Topical composition for promoting hair growth

Abstract

The present invention relates to a topical composition for the prevention of hair loss in women or men and / or the promotion of hair growth, a cosmetic or therapeutic treatment in which such a composition is used, and related formulations or dosage units comprising such a composition.

Description

[Technical Field]

[0001] The present invention relates to topical compositions for preventing hair loss and / or promoting hair growth, cosmetic or therapeutic treatments in which such compositions are used, and related formulations or dosage units comprising such compositions. [Background technology]

[0002] As we age, the rate of hair growth decreases, accompanied by a reduction in hair thickness. Gray hair appears as a result of decreased melanin production in the hair matrix. Generally, the hair follicle cycle is characterized by the growth phase (anagen phase), the subsequent regression and reconstruction phase (catagen phase), and finally the resting phase (telogen phase).

[0003] Estrogen levels have been linked to both hair growth and hair loss. For example, during pregnancy, a woman's estrogen levels are higher than normal, which increases hair follicle growth and leads to thick, full hair. Conversely, when estrogen levels decrease after pregnancy or during and after menopause, more hair follicles enter a "resting" phase, which leads to hair loss, reduced hair volume, and even partial baldness. Changes in hormone balance can affect skin composition and hair follicle function. Also, sebum secretions from the sebaceous glands provide a softening agent to hair and skin and are under the influence of androgen hormones. Sebum secretion decreases with age and can also be affected by estrogen levels. These effects are exacerbated in menopausal women (i.e., perimenopause and postmenopausal women) due to hormonal imbalances and often lead to negative self-image.

[0004] In men, hair loss is a common phenomenon, and numerous different forms and causes have been documented in the art. For example, androgenetic alopecia (AGA) in men is characterized by the shrinking of hair follicles in patterned hair loss resulting from systemic androgens and genetic factors. The prevalence and severity of AGA are thought to be specific to ethnic groups, with the prevalence in Caucasian men (up to 50% of the population) being higher than in Asian and Black men (Non-Patent Literature 1). Onset typically coincides with the end of puberty and progresses progressively throughout life. In some cases, hair loss in men can affect their satisfaction with their body image and self-esteem.

[0005] Considering the above, intensive research has been conducted over the past decade to prevent, reduce, or even reverse both female and male alopecia. Treatment strategies with several drugs (e.g., minoxidil, finasteride) and surgical treatment strategies (e.g., transplantation) have been widely used. Both systemic units and topical compositions are available for the treatment of female or male alopecia, and each approach has its own set of advantages and disadvantages. However, applicability and / or efficacy remain highly patient-specific, and therefore any additional treatments still hold great value in this field.

[0006] Hormonal imbalances have been reported to affect hair loss in both women and men (Non-Patent Document 2). The potential effects of estrogen on hair growth in women have been documented in the art (Non-Patent Document 3). Furthermore, the potential effects of estrogen on hair loss in men have also been extensively studied, leading to the conclusion that elevated estrogen levels may contribute to male pattern baldness (Non-Patent Document 4). In this context, see, for example, Patent Document 1, which reports on the topical application of a cosmetic composition containing estetrol. Notably, this document suggests that one of the effects of applying estrogen to the skin is a slowing of hair growth rate, which is considered beneficial, for example, to avoid facial hair growth in menopausal women.

[0007] Topical compositions have long been preferred by users. Furthermore, topical application, regardless of whether it contains multiple active pharmaceutical ingredients, generally reduces the risk of systemic (adverse) effects. Additionally, topical compositions allow for the convenient combination of several active ingredients within a single composition, thereby enabling the formulation of enhanced personal care products (e.g., shampoos that reduce hair loss).

[0008] Topical compositions and formulations containing estrogen have been documented in the art. An example of a commercially available topical formulation is EstroGel®, which is a gel containing estradiol. EstroGel® is indicated for the treatment of moderate to severe vasomotor symptoms and moderate to severe symptoms of vulvar and vaginal atrophy due to menopause. While its potential use for wound healing is conceivable, its use has not been pursued due to undesirable systemic side effects. In fact, conventional estrogen formulations are known to be suboptimal for direct application to individuals and are characterized by a list of adverse effects that may be associated with the use and / or unintended systemic exposure to estradiol. Such adverse effects may include nausea, vomiting, gastric cramps, flatulence, swelling, weight gain, breast pain, breast tenderness, headache, vaginal itching, vaginal discharge, menstrual dysregulation, irregular bleeding (spotting), and hair loss (see Non-Patent Literature 5). Thus, known pharmaceutical compositions are accompanied by a considerable amount of undesirable systemic effects on individuals.

[0009] Therefore, it is clear that there is a continuing need for new topical formulations that are effective in reducing, stopping, or even reversing hair loss in both women and men. The value of such formulations is considered to extend beyond merely reducing hair loss, as they can improve the psychological self-image of both women and men. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] PCT Application International Publication No. 03103685 [Non-patent literature]

[0011] [Non-Patent Document 1] Salman et al., An Bras Dermatol, 2017 [Non-Patent Document 2] Kische et al., JAMA Dermatol, 2017 [Non-Patent Document 3] Brough and Torgerson, Int J Womens Dermatol, 2017 [Non-Patent Document 4] Hu et al., PLoS One, 2012 [Non-Patent Document 5] Mayo Clinic report on transdermal estradiol: Estradiol (Transdermal Route) Side Effects - Mayo Clinic [Overview of the project]

[0012] The ability of hair follicles (HFs) to constantly regenerate and undergo repeated cycles depends on the interaction between the mesenchymal and epithelial segments, known as epithelial-mesenchymal interactions (EMI). Therefore, effective strategies aimed at promoting HF regeneration / stimulation would be most successful if they targeted not only the combination of relevant cell populations but also their function / signaling and / or migration. The HF mesenchyme mainly contains specialized dermal papilla (DP) cells, while the epithelial portion typically contains keratinocytes (KCs) that can be present in various hair follicle sources, including the bulge, outer root sheath (ORS), or the hair bulb itself. Such epithelial cells can originate from stem-like epidermal KCs, which can belong to the hair follicle itself or be present in the skin, forming the HF epithelium, forming EMI with DP cells, supporting DP cell growth, and promoting the formation of HFs and sebaceous gland-like structures (Abreu et al., 2021, Stem Cell Research & Therapy volume 12, Article number: 62).

[0013] In the postnatal period, hair follicles undergo cyclical growth, including a resting phase, a growth phase, and a regression phase. During the regression phase, epithelial (keratinocyte) cells at the base of the hair follicle undergo apoptosis, but DP cells (fibroblasts) remain intact and are pulled upward or migrate until they rest adjacent to the stem cells of the hair follicle bulge. This state continues during the resting phase. In the growth phase, cells at the base of the hair follicle begin to proliferate, leading to downward growth of the hair follicle and the encirclement of DP cells. Although DP cells themselves are thought not to (practically) divide, the number of cells in the DP increases during the growth phase, probably due to replenishment from adjacent cells (mesenchymal cells - fibroblasts) in the dermal sheath. At the start of the growth phase, DP cells activate the stem cells of the secondary hair bud, which leads to new downward growth of the hair follicle. During the regression phase, the hair follicle is separated from its blood supply, and the hair follicle cells undergo apoptosis.

[0014] Against this backdrop, and in contrast to previous reports in the art, the inventors have surprisingly found that topical administration of estetrol within a specific dosage range may be beneficial for preventing, inhibiting, or even reversing hair loss. Topical treatment of the scalp leads to improvements in hair texture, hair quality, and hair appearance. More specifically, the treatment leads to improvements in certain parameters such as target area hair count ("TAHC") and target area hair width ("TAHW"), but is not limited to these.

[0015] Unlike estradiol, estetrol can act on all of the following: epidermal keratinocytes, including, for example, bulge, outer root sheath (ORS), or hair bulb cells; dermal fibroblasts, such as dermal papilla (DP) cells; and sebaceous glands. Estetrol has been shown to have several beneficial effects in preventing or reducing hair loss, ranging from its effects on fibroblast and keratinocyte growth, migration, and epithelial-mesenchymal interactions to the activation of hair follicles and increased sebum secretion. Furthermore, estetrol has been shown to reduce skin inflammation, which contributes to hair follicle health. These characteristics make estetrol particularly interesting for inclusion in topical compositions. In addition, topical administration of estetrol has been found to be safe, with no or limited impact on important safety parameters (both locally at the administration site and at a systemic level).

[0016] While we do not wish to be bound by any theory, it is thought that estetrol (unlike estradiol, for example) can act on keratinocytes to promote and / or extend the growth phase and therefore the growth of hair follicles, and / or delay the onset of the regression phase by increasing the proliferation of keratinocytes and / or reducing their apoptosis, thereby reducing hair loss. Furthermore, by acting on fibroblasts (i.e., DP cells), estetrol is thought to improve epithelial-mesenchymal interactions, which leads to the activation of DP cells and hair growth.

[0017] These findings are, but are not limited to, directly applicable to all kinds of topical compositions, such as therapeutic topical compositions, preventive topical compositions, and cosmetic topical compositions. Several specific embodiments of the present invention are described in the following numbered embodiments.

[0018] Embodiment 1. A composition containing an effective amount of estetrol component for topical use in the prevention or treatment of hair loss in a subject, or for topical application such as topical application to the scalp. Accordingly, the present invention relates to a topical composition for use in the prevention or treatment of hair loss in a subject.

[0019] Embodiment 2. An external composition for use according to Embodiment 1, wherein the target is female or male. Optionally, the target is female. Alternatively, the target is male.

[0020] Embodiment 3. The external composition for use according to Embodiment 1 or 2, wherein the target is males aged 18 to 49 years.

[0021] Embodiment 4. A topical composition for use according to any one of the preceding embodiments, comprising about 0.01% to about 5% by weight of an estrol component, preferably about 0.02% to about 1% by weight of an estrol component, more preferably about 0.03% to about 0.75% by weight of an estrol component, even more preferably about 0.04% to about 0.5% by weight of an estrol component, and most preferably about 0.06% by weight of an estrol component. Preferably, the topical composition is configured and / or formulated for topical application to the scalp.

[0022] Embodiment 5.0.02 A topical composition for use according to any one of the preceding embodiments, comprising an estrol component in an amount from 0.02% by weight to 1.5% by weight, preferably an estrol component from 0.05% by weight to 1.2% by weight, an estrol component from about 0.02% by weight to about 1% by weight, an estrol component from about 0.03% by weight to about 1% by weight, preferably an estrol component from about 0.04% by weight to about 1% by weight, more preferably an estrol component from about 0.05% by weight to about 1% by weight, most preferably an estrol component from about 0.06% by weight to about 0.5% by weight, more preferably an estrol component from about 0.09% by weight to about 1.1% by weight, and further, an estrol component from 0.1% by weight to 1% by weight, most preferably an estrol component from 0.3% by weight to 0.7% by weight.

[0023] Embodiment 6. A topical composition for use according to any one of the preceding embodiments, which does not produce any significant systemic effect in the subject at the time of or after topical application. The absence of systemic effects is beneficial for both female and male subjects, but is particularly important for use in male subjects in order to avoid disrupting hormone balance.

[0024] Embodiment 7. A topical composition for use according to any one of the preceding embodiments, further comprising a permeability enhancer that enables permeation through the stratum corneum.

[0025] Embodiment 8. A topical composition for use according to any one of the preceding embodiments, for use in the treatment and / or prevention of female and / or male hair loss, more particularly female and / or male androgenetic alopecia.

[0026] Embodiment 9. A topical composition for use according to any one of the preceding embodiments, comprising about 0.02% to about 1.5% by weight of an estetrol component, more particularly about 0.05% to 1.2% by weight, even more particularly about 0.09% to about 1.1% by weight, or about 0.1% to about 1% by weight of an estetrol component, for topical use in the prevention or treatment of hair loss, preferably female and / or male hair loss, more preferably female and / or male androgenetic alopecia.

[0027] Embodiment 10. A topical composition for use according to any one of the preceding embodiments, comprising an estrol component in an amount from about 0.05% by weight to about 1.3% by weight, more specifically from about 0.08% by weight to about 1.2% by weight, even more specifically from about 0.09% by weight to about 1.1% by weight, or from about 0.1% by weight to about 1% by weight. Alternatively, the topical composition comprises an estrol component in an amount from about 0.03% by weight to about 0.75% by weight, preferably from about 0.04% by weight to about 0.5% by weight, more preferably from about 0.05% by weight to about 0.25% by weight, most preferably about 0.06% by weight.

[0028] Aspect 11. An external composition for use according to any one of the preceding aspects, which is in the form of a preparation selected from the group consisting of solutions, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, creams, and sprays.

[0029] Aspect 12. An external composition for use according to any one of the preceding aspects, which is a hydrogel characterized by an advantageous release profile, for example, an advantageous release profile as compared to other preparations such as creams.

[0030] Aspect 13. The average cumulative amount of the estetrol component released into a receptor solution of ethanol:PEG400:water in a volume ratio of 40:30:30 through an isopore membrane is at least about 2.5 μg / cm per square root of an hour 2 , at least about 5 μg / cm 2 , at least about 7 μg / cm 2 , at least about 10 μg / cm 2 , at least about 15 μg / cm 2 , at least about 20 μg / cm 2 , at least about 25 μg / cm 2 , preferably at least about 50 μg / cm 2 , more preferably at least 100 μg / cm 2 , more preferably at least about 150 μg / cm 2 , more preferably at least about 200 μg / cm 2 and is characterized by being thus, and is an external composition for use according to any one of the preceding aspects.

[0031] Aspect 14. The average cumulative amount of the estetrol component released into a receptor solution of ethanol:PEG400:water in a volume ratio of 40:30:30 through an isopore membrane is at least about 50 μg / cm in 8 hours 2 , at least about 100 μg / cm 2 , at least about 150 μg / cm 2, at least about 200 μg / cm³ 2 , at least about 250 μg / cm³ 2 , at least about 300 μg / cm³ 2 , at least about 350 μg / cm³ 2 , at least about 400 μg / cm³ 2 , at least about 450 μg / cm³ 2 A topical composition for use as described in any one of the preceding embodiments, characterized by being such that 2 It is characterized by being such.

[0032] Embodiment 15. A topical composition for use according to any one of the preceding embodiments, characterized in that the average percentage of the estetrol component released in a 40:30:30 ethanol:PEG400:water receptor solution by volume is at least about 15%, preferably at least about 20%, after 1 hour, and / or the average percentage of the estetrol component released in a 40:30:30 ethanol:PEG400:water receptor solution by volume is at least about 40%, preferably at least about 50%, more preferably at least 80%, and most preferably at least 90% after 8 hours.

[0033] Embodiment 16. A topical composition for use according to any one of the preceding embodiments, comprising a penetration enhancer in an amount ranging from about 0.5% by weight to about 60% by weight.

[0034] Embodiment 17. The topical composition for use according to Embodiment 16, comprising a substance or molecule (i.e., a permeation-promoting molecule) that enables the permeation-promoting agent to permeate the stratum corneum, and a solvent.

[0035] Embodiment 18. The topical composition for use according to Embodiment 16 or 17, wherein the permeation enhancer is selected from the group consisting of ethanol, ethers such as diethylene glycol monoethyl ether (Transcutol®), benzyl alcohol, their fatty acids and esters, or any combination thereof.

[0036] Embodiment 19. A topical composition for use according to any one of Embodiments 16 to 18, wherein the permeation enhancer comprises a solvent containing one or more polyethylene glycols (PEG), propylene glycols (PG), or a combination thereof.

[0037] Embodiment 20. The topical composition for use according to Embodiment 19, wherein the PEG is a PEG selected from the group consisting of PEG having a molecular weight between about 200 g / mol and about 600 g / mol, for example, PEG200, PEG300, PEG400, PEG500, PEG600, or any combination thereof.

[0038] Embodiment 21. A topical composition for use according to Embodiment 19 or 20, wherein the permeation enhancer comprises a solvent containing PEG having a molecular weight of about 400 g / mol, for example, PEG400. In some embodiments, the permeation enhancer comprises up to 50% by weight of PEG400 and / or about 15% to 45% by weight of PEG400 and / or about 20% to 40% by weight of PEG400 and / or about 30% to 35% by weight of PEG400.

[0039] Embodiment 22. A topical composition for use according to any one of the preceding embodiments, comprising benzyl alcohol preferably in an amount of about 1% to about 3%.

[0040] Embodiment 23. A topical composition for use according to any one of the preceding embodiments, comprising a thickening agent.

[0041] Embodiment 24. The external composition for use according to Embodiment 23, comprising a thickening agent in an amount ranging from about 0.3% by weight to about 20% by weight.

[0042] Embodiment 25. A topical composition for use according to Embodiment 23 or 24, comprising a thickener selected from the group consisting of hydroxyethylcellulose (HEC), carboxymethylcellulose (CMC), a high molecular weight crosslinked acrylic-based polymer, a nonionic triblock copolymer, or any combination thereof.

[0043] Embodiment 26. The topical composition for use according to Embodiment 25, wherein the high molecular weight crosslinked acrylic-based polymer is Carbopol (trademark).

[0044] Embodiment 27. The topical composition for use according to Embodiment 25, wherein HEC is HEC250 HHX.

[0045] Embodiment 28. The topical composition for use according to Embodiment 25, wherein the approximate molecular weight of the nonionic triblock copolymer is between about 1800 and about 4000, and the polyoxyethylene content is between about 70% and about 80%.

[0046] Embodiment 29. A topical composition for use according to Embodiment 25, wherein the nonionic triblock copolymer is selected from poloxamers, for example, poloxamer 188, poloxamer 407, or a combination thereof.

[0047] Embodiment 30. A topical composition for use according to any one of the preceding embodiments, comprising a preservative.

[0048] Embodiment 31. The topical composition for use according to Embodiment 30, which may contain a preservative in an amount from about 1% to about 10% by weight, preferably from about 1% to about 3% by weight.

[0049] Embodiment 32. A topical composition for use according to Embodiment 30 or 31, wherein the preservative is selected from the group consisting of lysozyme, nisin, quaternary ammonium preservatives, parabens, phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, natural preservatives, and any combination thereof.

[0050] Embodiment 33. A topical composition for use according to any one of the preceding embodiments, comprising a emollient in an amount of about 2.5% to about 30% by weight, preferably about 8% to about 12% by weight, most preferably about 10% by weight.

[0051] Embodiment 34. A topical composition for use according to Embodiment 33, comprising a emollient selected from the group consisting of glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, petroleum, and any combination thereof.

[0052] Embodiment 35. A topical composition for use according to any one of the preceding embodiments, comprising, in addition to the estolol component, a permeation enhancer, a thickener, and, optionally, a preservative and / or emollient, each preferably selected from the group described herein.

[0053] Embodiment 36. An external composition for use according to any one of the preceding embodiments, which is supplemented with an aqueous solution such as water to a total concentration of 100% by weight.

[0054] Embodiment 37. In addition to the estetrol component (by weight), A permeation enhancer in an amount ranging from approximately 0.1% by weight to approximately 60% by weight, preferably a permeation enhancer comprising a permeation enhancer molecule and a solvent or solvent system. Thickening agent ranging from approximately 0.3% by weight to approximately 20% by weight, Optionally, preservatives and / or emollients, Water until it reaches 100% by weight, A topical composition for use according to any one of the preceding embodiments, including the above.

[0055] Embodiment 38. In addition to the estrol component (by weight), PEG400 in amounts ranging from approximately 16% to approximately 20% by weight. PG from approximately 18% to approximately 22% by weight, Glycerol in an amount of approximately 8% to 12% by weight, HEC from approximately 1% by weight to approximately 2% by weight, Approximately 1.5% to 2.5% by weight of benzyl alcohol, A topical composition for use according to embodiment 37, comprising:

[0056] Embodiment 39. In addition to the estrol component, PEG400 in amounts ranging from approximately 18% to approximately 22% by weight. Carbopol (trademark) in amounts ranging from approximately 0.1% to approximately 1% by weight, Transcutol (trademark) in amounts ranging from approximately 4% to 6% by weight. A topical composition for use according to embodiment 38, comprising:

[0057] Embodiment 40. A topical composition for use according to any one of the preceding embodiments, wherein the prevention or treatment includes or results in an improvement in the feel, texture and appearance of the hair, preferably the prevention or treatment includes or results in an improvement in the target area hair count (TAHC) and / or target area hair width (TAHW).

[0058] Embodiment 41. A topical composition for use according to any one of the preceding embodiments, wherein hair loss is caused by a hair disorder in an adult female or adult male.

[0059] Embodiment 42. A topical composition for use according to any one of the preceding embodiments, wherein the hair loss is female pattern hair loss or male pattern hair loss (abbreviated as FPHL and MPHL, respectively, the latter of which also refers to male pattern baldness in the art) or female androgenetic alopecia or male androgenetic alopecia (abbreviated as FAGA and MAGA, respectively).

[0060] Embodiment 43. A topical composition for use according to any one of the preceding embodiments, wherein male pattern baldness is classified as, or deemed to be, male pattern baldness selected from stages 1 to 7 on the Hamilton-Norwood scale, preferably stages 3 to 7 on the Hamilton-Norwood scale, more preferably stages 5, 6, or 7 on the Hamilton-Norwood scale.

[0061] Embodiment 44. A topical composition for use according to any one of the preceding embodiments, wherein the prevention or treatment includes preventing hair loss, reversing hair loss, slowing hair loss, reducing hair loss, increasing hair growth, or resulting in an improvement in the feel, texture and / or appearance of the hair.

[0062] Embodiment 45. A topical composition for use according to any one of the preceding embodiments, wherein the above prevention or treatment increases the function and / or growth of keratinocytes, and in connection therewith, preferably increases the function and / or growth of epidermal keratinocytes (KCs) such as those belonging to the bulge, outer root sheath (ORS), or hair bulb.

[0063] Aspect 46. A topical composition for use according to any one of the preceding aspects, wherein the prevention or treatment described above increases the function and / or growth of follicular mesenchymal fibroblasts, and in connection therewith increases the function and / or growth of dermal papilla (DP) cells (i.e., a form of fibroblast specialized for the induction of differentiated hair).

[0064] Embodiment 47. A topical composition for use according to any one of the preceding embodiments, wherein the prevention or treatment described above means delaying the onset of the regression phase or preventing the regression phase.

[0065] Embodiment 48. A topical composition for use according to any one of the preceding embodiments, wherein the prevention or treatment described above means the promotion and / or extension of the growth period.

[0066] Embodiment 49. A topical composition for use according to any one of the preceding embodiments, wherein the above prevention or treatment means the restoration and / or promotion of epithelial-mesenchymal interactions between hair follicle cells.

[0067] Embodiment 50. A topical composition for use according to any one of the preceding embodiments, wherein the prevention or treatment described above is characterized by the reduction of inflammation and / or oxidative stress in the hair follicle.

[0068] Embodiment 51. The topical composition for use according to any one of the preceding embodiments, wherein the estetrol component is estetrol or an ester thereof, preferably estetrol in an anhydrous form or (i) hydrate.

[0069] Embodiment 52. The topical composition for use according to any one of the preceding embodiments, wherein the estol component is estol monohydrate.

[0070] Embodiment 53. A topical composition for use according to any one of the preceding embodiments, wherein a further active ingredient suitable for the prevention or treatment of hair loss is present in the topical composition, or the further active ingredient suitable for the prevention or treatment of hair loss is administered simultaneously, or administered before or after treatment using the topical composition.

[0071] Embodiment 54. A packaging unit comprising a topical composition as described in any one of the preceding embodiments. The packaging unit preferably contains one or more dose units of the topical composition as described herein. A suitable packaging unit comprises any container capable of holding and storing a liquid. Optionally, the packaging unit is a box, display unit, ampoule, bottle, vial, tube, syringe, cartridge, bag, sachet, pouch, film, laminate, foil, can, cylinder, or pressurized container.

