, the application of Protocol B (Figures 3D-F) also confirmed the previous observations, with statistically significant and favorable results (p<0.05) compared with Protocol A.

[0055] This data confirms that THPF250523 maintains proper keratinocyte function during 12 days of treatment without inducing significant adverse effects.

[0056] First, cell proliferation was analyzed by crystal violet staining. The highest cell proliferation rate was detected after 12 days of treatment with undiluted THPF250523. This was confirmed for both protocols (p<0.05). Specifically, application of Protocol B showed results consistent with those observed previously, highlighting an increase in cell proliferation between days 8 and 12, despite treatment being discontinued on day 8 (p<0.05). In addition, we investigated the activity of CDK1, a protein that belongs to the class of protein kinases that regulate the cell cycle and is involved in nuclear disassembly and reassembly. Therefore, increased activity clearly indicates improved cell viability and proliferation. Indeed, the results show that after application of Protocol A, 12 days of treatment with undiluted THPF250523 induces more cell proliferation and supports hair development than the other formulations used (p<0.05). On the other hand, application of Protocol A showed inferior results compared to Protocol B (p<0.05). Finally, we characterized the activity of the WNT / beta-catenin system, which plays a central role in the generation of hair follicles during embryonic development and in the life cycle of hair follicles in adult individuals. Without this mechanism, there would be no regeneration of hair, eyelashes, or eyebrows, nor would there be activation of melanogenesis, which leads to hair pigmentation [doi:10.3390 / ijms21144915.]. The data show that after application of Protocol A, WNT activity was significantly higher after stimulation with undiluted THPF250523 compared to other formulations (p<0.05). Again, after application of Protocol A, there was an increase in WNT activity compared to the control (p<0.05), but this increase was less than with Protocol B (p<0.05). These results are in direct proportion to the data previously described. This indicates that undiluted THPF250523 supports and promotes the maintenance of the 3D organ system and compensatory mechanisms for hair bulb development and subsequent hair growth. Furthermore, both stimulation protocols are effective at the hair bulb level, and optimal results can be obtained even if treatment is discontinued after day 8.However, a more beneficial effect was found in protocol B.

[0057] Finally, we addressed the role of BAX and caspase-3, biological markers involved in triggering cell death mechanisms. As can be seen in Figure 4, after application of Protocol A, BAX and caspase-3 activity were significantly reduced after stimulation with the undiluted test product compared to the other concentrations tested (p<0.05). The data obtained also confirmed this after application of Protocol B, demonstrating a statistically significant suppression of apoptotic mechanisms between days 8 and 12 of treatment (p<0.05). These results directly correlate with the previously observed data, demonstrating that undiluted THPF250523 supports and promotes compensatory mechanisms for the maintenance of the 3D organ system and hair bulb development and subsequent hair growth. Furthermore, both stimulation protocols were effective at the hair bulb level, and optimal results could be obtained even if treatment was discontinued after day 8. However, more beneficial effects were observed with Protocol B.

[0058] The results obtained show that formulations based on undiluted THPF250523, in contrast to other formulations tested, provide the following benefits: - Positive effect of maintaining cell functionality and survival, and therefore hair health - Positive effect of limiting the occurrence of inflammation and oxidative stress at the level of the 3D organotypic hair bulb system organization, and consequently limiting cell death mechanisms - Beneficial effects after application of both treatment protocols, with optimal results being obtained with the second treatment protocol after discontinuing stimulation on day 8.

[0059] Therefore, THPF250523 represents a potential and viable alternative to prevent hair loss and support hair bulb development and maintenance after a 12-day treatment protocol.