[0072] The above and further aspects and embodiments of the present invention are described in the following sections and in the appended claims. The subject matter of the appended claims is thus specifically incorporated herein. [Brief explanation of the drawing]

[0073] [Figure 1]This graph shows the average cumulative amount (μg / cm²) per unit area (expressed as the square root of time) of estetrol monohydrate released by volume through an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution from 10 different formulations during the experimental period from 1 to 8 hours. Error bars represent the standard deviation of the mean (n=6). [Figure 2] This graph shows the average cumulative amount (μg / cm²) per unit area (expressed as the square root of time) of estetrol monohydrate released by volume through an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution from seven different aqueous gel formulations during the experimental period from 1 to 8 hours. Error bars represent the standard deviation of the mean (n=6). [Figure 3] This graph shows the average cumulative amount (μg / cm²) per unit area (expressed as the square root of time) of estetrol monohydrate released by volume through an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution from three different cream formulations during the experimental period from 1 to 8 hours. Error bars represent the standard deviation of the mean (n=6). [Figure 4] This graph shows the average percentage (%) of estetrol monohydrate released by volume into a 40:30:30 ethanol:PEG400:water receptor solution via an isopore membrane from four aqueous gel formulations during the experimental period from 1 to 8 hours (expressed as the square root of time). Error bars represent the standard deviation of the mean (n=5-6). [Figure 5] This graph shows the average cumulative amount (μg / cm²) per unit area (shown as the square root of time) of estetrol monohydrate released by volume through an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution from four different aqueous gel formulations during the experimental period from 1 to 8 hours. Error bars represent the standard deviation of the mean (n=5~6). [Figure 6]This figure shows the effects of topical placebo (PBO), EstroGel® (EG), AG24, AG25, and AG26 on uterine weight and morphology. Eight-week-old female mice were subcutaneously injected with LPS 24 hours and 2 hours prior to wound creation (6 mice per group). EstroGel®, AG24, AG25, AG26, or placebo was applied as a thin layer to the surface of the wound the day before wound creation, at the time of wound creation, and on days 1, 2, 3, and 4 post-wound creation. Uterine morphological changes on day 5 were evaluated by measuring uterine weight (A) and taking photographs of the uterus (B). Scale bar = 10 mm. Results are shown as mean ± sem. Differences from placebo were determined using paired t-tests; *P ≤ 0.05 and ***P ≤ 0.001. [Figure 7] This graph shows the effects of topical placebo (PBO), EstroGel® (EG), AG26, and AG28 on uterine weight and morphology. Eight-week-old female mice were subcutaneously injected with LPS 24 hours and 2 hours before wound creation (6 mice per group). EstroGel®, AG26, AG28, or their placebos were applied as a thin layer to the surface of the wound on day 0 only (single dose), or on days -1, 0, 1, and 2 (repeated doses). Uterine morphological changes on day 3 were evaluated by measuring uterine weight. Results are shown as mean ± sem. Differences from placebo were determined using paired t-tests; *P ≤ 0.05 and ***P ≤ 0.001. [Figure 8] This graph shows the antioxidant activity of E4 as measured using the Skin-Biosense™ sensor. Antioxidant activity was evaluated by mixing a 5 mM H2O2 solution with E4 solutions of various concentrations and measuring the residual peak area. A 5 mM EUK-134 solution was used as the reference compound. [Figure 9]This figure shows the experimental design for Example 7. As previously described (Edelkamp et al., Molecular Dermatology, 2020 and Langan et al., Exp Dermatol, 2015), microscopically dissected human growing VI scalp HF was cultured at 37°C and 5CO2 in minimal Williams's E medium (Gibco, Life Technologies), which was converted to Williams Complete Media (WCM) by supplementing with 10 ng / ml hydrocortisone (Sigma Aldrich), 10 μg / ml insulin (Sigma Aldrich), and 1% penicillin / streptomycin mix (Gibco). [Figure 10] This diagram shows the stages of the hair cycle. The top two images: growth phase. The two middle images: early regression phase. DP: dermal papilla; DPst: dermal papilla stalk; CTS: connective tissue sheath; HM: hair matrix. The bottom image: a representative image of a dystrophic hair follicle (Keyence VHX900). [Figure 11] This is a representative image for the quantification of proliferation and apoptosis of hair matrix keratinocytes. Quantitative evaluation of germinal hair matrix keratinocytes is calculated as the percentage of Ki-67-positive nuclei below Auber's line. Intrafollicular apoptosis is analyzed in germinal and precortical hair matrix cells using TUNEL (TdT-mediated dUTP-biotin-nick-terminal labeling). Ki-67 staining is labeled red, TUNEL is labeled green, and nuclei (DAPI) are labeled blue. gHM: germinal hair matrix; pcHM: precortical hair matrix. [Figure 12] Estetrol does not induce LDH release into the culture medium. Culture medium was pooled from n=7-8 HF cells / group cultured from donor 2 up to day 6 (days 1, 3, 5, and 6); mean, GraphPad Prism 9, D'Agostino & Pearson omnibus normality test. Nonparametric analysis was applied (Kruskal-Wallis test, Dunn's multiple comparison test - vehicle fixed), no significant differences. [Figure 13]Estetrole does not affect hair shaft formation. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test p=0.4856; Dunn's multiple comparison test (vehicle fixed) ns; Mann-Whitney vs. vehicle *p<0.05. Samples with different gray intensities represent different donors. [Figure 14] Microscopically, HF is maintained in the growth phase longer at all estetrol concentrations (especially 3 μM). (A) Microscopic hair cycle stage classification. (B) Microscopic hair cycle score. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test *p=0.0304; Dunn's multiple comparison test (vehicle fixed) ns; Mann-Whitney vs. vehicle *p<0.05. Samples with different gray intensities represent different donors. (C, D) Microscopic images: DP: dermal papilla; HMx: hair matrix; DC: dermal cup. Display scale bar indicates 100 μm. [Figure 15] Estetrol (3 μM) increased the proliferation of hair matrix keratinocytes but did not affect apoptosis (A, B). Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. (A): One-way ANOVA p=0.2115; Holm-Sidak multiple comparison test vehicle-fixed ns; unpaired Student's t-test vs vehicle *p<0.05. Samples with different gray intensities represent different donors. (B): Kruskal-Wallis test p=0.6460; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs vehicle; ns. (C) Microscopic images: Ki-67+ cells were counted in the delimiting region below Ober's line (germ hair matrix, gHM), and TUNEL+ cells were counted in the delimiting region below Ober's line (gHM) and above (precortical hair matrix, pcHM). [Figure 16]All concentrations of E4 tested showed a trend of increasing versican expression and alkaline phosphatase activity in dermal papilla (DP) cells. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. (A) Versican expression in dermal papilla. Kruskal-Wallis test p=0.3529; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs. vehicle ns. Samples with different gray intensities represent different donors. (B) Alkaline phosphatase activity in dermal papilla. Kruskal-Wallis test p=0.5508; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs. vehicle ns. (C) Microscopic images. Top: Versican is labeled green and nucleus (DAPI) is labeled blue; Bottom: Alkaline phosphatase is labeled blue. DP = dermal papilla. [Figure 17] Analysis of DP fibroblast migration revealed that all concentrations of E4 tested did not significantly affect cell density in DP. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test p=0.2081, Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs. vehicle ns. Samples with different gray intensities represent different donors. [Figure 18] All estrol concentrations tended to decrease cell density, with 300 nM and 3 μM E4 significantly reducing the total number of cells in the DP stem. Pooled data from n=4 female donors. Mean ± SEM, GraphPad Prism 9. (A) Density of dermal papilla stem cells. One-way ANOVA p=0.5092; Holm-Sidak multiple comparison test vehicle-fixed ns; unpaired student t-test versus vehicle ns. Samples with different gray intensities represent different donors. (B) Total number of dermal papilla stem cells. One-way ANOVA p=0.0296; Holm-Sidak multiple comparison test vehicle-fixed p=0.0177; unpaired student t-test versus vehicle, p<0.05. [Figure 19]Estetrol did not significantly affect the cell density of induced dermal cups. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. One-way ANOVA p=0.7616; Holm-Sidak multiple comparison test vehicle-fixed ns; unpaired Student's t-test versus vehicle ns. Samples with different gray intensities represent different donors. [Figure 20] In the bulge basal layer, 3 μM E4 significantly decreased the percentage of K15-positive cells, while 30 μM (significantly) and 3 μM and 300 nM (trending) E4 increased their proliferation. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. (A) Percentage of K15+ cells in the bulge basal layer. One-way ANOVA p=0.0641; Holm-Sidak multiple comparison test vehicle-fixed #p=0288; Unpaired Student's t-test vs vehicle *p<0.05. Samples with different gray intensities represent different donors. (B) Percentage of Ki-67+ cells in the bulge basal layer. Kruskal-Wallis test p=0.1302; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs vehicle **p<0.01. [Figure 21] In suprabulbar ORS, estetrol had no effect on the percentage of K15-positive cells, but E4 at 300 nM (significant) and 3 μM (tendency) reduced their proliferation. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9. (A) Percentage of K15+ cells in the suprabulbar basal layer. Kruskal-Wallis test p=0.9375; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs. vehicle ns. Samples with different gray intensities represent different donors. (B) Percentage of Ki-67+ cells in the suprabulbar basal layer. Kruskal-Wallis test p=0.7462; Dunn's multiple comparison test (vehicle-fixed) ns; Mann-Whitney vs. vehicle *p<0.05. (C) Microscopic images. The orthotropic sclerosing (ORS) on the hair follicle is defined by a dotted line. Scale bar = 100 μm. [Figure 22]Estetrole at 3 μM (significantly) and 300 nM and 30 μM (tendency) increased the percentage of CD34-positive cells in the orthogonal root sheath (ORS) on the hair bulb. (A) Percentage of CD34+ cells in the bulge basal layer. Pooled data from n=4 female donors, mean ± SEM, GraphPad Prism 9 one-way ANOVA p=0.1011; Holm-Sidak multiple comparison test vehicle-fixed *p=0.0392; unpaired Student's t-test versus vehicle *p<0.05. Samples with different gray intensities represent different donors. (B) Microscopic image. Orthogonal root sheath (ORS) on the hair bulb is demarcated by a dotted line. Scale bar = 100 μm (ORS: outer root sheath). [Figure 23] This figure shows the experimental design for Example 8. Micro-dissected human growing VI scalp HF was supplemented with 10 ng / ml hydrocortisone (Sigma Aldrich), 10 μg / ml insulin (Sigma Aldrich), and 1% penicillin / streptomycin mix (Gibco), and cultured at 37°C and 5% CO2 in minimal Williams's E medium (Gibco, Life Technologies), which was modified Williams Complete Media (WCM) as previously described (Edelkamp et al., Molecular Dermatology 2020; Langan et al., Exp Dermatol 2015). [Figure 24] This graph shows the decrease in hair shaft formation due to estradiol and the trend decrease due to 300 nM and 3 μM estitol. Top line: vehicle; two middle lines: 300 nM estradiol and 300 nM estitol; bottom line: 3 μM estitol. n=5-6 HF / group (all HF) from one healthy male donor; mean ± SEM GraphPad Prism 9; Mann-Whitney test, **p<0.01. Dunn's multiple comparison test - vehicle fixed, ns. [Figure 25]The tested concentrations of estrol maintained hair follicles in the growth phase, which was even more pronounced with 3 μM estrol, but estradiol did not affect the hair cycle. n=5-6 HF / group from one healthy male donor, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test p=0.1723; Dunn's multiple comparison test (vehicle fixed); ns Mann-Whitney vs vehicle, ns. [Figure 26] Hair matrix keratinocyte proliferation is increased by 300 nM estradiol and 3 μM estetrol. 4–6 HF / group (all HF) cells from one healthy male donor (mean ± SEM, GraphPad Prism 9). Ki-67+ cells were counted in the area below Ober's line (germ matrix), and TUNEL+ cells were counted in the areas below and above Ober's line (precortical matrix). Kruskal-Wallis test p=0.2571; Dunn's multiple comparison test (vehicle-fixed), ns; Mann-Whitney vs. vehicle, ns. [Figure 27] Estrogen reduces versican expression in the hair papilla. Five to six HFs per group (all HFs) from one healthy male donor, mean ± SEM, Graph Pad Prism 9. Kruskal-Wallis test p=0.3137; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. [Figure 28] Estrogen reduces alkaline phosphatase activity in hair papillae. Five to six HF / group (all HF) from one healthy male donor, mean ± SEM, Graph Pad Prism 9. Kruskal-Wallis test p=0.1395; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. [Figure 29]3 μM estetrol significantly reduces cell density (upper panel) and total cell number (lower panel) in induced dermal cups in hair follicles during the growth phase. Estradiol shows no significant effect. n=2-6 HF / group (growth phase only) from one healthy male donor, mean ± SEM, GraphPad Prism 9. Upper graph: Kruskal-Wallis test *p=0.0335; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, *p<0.05. Lower graph: Kruskal-Wallis test p=0.0826; Dunn's multiple comparison test vehicle-fixed, #p<0.05; Mann-Whitney vs vehicle, *p<0.05. [Figure 30] Estrol tends to decrease cell density (top panel) and total number of cells (middle panel) in the dermal papilla (3 μM), but increases the area (300 nM and 3 μM) (bottom panel). Estradiol (E2) shows no significant effect. 5-6 HF / group (all hair follicles) from one healthy male donor, mean ± SEM, GraphPad Prism 9. Top graph: Kruskal-Wallis test p=0.0514; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. Middle graph: Kruskal-Wallis test p=0.1671; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. Bottom graph: Kruskal-Wallis test p=0.2226; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. [Figure 31]3 μM estol reduces cell density in the dermal papilla stalk (upper panel) and also shows a trend towards a decrease in the total number of dermal papilla stalk cells (lower panel). Estradiol shows no significant effect. 5-6 HF / group (all hair follicles) from one healthy male donor, mean ± SEM, GraphPad Prism 9. Upper graph: Kruskal-Wallis test *p=0.0190; Dunn's multiple comparison test vehicle-fixed, #p<0.05; Mann-Whitney vs vehicle, *p<0.05. Lower graph: Kruskal-Wallis test p=0.3056; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. [Figure 32] Estrol reduces the number of CD34+ cells in the epidermal root sheath, while 300 nM estradiol has no effect. 5-6 HF / group (all HF) from one healthy male donor, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test *p=0.0401; Dunn's multiple comparison test vehicle-fixed, #p<0.05; Mann-Whitney vs vehicle, *p<0.05. [Figure 33] 300 nM estradiol and estetrol tend to increase K15 expression in the outer root sheath of the hair bulb (upper panel) and also increase cell count (lower panel). n=5-6 HF / group from one healthy male donor, mean ± SEM, GraphPad Prism 9. ORS: outer root sheath. Upper graph: Kruskal-Wallis test p=0.1435; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. Lower graph: Kruskal-Wallis test p=0.5110; Dunn's multiple comparison test vehicle-fixed, ns; Mann-Whitney vs vehicle, ns. [Figure 34] These are representative bright-field images of terminal and intermediate hair follicles from affected and non-affected areas. DP = dermal papilla, FUE = hair follicle unit extraction. [Figure 35]This figure shows the experimental design for Example 9. As previously described (Edelkamp et al., Molecular Dermatology 2020; Langan et al., Exp Dermatol 2015), human growing stage VI hair follicles microdissected from FUE were cultured at 37°C and 5% CO2 in Williams's E medium (Gibco, Life Technologies) minimal medium, which was made into Williams Complete Media (WCM) supplemented with 2 mM L-glutamine (Gibco), 10 ng / ml hydrocortisone (Sigma Aldrich), 10 μg / ml insulin (Sigma Aldrich), and 1% penicillin / streptomycin mix (Gibco). [Figure 36] 3 μM estetrol prolongs the growth phase in hair follicles from both the non-affected (terminal) and affected (terminal and intermediate) scalp areas of FPHL patients. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, GraphPad Prism 9. Microscopic hair cycle stage classification was performed according to the criteria published in Langan et al., Exp Dermatol 2015. Graph below: Kruskal-Wallis test multiple comparison, p=0.5532; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 37]3 μM estetrol tended to increase the proliferation of matrix keratinocytes in terminal hair follicles from both the non-affected and affected scalps of FPHL patients, but did not affect apoptosis of matrix keratinocytes. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, GraphPad Prism 9. Ki-67+ cells (upper panel) were counted in the demarcation region below Ober's line (germ matrix), and TUNEL+ cells (lower panel) were counted in the demarcation region below Ober's line and the upper demarcation region (precortical matrix). Upper graph: Kruskal-Wallis test multiple comparison, p=0.2859; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Graph below: Kruskal-Wallis test for multiple comparisons, p=0.5686; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 38] 3 μM estetrol tends to increase versican expression in terminal and intermediate hair follicles from non-affected scalp. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test multiple comparison, p=0.2620; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 39] 3 μM estetrol significantly reduces the total number of cells in the dermal cup of terminal hair follicles from non-affected areas, but increases the total number of cells in the dermal cup of intermediate hair follicles. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, GraphPad Prism 9. Kruskal-Wallis test for multiple comparisons, p=0.7056; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, *p<0.05. [Figure 40]Treatment with 3 μM Estrol tends to decrease the density of dermal papilla (DP) cells in intermediate follicles from the non-affected area (bottom panel), but increases the total number of DP cells (top panel) and DP area size (middle panel). The density and total number of DP cells in intermediate follicles from the affected area tend to increase with 3 μM E4. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, GraphPad Prism 9. Top graph: Kruskal-Wallis test multiple comparison, p=0.4256; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Middle graph: Kruskal-Wallis test multiple comparison, p=0.9683; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Graph below: Kruskal-Wallis test for multiple comparisons, p=0.2878; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 41] 3 μM estetrol significantly reduced the total number of dermal papilla cells (top panel) and area size (middle panel) in terminal hair follicles in the non-affected area, but did not affect their density (bottom panel). In intermediate hair follicles in the non-affected area, the density of dermal papilla cells tended to decrease. Pooled data from n=2 FPHL donors, n=2-6 HF / group; n=1-4 nascent HF / group, mean ± SEM, Graph Pad Prism 9. Top graph: Kruskal-Wallis test multiple comparison, p=0.2890; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, *p<0.05. Middle graph: Kruskal-Wallis test multiple comparison, p=0.4812; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, **p<0.01. Graph below: Kruskal-Wallis test for multiple comparisons, p=0.6340; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 42]Estetrol tended to increase K15 expression and the number of positive cells in the extraroot sheath above the hair bulb in terminal HF from non-affected scalp, and also increased the proliferation of K15+ cells in terminal HF from non-affected scalp and intermediate HF from affected scalp. Pooled data from n=2 FPHL donors, n=2 to 6 HF / group, mean ± SEM, Graph Pad Prism 9. Top graph: Kruskal-Wallis test multiple comparison, p=0.6601; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Middle graph: Kruskal-Wallis test multiple comparison, p=0.8527; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Graph below: Kruskal-Wallis test for multiple comparisons, p=0.7225; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Figure 43] Estetrol tended to increase K15 expression in intermediate HF from non-affected scalp and increased the proliferation of K15+ cells in terminal HF from affected scalp. Pooled data from n=2 FPHL donors, n=1 to 6 HF / group, mean ± SEM, Graph Pad Prism 9. Top graph: Kruskal-Wallis test multiple comparison, p=0.6091; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. Bottom graph: Kruskal-Wallis test multiple comparison, p=0.8852; Dunn's multiple comparison test - vehicle fixed, ns; Mann-Whitney test - vehicle fixed, ns. [Modes for carrying out the invention]

[0074] As used herein, the singular forms "a," "an," and "the" refer to both singular and plural objects unless otherwise indicated by the context.

[0075] The terms “comprising,” “comprises,” and “comprised of,” as used herein, are synonymous with “including,” “includes,” or “containing,” and these terms are inclusive or non-restrictive and do not exclude any additionally unmentioned components, elements, or process steps. These terms also encompass “consisting of” and “consisting essentially of,” which enjoy well-established meanings in patent terminology.

[0076] A reference to a numerical range by an endpoint includes all numbers and fractions belonging to the individual range, as well as the endpoint being referenced. This applies to numerical ranges regardless of whether they are introduced using expressions such as "from... to...", "between... and...", or other expressions.

[0077] When used herein, the terms “about” or “approximately” refer to measurable values ​​such as parameters, quantities, and time intervals, and mean to include variations from a specified value relative to that value, such as variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1%, from a specified value relative to that value, but only if such variations are appropriate for carrying out the disclosed invention. Naturally, the values ​​to which the modifiers “about” or “approximately” apply are themselves specifically and preferably disclosed.

[0078] On the other hand, the terms “one or more” or “at least one,” for example, one or more members or at least one member of a group of members, etc., are self-evident, and as further examples, this term encompasses, in particular, references to any one of the aforementioned members, or any two or more of the aforementioned members, for example, any three or more, four or more, five or more, six or seven or more of the aforementioned members, etc., and references to all of the aforementioned members. In another example, “one or more” or “at least one” may refer to one, two, three, four, five, six, seven, or more.

[0079] The background discussion to the present invention described herein is included to illustrate the context of the invention. This does not imply that any of the materials referred to were publicly disclosed, known, or in common general knowledge in any country as of the priority date of any of the claims.

[0080] Throughout this disclosure, various publications, patents, and published patent specifications are referenced in a manner that identifies the source. All documents cited herein constitute part of this specification by reference. In particular, any teachings or sections of such documents specifically mentioned herein constitute part of this specification by reference.

[0081] Unless otherwise defined, all terms used in the disclosure of this invention, including technical and scientific terms, have the meanings generally understood by those skilled in the art to which this invention pertains. For further guidance, definitions of terms are included so that the teachings of this invention may be better understood. Where a particular term is defined in relation to a particular aspect or embodiment of this invention, such relevance or meaning is to apply throughout this specification, i.e., in the context of other aspects or embodiments of this invention, unless otherwise defined. For example, embodiments relating to products are also applicable to corresponding features of methods and uses.

[0082] Different aspects or embodiments of the present invention are defined in more detail in the following sections. Each of the aspects or embodiments thus defined may be combined with any other aspect(s) or embodiment(s) unless otherwise expressly contradictory. In particular, any feature indicated as preferred or advantageous may be combined with any other one or more features indicated as preferred or advantageous.

[0083] Throughout this specification, any reference to “one embodiment” or “embodiment” means that a particular feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment of the present invention. Therefore, the phrases “in one embodiment” or “in an embodiment” found in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, certain features, structures, or characteristics can be combined in one or more embodiments in any suitable manner, as would be apparent to those skilled in the art from this disclosure. Moreover, even if some embodiments described herein include some features but not others included in other embodiments, combinations of features from different embodiments are considered to be within the scope of the present invention and constitute different embodiments, as would be understood to those skilled in the art. For example, the appended claims include alternative combinations of the claimed embodiments, as would be understood to those skilled in the art.

[0084] As used throughout this Specification, the term “estetrol component” includes substances selected from the group consisting of estetrol, esters of estetrol, esters of estetrol in which at least one hydrogen atom of the hydroxyl group is substituted with an acyl radical of a hydrocarbon carboxylic acid, sulfonic acid, or sulfamic acid with 1 to 25 carbon atoms, anhydrous estetrol, estetrol hydrate, e.g., estetrol monohydrate, and combinations thereof. Wherever estetrol is referred to throughout any part of this Specification, it should be understood that any estetrol-containing component (i.e., compound) and / or estetrol derivative (e.g., estetrol ester or hydrate) are also assumed. More preferably, in the context of this Disclosure, the particularly preferred estetrol component suitable for the dosage units or cosmetic or medical use and therapeutic methods described herein is estetrol (including anhydrous estetrol or estetrol hydrate). Most preferably, the estetrol component is estetrol monohydrate.

[0085] As used herein, the term “estetrol” refers to 1,3,5(10)-estratriene-3,15α,16α,17β-tetrol or 15α-hydroxyestriol, as well as estetrol hydrate, e.g., estetrol monohydrate. “Estetrol” or abbreviated as “E4” is an estrogen steroid produced by the fetal human liver (PubChem CID:27125). Estetrol can be described as a 3-hydroxysteroid corresponding to 17β-estradiol, in which the 15α and 16α positions are substituted with two additional hydroxyl groups. Estetrol is known to be an estrogen receptor agonist (Coelingh Bennink et al., Estetrol review: profile and potential clinical applications, Climacteric, 2008). When an estetrol component described herein refers to estetrol, it may refer to endogenous estetrol. Alternatively, estetrol may be synthesized chemically, by the use of (mutant) recombinant enzymes, or by any combination thereof. Alternatively, in the art, estetrol may have the molecular formula: C 18 H 24 It can also be represented by O4, or by structural formula (I): [ka]

[0086] In preferred embodiments, estetrol exists or is used herein as estetrol monohydrate or anhydrous estetrol, more preferably as estetrol monohydrate. While estetrol monohydrate may be used as an ingredient in the preparation of the topical compositions described herein, those skilled in the art will understand that in the resulting topical compositions, the estetrol monohydrate may be solubilized and no longer exist in monohydrate form.

[0087] In some embodiments, the estetrol component may be used as a single active ingredient or in combination with any other cosmetic or pharmaceutical active agent known in the art for the mitigation or prevention of hair loss.

[0088] "Weight (w / w)" is also, alternatively, expressed throughout the art as "weight by weight" or "weight for weight," and refers to the contribution of a particular molecule or substance in a composition or mixture, measured by weight (i.e., mass).

[0089] The term “pharmaceutical active ingredient” is used throughout this disclosure without distinction from “pharmaceutical active drug” and should be interpreted in accordance with the World Health Organization’s definition of the term: “a substance used in a final product (FPP) of a medicinal product intended to exert pharmacological activity in humans or otherwise directly affect the diagnosis, cure, reduction, treatment or prevention of a disease or directly affect the restoration, correction or modification of a physiological function.” Physiological effects may be achieved by a single dose or by repeated doses. Unless otherwise specified, in the context of this disclosure, the term refers to the estetrol component, preferably estetrol.

[0090] Where used throughout this disclosure, the terms “therapy” or “treatment” mean the reduction or measurable decrease of one or more symptoms or measurable markers of a pathological condition, in the context of the Invention, female or male hair loss (e.g., male pattern baldness, female pattern hair loss, androgenic alopecia). Hair loss may also be hair loss caused by a certain medical condition or resulting from a medical treatment (e.g., chemotherapy) without any other underlying medical condition. Optionally, hair loss may be caused by, considered to be caused by, or deemed to be caused by alopecia areata. The term encompasses both the treatment of an already established pattern of hair loss (i.e., thinning of hair mass has already begun), as well as prevention or preventive measures, wherein the objective of treatment is to prevent the onset and / or recurrence, onset and progression of hair loss in a female or male subject. For illustrative and limited purposes, the prophylactic use of the topical compositions described herein may be administered to the scalp of a female or male subject who has initiated a medical treatment known to cause hair loss. An alternative example, not an exhaustive one but illustrative, could be the application to the scalp of a person who is expected to develop female-pattern baldness or male-pattern baldness in the near future, which can be predicted by observing female or male hair loss in family members or by performing trichogram analysis.

[0091] Measurable reductions include any statistically significant decrease in the rate of hair thinning, hairline recession, hair loss, or any combination thereof. Statistically significant, as used herein, means a p-value less than 0.05, which is the generally accepted cutoff score in statistical analysis, as will be understood by those skilled in the art. More specific indications for the prevention or treatment of hair loss are described in further detail below.

[0092] Those skilled in the art will recognize that in order to achieve effective therapeutic treatment, it is necessary to administer a therapeutically effective dose to the subject. Therefore, in the context of this disclosure, “effective dose” means the amount necessary to obtain a physiological effect. The physiological effect may be obtained by a single dose or multiple doses. “Therapeutably effective dose” or “therapeutably effective dose” indicates the amount of estetrol component that, when administered, produces a clinically advantageous response with respect to the treatment of the subject. Similarly, “effective dose for prevention” or “effective dose for prevention” means the amount of estetrol component that inhibits or delays female or male(er) hair loss or thinning hair. Those skilled in the art will recognize that terms such as “quantity,” “amount,” and “level” are synonymous and have well-defined meanings in the art, and will understand that in the context of this application, they refer to, but are not limited to, the relative quantification of the estetrol component in a topical composition such as a hydrogel, or, where indicated, the absolute quantification of the estetrol component that is considered an effective dose for the application described herein when applied to the skin of the subject.

[0093] External composition As described in the abstract of this disclosure, a topical composition comprising estolol is provided which has at least the same efficacy as topical compositions described in the literature in the art for the prevention and treatment of hair loss in female and / or male subjects. However, the topical compositions of the subject of the present invention exhibit significantly attenuated systemic effects, regardless of the exact formulation used. Accordingly, attenuation, and even almost certainly absence, of systemic effects can be observed with respect to solutions, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, creams, and sprays. This observation remains even when permeability enhancers are included in the composition. This allows for the formulation of topical compositions containing relatively low estrogen doses. Given that estol has historically been considered a weaker estrogen compared to other estrogens such as estradiol, the anti-hair loss properties of topical compositions using estolol at such low doses are even more noteworthy (Gerard et al., J Endocrinol, 2015). Therefore, topical compositions disclosed in this application for use in the prevention and / or treatment of hair loss have not been conceived.

[0094] Any reference to “topical compositions” is understood to include, and vice versa, any solutions, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, creams, and sprays described herein.

[0095] Accordingly, a first aspect of the present invention relates to a composition comprising an effective amount of estetrol component for topical use in the prevention or treatment of hair loss in a subject, or for topical application such as topical application to the scalp. Accordingly, in such an aspect, the present invention relates to a topical composition for use in the prevention or treatment of hair loss in a subject. In other words, the present invention envisions a topical composition comprising an effective amount of estetrol component for the prevention or treatment of hair loss in a subject. In further other words, the present invention envisions the use of a composition comprising an effective amount of estetrol component for use in the manufacture of a topical agent for the prevention or treatment of hair loss. Those skilled in the art will readily understand that, unless otherwise shown, all embodiments described herein are envisioned in the context of use for the prevention and treatment of hair loss. Accordingly, any combination of any embodiment and an indication of use for the prevention and / or treatment of hair loss should not be considered to constitute a combination of separate embodiments herein.

[0096] In certain embodiments, the topical composition contains an estetrol component in an amount of about 0.01% to about 0.18% by weight, for example, estetrol. In preferred embodiments, the topical composition contains an estetrol component in an amount of about 0.02% to about 0.16% by weight, for example, estetrol, preferably an estetrol component in an amount of about 0.03% to about 0.14% by weight, for example, estetrol, more preferably an estetrol component in an amount of about 0.04% to about 0.12% by weight, for example, estetrol, even more preferably an estetrol component in an amount of about 0.05% to about 0.10% by weight, for example, estetrol, and even more preferably an estetrol component in an amount of about 0.05% to about 0.08% by weight, for example, estetrol.

[0097] In alternative, specific embodiments, the topical composition comprises about 0.18% by weight or less of an estetrol component, for example, estetrol. In preferred embodiments, the topical composition comprises about 0.16% by weight or less of an estetrol component, for example, estetrol, preferably about 0.14% by weight or less of an estetrol component, for example, estetrol, more preferably about 0.12% by weight or less of an estetrol component, for example, estetrol, even more preferably about 0.10% by weight or less of an estetrol component, for example, estetrol, and even more preferably about 0.08% by weight or less of an estetrol component, for example, estetrol.

[0098] In preferred embodiments, estetrol exists or is used as a monohydrate herein. Therefore, in certain embodiments, the topical composition contains about 0.01% to about 0.18% by weight of estetrol monohydrate. In preferred embodiments, the topical composition contains about 0.02% to about 0.16% by weight of estetrol monohydrate, preferably about 0.03% to about 0.14% by weight of estetrol monohydrate, more preferably about 0.04% to about 0.12% by weight of estetrol monohydrate, even more preferably about 0.05% to about 0.10% by weight of estetrol monohydrate, and even more preferably about 0.05% to about 0.08% by weight of estetrol monohydrate.

[0099] In alternative, specific embodiments, the topical composition comprises about 0.18% by weight or less of estetrol monohydrate. In preferred embodiments, the topical composition comprises about 0.16% by weight or less of estetrol monohydrate, preferably about 0.14% by weight or less of estetrol monohydrate, more preferably about 0.12% by weight or less of estetrol monohydrate, even more preferably about 0.10% by weight or less of estetrol monohydrate, and even more preferably about 0.08% by weight or less of estetrol monohydrate.

[0100] In further alternative specific embodiments, the topical composition comprises 0.02% to 1.5% by weight of an estrol component, preferably 0.05% to 1.2% by weight of an estrol component, about 0.02% to about 1% by weight of an estrol component, about 0.03% to about 1% by weight of an estrol component, preferably about 0.04% to about 1% by weight of an estrol component, more preferably about 0.05% to about 1% by weight of an estrol component, most preferably about 0.06% to about 0.5% by weight of an estrol component, more preferably about 0.09% to about 1.1% by weight, even more preferably 0.1% to 1% by weight of an estrol component, and most preferably 0.3% to 0.7% by weight of an estrol component.

[0101] In a further alternative embodiment, the topical composition contains about 0.01% to about 5% by weight of estetrol components, preferably about 0.02% to about 1% by weight of estetrol components, more preferably about 0.03% to about 0.75% by weight of estetrol components, even more preferably about 0.04% to about 0.5% by weight of estetrol components, and most preferably about 0.06% by weight of estetrol components.

[0102] The embodiments described above do not exclude the presence of estrogen components other than estetrol in the topical composition. It is conceivable that different estetrol components may be present in a single composition. In such embodiments, the topical composition may, but not be limited, contain estetrol, or more specifically, both estetrol monohydrate and estetrol esters.

[0103] "Topical application" means application to a specific part of the body. In particular, application to the surface of the body, such as the skin, is envisioned. When used topically, a local effect, especially a local effect on the skin, is usually desired. Topical application is preferably understood in this context as application to the skin of the scalp, and may include application to the occipital and / or frontotemporal regions.

[0104] Those skilled in the art will understand that the “stratum corneum” as used herein refers to the outer layer of the epidermis, which consists of multiple layers of terminally differentiated keratinocytes, primarily made from keratin, a proteinaceous material. The composition, function, and characteristics of the stratum corneum are described in great detail in the art (e.g., Matsui and Amagai, Int Immunol, 2015). It is generally believed that the stratum corneum is the rate-limiting factor in the permeation of pharmaceutically active drugs through the skin. Therefore, the permeation enhancers described herein assist in the permeation of at least the estrol component through the stratum corneum. In certain embodiments, the topical composition includes a permeation enhancer that enables permeation through the stratum corneum. In the context of the present invention, the term “permeation enhancer” is used without distinction from, but is not limited to, terms including “permeability enhancer,” “permeability increaser,” “permeability inducer,” and even “skin enhancer.” When the term “permeation accelerator” is used throughout this disclosure, it will be understood by those skilled in the art to refer to a permeation accelerator molecule as part of a solvent or solvent system. Different permeation accelerators are described in detail in the art, including, but not limited to, those listed in the CPE database (Vasyuchenko et al., Pharmaceutics, 2021). Exemplary and non-limiting permeation accelerators are further described herein.

[0105] In a preferred embodiment, the topical composition comprises an estetrol component (e.g., estetrol, preferably estetrol monohydrate) in an amount from about 0.01% to about 5% by weight, for example, from about 0.08% to about 1.2% by weight, from about 0.09% to about 1.1% by weight, or from about 0.1% to about 1% by weight, and a penetration enhancer, preferably comprising an estetrol component in an amount from about 0.02% to about 2.5% by weight, and a penetration enhancer, more preferably comprising an estetrol component in an amount from about 0.02% to about 2% by weight, and a penetration enhancer. It contains, more preferably, about 0.03% to about 1.5% by weight of an estetrol component and a permeation enhancer, even more preferably, about 0.03% to about 1% by weight of an estetrol component and a permeation enhancer, even more preferably, about 0.03% to about 0.75% by weight of an estetrol component and a permeation enhancer, even more preferably, about 0.04% to about 0.5% by weight of an estetrol component and a permeation enhancer, and most preferably, about 0.06% by weight of an estetrol component and a permeation enhancer.

[0106] In alternative embodiments, the topical composition comprises an estetrol component (e.g., estetrol, preferably estetrol monohydrate) in an amount from about 0.01% to about 5% by weight, for example, from about 0.08% to about 1.2% by weight, from about 0.09% to about 1.1% by weight, or from about 0.1% to about 1% by weight, and a penetration enhancer, preferably comprising about 2.5% by weight or less of the estetrol component and a penetration enhancer, more preferably comprising about 2% by weight or less of the estetrol component and a penetration enhancer, more preferably comprising about 1.5% by weight or less of the estetrol component and a penetration enhancer, even more preferably comprising about 1% by weight or less of the estetrol component and a penetration enhancer, even more preferably comprising 0.75% by weight or less of the estetrol component and a penetration enhancer, even more preferably comprising about 0.5% by weight or less of the estetrol component and a penetration enhancer, and most preferably comprising about 0.1% by weight or less of the estetrol component and a penetration enhancer.

[0107] As will be apparent to those skilled in the art, this disclosure encompasses the use of each specific form of the topical compositions disclosed herein for use in the prevention or treatment of hair loss. Accordingly, suitable topical compositions and formulations include, but are not limited to, solutions, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, creams, and sprays. Each of these terms is intended to correspond to its generally accepted meaning. Similarly, these topical compositions may be applied directly to the skin of the scalp, or in combination with wound dressings, patches, bandages, adhesive bandages, tampons, transdermal patches, etc., to prevent the topical composition from being removed from the skin, and in some embodiments to shield the treatment area from external influences such as dirt, microorganisms, and direct sunlight. Furthermore, it should be understood that in topical compositions such as solutions or foams, estetrol may not be present in monohydrate form or may only be present as monohydrate in the composition due to solubilization.

[0108] A "solution" refers to a liquid in which two or more different substances are uniformly distributed amongst themselves. A distinction is made between true solutions and colloidal solutions. In a true solution, the dissolved substances are molecularly dispersed in the solvent, that is, they exist as distinct single molecules. In the case of a colloidal solution, the distributed particles are colloidal in size. In the solution according to the present invention, one or more pharmaceutical active ingredients are dissolved in a suitable solvent or a mixture of mutually miscible solvents (aqueous or non-aqueous).

[0109] "Emulsion" broadly refers to any mixture of at least two immiscible (i.e., incompatible) liquids, where the first liquid is distributed throughout the second liquid (dispersion medium) as small droplets (dispersed phase). Therefore, in certain embodiments, the pharmaceutical compositions described herein are oil-in-water emulsions or water-in-oil emulsions. Emulsions are widely used in skin care formulations and can be classified into creams and lotions. In this context, "suspension" broadly refers to a heterogeneous mixture containing undissolved solids dispersed in a liquid phase, having a size large enough to settle.

[0110] The term "cream" generally refers to a water-in-oil emulsion in which an aqueous phase is dispersed in an oil phase, but it may also refer to an oil-in-water emulsion in which oil is dispersed in an aqueous base. While an emulsion is a stable suspension of small, immiscible droplets of fluid that are immiscible with other fluid portions of the emulsion, creams are generally recognized as different from emulsions in that they do not, and instead represent a specific subset of emulsions that are more viscous and typically contain more lipophilic and / or surfactant components.

[0111] A "lotion" is a liquid composition with low to medium viscosity. Generally, lotions have a lower viscosity than creams, although the viscosity may be similar for both. Lotions can contain finely powdered substances insoluble in the dispersion medium by using suspending and dispersing agents. Alternatively, a lotion may have a liquid substance as the dispersion phase that is immiscible with the vehicle and is usually dispersed by an emulsifier or other suitable stabilizer. In one embodiment, a lotion is in the form of an emulsion with a viscosity between 100 centistokes and 1000 centistokes. The fluidity of a lotion allows for rapid and uniform application to a large surface area. Lotions are typically intended to dry on the skin, leaving their medicinal components on the skin's surface.

[0112] "Ointment" generally refers to a broad range of high-viscosity oil-in-water cream formulations, i.e., semi-solid substances containing an ointment base and optionally one or more pharmaceutically active ingredients (estetrol ingredients in the context of this invention). Examples of suitable ointment bases include hydrocarbon bases, absorbent bases, water-removable bases, and water-soluble bases. "Paste" generally differs from ointments in that it has a higher percentage of solids. Overall, pastes are more absorbent and less greasy compared to ointments based on the same set of ingredients / excipients.

[0113] As used herein, "foam" refers to a product consisting of a large volume of gas dispersed in a liquid containing one or more active substances, a surfactant to ensure its formation, and other components. Generally, foams are gas dispersed in a liquid (liquid foams) or gas dispersed in a solid (solid foams). Based on the size of the bubbles, foams can be classified into the following structures: microporous (pore size less than 2 nm), mesoporous (pore size 2 nm to 50 nm), and macroporous (pore size greater than 50 nm). Furthermore, they can be classified into monodisperse foams or polydisperse foams based on the pore size distribution. Foams as a drug form are listed in the monograph "Foams, Medicated" in the European Pharmacopoeia [Eur. Ph. 11.0]. Medicinal foams are usually preparations intended for application to the skin or mucous membranes, and are typically formed by dispersing a large volume of gas in a liquid at the time of administration. This liquid contains one or more active substances, and generally surfactants that assist in foam formation, along with various other excipients. This preparation is usually supplied in a pressurized multi-dose container equipped with a device consisting of a valve and a push button suitable for delivering the foam. While the aforementioned pharmacopoeia monograph focuses primarily on liquid foams for topical application, those skilled in the art understand that the use of carriers in the form of solid foams is increasing in pharmaceutical technology. The high gas content and small fluid volume allow a small amount of fluid to cover a large area of ​​skin, thereby resulting in the acceptability of the foam. Furthermore, the delicate structure does not cause irritation during diffusion. The large amount of gas lowers the specific gravity of the foam, allowing it to sit lightly on the surface of the scalp. The components contained (e.g., emulsifiers) increase skin permeability. Absorption into the skin usually occurs rapidly, leaving a non-oily or non-greasy thin film. Applying estetrol, formulated as a foam, topically enhances the effectiveness of the treatment while also increasing patient comfort. This application method increases the absorption of estetrol by hair follicles, leading to improved therapeutic effects.Using a foam formulation can improve medication adherence, which in turn impacts the effectiveness of treatment.

[0114] The term "mousse" refers to a substance that is very similar to a foam, but is generally used to refer to a substance with less water content.

[0115] A "spray" is a preparation of a drug in an aqueous, alcoholic, or glycerin-containing medium. Sprays are applied to mucous membranes or skin using an atomizer or nebulizer. "Spray" means that the drug-containing solution is filled into a device suitable for using pressure to atomize and release the drug-containing solution as a mist.

[0116] A "gel agent" is a semi-solid system in which a liquid solidifies due to a gel-background agent. In the case of hydrogels, the liquid forming the gel is water or an aqueous solution. As used herein, the term "hydrogel" primarily refers to an aqueous solution of an active ingredient that solidifies using a polymeric hydrophilic substance to form a gel. Polymeric hydrophilic substances, and therefore polymer materials, swell upon contact with water, thereby resulting in a solution with pseudoplastic fluid behavior or a plastic structure containing the necessary aqueous components, depending on the concentration. Therefore, hydrophilic gel agents typically consist of water or an aqueous solution that gels with a hydrophilic polymer compound. Gels constructed using a hydrophilic polymer scaffold are generally thixotropic (i.e., their viscosity decreases when pressure is applied). In contrast to creams, gel agents are referred to as true single-phase systems. In preferred embodiments, the topical compositions referred to herein may be or contain hydrogels.

[0117] The term "hydrogel" as used herein may include, but is not limited to, hydrophilic polymers, acrylic acid, acrylamide, and any suitable polymer or combination of polymers such as 2-hydroxyethyl methacrylate. Examples of hydrogels, but are not limited to, synthetic hydrogels, stimulus-responsive hydrogels, (poly)peptide-based hydrogels, hybrid hydrogels, and DNA-based hydrogels. Methods for producing each of these hydrogel categories have been described in the art and are therefore known to those skilled in the art.

[0118] Examples of synthetic hydrogels, though not limiting, include double-network hydrogels. Nanocomposite hydrogels are also examples. Stimulus-responsive hydrogels are characterized by their ability to undergo swelling changes mediated by external stimuli (e.g., pH, temperature, ionic strength, solvent type, electric field, magnetic field, light, and chelated species). Examples of stimulus-responsive hydrogels, though not limiting, include recombinant segments of block copolypeptides of native structural proteins, such as elastin, silk, silky, and elastin-like peptide blocks, and hydrogels formed from recombinant triblock copolymers of one or more polypeptide sequences.

[0119] The term "hybrid hydrogel" is used herein to refer to a hydrogel comprising at least two distinct classes of molecules, e.g., a synthetic polymer and a component derived from a biological macromolecule, which are interconnected by either covalent or non-covalent bonds.

[0120] With respect to the (hydro)gel compositions described herein, the presence of several excipients, such as propylene glycol, glycerol, Transcutol®, benzyl alcohol, and PEG400, can increase the solubility of estetrol. The use of these excipients in the gel composition makes it possible to make estetrol soluble and fully available for permeation through the membrane, or similarly, through the stratum corneum. This is because the increased estetrol solubility creates a larger estetrol concentration gradient on both sides of the membrane, thus contributing to an increased diffusion rate across the membrane, or similarly through the stratum corneum, as predicted by Fick's law of diffusion.

[0121] In embodiments where the topical composition is a hydrogel, the hydrogel preferably contains a permeability enhancer that enables permeation through the stratum corneum. More preferably, the hydrogels referred to herein are hydrogels containing about 0.02% to about 10% by weight of an estol component, such as estol, preferably estol monohydrate. Preferably, the hydrogels referred to herein are hydrogels containing an estrol component in an amount from about 0.02% by weight to about 5% by weight; more preferably, the hydrogels referred to herein are hydrogels containing an estrol component in an amount from about 0.02% by weight to about 2.5% by weight; more preferably, the hydrogels contain an estrol component in an amount from about 0.03% by weight to about 0.75% by weight, preferably from about 0.04% by weight to about 0.5% by weight, more preferably from about 0.05% by weight to about 0.25% by weight, for example, from about 0.08% by weight to about 1.2% by weight, from about 0.09% by weight to about 1.1% by weight, or from about 0.1% by weight to about 1% by weight; most preferably, the hydrogels contain an estrol component in an amount from about 0.06% by weight.

[0122] Alternatively, the hydrogels referred to herein are hydrogels containing about 10% by weight or less of an estrol component. Preferably, the hydrogels referred to herein are hydrogels containing about 5% by weight or less of an estrol component; more preferably, the hydrogels referred to herein are hydrogels containing about 2.5% by weight or less of an estrol component; more preferably, the hydrogels contain about 0.75% by weight or less of an estrol component; preferably, about 0.5% by weight or less of an estrol component; more preferably, about 0.25% by weight or less of an estrol component; more preferably, about 0.1% by weight or less of an estrol component; and most preferably, about 0.08% by weight or less of an estrol component.

[0123] In certain embodiments where the topical composition is a hydrogel, the hydrogel is characterized by a release profile that is advantageous compared to other compositions, such as creams, for example, but not limited to these. In preferred embodiments, the hydrogel described herein releases an average cumulative amount of estetrol component within 1 hour into a 40:30:30 ethanol:PEG400:water receptor solution by volume via an isopore membrane, at least about 2.5 μg / cm³. 2 at least about 5 μg / cm³ 2 at least about 7 μg / cm³ 2 at least about 10 μg / cm³ 2 at least about 15 μg / cm³ 2 at least about 20 μg / cm³ 2 at least approximately 25 μg / cm³ 2 Preferably, at least about 50 μg / cm³ 2 More preferably, at least 100 μg / cm³ 2 More preferably, at least about 150 μg / cm³ 2 More preferably, at least about 200 μg / cm³ 2It is characterized by the following: In an alternative embodiment, the hydrogel described herein releases an average cumulative amount of estetrol component within 1 hour into a 40:30:30 ethanol:PEG400:water receptor solution by volume via an isopore membrane, with an average cumulative amount of estetrol component of about 25 μg / cm³. 2 From approximately 200 μg / cm³ 2 It is characterized by being up to [a certain value]. In a preferred alternative embodiment, the hydrogel described herein releases an average cumulative amount of estetrol component within 1 hour into a 40:30:30 ethanol:PEG400:water receptor solution by volume via an isopore membrane, with an average cumulative amount of estetrol component of about 25 μg / cm³. 2 From approximately 100 μg / cm³ 2 It is characterized by being up to [a certain value]. In a further preferred alternative embodiment, the hydrogel described herein is characterized by an average cumulative amount of estetrol component released by volume within 1 hour into a 40:30:30 ethanol:PEG400:water receptor solution via an isopore membrane of about 25 μg / cm³. 2 From approximately 50 μg / cm³ 2 It is characterized by being such.