[0060] Ex vivo evaluation of Formulation 9 (experimental code THPF250523) diluted 1 / 4 in 0.9% NaCl on scalp hair follicles using two different administration protocols Isolation and culture of scalp hair follicles To create the ex vivo model, a portion of skin tissue removed from the back of a mouse was cut into small pieces (4 mm x 4 mm). The resulting skin pieces were processed according to standard protocols reported in [Molina, B. et al., Microscopy Research, 2020:8, 9-30] to recapitulate the epithelial compartment surrounding the hair bulb ex vivo. Specifically, newborn mice were sacrificed, placed in 70% alcohol for 10 minutes, and washed twice with PBS for 5 minutes. The tissue was then cut into small pieces (4 mm x 4 mm). The skin pieces were then incubated in 0.25% dispase II (Bacillus polymyxa, Gibco, BRL) in DMEM / F12 (1:1; Gibco-BRL, Thermo Fisher Scientific, Waltham, Massachusetts, USA) at 4°C for 12–18 hours. The tissue was then first rinsed with saline to remove excess adipose tissue. Hair follicles were gently removed and collected using a dissecting microscope. After two additional rinses, hair bulbs were transferred to Dulbecco's modified Eagle's medium (DMEM, Gibco, Thermo Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Thermo Scientific, Waltham, Massachusetts, USA), 10 ng / ml epidermal growth factor (Invitrogen), 5 g / ml hydrocortisone, 5 g / ml insulin (Sigma-Aldrich), 200 mmol / L L-glutamine (Gibco, Thermo Scientific, Waltham, Massachusetts, USA), and antibiotics (100 U / ml penicillin and 100 g / ml streptomycin). Culture was performed at 37°C, 95% humidity, and 5% CO2.

[0061] Experimental Protocol The beneficial effects of products modulating hair bulb growth were evaluated using sections of skin tissue excised from the back of mice. Therefore, using an ex vivo model, we analyzed the key mechanisms responsible for hair bulb death by studying the activation of oxidative stress, antioxidant mechanisms (e.g., SOD), and inflammatory processes (e.g., NFKb, TNFa, and related key cytokines). Furthermore, we evaluated the main mechanisms involved in apoptosis and cell survival processes, such as the activation of cytochrome C and the involvement of ERK / MAPK. Finally, we investigated the role of novel substances in activating mechanisms involved in hair bulb regeneration, such as cell proliferation, cell cycle control proteins, and the WNT / beta-catenin pathway.

[0062] MTT tests, reactive oxygen species (ROS) production, ELISA assays for TNFα, ELISA assays for interleukin-1β (IL-1β), assays for Wnt activity, and assays for BAX activity were performed using the same materials and methods as those utilized for the in vitro tests.

[0063] ELISA assay for superoxide dismutase (SOD) SOD levels were measured according to the manufacturer's instructions (Cayman's Superoxide Dismutase Assay Kit). This kit detects all three types of SOD (Cu / Zn, Mn, and FeSOD)

[13] . Briefly, the levels of SOD present in the cell lysates were measured by comparing the data with a standard curve (0.05–0.005 U / mL). Whole cell lysates were prepared in chilled 1x PBS. The absorbance of all samples was measured at 480 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland), and the results were expressed as the mean (%) relative to the control. Concentrations were expressed in ng / mL relative to the standard curve (range 0–0.005 U / mL), and the results were expressed as a percentage of the control (0 line).

[0064] ELISA assay for NFKB The DNA-binding activity of NFKB was analyzed by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (Cayman Chemical Company, Ann Arbor, MI, USA). Nuclear extracts were prepared using a nuclear extraction protocol [Uberti, F et al., J. Ovarian Res. 2017, 10, 61], and NFKB in these extracts was detected by adding a specific primary antibody. An HRP-conjugated secondary antibody was added to provide a highly sensitive colorimetric assay measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Concentrations were calculated by comparing the results to a standard curve. Concentrations were expressed in ng / mL relative to the standard curve (range 0.05–0.005 U / mL), and results were expressed as a percentage of the control (0 line).

[0065] Assay for cytochrome c activity The amount of cytochrome c in the cell lysates was measured using a cytochrome c ELISA kit (MyBiosource, San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 100 μL of each sample was added and incubated at 37°C for 90 minutes. The material was then removed, and 100 μL of detection solution A was added and incubated at 37°C for 45 minutes. At the end of the incubation period, the wells were washed, and 100 μL of detection solution B was added to each well, followed by incubation at 37°C for 45 minutes. Then, 90 μL of substrate solution was added to each well, and the plate was incubated at 37°C in the dark for 20 minutes. The reaction was stopped using 50 μL of stop solution, the absorbance was analyzed at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland), and the concentration was expressed in ng / mL by comparing the data with a calibration curve (range 15.6 nmol / L to 500 nmol / L).

[0066] Assay for ERK / MAPK activity ERK / MAPK activity was analyzed using InstantOne™ ELISA (Thermo Fisher, Milan, Italy) on chondrocyte lysates [Molinari C et al., Oxid Med Cell Longev.;2019:2843121]. Cells at the end of treatment were lysed with 100 μL of cell lysis buffer, and 50 μL / well of each sample was tested in an InstantOne ELISA microplate. Antibody cocktail was added to each well and incubated for 1 hour at room temperature under agitation. At the end of the time period, detection reagent was added to each well, and after 20 minutes, the reaction was stopped by adding stop solution. Absorbance was measured at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were expressed as the mean absorbance (%) compared to the control. Concentrations were expressed in ng / mL relative to a standard curve (range 62.5-2000 pg / mL) and results were expressed as a percentage of the control (0 line).

[0067] ELISA assay for CDK1 A CDK1 ELISA kit (MyBiosource, San Diego, CA, USA) was used according to the manufacturer's instructions [doi:10.1016 / j.stemcr.2015.01.019]. HOSEpiC cells were lysed in cold 1x phosphate-buffered saline (PBS, Merck Life Science, Rome, Italy) and centrifuged at 5000 x g for 5 minutes. 100 μL of each sample was analyzed by reading the sample absorbance at 450 nm using a spectrophotometer (Infinite 200 Pro MPlex, Tecan, Maennedorf, Switzerland). Results were obtained by comparing the data to a standard curve (range 0.156–10 ng / mL) and expressed as a percentage compared to the control (0 line) in five independent experiments performed in triplicate.

[0068] result Figures 5A and 5B show that after Protocol A, cell viability was statistically significantly higher (p<0.05) after stimulation with undiluted test product (100%) than with the other concentrations tested. The data also confirmed that after Protocol B (Figure 5B), Protocol B statistically significantly improved cell viability (p<0.05) compared with the other concentrations tested between days 8 and 12 of treatment. This confirmed the results obtained after applying both protocols in the in vitro model. In fact, in the ex-vivo model, Protocol B also showed better results in maintaining cell viability and functionality (p<0.05), even after stimulation was discontinued after day 8 of treatment.

[0069] Regarding oxidative stress, Figure 6 further demonstrates that ROS production was dramatically reduced (p<0.05) after treatment with undiluted THPF250523, consistent with that determined by cell viability. Protocol B (Figure 6 / D) demonstrated lower ROS levels from day 8 of treatment onward. These data were further confirmed by determining SOD levels, which limit oxidative stress-induced cellular damage, protect functionality, and limit cellular senescence [Prie BE, et al., J Med Life. 2016;9(1):79-83]. For both protocols, SOD activity was statistically significantly lower (p<0.05) after treatment with undiluted THPF250523 compared to other concentrations examined. This data confirms the results obtained in in vitro models, suggesting that undiluted THPF250523 has strong potential to maintain cellular function and viability, indicating its potential as an anti-hair loss agent.

[0070] To further confirm the positive effects promoted by THPF250523 on hair bulb health, key biological markers involved in the induction of inflammatory processes were also investigated in the ex-vivo model. The data shown in Figure 7 demonstrate that 100% THPF250523 promoted the maintenance of reduced levels of TNFα, NFkB, and IL-1β during Protocol A treatment compared with other formulations (p<0.05). Indeed, the data demonstrate a favorable preventative and protective effect against inflammatory mechanisms, confirming the results observed in the in vitro model. Similarly, application of Protocol B (Figures 7D-7F) confirmed the previous results with a statistically significant (p<0.05) improvement over Protocol A.