[0124] Preferably, the hydrogel described herein releases an average cumulative amount of estrol components via an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution by volume within 8 hours, with an average cumulative amount of at least about 350 μg / cm³. 2 Preferably, at least about 400 μg / cm³ 2 More preferably, at least about 450 μg / cm³ 2 It is characterized by being such.

[0125] Preferably, the hydrogels described herein are characterized by the fact that the average percentage of the estetrol component released by volume into a 40:30:30 ethanol:PEG400:water receptor solution is at least about 15%, preferably at least about 20%, after 1 hour and / or the average percentage of the estetrol component released by volume into a 40:30:30 ethanol:PEG400:water receptor solution is at least about 40%, preferably at least about 50%, more preferably at least 80%, after 8 hours. In an alternative embodiment, the hydrogel described herein is characterized by the average percentage of the estetrol component released by volume into a 40:30:30 ethanol:PEG400:water receptor solution being about 15% to about 30% after 1 hour, and / or the average percentage of the estetrol component released by volume into a 40:30:30 ethanol:PEG400:water receptor solution being about 45% to about 90% after 8 hours.

[0126] In a very preferred embodiment, the hydrogel described herein has an estrol component release rate (i.e., gradient) of approximately 2.5 μg / cm². 2 / √hour, at least about 5 μg / cm³ 2 / √hour, at least about 7 μg / cm³ 2 / √hour, at least about 10 μg / cm³ 2 / √hour, at least about 15 μg / cm³ 2 / √hour, at least about 20 μg / cm³ 2 / √hour, at least about 25 μg / cm³ 2 / √ time ~ approx. 50μg / cm 2 The average cumulative amount of estetrol released is approximately 50 μg / cm³ after 8 hours. 2 From approximately 100 μg / cm³ 2The hydrogels described herein are characterized by the fact that the percentage of estitol released by volume into a 40:30:30 ethanol:PEG400:water receptor solution via the isopore membrane is between approximately 75% and approximately 95%. More preferably, the hydrogels described herein have an estitol component release rate (i.e., gradient) of approximately 30 μg / cm². 2 Approximately 35 μg / cm³ based on / √ hours. 2 Up to √ hours, the average cumulative amount of released estetrol component is approximately 75 μg / cm³ after 8 hours. 2 From approximately 90 μg / cm³ 2 The hydrogel is characterized by the fact that the percentage of estitol released by volume into a 40:30:30 ethanol:PEG400:water receptor solution via the isopore membrane is between approximately 85% and approximately 90%. Most preferably, the hydrogel described herein has an estitol component release rate (i.e., gradient) of approximately 33 μg / cm². 2 From / √ hours, approximately 34 μg / cm³ 2 The average cumulative amount of estetrol released over 8 hours is approximately 82 μg / cm³. 2 From approximately 83 μg / cm³ 2 This is characterized by the fact that, optionally, the percentage of estitol released by volume into a 40:30:30 ethanol:PEG400:water receptor solution via an isopore membrane is between approximately 86% and approximately 88%.

[0127] In embodiments where the topical composition is a cream, the release rate (i.e., gradient) of the estrol component into a 40:30:30 ethanol:PEG400:water receptor solution by volume via an isopore membrane is approximately 2.5 μg / cm². 2 From / √time, we can deduce at least approximately 5 μg / cm³. 2 / √hour, at least about 7 μg / cm³ 2 / √hour, at least about 10 μg / cm³ 2 / √hour, at least about 15 μg / cm³ 2 / √hour, at least about 20 μg / cm³ 2 / √hour, or 15 μg / cm³ 2 From / √ hours, approximately 75 μg / cm³ 2 The cream formulation may be characterized by having a release rate (i.e., slope) of the estrol component into a 40:30:30 ethanol:PEG400:water receptor solution by volume via an isopore membrane of approximately 25 μg / cm². 2 From / √ hours, approximately 55 μg / cm³ 2 It can be characterized by being up to / √time.

[0128] The cream formulation releases an average cumulative amount of estetrol component via an isopore membrane into a 40:30:30 ethanol:PEG400:water receptor solution by volume, with an average cumulative amount of estetrol component of approximately 1 μg / cm³ after 1 hour. 2 From approximately 100 μg / cm³ 2 It can be further characterized by being up to [a certain value]. Preferably, the cream is released via an isopore membrane into a receptor solution of 40:30:30 ethanol:PEG400:water by volume, and the average cumulative amount of estrol components released is about 5 μg / cm³ after 1 hour. 2 From approximately 80 μg / cm³ 2 It can be characterized by being up to . The average cumulative amount of estetrol component released by volume into a 40:30:30 ethanol:PEG400:water receptor solution via the isopore membrane is approximately 25 μg / cm³ after 8 hours. 2 From approximately 150 μg / cm³ 2 It can be further characterized by being up to [a certain value]. Preferably, the cream is released via an isopore membrane into a receptor solution of 40:30:30 ethanol:PEG400:water by volume, and the average cumulative amount of estrol components released is about 50 μg / cm³ after 8 hours. 2 From approximately 120 μg / cm³ 2 It can be characterized by being up to that point.

[0129] In embodiments where the topical composition is a foam formulation, the foam formulation may contain one or more excipients selected from the group consisting of butane, butylated hydroxytoluene (optionally butylated hydroxytoluene E321), cetyl alcohol, citric acid (optionally anhydrous citric acid), glycerol, isobutane, lactic acid, polysorbate 60, propane, purified water, stearyl alcohol, and anhydrous ethanol. An exemplary foam formulation may contain, in addition to the estrol component, butane, butylated hydroxytoluene, cetyl alcohol, citric acid, glycerol, isobutane, lactic acid, polysorbate 60, propane, purified water, stearyl alcohol, and anhydrous ethanol. An alternative exemplary foam formulation may contain, in addition to the estrol component, anhydrous ethanol, purified water, butylated hydroxytoluene (E321), lactic acid, anhydrous citric acid, glycerol, cetyl alcohol, stearyl alcohol, polysorbate 60, propane, butane, and isobutane.

[0130] In embodiments where the topical composition is a solution, the solution may contain ethanol (alcohol), propylene glycol, water, or any combination thereof as excipients.

[0131] In certain embodiments, the topical composition is formulated as a shampoo. Preferably, the topical composition is formulated as a shampoo for daily use.

[0132] As described above, any topical composition disclosed herein may contain a penetration enhancer. Preferably, the penetration enhancer comprises a molecule that enables penetration through the stratum corneum (i.e., a penetration-enhancing molecule) and a solvent or solvent system. Optionally, the penetration enhancer is present in the topical composition disclosed herein in an amount from about 0.5% to about 60% by weight, preferably from about 1% to about 50% by weight, more preferably from about 2.5% to about 45% by weight, more preferably from about 5% to about 40% by weight, and more preferably from 10% to about 30% by weight. Optionally, permeation-promoting molecules are present in the topical compositions disclosed herein in amounts ranging from about 0.1% to about 25% by weight, preferably from about 0.5% to about 15% by weight, more preferably from about 1% to about 10% by weight, more preferably from about 2.5% to about 7.5% by weight, and more preferably from 3.5% to about 5% by weight. Optionally, solvents(s) are present in the topical compositions disclosed herein in amounts ranging from about 1% to about 60% by weight, preferably from about 5% to about 50% by weight, more preferably from about 10% to about 40% by weight, more preferably from about 15% to about 30% by weight, and more preferably from 18% to about 25% by weight.

[0133] The permeation enhancers described herein are not particularly limited with respect to the present invention and therefore may include or consist of molecules selected from the group consisting of suberin, lignin, kuchin, dimethyl sulfoxide, ethanol, propylene glycol, glycerin, propyl ethylene glycol, urea, dimethylacetamide, sodium lauryl sulfate, poloxamer, span, tween, lecithin, terpenes, and combinations thereof. Preferred permeation enhancers in the context of the present invention include ethanol, ether, benzyl alcohol, their fatty acids and esters, or any combination thereof. Particularly preferred permeation enhancer molecules in the context of the present invention include Transcutol®, benzyl alcohol, and any combination thereof. Benzyl alcohol (C6H5CH2OH) may indiscriminately refer to "phenylmethanol," "phenylcarbinol," and "benzenemethanol" in the art. Transcutol® is a commonly recognized trade name for 2-(2-ethoxyethoxy)ethanol and is also known in the art as diethylene glycol monoethyl ether (C6H 14 They are also annotated without distinction by O3). Optionally, the topical compositions described herein include, as a permeation enhancer, about 1% to about 20% by weight of Transcutol®, preferably about 2% to about 10% by weight of Transcutol®, more preferably about 3% to about 8% by weight of Transcutol®, and most preferably about 4% to about 6% by weight of Transcutol®.

[0134] The permeation accelerator of the present invention comprises a permeation accelerator molecule and a solvent or solvent system. Preferred solvents for the permeation accelerator include, but are not limited to, polyethylene glycol (PEG), propylene glycol (PG), and combinations thereof. "Polyethylene glycol" may also be referred to without distinction by terms such as polyethylene oxide, poly(oxyethylene), poly(ethylene oxide), etc. Polyoxyethylene has been described in detail in the art and is therefore known to those skilled in the art, and those skilled in the art will know that polyethylene glycol has the chemical formula H-(O-CH2-CH2) n It is understood that it is characterized by -OH (wherein n is an integer). Polyethylene glycol is a polyether compound derived from petroleum. Preferred PEGs are those characterized by molecular weights of about 150 g / mol to about 5000 g / mol, more preferably about 200 g / mol to about 2500 g / mol, even more preferably about 250 g / mol to about 1000 g / mol, and most preferably about 300 g / mol to about 600 g / mol. Thus, the PEGs referred to herein can be selected from the group consisting of PEG200, PEG300, PEG400, PEG500, PEG600, and any combination thereof. Most preferably, the permeation enhancer contains, as a solvent or as part of a solvent, PEG having a molecular weight of about 400 g / mol, such as PEG400, but not limited to this. Propylene glycol is typically annotated in the art as propane-1,2-diol, α-propylene glycol, 1,2-propanediol, or 1,2-dihydroxypropane. Propylene glycol is characterized by the chemical formula CH3CH(OH)CH2OH.

[0135] Preferably, the permeation accelerator contains about 5% to about 35% by weight of PEG, preferably PEG400, and / or about 10% to about 35% by weight of PG. More preferably, the permeation accelerator contains about 10% to about 30% by weight of PEG, preferably PEG400, and / or about 12% to about 30% by weight of PG. More preferably, the permeation accelerator contains about 12% to about 25% by weight of PEG, preferably PEG400, and / or about 15% to about 25% by weight of PG. More preferably, the permeation accelerator contains about 14% to about 23% by weight of PEG, preferably PEG400, and / or about 16% to about 23% by weight of PG. More preferably, the permeation enhancer comprises about 16% to about 22% by weight of PEG, preferably PEG400, for example, about 18% to about 22% by weight of PEG or about 16% to about 20% by weight of PEG, and / or about 18% to about 22% by weight of PG.

[0136] Optionally, the topical composition contains benzyl alcohol. The term "benzyl alcohol" should be interpreted in accordance with the general interpretation in the art and therefore refers to an aromatic alcohol characterized by the chemical formula C6H5CH2OH. If present, the amount of benzyl alcohol in the topical composition is not particularly limited. However, preferably, the amount of benzyl alcohol is from about 0.1% by weight to about 10% by weight, more preferably from about 0.5% by weight to about 5% by weight, more preferably from about 1% by weight to about 3% by weight, and even more preferably from about 1.5% by weight to about 2.5% by weight.

[0137] Optionally, the topical composition includes a thickener. The term "thickener" may also be expressed by alternative terms such as "thickening agent" and "viscosity agent," although these terms are not limited to the above. When used in the context of the present invention, "thickener" refers to any substance or molecule that, when added to a liquid or semi-solid composition, increases the viscosity and / or texture of the composition. Therefore, if present, the thickener may be selected from the group consisting of carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, diblock polymers, triblock polymers, gums, and any combination thereof. In embodiments in which the thickening agent is a polysaccharide or contains a polysaccharide, the polysaccharide can be selected from the group consisting of cellulose, cellulose derivatives, carboxymethylcellulose, cellulose acetate propionate carboxylate, hydroxyethylcellulose, hydroxyethylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylhydroxyethylcellulose, hydroxyalkylated cellulose, lignin, kutin, suberin, microcrystalline cellulose, sodium cellulose sulfate, scleroglucan, and any combination thereof. Suitable gums that can act as thickeners include gum arabic, agar, algin, alginic acid, cetyl alcohol, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, sodium carboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and any combination thereof.

[0138] In certain embodiments, the topical compositions described herein contain the thickeners described herein in an amount from about 0.1% to about 25% by weight, preferably from about 0.3% to about 20% by weight, more preferably from about 0.4% to about 15%, more preferably from about 0.5% to about 10% by weight, and more preferably from about 0.75% to about 5% by weight. Preferred thickeners in the context of the present invention include, but are not limited to, hydroxyethylcellulose (HEC), carboxymethylcellulose (CMC), high molecular weight crosslinked acrylic base polymers, nonionic triblock copolymers, or any combination thereof. More preferred thickeners in the context of the present invention include, but are not limited to, hydroxyethylcellulose, high molecular weight crosslinked acrylic base polymers, nonionic triblock copolymers, and any combination thereof. A preferred high molecular weight crosslinked acrylic base polymer is a carbomer, which is undifferentiated throughout the art by the trade name Carbopol®. Any of the following are envisioned: Carbopol® homopolymer (i.e., acrylic acid crosslinked with allyl pentaerythritol), Carbopol® copolymer (acrylic acid and C10-C30 alkyl acrylates crosslinked with allyl pentaerythritol), or Carbopol® interpolymer (carbomer homopolymer or copolymer containing a block copolymer of polyethylene glycol and a long-chain alkyl acid ester). Particularly preferred in the context of the present invention is Carbopol® 980, which is alternatively referred to throughout the art as "Carbomer Homopolymer Type C USP NF" and is a homopolymer of acrylic acid crosslinked with allyl pentaerythritol in a cosolvent of cyclohexane and ethyl acetate with a viscosity of 40,000 cP to 60,000 cP. A preferred hydroxyethylcellulose is HEC250 HHX.Preferred nonionic triblock copolymers are those having a molecular weight of about 1800 g / mol to about 4000 g / mol and a polyoxyethylene content of about 70% to about 80%. Highly preferred nonionic triblock copolymers include poloxamer 188, poloxamer 407, or combinations thereof.

[0139] In certain embodiments, the thickener is HEC or contains the same and is present in the topical composition in an amount from about 0.1% to about 10% by weight, preferably from 0.2% to about 7.5% by weight, more preferably from about 0.5% to about 5% by weight, more preferably from about 0.75% to about 2.5% by weight, and most preferably from about 1% to about 2% by weight. In certain embodiments, the thickener is Carbopol® or contains the same and is present in the topical composition in an amount from about 0.1% to about 10% by weight, preferably from 0.2% to about 7.5% by weight, more preferably from about 0.5% to about 5% by weight, more preferably from about 0.75% to about 2.5% by weight, and most preferably from about 0.75% to about 1.5% by weight. In alternative embodiments, Carbopol® is present in the topical composition in an amount ranging from about 0.1% to about 1% by weight. In certain embodiments, the thickener is or comprises poloxamer 188 and is present in the topical composition in an amount ranging from about 0.1% to about 30% by weight, preferably from about 1% to about 15% by weight, and more preferably from about 2.5% to about 10% by weight. In certain embodiments, the thickener is or comprises poloxamer 407 and is present in the topical composition in an amount ranging from about 0.1% to about 30% by weight, preferably from about 5% to about 25% by weight, and more preferably from about 10% to about 20% by weight.

[0140] In certain embodiments, the thickener is or contains CMC and is present in the topical composition in an amount of about 0.1% to about 10% by weight, preferably 0.2% to about 7.5% by weight, more preferably about 0.5% to about 5% by weight, more preferably about 0.75% to about 2.5% by weight, most preferably about 1% to about 2% by weight or about 1.5% by weight.

[0141] Optionally, the topical composition includes a preservative. In certain embodiments, the topical composition contains a preservative in an amount from 0.5% to 20% by weight, preferably 1% to about 10% by weight, and more preferably about 1% to about 3% by weight. The exact preservative is not particularly limited with respect to the present invention and can therefore be selected from the group consisting of lysozyme, nisin, quaternary ammonium preservatives, parabens, phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, natural preservatives, and any combination thereof. A preferred preservative in the context of the present invention is benzyl alcohol.

[0142] Optionally, the topical composition (which may be a hydrogel) contains a emollient. “Emulsifying agent,” when used throughout this disclosure, refers to a material useful for preventing and / or treating dry skin conditions and for providing special protection to the skin. Specific emollient agents are not particularly limited with respect to the present invention and can therefore be selected from the group consisting of glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, petroleum, and any combination thereof. A particularly preferred emollient agent in the context of the present invention is glycerol. The emollient may be present in the topical composition in an amount from about 2.5% to about 30% by weight, preferably from about 5% to about 25%, more preferably from 7.5% to about 20% by weight, and more preferably from about 8% to about 12% by weight. In further embodiments, the emollient may be glycerol present in the topical composition in an amount of about 2.5% to about 30% by weight, preferably about 5% to about 25% by weight, more preferably 7.5% to about 20% by weight, more preferably about 8% to about 12% by weight, and most preferably about 10% by weight.

[0143] As described in detail throughout this disclosure, any topical composition described herein may also contain certain amounts of other components (i.e., pharmaceutically active drugs and / or excipients) in addition to the estolol component. In any embodiment of the present invention, a solvent may be added until the components reach a certain concentration. In further embodiments, an aqueous solution is used to complement the topical composition. The term “aqueous solution” refers to any solution containing water or in which the solvent is water. Furthermore, “aqueous solution” is used to describe a solution that exhibits commonalities with respect to water or a water-heavy solution, not limited to characteristics such as appearance, odor, color, taste, viscosity, pH, absorbance, or physical state at a particular temperature. In such embodiments, the aqueous solution may be water. In alternative further embodiments, a non-aqueous solution is used to complement the topical composition. In further alternative embodiments, a mixture of a non-aqueous solution and an aqueous solution is used to complement the topical composition.

[0144] As defined herein, the pH of a topical composition, solution, or formulation can be measured using various methods known to those skilled in the art. + A pH indicator that changes color upon ion uptake or release can be used, in which case the resulting color indicates a specific pH value. Alternatively, a pH meter may be used, which measures the potential difference between a pH electrode and a reference electrode. The potential difference relates to the acidity or pH of the solution.

[0145] Taking the above into consideration, an exemplary topical composition according to the present invention, in addition to an estrol component at any one of the concentrations listed herein, Permeation enhancer from approximately 0.1% by weight to approximately 60% by weight, Thickening agent ranging from approximately 0.3% by weight to approximately 20% by weight, Optionally, preservatives and / or emollients, Water until it reaches 100% by weight, Includes.

[0146] In certain embodiments, the topical composition essentially consists of, or comprises (in addition to the estetrol component) a penetration enhancer, a thickener, a preservative, and a skin emollient. In preferred embodiments, the topical composition according to the present invention, in addition to the estetrol component, Permeation enhancer in amounts ranging from approximately 0.1% by weight to approximately 10% by weight. Permeation-enhancing solvent in a concentration of approximately 10% to 60% by weight. Thickening agent ranging from approximately 0.3% by weight to approximately 20% by weight, Optionally, preservatives and / or emollients, Water until it reaches 100% by weight, Includes.

[0147] In a more preferred embodiment, the topical composition according to the present invention includes, in addition to one of the concentrations of estrol component listed herein, Permeation enhancer in amounts ranging from approximately 1% by weight to approximately 7.5% by weight. Permeation-enhancing solvent in a concentration of approximately 15% to 45% by weight. Thickening agent ranging from approximately 0.3% by weight to approximately 20% by weight, Optionally, preservatives and / or emollients, Water until it reaches 100% by weight, Includes.

[0148] In a particular embodiment, the topical composition contains, in addition to one of the concentrations of estrol component listed herein, PEG400 in an amount of approximately 8% to approximately 40% by weight, preferably approximately 16% to approximately 20% by weight, most preferably approximately 18% by weight. Approximately 9% by weight to approximately 44% by weight, preferably approximately 18% by weight to approximately 22% by weight, most preferably approximately 20% by weight of PG. Glycerol, preferably about 4% by weight to about 24% by weight, preferably about 8% by weight to about 12% by weight, most preferably about 10% by weight. HEC in an amount of approximately 0.5% to approximately 4% by weight, preferably approximately 1% to approximately 2% by weight, most preferably approximately 1.5% by weight, and Approximately 0.75% by weight to approximately 1.25% by weight, preferably approximately 1.5% by weight to approximately 2.5% by weight, most preferably approximately 2% by weight of benzyl alcohol. It includes, is essentially derived from, or consists of.

[0149] In a particular embodiment, the topical composition contains, in addition to one of the concentrations of estrol component listed herein, PEG400 in an amount of approximately 9% by weight to approximately 44% by weight, preferably approximately 18% by weight to approximately 22% by weight, most preferably approximately 20% by weight. Carbopol® in an amount of approximately 0.01% to approximately 2% by weight, preferably approximately 0.1% to approximately 1% by weight, most preferably approximately 0.5% by weight, and Transcutol® in an amount of approximately 2% to approximately 12% by weight, preferably approximately 4% to approximately 6% by weight, most preferably approximately 5% by weight. It includes, is essentially derived from, or consists of.

[0150] The terms “preparation” and “composition” are to be used herein without distinction. It is apparent that in any embodiment relating to a topical composition described herein, such composition may contain one or more pharmaceutically or cosmetically acceptable carriers (i.e., excipients) not described in detail throughout this disclosure. As used herein, the term “pharmaceutically acceptable” is consistent with the art and means compatible with other components of a pharmaceutical or cosmetic topical composition and not harmful to its recipient. In a particularly preferred embodiment of the present invention, the topical composition according to the present invention is designed for daily administration, i.e., represents a daily dose unit. Excipients that can be used in topical compositions are not particularly limited, and therefore include pharmaceutically active ingredient excipients, binder excipients, carrier excipients, co-processing excipients, coating excipients, controlled-release excipients, diluent excipients, disintegrant excipients, dry powder inhalation excipients, foaming excipients, emulsifier excipients, lipid excipients, lubricant excipients, sustained-release excipients, penetration enhancer excipients, permeation enhancer excipients, pH adjuster excipients, plasticizer excipients, preservative excipients, and other preservative excipients. The excipient may be one or more excipients selected from the group consisting of solubilizing excipients, solvent excipients, sustained-release excipients, sweetener excipients, flavoring excipients, thickener excipients, viscosity modifier excipients, filler excipients, compression excipients, dry granulation excipients, hot-melt extrusion excipients, wet granulation excipients, rapid-release excipients, high bioavailability excipients, dispersing excipients, solubilization-promoting excipients, stabilizing excipients, capsule volume-enhancing excipients, or any combination thereof. Those skilled in the art will recognize that the use of such media and agents with pharmaceutically active substances is common practice, and therefore the incorporation of these excipients is well known in the art. It is clear that all components used should be non-toxic at the concentrations contained in the final topical composition and should not adversely interfere with the activity of the estetrol component (which preferably exists as the main pharmaceutically active ingredient in the topical composition). In a particular embodiment, two or more excipients, which those skilled in the art would classify as belonging to the same group of excipients, are added to the topical composition. In a further embodiment, two or more excipients, which are different excipients belonging to different groups, are added to the topical composition.In certain embodiments, the excipient may perform two or more functions and / or may be classified by those skilled in the art as belonging to different groups or classes of excipients.

[0151] Optionally, the topical compositions envisioned by the present invention may contain further skin-active ingredients capable of providing skincare and / or haircare benefits. Skincare benefits include, but are not limited to, benefits related to the cosmetic appearance of the skin. Haircare benefits include, but are not limited to, benefits related to the cosmetic appearance of the hair. Further skin-active ingredients may provide immediate, short-term (i.e., acute) benefits and / or long-term, sustained (i.e., chronic) benefits.

[0152] Optionally, the topical compositions envisioned by the present invention may contain at least one further pharmaceutically active ingredient in addition to the estolol component. In certain embodiments, the at least one further pharmaceutically active ingredient is selected from the group consisting of anti-inflammatory agents, analgesics, and anti-infective agents. The anti-inflammatory component may be a steroidal anti-inflammatory component, a nonsteroidal anti-inflammatory component, or a combination thereof.

[0153] In certain embodiments, further pharmaceutically active ingredients may also have properties that reduce female or male pattern baldness. Non-limiting examples of such ingredients include, but are not limited to, minoxidil, finasteride, spironolactone, and dutasteride.

[0154] The topical compositions described herein may be part of any means suitable for application to the scalp of a subject, i.e., they may be included in such means. Alternatively, the topical compositions described herein may be applied to the subject, another subject, or any means suitable for application to the scalp of a subject by a skilled medical practitioner before applying the compositions to the scalp of the subject (i.e., they may be used in conjunction with means for applying the compositions to the scalp).

[0155] Alternatively, the topical composition may be contained in any other suitable means for application to the scalp in question, and therefore, not limited to, means such as bandages, adhesive bandages, patches, and transdermal dressings.

[0156] Those skilled in the art will understand that the topical compositions and formulations described herein can be used either as cosmetic compositions (i.e., for cosmetic purposes) or as therapeutic compositions (i.e., for therapeutic purposes). Those skilled in the art will understand that the purpose of cosmetic application is to improve the appearance of a subject who is considered to be in good health. In contrast, therapeutic application includes situations in which hair loss in a subject is caused by or related to a medical abnormality. To the extent that it is illustrative and not limited to, a medical abnormality may be a hormonal dysregulation, or may be related to certain medical treatments such as chemotherapy, or may occur in a situation of autoimmune disorder.

[0157] Hair removal As used herein, the term “alopecia” refers to all types of hair loss in subjects (female or male), preferably adult subjects, more preferably adult male subjects. As used herein, “alopecia” broadly refers to all types of alopecia. Thus, as used herein, “alopecia” encompasses clinical conditions better known as androgenetic alopecia (AGA), or alopecia areata (AA), fibrous alopecia, diffuse alopecia, scarring alopecia, and generalized alopecia. Accordingly, the topical compositions described herein can be used to treat alopecia, in particular androgenetic alopecia. Androgenetic alopecia has been described in great detail in the literature on numerous occasions in the art (e.g., in Chin et al., Androgenetic alopecia, StatPearls, 2022) and is therefore known to those skilled in the art. Male pattern androgenetic alopecia (indicated without distinction from the term “pattern alopecia”) is a condition of hair loss that primarily affects the crown and anterior part of the scalp. Male pattern baldness is generally described as a receding hairline at the front, hair loss on the crown, or a combination thereof. Female pattern androgenic alopecia is a hair condition that primarily affects the crown and frontal areas of the scalp and is generally a more diffuse pattern compared to male pattern androgenic alopecia.

[0158] Accordingly, in one embodiment, the present invention provides a topical composition comprising the estetrol component for use in the prevention or treatment of hair loss as defined elsewhere in this specification. In other words, the present invention envisions the use of a topical composition comprising the estetrol component for the prevention or treatment of hair loss, and for improving the feel, texture and appearance of hair. In further other words, the present invention envisions the use of the estetrol component (or a composition comprising the estetrol component) for the manufacture of a drug for the prevention or treatment of hair loss by topical administration. Finally, the present invention further provides a therapeutic method for the prevention or reduction of hair loss, comprising the administration of a composition comprising topical administration of the estetrol component to a subject. Numerous methods and approaches for measuring and / or monitoring hair loss are described in the literature in the art, such as target area hair count ("TAHC") and target area hair width ("TAHW") (e.g., reviewed by Dhurat and Saraogi, Int J Trichology, 2009). Throughout this specification and the art, the terms target area hair width and target area hair thickness are used interchangeably.

[0159] In certain embodiments, in a subject (female or male), the use of the topical composition results in a reduction of TAHC and / or TAHW that is smaller than the reduction of TAHC and / or TAHW, respectively, in the subject (female or male) before use of the topical composition of the subject of the present invention. Optionally, the reduction is at least 10% less, preferably at least 20% less, preferably at least 30% less, preferably at least 40% less, and preferably at least 50% less than the reduction in the subject before use of the topical composition of the subject of the present invention.

[0160] The terms "subject," "patient," and "individual" can be used interchangeably in this specification and refer to human subjects. Preferred subjects are human male subjects. In certain embodiments, subjects are considered to be male subjects of adult age (i.e., at least 18 years of age). In preferred embodiments, male subjects are post-pubescent male subjects or subjects considered to be post-pubescent male subjects. In preferred embodiments, subjects are male subjects who have been diagnosed with or are considered to be experiencing hair loss, thinning hair, or a receding hairline. In even more preferred embodiments, male subjects are adult male subjects between the ages of 19 and 49.

[0161] Alternatively, the subjects are female subjects. Optionally, the subjects are female subjects of adult age (i.e., at least 18 years of age). In certain embodiments, the subjects are female subjects in menopause, perimenopause, and / or postmenopausal periods. In certain embodiments, the subjects are menopausal, perimenopause, or postmenopausal subjects having an estradiol level of less than 100 pg / ml, preferably less than 50 pg / ml, preferably less than 30 pg / ml, more preferably less than 20 pg / ml, more preferably less than 20 pg / ml, and most preferably less than 10 pg / ml. In alternative embodiments, the subjects are menopausal, perimenopause, or postmenopausal subjects characterized by a follicle-stimulating hormone concentration of at least 20 milliinternational units (mIU / ml) per milliliter, preferably at least 25 mIU / ml, more preferably at least 30 mIU / ml, more preferably at least 35 mIU / ml, and most preferably at least 40 mIU / ml.

[0162] In the art, the term "menopausal subjects" is used without distinction from "postmenopausal subjects" or "perimenopausal subjects," and refers to women who have not had menstrual bleeding for one year, accompanied by a decrease or cessation of ovarian hormone (e.g., estradiol) production. In other words, "menopause" can be described as a biological state characterized by impairment or cessation of major ovarian functions. Menopause may be accompanied by a wide range of clinical symptoms of varying severity, including, but not limited to, vasomotor dysfunction, vaginal dryness, mood changes, sleep disturbances, urinary incontinence, cognitive changes, physical complaints, and sexual dysfunction. The diagnostic methods for menopause are described in the art and are therefore known to those skilled in the art (Nelson, Menopause, Lancet, 2008).

[0163] The “perimenopausal period” refers to the period in a woman’s life that begins approximately 3 to 4 years before menopause and ends one year after the last menstrual period, characterized by persistent irregular menstrual cycles, extreme fluctuations in hormone levels, frequent anovulation, and the appearance of vasomotor symptoms (Harlow et al., Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging, Menopause, 2012). The terms “postmenopausal” or “postmenopausal” refer to women characterized by the permanent cessation of menstruation. This permanent cessation is determined retrospectively after the observation of 12 months of amenorrhea without any other apparent pathological or physiological cause. The term “postmenopausal” also includes menopause as a result of early ovarian failure, surgery (e.g., oophorectomy), chemotherapy or radiotherapy for cancer, and certain diseases (e.g., infection or hypothyroidism).

[0164] In certain embodiments where the subject is male, male pattern baldness is classified, or considered to be, male pattern baldness, selected from stages 1 to 7 on the Hamilton-Norwood scale, preferably stages 3 to 7, more preferably stages 5, 6, or 7 on the Hamilton-Norwood scale. Those skilled in the art are familiar with the Hamilton-Norwood scale and understand that this scale is a widely used measuring instrument for categorizing hair loss (i.e., male pattern androgenic baldness in men) (Gupta and Mysore, J Cutan Aesthet Surg, 2016).

[0165] While certain preferred forms of alopecia are described above, it should be understood that the present invention is intended to treat all forms of alopecia. Non-limited examples of specific forms of alopecia include, but are not limited to, keloid folliculitis (acne keloidalis), alopecia areata, alopecia totalis, alopecia universalis, anagenous alopecia, androgenic alopecia, Brocq's alopecia, parietal centrifugal scarring alopecia, scarring alopecia, congenital alopecia, congenital hypotrichosis, alopecia areata widespread, discoid lupus erythematosus, dissociative cellulitis, "terminal" scarring alopecia, bald folliculitis, frontal fibrous alopecia, hair shaft abnormalities, androgenic hypertrichosis, hypotrichosis, hereditary alopecia, lichen planus, keratosis pilaris planus, lipedematous alopecia, male pattern baldness, non-scarring alopecia, psoriasiform alopecia Examples include alopecia, pressure alopecia, seborrheic dermatitis-like psoriasis, telogen effluvium, temporal triangular alopecia, tinea capitis, TNF-α inhibitor-induced psoriasis, traction alopecia, trichorrhizosis nodosa, trichotillomania, and fasciculitis.

[0166] The inventors unexpectedly discovered that estetrol has beneficial effects on the proliferation and function of keratinocytes.

[0167] The term "keratinocyte" encompasses a type of cell found in hair follicle cells of the epidermis and epithelium, including bulge keratinocytes, outer root sheath (ORS) keratinocytes, and matrix keratinocytes. In the outer layers of the skin, keratinocytes make up approximately 90% of the cells. The term "enhanced keratinocyte function" means both increased proliferation of keratinocytes in the epidermis and hair follicles, and enhanced, improved, or restored function of keratinocytes in the epidermis and hair follicles. These functions include, but are not limited to, skin thickness and hydration, providing a sufficient skin barrier and ensuring proper wound healing, promoting the growth of hair follicle cells in the growth phase, and epithelial-mesenchymal interactions, i.e., interactions with mesenchymal cells such as fibroblasts in the hair follicle, including dermal papilla cells.

[0168] Keratinocytes also play a role in oxidation and inflammation. Increased ROS production (particularly in keratinocytes) and oxidative stress can increase damage to DNA, proteins, and lipids, potentially leading to premature skin aging and hair loss. Chronic, low-level inflammation damages the skin by increasing the expression of pro-inflammatory cytokines by keratinocytes, which can lead to adverse changes and premature skin aging and hair loss.

[0169] Furthermore, skin pigmentation is influenced by keratinocytes and melanocytes, whose primary function is the production of melanin pigment. Melanocytes are surrounded by keratinocytes, and melanocytes transmit their melanin pigment to keratinocytes. Due to the close interaction and communication between melanocytes and keratinocytes, a single melanocyte can be surrounded by 36 keratinocytes, thereby influencing keratinocytes, for example, estetrol, which in turn can affect melanocytes and, consequently, pigmentation. As used in this disclosure, the term “melanin” refers to a group of natural pigments produced by melanocytes localized in the epidermis. Various types of melanin have been described in the literature in the art (e.g., Wei et al., Unraveling the structure and function of melanin through synthesis, J Am Chem Soc, 2021). “Melaninogenesis” is a commonly used term in the art and relates to the process of melanin production. Melanin formation is upregulated, for example, when the skin is exposed to ultraviolet radiation, resulting in a darkening of the skin tone.

[0170] The term "reactive oxygen species," commonly abbreviated as "ROS," is a general term for molecules formed from oxygen (O2). Examples for understanding include peroxides, superoxide, hydroxyl radicals, singlet oxygen, and alpha oxygen. Reactive oxygen species are produced, in particular, in fibroblasts and keratinocytes. While both the dermis and epidermis can protect the skin from oxidation, the epidermis contains the highest concentration of antioxidants. ROS are a negative or aggravating factor in female pattern hair loss because they cause damage to hair follicles. Therefore, reducing ROS is important to avoid female pattern hair loss.

[0171] Keratinocytes are an important cell type for promoting wound healing, as they play a role not only in wound closure and re-epithelialization, but also in regulating inflammation in wounds alongside immune cells. An imbalance in keratinocyte function can lead to unbalanced inflammation.

[0172] Furthermore, the inventors have found that estetrol also has a beneficial effect on the proliferation and function of fibroblasts.

[0173] The term "fibroblast" encompasses a type of cell found in the dermis and in mesenchymal hair follicle cells, including dermal papilla cells. These fibroblasts, including dermal papilla cells within the hair follicle, do not actually proliferate but increase in number through interactions with surrounding keratinocytes resulting from epithelial-mesenchymal interactions (EMI). Skin fibroblasts are the primary cell type found in skin connective tissue (dermis). During hair growth, fibroblasts interact with epidermal cells in the skin between hair follicles.

[0174] Treatment and Prevention The terms “treatment” or “to treat” are interpreted to mean both therapeutic treatment of symptoms, diseases, or conditions that have already developed and are (clinically) manifest, and preventive or preventive measures in which the purpose of treatment is to prevent, reduce, or lessen the likelihood of unwanted distress occurring, for example, by preventing the onset, progression, and advancement of symptoms, (clinical) conditions, or diseases associated with hair loss. Accordingly, further aspects of the present invention relate to an effective amount of estetrol component used for the prevention of hair loss in a subject. Beneficial or desirable clinical outcomes may include, without limitation, relief of one or more symptoms, improvement of one or more biological markers, reduction of the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or relief of the disease state, etc.

[0175] In the context of this invention, "prevention" or "preventing" refers to avoiding the manifestation of a condition or disease in the subject, i.e., establishing preventive or deterrent measures. Preventive treatment refers to treatment aimed at preventing the subject's body or its components from exhibiting (or worsening) symptoms of undesirable physiological changes.

[0176] As used herein, the terms “therapy” or “therapy” refer to treatment aimed at restoring the body or part of the body of a subject from an undesirable physiological condition, disease, or disorder caused by aging to a desirable condition, such as a less severe condition (e.g., improvement), or even to a normal state of health (e.g., restoration of the subject’s health, physical integrity, and physical health), maintaining the undesirable physiological condition (i.e., not worsening it) (e.g., stabilizing it), or slowing the progression to a more severe or worsened condition compared to the undesirable physiological change or disorder. Measurable relief includes any statistically significant reduction in a measurable marker or symptom. As used herein, statistically significant means a p-value less than 0.05, which is a cutoff score generally accepted in statistical analysis as understood by those skilled in the art. “Therapy” encompasses both curative treatments and treatments that reduce symptoms and / or slow their progression and / or stabilize a condition or disease.

[0177] Those skilled in the art will recognize that in order to achieve effective therapeutic treatment, it is necessary to administer a therapeutically effective dose to the subject. Therefore, in the context of this disclosure, “effective dose” means the amount required to obtain a physiological effect. The physiological effect may be obtained by a single dose or multiple doses. “Therapeutably effective dose” refers to the amount of estetrol component that, when administered, produces a clinically favorable response with respect to the treatment of a subject suffering from hair loss. Similarly, “effective dose for prevention” refers to the amount of estetrol component that inhibits or delays the onset of (clinically) manifestation of hair loss. Those skilled in the art will recognize that terms such as “quantity,” “amount,” and “level” are synonymous and have well-defined meanings in the art, and will understand that these may refer to an absolute quantification of the estetrol component considered to be an effective dose for the uses described herein, or a relative quantification of the estetrol component, such as the concentration of the estetrol component relative to the subject’s body weight. Appropriate values ​​or ranges of values ​​can be obtained from one subject or a group of subjects (i.e., at least two subjects).

[0178] While it is emphasized that any values ​​and ranges of estetrol components disclosed herein are suitable for different medical indications or purposes, those skilled in the art will recognize that certain individuals may experience further improved efficacy from treatment with estetrol components by further optimizing the optimal dosage of the component by considering a wide range of parameters, including, but not limited to, the nature and severity of the condition or disease to be treated, the sex of the subject, the age of the subject, weight, other medical indications, nutritional status, method of administration, metabolic status, interference or influence or efficacy of other pharmaceutically active ingredients, etc. Furthermore, individuals may have specific and unique responsiveness to the estetrol component used.

[0179] Administration The subject composition of the present invention is particularly configured for topical administration, i.e., local application to the scalp of the subject.

[0180] As an alternative to the medical treatment methods or medical uses as defined herein, the present invention also envisions a cosmetic product or method for preventing or reducing hair loss, comprising the step of topically administering a composition containing the estetrol component. In other words, the present invention also envisions a cosmetic use of a topical composition containing the estetrol component for preventing or reducing hair loss. Those skilled in the art will understand that “cosmetic use” means “non-medical, non-therapeutic use,” and therefore use not intended to improve, prevent or treat a clinical pathology.

[0181] The therapeutic or cosmetic treatment of the present invention typically involves the continuous administration of the estetrol component for a period of at least 10 days, preferably at least 20 days.

[0182] As used herein, the terms “continuous” and “sequentially” mean administering the components at relatively regular intervals without significant (therapeutic) interruptions. Naturally, minor interruptions that do not affect the overall effectiveness of the method of the present invention may occur, and in fact, such deviations are also included in the present invention. In preferred embodiments, more mathematically, an administration regimen is considered continuous if the longest interval between two consecutive administrations is 3.5 times or less the average interval. More preferably, the longest interval is 2.5 times or less the average interval, most preferably 1.5 times or less.

[0183] Therefore, those skilled in the art will understand that the estetrol component can be administered together with any other cosmetic or pharmaceutical active agent known in the art for reducing or preventing hair loss.

[0184] In the therapeutic or cosmetic method according to the present invention, the estetrol component is administered continuously and appropriately for a period of at least 10 days.

[0185] The present invention can be appropriately simplified to be implemented in various forms of administration known to those skilled in the art. Among these methods is one that uses a single-phase preparation containing a dosage unit containing a fixed amount of estolol component.

[0186] In a particularly preferred embodiment of the present invention, the topical composition according to the present invention is designed for daily administration; that is, the topical composition according to the present invention represents a topical composition that is applied topically, for example, to the scalp of a subject, once every 24 hours.

[0187] Topical compositions may be applied by pouring, dropping, spraying, rubbing, or by any other suitable means. Optionally, the composition is administered to the scalp once. Alternatively, the composition is administered to the scalp at multiple points in time, preferably at substantially regularly spaced intervals. In certain embodiments, the composition is administered to the scalp daily. Optionally, the topical composition is administered and remains there (i.e., not removed from the scalp) for a sustained period corresponding to at least 30 minutes, preferably at least 1 hour, more preferably at least 2 hours, more preferably at least 4 hours, more preferably at least 8 hours, more preferably at least 1 day, more preferably at least 1 week, more preferably at least 1 month. In such embodiments, it is understood that the scalp is continuously exposed to the composition over the indicated time periods.

[0188] In certain embodiments, the estetrol component is included in an immediate-release topical composition. In alternative embodiments, the estetrol component is included in a delayed-release, sustained-release, or controlled-release topical composition. The terms “immediate-release,” “delayed-release,” and “sustained-release” or “controlled-release” are obvious to those skilled in the art and describe the release profile of the topical composition. In immediate-release, the topical composition is released from the dose unit to the subject or patient's body almost immediately. In delayed-release, the composition is delivered into the body with a delay after administration. In sustained-release or controlled-release dose units, the composition is designed to release the pharmaceutically active ingredient(s) at a predetermined rate to maintain a constant drug concentration over a specific period. The release profile of a dose unit can be evaluated as described in major pharmacopoeias. For example, according to the European Medicines Agency, immediate-release is defined as the dissolution of at least 75% of the active substance within 45 minutes (European Pharmacopeia (Ph. Eur.) 11). th(edition). However, it is a minor point to those skilled in the art that the appropriate tests and timeframes may vary depending on the therapeutic range, solubility, and permeability factors of the active pharmaceutical ingredient.

[0189] The topical compositions envisioned by the present invention may contain further skin-active ingredients capable of providing skincare benefits. These skincare benefits are not limited to, but may include benefits related to the cosmetic appearance of the skin. Further skin-active ingredients may provide immediate, short-term (i.e., acute) benefits, and / or long-term, sustained (i.e., chronic) benefits.

[0190] The topical compositions envisioned by the present invention may contain further hair loss prevention or hair care promoting active ingredients capable of providing hair care benefits known in the art. These hair care benefits include, but are not limited to, benefits related to the aesthetic appearance of hair and its growth. Further hair care active ingredients may provide immediate, short-term (i.e., acute) benefits and / or long-term, sustained (i.e., chronic) benefits.

[0191] The topical compositions described herein are also intended for use as supportive treatment for subjects undergoing hair transplantation surgery. In such embodiments, the topical compositions may be applied after, before, or both of the hair transplantation procedures. The topical compositions may be applied to the transplantation area of ​​the scalp (donor and / or recipient area) or to the entire scalp of the subject.

[0192] Although the present invention has been described in detail along with its embodiments, it will be apparent from the above description that many alternative forms, modifications, and variations will be obvious to those skilled in the art. Therefore, it is intended to encompass all such alternative forms, modifications, and variations that fall within the spirit and broad scope of the appended claims. The aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting examples.

[0193] The following specific experimental examples are provided to support the claimed invention, but should not be considered to limit the scope of the invention. [Examples]

[0194] Example 1. Manufacturing Process Aqueous gel formulation Batch AG23, AG24, AG25, AG26 i. Weighed the water (first addition) and put it into Duran (container 1). ii. The sample from step (i) was placed on a hot plate stirrer and a vortex was formed at 800 rpm. iii. The polymer (Carbopol®) was weighed using a weighing boat and dispersed in the contents of step (ii), maintaining a vortex. The sample was left to stand with stirring for at least 1 hour to disperse the polymer. iv. The weight of the polymer dispersion in step (iii) was recorded. v. Samples from step (iv) are processed under standard conditions (121°C ± 2°C, 2 × 10⁻⁶). 5 It was autoclaved at Pa for 15 minutes. vi. Once the autoclaved sample had cooled, the sample from step (v) was weighed again, and additional water was added to compensate for the loss due to evaporation. vii. The solvents (PEG400, Transcutol® P (diethylene glycol monoethyl ether)) were weighed and placed in a separate container. viii. Estrol monohydrate was weighed and added to the contents of step (vii), and the mixture was stirred at 500 rpm for 2.5 hours (in a hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug. ix. In a biosafety laminar flow hood, the contents of process (viii) were filtered through multiple 0.2 μm sterile Spartan (regenerated cellulose) filters and placed into multiple 20 mL autoclaved vials. The reason for dividing the contents into multiple vials was to avoid contamination of the bulk in case of filter failure. The contents of step (ix) were combined, weighed, and placed into a Duran container that had been autoclaved beforehand. xi. The contents of step (x) were poured into the contents of step (vi) and mixed using an overhead stirrer and a spatula; the mixing speed and time were recorded in a laboratory notebook. xii. The container from step (x) was rinsed with water (about 4% of the total amount) and poured into Container 1. xiii. The formulation was allowed to rest for at least overnight. xiv. The pH of the sample was adjusted to the target pH of 6 - 6.5 using a sodium hydroxide solution and the weight was adjusted with water. xv. It should be noted that manufacturing steps (ix) to (xiv) were carried out under aseptic conditions in a laminar flow hood.