[0071] Consistent with what was studied in the in vitro model, analysis of the mechanisms involved in cell proliferation was crucial to explain the maintenance of functional status and hair growth by THPF250523. Furthermore, in the ex-vivo model, CDK1 activity (Figures 8A and 8C) was also increased after application of Protocol A (p<0.05). Indeed, similar to the in vivo model, 12-day treatment with undiluted THPF250523 induced more cell proliferation and supported hair growth than the other formulations used (p<0.05). Application of Protocol B demonstrated better results than Protocol A (p<0.05), increasing CDK1 activity between days 8 and 12 after treatment was discontinued after day 8. Finally, the activity of the WNT / β-catenin system was characterized in parallel. The data show that after application of Protocol A, WNT activity was better (p<0.05) after stimulation with undiluted THPF250523 compared to the other formulations. Also, in this case, after application of Protocol B, the improvement in WNT activity was found to be higher (p<0.05) than with Protocol A (Figures 8B and 8D). These results are directly proportional to the previously observed data, indicating that undiluted THPF250523 supports and promotes compensatory mechanisms for maintaining hair bulb development and subsequent hair growth, even in an ex-vivo model. Both stimulation protocols were effective at the hair bulb level, with optimal results obtained even when treatment was discontinued after day 8. Better performance data was found using Protocol B.

[0072] Finally, we described the role of cytochrome C, a biological marker involved in triggering the programmed cell death mechanism (Figure 9). The initiation of the apoptotic mechanism directly correlates with the activation of BAX, which stimulates the release of cytochrome C from mitochondria, which acts as a downstream cell death signal.

[0073] The results obtained show that undiluted THPF250523, in contrast to diluted formulations, provides beneficial effects, maintaining cell function and viability and, consequently, hair health.

[0074] Furthermore, undiluted THPF250523 inhibits the development of inflammatory and oxidative stress in an ex-vivo model of the hair bulb mechanism, thereby limiting cell death mechanisms. The beneficial effects of undiluted THPF250523 after both treatment protocols were confirmed by a second treatment protocol after cessation of stimulation on day 8.

Claims

1. A topical formulation containing peroxyacetic acid or peroxyhalogenated acetic acid or a combination of acetic acid or halogenated acid and hydrogen peroxide solution in admixture with a suitable carrier, vehicle or supplemental beneficial ingredient, for use in preventing hair loss or improving hair growth.

2. 2. The formulation of claim 1, wherein the peroxyacetic acid or peroxyhalogenated acetic acid is selected from peroxytrichloroacetic acid, peroxydichloroacetic acid, peroxymonochloroacetic acid, or peracetic acid.

3. 2. The formulation of claim 1, comprising trichloroacetic acid, dichloroacetic acid, monochloroacetic acid or acetic acid and 28-30% hydrogen peroxide.

4. 4. The preparation according to claim 3, wherein the weight ratio of acetic acid or halogenated acetic acid to 28-30% hydrogen peroxide is 22.0-5.

3.

5. 5. The formulation according to claim 3 or 4, which contains trichloroacetic acid, dichloroacetic acid, monochloroacetic acid or acetic acid in the range of 33.0 to 0.08% by weight.

6. A formulation according to any one of claims 3 to 5 in the form of a kit containing separate dosage forms for the hydrogen peroxide solution and for acetic acid or halogenated acetic acid.

7. 3. The formulation according to claim 2, which contains peroxytrichloroacetic acid, peroxydichloroacetic acid, or peroxymonochloroacetic acid, or peracetic acid in the range of 2.0 to 0.8% by weight.

8. 8. The formulation according to any one of claims 1 to 7, wherein the carrier, vehicle or additional useful ingredient is selected from glycerol in a concentration of 2.0 to 10.0% by weight, water or 0.9% aqueous NaCl solution (added individually in an amount of 52 to 94% by weight), 30% aqueous ammonia in a concentration range of 12.0 to 0.03% by weight.

9. 9. The formulation of any one of claims 1 to 5 and 7 to 8, having a pH of 2 to 3.

10. A cosmetic, cosmetic or therapeutic method for preventing hair loss or improving hair growth, comprising:

10. A method comprising topically applying an effective amount of the composition of any one of claims 1 to 9 to the skin of a subject in need thereof.

11. 11. The method of claim 10, wherein the composition is administered topically at T0 and treatment continues every other day for a total of 12 days.

12. 11. The method of claim 10, wherein the composition is administered topically at T0 and treatment continues every other day for 8 days.

13. 13. The method of claim 11 or 12, wherein the composition is diluted to 25, 1.25 and 0.25% weight / volume with demineralized water or physiological 0.9% NaCl / demineralized water before administration.

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