[0195] Batch AG30, AG22 (Carbopol (trademark) 980) i. Water (first addition) was weighed and placed into a 150 mL Duran (Container 1). ii. The sample from step (i) was placed on a hot plate stirrer and vortexed at 800 rpm. iii. The polymer (Carbopol (trademark) 980) was weighed using a weighing boat and dispersed into the contents of step (ii), maintaining the vortex. The sample was left to stir for at least 1 hour to disperse the polymer. iv. The weight of the polymer dispersion in step (iii) was recorded. v. PEG400 and glycerol were weighed and added to the contents of step (iv). vi. The sample from step (v) was autoclaved under standard conditions (121 °C ± 2 °C, 2 × 10 5 Pa, 15 minutes). vii. Once the autoclaved sample had cooled, the sample from step (v) was weighed again and additional water was added to compensate for the weight loss due to evaporation. viii. The solvents (propylene glycol (PG), Transcutol (trademark) P) were weighed and placed into a 100 mL Duran. ix. Weigh estetrol monohydrate and add it to the content of step (viii), and stir at 500 rpm (using a hot plate magnetic stirrer at the laboratory room temperature). x. In a biosafety laminar flow hood, filter the content of step (ix) through a plurality of Spartan 0.2 μm sterilizing filters to avoid bulk contamination in case of filter breakage, and place it in a plurality of 20 mL autoclaved vials. xi. Combine the content of step (x), weigh it, and place it in a 100 mL Duran that has been autoclaved in advance. xii. Pour 18% sodium hydroxide (previously filtered through a 0.2 μm sterilizing Spartan filter) into the content of step (xi) and mix. xiii. Quickly pour the content of step (xii) into the content of step (vii), and mix using a spatula. xiv. Rinse the container from step (x) with water (about 4% of the total amount) and place it in container Ⅰ. xv. Let the thickened sample rest for at least overnight. xvi. Measure the pH of the sample, and if necessary, add the remaining sterilized water together with a sodium hydroxide solution (also previously sterilized through a 0.2 μm Spartan filter) to adjust the pH to 7 - 7.5.

[0196] It should be noted that manufacturing steps (x) to manufacturing step (xvi) were carried out under aseptic conditions in a laminar flow hood.

[0197] Batch AG27 (Poloxamer) i. Weigh water (first addition) and place it in a 150 mL Duran, and cool it in a refrigerator maintained at 2℃ - 8℃ for 20 minutes. ii. Place the sample from step (i) on a hot plate stirrer, weigh poloxamer (P407, followed by P188), and add it above the vortex at 800 rpm. iii. Stir the sample from step (ii) at 200 rpm for 6 hours using an overhead stirrer in a water bath maintained at 2℃ - 8℃ until the poloxamer is dissolved. iv. PEG400, benzyl alcohol (BA), and PG were weighed and placed together in a separate container. v. Estol monohydrate was weighed and added to the contents of step (iv), and left overnight while being stirred at 500 rpm in a hot plate stirrer. vi. Once the drug was dissolved, the required amount of the solution from step (v) was weighed and placed in the first container containing the poloxamer solution. The sample was left overnight in a magnetic stirrer at 300 rpm. vii. The pH of the sample from step (vi) was adjusted to 7-7.5.

[0198] Batch AG28 (Hydroxyethylcellulose (HEC)), AG29 (Carboxymethylcellulose (CMC)) i. The solvent systems for the formulations were prepared by weighing all the solvents, except for the water / buffer solution in the active system. ii. The drug was added to the solvent system for the active formulation and stirred overnight at 500 rpm. iii. After the drug was dissolved, water / pH 7.0 buffer was added to the solvent system while stirring Duran. iv. Weigh out an appropriate amount of premix and place it in another 150 mL Duran. The polymer was dispersed while the mixture was stirred at 800 rpm to form a vortex, and the formulation was left to stand overnight while being stirred at 500 rpm. For vi.AG28, the pH was adjusted to pH 7-7.5. For AG29, since it contains phosphate buffer pH 7.0, there was no need to adjust the pH.

[0199] Batch AG15 (Poloxamer) i. Weigh the water (first addition) and place it in a 150 mL Duran container, then cool it in a refrigerator maintained at 2°C to 8°C for 20 minutes. ii. The sample from step (i) was placed on a hot plate stirrer, and poloxamer (P407, followed by P188) was weighed and added to the vortex at 800 rpm. iii. The samples from step (ii) were stirred at 200 rpm using an overhead stirrer for 6 hours in a water bath maintained at 2°C to 8°C until the poloxamer dissolved. iv. PEG400, BA, and PG were weighed and placed together in a separate container. v. Estolol was weighed and added to the contents of step (iv), and left overnight while being stirred at 500 rpm in a hot plate stirrer. This step was omitted for the production of the placebo. vi. Once the drug was dissolved, the required amount of the solution from step (v) was weighed and placed in the first container containing the poloxamer solution. The sample was left overnight in a magnetic stirrer at 300 rpm. vii. The pH of the sample from step (vi) was adjusted to 7-7.5.

[0200] Batch AG17 i. Weigh the water (first addition) and put it into a 150 mL Duran container (container 1). ii. The sample from step (i) was placed on a hot plate stirrer and a vortex was formed at 800 rpm. iii. The polymer (Carbopol®) was weighed using a weighing boat and dispersed in the contents of step (ii), maintaining a vortex. The sample was left to stand with stirring for at least 1 hour to disperse the polymer. iv. The weight of the polymer dispersion in step (iii) was recorded. v. Samples from step (iv) are processed under standard conditions (121°C ± 2°C, 2 × 10⁻⁶). 5 It was autoclaved at Pa for 15 minutes. vi. Once the autoclaved sample had cooled, the sample from step (v) was weighed again, and additional water was added to compensate for the loss due to evaporation. vii. Benzyl alcohol, PEG400, and PG were weighed and placed in another 100 mL of Duran. viii. Weigh estetrol monohydrate and add it to the content of step (vii). Stir at 500 rpm overnight (using a hot plate magnetic stirrer at the laboratory room temperature) to dissolve the drug. This step was omitted for the placebo formulation. ix. Pour 18% sodium hydroxide (1.38 g) into the content of step (vi) and mix. x. Quickly pour the content of step (ix) into the content of step (vi) and mix using a spatula. xi. Rinse the container from step (ix) with water (4.5 g) and pour it into container 1. xii. Let the thickened sample rest for at least overnight. xiii. Measure the pH of the sample and gradually add the remaining water while continuing to measure the pH.

[0201] Batch AG18 (HEC), AG19 (CMC) i. The solvent systems for the placebo and active formulations were prepared by weighing all the solvents, but for the active system, water / buffer was excluded. ii. Add the drug to the solvent system for the active formulation and stir at 500 rpm overnight. iii. After the drug was dissolved, while stirring the Duran, add water / pH 7.0 buffer to the solvent system. iv. Weigh an appropriate amount of premix and put it into another 150 mL Duran. While stirring at 800 rpm to form a vortex, disperse the polymer and let the formulation stand overnight while stirring at 500 rpm. v. For AG18, adjust the pH to 7 - 7.5. For AG19, since it contained phosphate buffer pH 7.0, there was no need to adjust the pH.

[0202] Batch AG20, AG23 i. Followed steps (i) - (vi) of AG17. ii. Weigh the solvents (PEG400, Transcutol™ P) and put them into another container (100 mL). iii. Estrol monohydrate was weighed and added to the contents of step (ii), and the mixture was stirred at 500 rpm for 2.5 hours (in a hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug. This step was omitted for the placebo formulation. iv. In a biosafety laminar flow hood, the contents of step (iii) were filtered through multiple 0.2 μm sterile Spartan (regenerated cellulose) filters and placed into multiple 20 mL autoclaved vials. The reason for dividing the sample into multiple vials was to avoid contamination of the bulk in case of filter failure. It should be noted that the sample was initially filtered through a Nalgene bottle system filter (PES) using a vacuum pump, but the filter failed, and therefore a Spartan syringe filter was used. v. The contents of step (iv) were combined, weighed, and placed into a 100 mL Duran container that had been autoclaved beforehand. vi. The contents of step (v) were poured into the contents of step (i) and mixed using a spatula. vii. Rinse the container from step (v) with water (4.5g) and pour it into container 1. viii. Since the formulation did not thicken, it was left to rest for at least one night. ix. The pH of the sample was adjusted to 6-6.5 with a sodium hydroxide solution, and the weight was adjusted with water. It should be noted that the process using 18% sodium hydroxide was initially performed, but Carbopol (trademark) either aggregated or could not be hydrated. Furthermore, although the original target pH was 7-7.5, polymer precipitation was observed above pH 6.5, so to avoid this problem, the sample was repeatedly adjusted and left to stand at pH 6-6.5.

[0203] It should be noted that manufacturing steps (iv) to (ix) were carried out under sterile conditions within a laminar flow hood.

[0204] Batch AG21, AG22 i. Followed steps (i) to (vi) of AG17. ii. The solvents (PEG400, PG, Trans P) were weighed and added to 100 ml of Duran. iii. Estol monohydrate was weighed and added to the contents of step (i), and stirred at 500 rpm (in a hot plate magnetic stirrer at laboratory room temperature). For AG22, the drug was dissolved within 2.5 hours of stirring, and for AG21, it was dissolved overnight. This step was omitted for the placebo formulation. iv. In a biosafety laminar flow hood, the contents of process (viii) were filtered through multiple Spartan 0.2 μm sterile filters to avoid bulk contamination in case of filter failure, and then placed into multiple 20 mL autoclaved vials. The contents of step (ix) were combined, weighed, and placed into a 100 mL Duran container that had been pre-autoclaved. vi. 18% sodium hydroxide (1.38 g) (pre-filtered through a 0.2 μm sterile Spartan filter) was poured into the contents of step (x) and mixed. vii. The contents of step (xi) were quickly poured into the contents of step (vi) and mixed using a spatula. viii. Rinse the container from step (x) with water (4.5g) and place it in container 1. ix. The sample thickened and was left to rest for at least one night. x. The pH of the sample was measured, and if necessary, the remaining sterile water was added along with a sodium hydroxide solution (also sterilized beforehand by passing it through a 0.2 μm Spartan filter) to adjust the pH to 7-7.5.

[0205] It should be noted that manufacturing steps (ix) through (xv) were carried out under sterile conditions within a laminar flow hood.

[0206] TIFF2026520082000002.tif255170

[0207] cream Batch CR01, CR05, CR12 i. Solvent systems for the placebo and active ingredients were prepared by weighing all solvents, except for the water / buffer solution in the active ingredient system. ii. The drug was added to the solvent system for the active cream and stirred overnight at 500 rpm. iii. After the drug was completely dissolved, water / pH 7.0 buffer was added to the solvent system while stirring the container (Duran). iv. Next, the oil phase was prepared. For CR01, the oil phase was placed in a 75°C water bath and left until it melted. For v.CR05 and CR12, the oil phase was placed in a drying oven and left at 160°C for 2 hours (to simulate the sterilization process). Once the oil phase cooled to room temperature and re-solidified, it was placed in a water bath (75°C) and left until the oil phase melted again. vi. Before processing the active cream and placebo cream, the required amount of solvent system premix was weighed and placed in a separate Duran container. vii. Duran with the premix was placed in a water bath with a homogenizer head and left for 5 minutes. viii. For CR01 and CR12, the cream formulations were processed using an Ultra-turrax equipped with a 25G dispersion head at 10,000 rpm for 2 minutes. For ix.CR05, instead, the cream was homogenized at 5000 rpm for 5 minutes to avoid overflow during homogenization. x. After processing, the cream was manually stirred with a metal spatula until it reached room temperature. xi. They were left to mature overnight. xii. The pH of the formulation was adjusted to pH 7-7.5.

[0208] Batch CR10, CR13, CR14, CR15, CR16 i. Solvent systems for the placebo and active ingredients were prepared by weighing all solvents, except for the water / buffer solution in the active ingredient system. ii. The drug was added to the solvent system for the active cream and stirred overnight at 500 rpm. iii. After the drug was completely dissolved, water / pH 7.0 buffer was added to the solvent system while stirring Duran. The oil phases of CR13, CR14, and CR15 were placed in a drying oven and left at 160°C for 2 hours (sterilization), and then left at room temperature until they re-solidified. v. The aqueous phase was sterilized by filtration using a 0.2 μm sterile PES syringe filter. vi. The required amount of aqueous phase was weighed and placed into a 250 mL Duran container that had been pre-sterilized by autoclaving. vii. The oil phase was placed in a 75°C water bath and left until it melted. viii. The aqueous phase was placed in a water bath along with the homogenizer head and allowed to equilibrate for 5 minutes. ix. For each cream formulation, the oil phase was added to the respective aqueous phase and homogenized for 2 minutes at 10,000 rpm using an Ultra-turrax equipped with a 25G dispersion head. x. After processing, the cream was manually stirred with a metal spatula until it reached room temperature. xi. They were left to mature overnight. xii. The pH of the formulation was adjusted to pH 7-7.5.

[0209] It should be noted that manufacturing steps (v) to (xii) were carried out under sterile conditions within a laminar flow hood, and that sterilized containers were opened only within the sterile laminar flow hood.

[0210] TIFF2026520082000003.tif247170

[0211] Example 2. In vitro release experiment 1 Following method development and small-scale preliminary in vitro release (IVRT) experiments, large-scale IVRT experiments were conducted using the 10 test formulations described in Example 1. The experimental conditions used are shown in Table 3.

[0212] TIFF2026520082000004.tif60170

[0213] Large-scale in vitro release experiments were carried out using the experimental conditions developed during method development and feasibility experiments. The results of all the tested gel formulations and cream formulations are shown in Figure 1, and those presented in a table are shown in Table 4.

[0214] Aqueous gel formulation The release rates of the aqueous gel formulations are shown in Figure 2 and Table 4. The highest release rates were brought about for the AG18 formulation, AG19 formulation, and AG23 formulation (161 μg / cm 2 / √hour ~ 221 μg / cm 2 / √hour), followed by AG21 (51 μg / cm 2 / √hour), AG22 (sterilized), AG22 (autoclaved), and the release rate of AG15 was the lowest (3 μg / cm 2 / √hour ~ 10 μg / cm 2 / √hour). As a general trend, the observed release rates correlated with the concentration of the API, and the formulations with the highest release rates (AG18, AG19, and AG23) also contained the highest concentration of estetrol monohydrate (0.50% by weight).

[0215] For the aqueous gel formulations with the lowest release rates (AG21, AG22 (sterilized), AG22 (autoclaved), and AG15), it was found that the administered API was rapidly released in large amounts into the receptor solution (more than about 50% release at t = 0.5 hours). For many of the formulations, as a result of the large release of the API, linear steady-state drug release was not achieved (r2 < 0.9). Therefore, no conclusions and statistical comparisons were made when using aqueous gel formulations.

[0216] Cream formulation The release rates of the cream formulations are shown in Figure 3 and complemented by Table 4. The highest release rate was brought about for CR16 (0.50% by weight), followed by CR14 (0.35% by weight), and finally CR01 (0.24% by weight), and the average release rates were from 32 μg / cm 2 / √hour to 53 μg / cm 2The release rate lasted up to / √ hours. Following the same trend as the aqueous gel formulation, the release rate correlated with the concentration of API present in the formulation, and the highest release rate was observed for the formulation with the highest drug load (CR16, containing 0.50% API, with a release rate of 52.20 μg / cm³). 2 (This was / √ hours). Average drug release of cream formulation (μg / cm³) 2 A one-way statistical analysis of the release rate (√time) was performed using the Tukey-Kramer method (n=6). No statistically significant difference was found between the release rates of CR14 and CR16 (p>0.05), but the release rate of CR01 was observed to be significantly lower (p<0.05).

[0217] TIFF2026520082000005.tif85170

[0218] Example 3. In vitro release experiment 2 Following the results of the first in vitro release experiment (Example 2), a second IVRT experiment was conducted using four formulations and the experimental parameters outlined in Table 5.

[0219] TIFF2026520082000006.tif79170

[0220] A second large-scale in vitro release experiment was conducted using the experimental parameters detailed in Table 5 and four aqueous gel formulations. The results for all tested formulations are shown in Figures 4 and 5, and the results are summarized in Tables 6 and 7.

[0221] The objective of this part of the study was to determine the effect of thermodynamic activity on the release rate of estolol monohydrate from the formulation by comparing an unoptimized formulation (AG23, 0.5 wt% API) with optimized formulations of various strengths (AG24, 0.5 wt% API, AG25, 0.22 wt% API, and AG26, 0.06 wt% API).

[0222] When these four formulations were considered with respect to the percentage of release with respect to the applied dose of the API (Table 7), 87.95 ± 10.88% of the API was released from the AG26 formulation (0.06 wt% of the API) after 8 hours, which was significantly higher than the other three formulations (p, 0.05). The other three formulations were not statistically different from each other (p>0.05) (AG25 (0.22 wt% of the API) was 61.51 ± 4.91%, AG24 (0.5 wt% of the API) was 60.67 ± 4.86% and AG23 (0.5 wt% of the API) was 56.88 ± 5.90%).

[0223] When considering the percentage (slope) of the release rate with respect to the applied dose over time, a significant difference (p<0.05) was observed between the AG26 formulation and the other three formulations (AG23, AG24 and AG25). No statistically significant difference was seen between AG23, AG24 and AG25. The statistical analysis was performed by Tukey's Kramer test (n = 5 - 6).

[0224] The release rate shown by the AG23 formulation during this test was approximately 167 ± 13 μg / cm 2 / hour compared to 195 ± 20 μg / cm 2 / hour in the first IVRT large-scale experiment. In this test, the AG24 formulation had the highest release rate (approximately 182 ± 8 μg / cm 2 / hour), followed by AG23, AG25 (approximately 81 ± 5 μg / cm 2 / hour) and finally AG26 (approximately 34 ± 3 μg / cm 2The release rate of each formulation differed significantly from that of the following formulations (p<0.05), which in the case of AG23 and AG24, both containing 0.5 wt% estetrol monohydrate, may be due to problems encountered during AG23 administration, batch variability compared to a batch of AG23 used in a previous experiment (which had a release rate much closer to that observed for AG24 in this study), or simply differences between the AG23 and AG24 formulations. For the other two formulations, the differences in release rates were due to the lower API content of these formulations (0.5 wt% for AG23 and AG24, 0.22 wt% for AG25, and 0.06 wt% for AG26), as expected.

[0225] To evaluate the differences between the aqueous gel formulations reported above, a one-way statistical analysis was performed using Tukey-Kramer (n=5-6).

[0226] TIFF2026520082000007.tif70170

[0227] TIFF2026520082000008.tif60170

[0228] Example 4. In vivo LPS treatment protocol (wound healing delay model) To evaluate the systemic effects of topical application of estrogen-containing compositions, we used a previously described in vivo LPS-induced delayed wound healing model (Crompton R, Williams H, Ansell D, Campbell L, Holden K, Cruickshank S, Hardman MJ. Oestrogen promotes healing in a bacterial LPS model of delayed cutaneous wound repair. Lab Invest. 2016 Apr;96(4):439-49).

[0229] Female wild-type (C57BL / 6J) 8-week-old mice were divided into 6 groups (6 animals per experimental group): Double placebo control (no LPS, placebo treatment), LPS control (LPS, placebo treatment), LPS and topical EstroGel (trademark) (estradiol gel, containing 0.06% 17β-estradiol hemihydrate in water-alcohol gel), LPS and topical AG24 (0.5% E4), LPS and topical AG25 (0.22% E4), LPS and topical AG26 (0.06% E4).

[0230] On the day before wound creation (-1 day), all animals were weighed and anesthetized with oxygen and isoflurane (2%-2.5% isoflurane, flow rate 1.25L-2L depending on clinical signs). The animals were shaved and prepared on their dorsal regions. The wound locations (two wounds) were marked on the back of each animal. Except for the double placebo group which received DPBS only, the animals received an initial subcutaneous injection of 2 μg of K. pneumoniae-derived LPS diluted in DPBS (1 μg per wound; Sigma Aldrich, UK: L4268) at the wound site. Subsequently, a thin layer of EstroGel®, AG24, AG25, AG26, or placebo was applied to the back of each animal in a total of 60 μl (30 μl / wound). The animals were allowed to recover in a heated cabinet and then individually housed in new cages containing AlphaPad, RO water, feed, mash feed, and a house. On day 0 (24 hours after the first anesthesia), the mice were anesthetized again and the skin on their backs was cleaned with a chlorhexidine wipe. Two 6 mm excision wounds were created on the back. Except for the double placebo mice injected with DPBS, 2 μg of LPS (1 μg per wound) was subcutaneously injected into the wound sites. Then, EstroGel®, AG24, AG25, AG26, or AG23 as a placebo was carefully applied as a thin layer to the surface of the wounds, taking care not to damage the "LPS bleb" (60 μl in total; 30 μl / wound). Buprenorphine (0.1 mg / kg), an analgesic, was administered by subcutaneous injection into the neck postoperatively, and each animal was imaged. The mice were allowed to recover in a heated cabinet and returned to individual housing. Postoperative observations were performed.

[0231] On days 1, 2, 3, and 4, the treated group was again subjected to a thin layer application of topical EstroGel®, AG24, AG25, AG26, or placebo to the surface of the wound while being observed (60 μl in total; 30 μl per wound).

[0232] On the fifth day, the mice were humanely euthanized by gradually increasing CO2 concentrations and cervical vertebral dislocation. The uterus was removed and weighed.

[0233] Figure 6 clearly shows that topical application of EstroGel® promoted an increase in uterine weight and hypertrophy. Topical application of E4 affected uterine weight in a dose-dependent manner; AG26 showed no effect, while AG24 induced an increase in uterine weight. The data confirm that topical application of a low-concentration E4 formulation (AG26) to an open wound for 6 days did not result in systemic side effects, such as an increase in uterine weight, in contrast to EstroGel® (E2) and high-level E4 formulations (AG24, AG25).

[0234] Based on the data shown in Figure 6, it was concluded that topically applied AG26 formulations do not have systemic effects such as increased uterine weight. This indicates a preferred dosage range when systemic effects should be completely avoided. However, in some cases, higher dosages may be considered based on the current favorable risk / benefit ratio for the patient.

[0235] Example 5. In vivo LPS treatment protocol (delayed wound healing model): Comparison of variations in different formulations and treatment durations. The in vivo LPS treatment protocol (wound healing delay model) presented in Example 4 was repeated using the following animal group: Double placebo group (untreated control), LPS control (LPS, placebo treatment), LPS and topical EstroGel® (estradiol gel containing 0.06% 17β-estradiol hemihydrate in water-alcohol gel), repeated administration for 4 days. LPS and topical AG26 (0.06% E4), single dose. LPS and topical AG26 (0.06% E4), repeated administration for 4 days. LPS and topical AG28 (0.06% E4), single dose. LPS and topical AG28 (0.06% E4), repeated administration for 4 days.

[0236] Topical EstroGel®, AG26, AG28, or the corresponding placebo was applied as a thin layer to the surface of the wound while observing the patient, either on day 0 (single dose) or on days -1, 0, 1, and 2 (repeated doses) (60 μl total; 30 μl / wound).

[0237] On day 3, the mice were humanely euthanized by gradually increasing concentrations of CO2 and cervical dislocation. The uterus was removed and weighed as described in Example 4. In contrast to EstroGel®, topical application of AG26 and AG28 did not affect the weight of the uterus (Figure 7).

[0238] Example 6: Evidence for the antioxidant properties of estetrol The antioxidant activity of E4 was evaluated using the Skin-Biosense™ sensor, a technology developed by Qima Life Sciences (Gencay, France). This electrochemical sensor measures electron transfer between two conductive elements: the sensor and an H2O2 solution. The H2O2 solution is used as a marker to determine detoxification power: since H2O2 is one of the most oxidative non-radical species, any molecule capable of reducing its concentration can be considered an antioxidant. In the presence of H2O2, all of the antioxidant species in the sample spontaneously react with H2O2, reducing their quantity. The peak area measured by the sensor is directly proportional to the H2O2 concentration, so the detoxification activity of a compound is evaluated by the modulation of the peak area by the compound.

[0239] 5 mM H2O2 in an ethanol solution containing an electrolyte was mixed with E4 at several concentrations (0.16 mM, 0.5 mM, 1.6 mM, 5 mM, 16 mM, 50 mM) in an ethanol solution containing an electrolyte, and the H2O2 oxidation peak area was measured. Antioxidant activity was calculated by comparing the peak area of ​​the H2O2 solution with the peak area of ​​the H2O2 solution mixed with the E4 solution.

[0240] A potent antioxidant activity of E4 was observed, following a positive dose-response. The average capacity was 77.5% when the E4 / H2O2 molar ratio reached 1, which was similar to that of the reference compound (EUK-134). 100% antioxidant activity was observed at the highest E4 concentration (Figure 8).

[0241] Example 7: Culture of hair follicle organs from a healthy female donor (ex vivo model) Twelve to forty-four full-length frontotemporal hair follicles (HFs) were obtained from four healthy human female donors over 50 years of age. The donors were Caucasian women aged 59, 62, 59, and 67 years, respectively (Donor 1: 12 temporal growth HFs; Donor 2: 29; Donor 3: 44; Donor 4: 32). As described in the literature in the art (Edelkamp et al., Methods Mol Biol, 2020, 2154:105-119 and Langan et al., Exp Dermatol, 2015, Dec;24(12):903-1), the HFs were micro-dissected from the skin, placed in appropriate culture media, and allowed a 24-hour resting period to allow for recovery from the trauma of micro-dissection. The test substance (vehicle (0.3% EtOH) or E4 at concentrations of 30 μM, 3 μM, or 300 nM) was added to the culture medium at the end of the 24-hour period. The total culture time ranged from 5 to 9 days, depending on the HF stage of the vehicle group. The experimental design for the 6-day HF organ culture experiment is shown in Figure 9.

[0242] This model allows us to evaluate whether treatment with E4 can prolong the growth phase and / or delay the onset of the regression phase in healthy women. Clinically, hair loss is associated with a decrease in HF in the growth phase or a shortening of the growth phase duration. Therefore, promoting the growth phase may limit hair loss.

[0243] The following parameters were investigated: Release of lactate dehydrogenase (LDH) into the culture medium LDH is released from the cytoplasm of damaged cells into the culture medium. Therefore, the lipotoxicity of a drug can be estimated based on the activity of LDH present in the culture medium using a colorimetric quantitative assay (Cytotoxicity Detection Kit [LDH], Roche) as described in the literature (Gherardini et al Int J Cosmet Sci, 2019, Apr;41(2):164-182). Measurements were performed on the supernatant collected on the day corresponding to each culture and normalized to the blank.

[0244] Regarding maximal release, HF was treated with 1% Triton X100 in vehicle medium at 37°C for 30 minutes to induce maximum release of LDH into the culture medium.

[0245] Hair follicle elongation in ex vivo (correlated with hair shaft formation) To determine the length of the hair follicle (HF), each HF was measured from the end of the hair bulb connective tissue sheath to the distal end of the outer root sheath (ORS) using a 50x digital optical microscope (VHX 900, Keyence Corporation, Osaka, Japan) and associated software (Edelkamp et al., Methods Mol Biol, 2020, 2154:105-119, and Langan et al., Exp Dermatol, 2015, Dec;24(12):903-1). Measurements were taken at baseline, after a 24-hour rest period, and thereafter every 24-48 hours throughout the entire duration of the experiment. Elongation is expressed as growth in millimeters and / or as percentage elongation from baseline measurement.

[0246] Microscopic hair cycle stage classification and hair cycle scoring Hair cycle stage classification and hair cycle score calculation are indicators of hair cycle progression and transition from the growth phase to the regression phase (Langan et al., Exp Dermatol, 2015). Hair cycle stage classification and hair cycle scoring were performed at the end of culture. The hair cycle stage for each HF was determined based on several established macroscopic and microscopic parameters (Langan et al., Exp Dermatol, 2015). Microscopic hair cycle stage classification was determined using Ki-67 / TUNEL immunohistochemistry and Masson-Fontana histochemistry. Hair cycle stage classification was analyzed by calculating the percentage of HF in each hair cycle stage. Hair cycle scores were determined by assigning arbitrary scores to each hair cycle stage as follows: growth phase: 100, early regression phase: 200, mid-regression phase: 300, late regression phase: 400, dystrophic: 500.

[0247] TIFF2026520082000009.tif180170

[0248] Figure 10 shows representative images of dystrophic hair follicles, as well as those in the growth and early regression phases.

[0249] Proliferation and apoptosis of hair matrix keratinocytes Germ matrix keratinocytes proliferate primarily during the growth phase. Their proliferation dramatically decreases during the transition between the growth and regression phases and disappears during the late regression phase (Langan et al., Exp Dermatol, 2015). Apoptosis of matrix keratinocytes is used as an additional indicator of the growth / regression phase transition.

[0250] Proliferation was evaluated by quantifying the percentage of hair matrix keratinocytes (germ hair matrix keratinocytes, below Ober's line) that were positive for the proliferation marker Ki67 in clearly identified regions. Apoptosis was evaluated using TUNEL (TdT-mediated dUTP-biotin-nick-terminal labeling) immunofluorescence for the same keratinocyte population (below Ober's line) and precortical hair matrix keratinocytes (above Ober's line) (Langan et al., Exp Dermatol 2015) (Figure 11).

[0251] Alkaline phosphatase activity - Dermal papilla (DP) induction Alkaline phosphatase is an indicator of DP induction (Yang, J Dermatol Sci, 2010, Jan; 57(1):2-11). The in situ activity of alkaline phosphatase was measured in DP.

[0252] Versican expression-DP induction Versican is a large chondroitin sulfate proteoglycan molecule that is expressed in DP during the growth phase of HF and decreases during the regression phase of HF, with its expression correlating with the inducible properties of hair (Yang, J Dermatol Sci 2010). The intensity of versican was analyzed in DP.

[0253] CD34-expressing stem cell offspring CD34 is a marker expressed by the offspring of HF bulge stem cells. It is expressed in the outermost layer of the outer root sheath (ORS) below the isthmus (the region from below the bulge to above the hair bulb) (Purba et al., BioEssays, 2014, May; 36(5):513-25). CD34 expression and CD34+ The percentage of cells was analyzed using ORS on hair follicles.

[0254] DP fibroblast migration Fibroblasts belonging to the DP migrate during the transition between the growth phase and the regression phase (Hawkshaw et al., Journ of Invest Dermatol, 2015, 135(8):2129-2132). At the start of the regression phase, DP fibroblasts migrate through the DP stalk to the dermal cup, and then migrate back to the DP during the induction of the growth phase. Therefore, quantifying the number of DP fibroblasts present in the DP stalk and DP (Kloepper et al., Exp Dermatol, 2010, Mar; 19(3):305-12) provides a direct indication of growth phase induction or regression phase acceleration.

[0255] K15-expressing stem cells K15 is a type I cytokeratin expressed in HF bulge stem cells and their offspring (Purba et al., BioEssays, 2014). It is expressed in the bulge, infra-bulge, and epidermal orthosteroidal receptacles (ORS). Bulge stem cells are identified using K15 expression, cell morphology, and anatomical location (below the sebaceous glands, in the region where the arrector pili muscle attaches to HF). K15 expression was analyzed in the bulge basal layer and epidermal ORS, and the proliferation of K15-positive cells was evaluated in combination with Ki67 immunofluorescence staining.

[0256] statistics Each dot represents a single hair follicle; mean ± SEM; GraphPad Prism 9; D'Agostino & Pearson omnibus normality test; for group comparisons (#p<0.05, ##p<0.01, ###p<0.001, ####p<0.0001), non-Gaussian distribution (Kruskal-Wallis test, Dunn's multiple comparison test - each group vs. vehicle), Gaussian distribution (one-way ANOVA, Dunett's or Holm-Sidak multiple comparison test - each group vs. vehicle); pairwise comparisons ( *p<0.05), non-Gaussian distribution (Mann-Whitney), Gaussian distribution (t-test); ns, no significance. If at least one group does not exhibit a Gaussian distribution in a given group comparison or pairwise comparison, statistics for non-Gaussian distributions were followed.

[0257] n=3-4 donors, each with individual HF. The data were first analyzed for Gaussian distribution using the D'Agostino & Pearson omnibus normality test, and outliers were identified and removed using the ROUT method (0=10 / 0). If the data followed a Gaussian distribution, a one-way ANOVA analysis was performed, followed by the Holm-Sidak multiple comparison test (vehicle-fixed and unpaired Student's t-test vs. vehicle). If the data was too small to determine whether it followed a Gaussian distribution, or if the data did not follow a Gaussian distribution, the Kruskal-Wallis test was performed, followed by Dunn's multiple comparison test (vehicle-fixed and Mann-Whitney test vs. vehicle). Note that the evaluation of "growing only" includes data from only n=3 patients. Donor 1 does not have any growing HFs in the vehicle group.

[0258] result None of the E4 concentrations tested induced HF cytotoxicity (LDH release into the culture medium) (Figure 12). E4 did not alter hair shaft formation (Figure 13). Microscopically, E4 at 3 μM (significant) and 300 nM and 30 μM (tendency) prolonged the growth phase (Figure 14). E4 at 3 μM (significant) and 300 nM and 30 μM (tendency) increased the proliferation of hair matrix keratinocytes without affecting apoptosis (Figure 15). All concentrations of E4 tested tended to increase versican expression and alkaline phosphatase activity in DP cells (Figure 16). Analysis of DP fibroblast migration revealed that all concentrations of E4 tested did not significantly affect cell density in DP (Figure 17), and all concentrations tended to decrease cell density. E4 at 300 nM and 3 μM significantly reduced the total number of cells in DP stems (Figure 18), but E4 did not significantly affect cell density in induced dermal cups (Figure 19). In the bulge basal layer, 3 μM E4 significantly reduced the percentage of K15-positive cells, while 30 μM (significant) and 3 μM and 300 nM (tendency) E4 increased their proliferation (Figure 20). In hair follicle-associated orthosteroids (ORS), E4 had no effect on the percentage of K15-positive cells, however, E4 at 300 nM (significant) and 3 μM (tendency) reduced their proliferation (Figure 21). E4 at 3 μM (significant) and 300 nM and 30 μM (tendency) increased the percentage of CD34-positive cells in the hair follicle-derived orthosteroid (ORS) (Figure 22).

[0259] Discussion and Conclusion The experiments reported here investigated the potential effects of the estrogen compound E4 on the hair cycle, hair matrix keratinocytes, DP induction and DP fibroblast migration, stem cell maintenance, and fertility in the context of promoting hair growth in healthy female subjects. The experiments confirmed that the tested concentrations of E4 did not induce HF cytotoxicity (release of LDH into the culture medium). Most importantly, data from four individual donors revealed that E4 tended to prolong the anagen phase (at 300 nM and 30 μM) and significantly (at 3 μM) ex vivo. The significant anagen phase prolongation effect of 3 μM E4 was further supported by a decrease in cell density in the DP stem, as DP fibroblasts accumulate in this particular segment during their migration from the DP during the catagen phase (Kloepper et al., Exp Dermatol. 2010). The growth phase extension effect was also supported by a significant increase in the proliferation of hair matrix keratinocytes after treatment with 3 μM E4.

[0260] The data suggested that the growth phase extension effect of E4 may have been mediated, at least partially, by DP fibroblast-inducible stimulation. Indeed, all concentrations of E4 tested appeared to favorably upmodulate alkaline phosphatase activity and versican expression in DP.

[0261] In the bulge, 3 μM E4 appeared to trend-increase the proliferation rate of pluripotent HF stem cells, while this significantly reduced the percentage of K15-positive cells. However, this treatment had a beneficial effect on the generation and / or activity of stem cell progeny in the hair follicle-associated orthosteroids (ORS). Specifically, 3 μM E4 trend-increased the number of proliferative K15-positive cells and significantly increased the percentage of CD34-positive cells, the latter further supporting the growth phase extension effect of E4 (Purba et al., 2014).

[0262] In summary, these experiments further encouraged the exploration of 3 μM E4 treatment for promoting hair growth in healthy women. E4 may have the ability to maintain the growth phase and prevent HF reduction. The above experiments can be repeated in Examples 8 to 10 for separate control groups (healthy men, women with female pattern baldness, and men with male pattern baldness).

[0263] Example 8: Hair follicle organ culture from a healthy male donor (ex vivo model) Example 7 was repeated with a healthy male donor, with only minor modifications. Briefly, 24 fully-length occipital hair follicles (HFs) were obtained from a healthy 47-year-old Caucasian male donor. After microdissection from the skin as described in the art (Edelkamp et al., Methods Mol Biol, 2020, 2154:105-119 and Langan et al., Exp Dermatol, 2015, Dec;24(12):903-1), the HFs were placed in appropriate culture media and allowed a 24-hour rest period to allow recovery from the trauma of microdissection. The test substance (either vehicle (0.03% EtOH), E2 at a concentration of 300 nM, or E4 at a concentration of 3 μM or 300 nM) was added to the medium at the end of the 24-hour period. The total culture time was 5 days, depending on the stage of the HFs in the vehicle group. The experimental design is shown in Figure 23. The same parameters as those described in Example 7 were investigated.

[0264] result Hair shaft formation was significantly reduced by E2 and showed a trend of reduction by E4 at 300 nM and 3 μM (Figure 24). Microscopically, E4 more significantly maintained HF in the growth phase at 3 μM, while E2 did not affect the hair cycle (Figure 25). The proliferation of hair matrix keratinocytes tended to increase with 3 μM E4 and E2 (Figure 26). Versican expression and alkaline phosphatase activity in the hair papilla appeared to decrease in a general trend with all treatments (Figures 27 and 28). The density and total cell number of dermal cups in growing HF cells were significantly reduced by 3 μM E4, but E2 had no effect (Figure 29). The density and total cell number of dermal papillae tended to decrease with 3 μM E4, and the area of ​​the dermal papillae increased with 300 nM E4 and 3 μM E4, but E2 had no effect (Figure 30). The density and total cell number of hair papillae were reduced by 3 μM E4, but E2 had no effect (Figure 31). The number of CD34+ cells in the epidermal root sheath of the hair bulb was reduced by 300 nM E4 and 3 μM E4, but E2 had no effect (Figure 32). K15 expression and cell number in the outer root sheath above the hair bulb tended to increase with 300 nM E4 and E2 (Figure 33).

[0265] Discussion and Conclusion The purpose of this experiment was to determine whether findings regarding hair growth (HF) in healthy women can be extrapolated to HF in healthy men, and therefore to provide motivation for further exploration of estetrol treatment for promoting hair growth in healthy men.

[0266] This experiment confirmed the ex vivo growth-prolonging effect of E4 on male HF, similar to that previously observed in healthy female HF, for all concentrations tested. However, 300 nM E2 showed no effect on this particular donor.

[0267] The promising growth phase extension effect of 3 μM E4 was further supported by a significant reduction in cell accumulation in the dermal papilla stalk. This is because fibroblasts accumulate in this specific compartment as they migrate from the dermal papilla and enter the dermal cup as a result of the transition to the regression phase (Kloepper et al., Exp Dermatol, 2010, Mar;19(3):305-12). Furthermore, the observed growth phase extension effect was also supported by increased proliferation of matrix keratinocytes when using 3 μM E4.

[0268] In summary, these results are promising and encourage further exploration of E4 treatment for additional male donors. E4 is suggested to have the potential to maintain the growth phase and prevent HF reduction, and should be considered in the management of male pattern baldness.

[0269] Example 9: Culture of hair follicle organs from female pattern hair loss (FPHL) donors (ex vivo model) Example 7 was repeated with minor modifications using a female donor suffering from pattern alopecia (FPHL). Briefly, terminal and intermediate hair follicles (HF) were obtained from the affected (vertex) and non-affected (occipital) regions of a female donor diagnosed with FPHL (Figure 34). After microdissection of the skin as described in the literature in the art (Edelkamp et al., Methods Mol Biol, 2020, 2154:105-119 and Langan et al., Exp Dermatol, 2015, Dec;24(12):903-1), the HF were placed in a suitable culture medium and allowed to rest for 24 hours to recover from the trauma of microdissection. The test substance (either vehicle (0.03% EtOH) or E4 at a concentration of 3 μM) was added to the medium at the end of the 24-hour period. The total culture time ranged from 3 to 5 days, depending on the HF stage of the vehicle group. The experimental design is shown in Figure 35. The same parameters as those described in Example 7 were investigated.

[0270] Preliminary results are available for two donors: a 60-year-old Caucasian woman with a diffuse FPHL pattern and a 31-year-old Middle Eastern woman with a classic FPHL pattern. Three additional donors are still needed.

[0271] Preliminary results Microscopically, E4 extended the growth phase of HF (terminal hair) from the non-affected scalp and HF (terminal and intermediate hair) from the affected scalp (Figure 36). E4 increased the proliferation of hair matrix keratinocytes in terminal HFs from both the non-affected and affected scalp areas without affecting apoptosis of hair matrix keratinocytes (Figure 37). E4 tended to increase versican expression in terminal and intermediate HF from non-affected scalp (Figure 38). E4 significantly reduced the total number of cells in the dermal cups of terminal HF from non-affected scalp (Figure 39). E4 tended to increase the total number of dermal papilla cells and area size in the intermediate HF from the non-affected scalp, and tended to increase the density and total number of cells in the intermediate HF from the affected scalp (Figure 40). E4 significantly reduced the total number and area size of dermal papilla cells in terminal HF from non-affected scalp, and showed a trend of decreasing dermal papilla cell density in intermediate HF from non-affected scalp (Figure 41). In the outer root sheath above the hair bulb, E4 tended to increase K15 expression and the number of K15-positive cells in terminal HF from non-affected scalp, and increased the proliferation of K15+ cells in terminal HF from non-affected scalp and intermediate HF from affected scalp (Figure 42). In the bulge basal layer, E4 tended to increase K15 expression in intermediate HF from non-affected scalp and increased the proliferation of K15+ cells in terminal HF from affected scalp (Figure 43).

[0272] Discussion and Conclusion The purpose of this experiment was to determine whether findings regarding HF in healthy women and healthy men can be extrapolated to HF in women with FPHL, and therefore to provide motivation for further exploration of estetrol treatment for promoting hair growth in women facing hair loss.

[0273] Preliminary data from two individual patients confirmed the growth phase extension effect of E4 on terminal HF from non-affected scalp, consistent with an increase in matrix keratinocytes. This beneficial effect was supported by a significant reduction in cell count in the dermal papilla and dermal cup. This is because fibroblasts accumulate in these specific compartments as they migrate from the dermal papilla and enter the dermal cup as a result of transitioning to the regression phase (Kloepper et al., 2010). Interestingly, E4 also appeared to maintain the growth phase of terminal HF from the affected area, reduce elongation of intermediate HF, and increase matrix keratinocytes only in terminal HF.

[0274] Furthermore, E4 increased dermal papilla induction in both terminal and intermediate HF from non-affected areas through a trend of increased versican expression and alkaline phosphatase activity, but there was no significant change in HF from affected areas.

[0275] Furthermore, the number of K15-positive cells in terminal fibrous hematophoresis (HF) from non-affected areas tended to increase with E4, and the proliferation of K15-positive cells also increased.

[0276] In summary, we were able to replicate the beneficial effects of E4 already observed in healthy female HF in terminal HF areas from non-affected areas, and partially replicate them in terminal HF areas from affected areas. Since the latter corresponds to HF that is more prone to shrinkage, this preliminary data encourages further exploration of E4 treatment for FPHL management, as it suggests the potential of E4 to maintain the growth phase and prevent HF shrinkage.

[0277] Example 10: Culture of hair follicle organs from male pattern baldness (MPHL) donors (ex vivo model) Example 7 was repeated for male donors suffering from pattern alopecia (MPHL). Briefly, full-length frontotemporal hair follicles (HF) were obtained from Caucasian male donors diagnosed with MPHL. After microdissection from the skin as described in the literature in the art (Edelkamp et al., Methods Mol Biol, 2020, 2154:105-119 and Langan et al., Exp Dermatol, 2015, Dec;24(12):903-1), the HF were placed in a suitable culture medium and allowed to rest for 24 hours to recover from the trauma of microdissection. The test substance (vehicle (0.3% EtOH) or E4 at concentrations of 30 μM, 3 μM, or 300 nM) was added to the medium at the end of the 24 hours. The total culture time was 5 to 9 days depending on the stage of the HF in the vehicle group. This model allows us to evaluate whether treatment with E4 can prolong the growth phase and / or delay the onset of regression in men with MPHL. The same parameters as those described in Example 7 will be investigated.

[0278] This experiment aims to confirm that findings regarding HF in healthy women, healthy men, and women with FPHL can be extrapolated to HF in men with MPHL, and therefore to provide motivation for further exploration of estetrol treatment for promoting hair growth in men facing hair loss.

[0279] The preliminary results described in Example 8 are promising and encourage further exploration.

[0280] Example 11: A clinical study to evaluate the effectiveness of Esthetrol treatment for hair loss in menopausal women (perimenopausal and postmenopausal). We will initiate a randomized, double-blind, placebo-controlled phase II (pilot) study to evaluate the effects of estetrol on hair parameters in postmenopausal women with androgenetic alopecia.

[0281] Research design: This randomized, double-blind, placebo-controlled phase II (pilot) study consists of a dermatological component.

[0282] Eligibility determination includes classification of hair loss patterns using the Savin Density Scale. Participants will be randomly assigned to receive either a topical foam formulation based on estetrol monohydrate (formulated as a foam using butane, butylated hydroxytoluene, cetyl alcohol, citric acid, glycerol, isobutane, lactic acid, polysorbate 60, propane, purified water, stearyl alcohol, and anhydrous ethanol, respectively) or a placebo topical foam formulation. It should be noted that due to solubilization during the formulation process, estetrol monohydrate may no longer exist in monohydrate form, and this should be evaluated separately for each topical composition.

[0283] Dermatological investigations include quantitative hair measurement, subject self-assessment (SSA) of hair growth, and investigator's global assessment (IGA). The safety of Esthetrol (E4) will be evaluated throughout the study for all subjects.

[0284] Number of participants (planned: 120 participants (30 per treatment regimen) This sample size is 20 hairs / cm² between groups in terms of target area hair count (TAHC). 2 To detect the difference, a standard deviation of 25 lines / cm is used. 2 Assuming this, and considering the use of a nonparametric approach (Sauerbronn, int J Gynaecol Obstet, 2000, Jan; 68(1):35-41), we determined the 5% two-sided type I error and 80% power.

[0285] Dermatological efficacy variables: Dermatological measurements and evaluations will be performed at baseline (at screening or the next visit) and at follow-up visits (e.g., at weeks 12 and 24, 4 months, 8 months, and 12 months). The top dermatological efficacy variables are as follows: Target area hair count (TAHC), Hair growth assessment (HGA). Other variables are considered secondary variables. Hair parameters are evaluated using the following measurements.

[0286] Digital image analysis from standardized close-up photography (HairMetrix Phototrichogram, Canfield) for quantitative hair measurement: Target area hair count (TAHC) and, For non-vellus hairs and vellus / vellus-like (reduced) hairs, the target area hair width (TAHW) is measured individually. To assist with standardized close-up photography, scalp microdot tattoos are performed at baseline and between one or more visits.

[0287] Standardized global photography for SSA and IGA using baseline global images of the subject's scalp taken via Canfield camera system software, and comparison with real-time global images immediately available using Canfield review software installed on a Canfield-supplied laptop.

[0288] Self-assessment of hair growth on the scalp is performed using the following three evaluations: Hair Growth Assessment (HGA) (Olsen EA, Whiting D, Bergfeld W, Miller J, Hordinsky M, Wanser R, et al. A versus multicenter, randomized, placebo-controlled, double-blind clinical trial of a novel formulation of 5% Minoxidil topical foam placebo in the treatment of androgenetic alopecia in men. J Am Acad Dermatol. 2007;57:767-74)

[0289] Scalp hair growth will be compared to baseline (Visit X) using the following 7-point scale: [-3] Significant decrease [-2] Moderately decreased [-1] Slightly decreased [0] No change [1] Slight increase [2] Moderate increase [3] A significant increase This is in response to the following statement: "My scalp hair growth is... compared to the baseline."

[0290] Effectiveness variable: Hair Growth Index (HGI): Hair growth index (HGI)(Gubelin Harcha W, Barboza Martinez J, Tsai TF, Katsuoka K, Kawashima M, Tsuboi R, et al. A randomized, active- and placebo-controlled study of the efficacy and safety of different doses of dutasteride versus placebo and finasteride in the treatment of male subjects with androgenetic alopecia. J Am Acad Dermatol. 2014;70:489-98 (see e3)

[0291] Scalp hair growth will be compared to baseline (visit X) using a health outcome questionnaire with the following 7-point scale: [-3] Very few [-2] Moderately low [-1] Slightly less [0] Same amount [1] Slightly more [2] Moderately frequent [3] Very many This addresses the following three questions: "Since starting treatment, when I look at areas where my hair volume has decreased, I can see...(the scalp)." "Since starting treatment, my hair is now covering my scalp." "Since starting treatment, the appearance (thickness / quality / quantity) of the areas on my scalp where hair volume has decreased has changed..."

[0292] Hair Growth Satisfaction Scale (HGSS) (See Gubelin Harcha W, Barboza Martinez J, Tsai TF, Katsuoka K, Kawashima M, Tsuboi R, et al. A randomized, active- and placebo-controlled study of the efficacy and safety of different doses of dutasteride versus placebo and finasteride in the treatment of male subjects with androgenetic alopecia. J Am Acad Dermatol. 2014;70:489-98 e3)

[0293] Compare scalp hair growth / appearance to baseline (visit X) using the following 7-point scale: [-3] Very dissatisfied [-2] Dissatisfaction [-1] Somewhat dissatisfied [0] Neutral / Neither satisfied nor dissatisfied [1] Somewhat satisfied [2] Satisfied [3] Very satisfied This is a five-part question: "How satisfied are you with how you feel about...?" The overall appearance of your hair. The appearance of areas (or multiple areas) of hair volume loss within the treatment area of ​​your scalp. The amount of scalp that can be observed in the treatment area. The amount of hair in the treatment area. Hair growth in the treatment area.

[0294] The overall assessment of scalp hair growth by a physician (IGA) is performed using the following 7-point scale: [-3] Significant decrease [-2] Moderately decreased [-1] Slightly decreased [0] No change [1] Slight increase [2] Moderate increase [3] A significant increase

[0295] Furthermore, the subject can respond to inquiries regarding DLQI and Hairdex (trademark) (see Fischer TW, Schmidt S, Strauss B, Elsner P. Hairdex Ein Instrument zur Untersuchung der krankheitsbezogenen Lebensqualitaet bei Patienten mit Haarerkrankungen. Hautarzt., 2001 Mar; 52 (3):219-27).

[0296] Safety variables: Medical history and gynecological history Physical examination Gynecological examinations Breast examination Dermatological examination of the skin Vital signs Electrocardiogram (ECG) Papa Nicolau (PAP) Test Transvaginal ultrasound (TVUS) mammography Laboratory testing: Fasting blood sampling for the following purposes Hematology / Chemistry Lipid / glucose parameters for participation Blood sampling for the following purposes: Follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH) for participation Urine pregnancy test Prior and concomitant medication use will be monitored and observed throughout the entire study period. Adverse events (AEs) will be monitored and collected throughout the entire study period.

[0297] Participation criteria: Patients will be assigned to treatment if they meet all of the following participation criteria: 1. A signed and dated informed consent form and all necessary privacy permissions, provided prior to the commencement of any test procedure and after the nature of the test has been explained in accordance with local regulatory requirements; 2. Women aged 40 to 65 at the time of screening; 3. Regarding the criteria for hysterectomy: It is documented that hysterectomy should have been performed at least 6 weeks before the start of screening. Hysterectomy can be a total hysterectomy or a subtotal hysterectomy (i.e., without removal of the cervix); 4. Eligibility criteria for non-hysterectomy: Endometrial thickness of two layers is 4 mm or less as measured by TVUS; 5. Seeking treatment aimed at alleviating VMS associated with menopause; 6. Body Mass Index is 18.5 kg / m² 2 More than 35.0kg / m 2 below; 7. No significant signs of disease are shown in a mammogram performed during or within nine months prior to the start of screening. 1 ; 8. Postmenopausal condition as defined by any of the following: a. Regarding the criteria for non-hysterectomy: At least 12 months of natural amenorrhea Alternatively, six months of spontaneous amenorrhea with a serum FSH level greater than 40 mIU / mL (value obtained after discontinuation of estrogen / progestin-containing medication (if applicable), see exclusion criteria 18 and 20). Alternatively, bilateral oophorectomy, regardless of whether a hysterectomy has been performed. 2 Six weeks post-surgery; b. Regarding who is eligible for hysterectomy: Serum FSH level greater than 40 mIU / mL (value obtained after discontinuation of estrogen / progestin-containing medication (if applicable); see exclusion criteria 18 and 20); Alternatively, bilateral oophorectomy 2 At least 6 weeks post-surgery; 9. A good physical and mental condition as determined by the attending physician, based on the medical history prior to the visit, physical and gynecological examinations, and clinical evaluation; 10. Can understand and comply with the requirements, instructions, and limitations set forth in the protocol; 11. I am able to complete the examination log and questionnaire and will cooperate. 12. Non-smoker for at least one year (including e-cigarettes)

[0298] Further participation criteria considering skin condition: 13. Decrease in scalp hair density in the parietal region compared to the temporal and occipital regions, with scalp hair density ranging from n.2 to n.6 on the Sabin scale within the relevant density region. 14. The subject is willing to maintain the same hairstyle, hair length, and hair color throughout the entire study period (meaning the same hair staining interval prior to the approved photograph at screening / baseline). 15. The subject agrees to continue using other overall hair care products and plans throughout the study. 16. The subject agrees to maintain the same dietary and supplemental pattern.

[0299] 1 To be enrolled in the study, participants must have a Breast Imaging-Reporting And Data System (BI-RADS) score of 1 or 2. Insufficient mammogram results, i.e., BI-RADS 0, are not acceptable and require further evaluation. The testing facility must obtain a copy of the official report regarding the participant's study file. If mammography is performed as part of this study, digitized images should be obtained. 2 A letterhead report or statement from the physician in question, confirming that both ovaries were removed, is required.

[0300] Exclusion criteria: Patients will not be allocated treatment if they meet any of the following exclusion criteria: 1. A history of malignant tumors, excluding basal cell carcinoma or non-metastatic squamous cell carcinoma, diagnosed more than one year prior to the screening visit. 3 ; 2. If the attending physician finds any clinically significant findings during a breast examination and / or mammography that suggest a malignant tumor of the breast that would require additional clinical trials to remove breast cancer (however, confirmation of a simple cyst by ultrasound is acceptable); 3. PAP test in patients undergoing partial or complete hysterectomy using atypical squamous cells of unspecified significance (ASC-US) or higher (low-grade squamous intraepithelial lesion [LSIL], atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion [HSIL][ASC-H], HSIL dysplasia, or malignant cells) 4 Note: If a Remeron Human Papillomavirus (HPV) test is performed and the result is negative for high-risk oncogenic HPV subtypes 16 and 18, ASC-US is acceptable. 4. Regarding those who will not undergo hysterectomy: a. Presence of uterine cancer or endometrial hyperplasia; b. Presence of endometrial polyps (sometimes multiple); c. Vaginal bleeding or abnormal uterine bleeding of unknown diagnosis; d. Endometrial ablation; e. Uterine / endometrial abnormalities that make the use of estrogen and / or progestin therapy contraindicated, as determined by the attending physician. This includes the presence or history of adenomyosis or prominent fibroids; 5. Systolic blood pressure (BP) greater than 139 mmHg and diastolic blood pressure greater than 89 mmHg during screening. 5 ; 6. A history of venous or arterial thromboembolism (e.g., superficial or deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, angina pectoris, etc.), or a family history of venous thromboembolism (VTE) within the first degree of kinship; 7. A history of known acquired or congenital coagulation disorders or abnormal coagulation factors (including known thrombotic diathesis); 8. In the laboratory, fasting glucose levels exceed 125 mg / dL and / or glycated hemoglobin levels exceed 7.5%. 6 ; 9. Abnormal lipoproteinemia (LDL greater than 190 mg / dL and / or triglycerides greater than 300 mg / dL) 7 ; 10. Presence or history of gallbladder disease (excluding cases where the cholecystectomy has been performed); 11. Systemic lupus erythematosus; 12. All malabsorption diseases, including gastric bypass surgery; 13. History of acute liver disease, or chronic or severe liver disease [alanine transaminase (ALT) or aspartate transaminase (AST) greater than twice the upper limit of normal (ULN), bilirubin greater than 1.5 times the ULN] or presence or history of liver tumors within the 12 months prior to the start of screening; 14. Chronic or current acute renal impairment (estimated glomerular filtration rate less than 60 mL / min); 15. Positive for human immunodeficiency virus (HIV), hepatitis B, or hepatitis C serological test; 16. Porphyria; 17. Diagnosis or treatment of major mental illnesses (e.g., schizophrenia, bipolar disorder, etc.) at the discretion of the attending physician; 18. Use of the following estrogen / progestin-containing medications (or multiple medications): a. For non-systemic vaginal hormone products (rings, creams, gels), use them one week before the start of screening; b. For vaginal or transdermal estrogen or estrogen / progestin products with systemic effects, four weeks prior to the start of screening; c. For oral estrogen and / or progestin products and / or selective estrogen receptor modulator therapies, 8 weeks prior to the start of screening; d. For intrauterine progestin therapy, 8 weeks before the start of screening; e. For progestin implantation or estrogen monotherapy, three months prior to the start of screening; f. For estrogen pellet therapy or progestin injection therapy, six months prior to the start of screening; 19. Use of androgen / dehydroepiandronesterone (DHEA)-containing medications: a. For oral, topical, vaginal, or transdermal androgens, 8 weeks prior to the start of screening; b. For implantable or injectable androgen therapies, six months prior to the start of screening; 20. Use of phytoestrogens or black cohosh for the treatment of VMS up to two weeks prior to the start of screening; 21. During participation in the study, fail to cooperate with the discontinuation of all hormone products listed in exclusion criteria 18, 19, and 20; 22. Inadequately treated thyroid dysfunction. If free thyroxine (T4) levels are within the normal range at screening, patients with low or high TSH levels are also acceptable. 8 . 23. A history or existence of allergy / intolerance to the product under investigation, or to any of the pharmaceuticals in this category or any of their components, or a history of pharmaceuticals or other allergies to which the participant's participation is contraindicated in the opinion of the attending physician; 24. A history of alcohol or drug abuse (including marijuana, even if legal) or dependence in the 12 months prior to the start of screening, as determined by the attending physician based on reported observations; 25. Employees of the sponsor or CRO, or employees under the direct supervision of the attending physician and / or directly involved in the trial; 26. Subjects who, in the opinion of the attending physician, have a clinically significant systemic disease, an unstable medical condition, a life-threatening disease, or a known or suspected history of current malignancy that would pose a risk to the subject; 27. Participated in a clinical trial of another drug under investigation within the past month (30 days), or received the drug under investigation within the past month (30 days) prior to the start of screening; 28. The attending physician determines that the patient is unsuitable for any reason.

[0301] Further exclusion criteria considering the impact on hair condition: 29. The subject has any dermatological disorder on the scalp of the target area that may interfere with the application of IMP or the diagnostic method, such as fungal or bacterial infection, seborrheic dermatitis, psoriasis, eczema, folliculitis, scarring, or scalp atrophy. 30. The subject has a local, systemic (e.g., uncontrolled thyroid disease, certain genetic disorders affecting hair growth or pattern), or skin condition requiring surgical treatment that, in the opinion of the attending physician, may interfere with the assessment of IMP. 31. The subject has a history of or signs of hyperandrogenism (e.g., excessive facial / genital / navel hair growth, severe acne, or a history of elevated testosterone levels). 32. The subject has a current or recent (within the last 6 months) history of using hair extensions, non-breathable wigs, or hair bonding. 33. The subjects underwent scalp hair transplantation at any time. 34. The subject has a history of diffuse telogen effluvium, alopecia areata, scarring alopecia, trichotillomania, or active hair loss due to a condition / disease other than AGA. 35. The subject has a current or recent history (within the last 6 months) of severe eating or weight changes, or has a diagnosis of an eating disorder(s), a history of any weight-loss surgery (gastric bypass, gastric sleeve, gastric stapling), macro or micro nutritional deficiency (i.e., clinically significant iron deficiency or protein deficiency confirmed by laboratory testing) within the last 6 months, and / or a current diagnosis of any malabsorption disorder (i.e., celiac syndrome, irritable bowel syndrome, etc.). 36. The subject received any of the following topical preparations or treatments on the scalp: a) Topical scalp treatment for hair growth, including minoxidil, hormone therapy, antiandrogenic substances, or other medications known to affect hair growth, within 12 weeks prior to baseline (visit 2). b) Use of over-the-counter (OTC) topical scalp medications or cosmetic treatments known or reasonably thought to affect hair growth (e.g., brands such as Maxilene, Nioxin, Foltene, etc.) or hair health or hair growth products containing saw palmetto, copper, etc., within four weeks prior to baseline (visit 2). c) Topical scalp treatments that may incidentally affect hair growth, including but not limited to corticosteroids, pimecrolimus, tacrolimus, and retinoids, within four weeks prior to baseline (visit 2). d) Scalp treatments (surgical, laser, light, or energy therapy, microneedling, etc.) within 6 months prior to baseline (visit 2). e) Platelet-rich plasma (PRP) treatment of the scalp within the past year. 37. The subject received the following systemic medications or treatments: a) Beta-blockers, cimetidine, diazoxide, or corticosteroids (including intramuscular and intrafocal injections) within 12 weeks of visit 2 / baseline. Inhaled, intranasal, or ocular corticosteroids are permitted as long as use is stable (defined as dose and frequency remaining unchanged for at least 4 weeks prior to baseline (visit 2)). b) Retinoids, isotretinoin, more than 10,000 IU of vitamin A per day, or cyclosporine therapy within the six months prior to baseline (visit 2). c) Any 5α-reductase medication (i.e., finasteride (e.g., Propecia®), dutasteride, or similar products (or multiple products)) within 12 months prior to baseline (visit 2). d) Chemotherapy or cytotoxic agents used within the past five years. e) Radiation therapy of the scalp at any time. f) Antiandrogenic substances within 6 months prior to baseline (visit 2); concomitant use of antiandrogenic substances (such as cyproterone acetate, chlormadinone acetate, etc.) with birth control pills is permitted as long as the use is stable (defined as the dosage and frequency remaining unchanged for at least 3 months prior to baseline (visit 2)). g) Any other systemic therapy, including but not limited to spironolactone, vitamins (including biotin intake of more than 5 mg / day), or homeopathic supplements for hair growth or hair health, or any other steroid hormone (any form) including anabolic steroids, within three months prior to baseline (visit 2) or during the study, which, in the opinion of the attending physician, may significantly affect the hair or hair growth of the subject.

[0302] 3 Only patients with basal cell carcinoma of the skin or non-metastatic squamous cell carcinoma of the skin, who were diagnosed with either of these conditions more than one year prior to the screening, are excluded. 4 As indicated in writing within 18 months prior to screening, or before the test is conducted at the time of screening. 5 If the blood pressure remains below the inclusion criteria even after sitting for an additional 5 to 10 minutes, blood pressure measurement can be repeated during screening. The last measurement will be used for eligibility. Individuals with mild to moderate hypertension managed with a stable antihypertensive regimen are eligible for enrollment if they meet all inclusion / exclusion criteria. Individuals receiving methyldopa or clonidine-containing antihypertensive medications are not included. 6 Laboratory values ​​of fasting glucose and glycated hemoglobin assessed over the past six months, as well as during drug withdrawal and screening, must be considered. 7 Patients receiving lipid-lowering therapy must be on a stable dose for at least one month prior to screening. 8 The Remeron T4 test should only be performed during screening if TSH levels are outside the normal range.

[0303] Statistical methods: The analysis will be performed on the treatment-initiated group and the protocol-adhering group. Classification factors will be summarized using frequency and / or percentages, and consecutive measurements will be described using mean and standard deviation.

[0304] For data following a normal distribution, the significance of within-group change from baseline is estimated using Student's t-distribution, and between-group changes from baseline are compared using independent t-tests. Results are presented as the mean and 95% confidence interval. For data not following a normal distribution, equivalent nonparametric methods are used: Wilcoxon signed-rank test is used for within-group change from baseline, and Mann-Whitney U test is used for between-group comparisons. Results are presented as the median, minimum, and maximum.

[0305] The analysis will be performed using SAS software (Stat version 15.2; SAS Institute, Cary, NC, USA) with a significance level of 0.05. No adjustment for multiple tests will be made in this exploratory proof-of-concept trial.

[0306] Example 12: A randomized, double-blind, placebo-controlled trial evaluating the effect of a topical estetrol (E4) formulation on hair parameters in men with excessive hair loss. Test products: Esthetrol monohydrate topical formulations at 0.06%, 0.22%, or 0.50%, formulated as solutions using ethanol, propylene glycol, and water. Note that due to solubilization during the formulation process, estetrol monohydrate may no longer exist in monohydrate form, and this should be evaluated separately for each topical composition.

[0307] Control product: Placebo formulation All test treatments will be administered to patients for self-administration by the research physician or designated staff. The four formulations will be provided in identical bottles numbered from 1 to 120. Patients will be instructed to apply the product topically once daily, preferably before bedtime, to dry hair and scalp, by gently massaging the scalp, throughout the entire 24-week study period.

[0308] the purpose Main purpose: In men with clinical signs of excessive hair loss, evaluate the hair growth-promoting ability of the E4 topical formulation compared to a placebo formulation by investigating hair loss, hair volume, hair thickness, hair density, hair reduction, extension of the growth phase, and patient self-assessment.

[0309] Secondary purpose: In men with clinical signs of excessive hair loss, the skin compatibility and tolerability of the E4 topical formulation will be evaluated compared to a placebo formulation by investigating patient self-assessments and clinical observations of adverse effects.

[0310] method system Twenty healthy male volunteers aged 18 to 65 years who complain of hair loss lasting more than four weeks will be asked to apply either the E4 topical formulation or a placebo formulation to their scalp over the entire 24-week trial period. Each patient will be randomly assigned to one of four groups: three treatment groups receiving different intensities of the E4 topical formulation and a placebo group (n=30 in each group). Patients will visit the clinic at weeks 0, 8, and 24.

[0311] Number of patients: 120 (30 per test group)

[0312] Duration of treatment: 24 weeks

[0313] Diagnostic and primary selection criteria This study will include patients with a confirmed diagnosis of male pattern androgenetic alopecia who have not received any topical or systemic therapy for hair loss (including oral nutritional supplements) in the two months prior to the start of the study.

[0314] Evaluation Criteria Main: Hair growth assessment: Visual scale of hair loss: After not washing their hair for two days, patients are asked to wash and comb their hair before coming to the clinic, collect all the hair, and place it in a plastic bag. The collected hair is then evaluated using an arbitrary scale from 1 to 9 according to the visual scale of hair loss described in the literature by Sinclair and recently validated by Tosti et al. (Tosti et al., Dermatol Ther, 2017). Cross-sectional trichometer (Hair Check®): The cross-sectional area of ​​a bundle of hair selected using a measurement template (numbered 10 each time) from a specific 2cm x 2cm area located in the central region of the scalp's crown, corresponding to approximately 12.5cm from the glabella. This is evaluated using the Hair Check® device. The quantitative result, known as the hair mass index, is an indirect measure of hair volume. Hair Thickness (Folliscope®): The diameter of 10 to 15 hairs in the same scalp area as the hair identification is measured using a Folliscope® device. The average hair diameter is calculated. Terminal hair / vellus hair ratio: Based on hair thickness measurements using a Folliscope® device, the number of terminal hairs (defined as hairs greater than 40 microns) and vellus or reduced hairs (defined as hairs less than 40 microns) are counted. Hair density: Number of hairs / cm 2 To measure hair density, a circular area with a diameter of 1.6 cm is shaved from the scalp, the area is stained, and then measured using a TrichoScan® device. The area adjacent to the right of the area used for hair identification is 0.6 cm. 2 Measure the hair density within the region. Phototrichogram (anagen / catagen-telogen ratio): The anagen / catagen-telogen ratio is determined using the same area used for hair density analysis with TrichoScan® digital video microscopy. The number of growing (anagen) hairs is manually counted 48 hours after shaving. Hairs that are not growing after 48 hours are considered to be in the catagen / telogen phase. Patient Self-Assessment Questionnaire: A self-assessment questionnaire containing the following questions will be given to all participants on days 60 and 180: P1: Noticeable decrease in hair loss, P2: Feeling that less hair remains on the comb / brush, P3: Feeling that hair is growing, P4: Increased resistance of hair after stress, P5: Improved hair quality, P6: Stronger hair, P7: Increased hair density, P8: Thicker hair, P9: Softer hair, P10: Shinier hair, P11: Increased hair volume, P12: Satisfied with the results.

[0315] secondary: Patient self-assessment questionnaire: A self-assessment questionnaire containing the following questions will be distributed to all participants on days 60 and 180: P13: Visual impression, P14: Odor impression, P15: Tolerability of the product. Clinical observation of adverse effects, namely redness and scalp desquamation.

[0316] Predicted conclusion We predict that the E4 topical formulation will dose-dependently and more efficiently improve the clinical signs of male pattern androgenic alopecia than placebo, and will protect against HF reduction (i.e., re-conversion from vellus to terminal hair).

[0317] The majority of the effects are already observed 8 weeks after treatment and are maintained even at 24 weeks.

[0318] Example 13. Preparation of aqueous gel formulation for stability testing Table 8 lists the hydrogels prepared for stability testing. The preparation was carried out as described for AG24 in Example 1.

[0319] TIFF2026520082000010.tif51170

Claims

1. A topical composition containing an effective amount of estetrol for use in the prevention or treatment of hair loss in the subject.

2. The topical composition for use according to claim 1, wherein the subject is female or male, preferably male.

3. A topical composition for use according to claim 1 or 2, comprising about 0.01% to about 5% by weight of an estrol component, preferably about 0.02% to about 1% by weight of an estrol component, preferably 0.1% to 1% by weight of an estrol component, and most preferably 0.3% to 0.7% by weight of an estrol component.

4. The composition for use according to any one of claims 1 to 3, further comprising a permeability enhancer that enables permeation through the stratum corneum of the target skin.

5. A composition for use according to any one of claims 1 to 4, which is a formulation selected from the group consisting of solutions, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, creams, and sprays.

6. The composition for use according to any one of claims 1 to 5, wherein the estetrol component is estetrol, and preferably the estetrol component is estetrol monohydrate.

7. A composition for use according to any one of claims 1 to 6, wherein the prevention or treatment results in an improvement or maintenance of the feel, quality and / or appearance of the hair, and preferably the treatment includes or results in an improvement in the number of target area hairs (TAHC) and / or the width of target area hairs (TAHW).

8. A composition for use according to any one of claims 1 to 7, wherein the prevention or treatment includes preventing hair loss, reversing hair loss, slowing hair loss, reducing hair loss, or increasing hair growth.

9. The composition for use according to any one of claims 1 to 8, wherein the hair loss is female pattern hair loss (female androgenetic alopecia (FAGA)) or male pattern hair loss (MAGA).

10. A composition for use according to any one of claims 1 to 9, wherein the prevention or treatment increases the function and / or growth of keratinocytes, and in connection therewith, preferably increases the function and / or growth of epidermal keratinocytes (KCs) such as those belonging to the bulge, outer root sheath (ORS), or hair bulb.

11. A composition for use according to any one of claims 1 to 10, wherein the prevention or treatment increases the function and / or growth of follicular mesenchymal fibroblasts, and in connection therewith, increases the function and / or growth of dermal papilla (DP) cells.

12. The composition for use according to any one of claims 1 to 11, wherein the prevention or treatment means delaying the onset of the regression phase or preventing the regression phase.

13. The composition for use according to any one of claims 1 to 12, wherein the prevention or treatment means promoting and / or extending the growth period.

14. The composition for use according to any one of claims 1 to 13, wherein the prevention or treatment means the restoration and / or promotion of epithelial-mesenchymal interactions between hair follicle cells.

15. A composition for use according to any one of claims 1 to 14, wherein the prevention or treatment is characterized by the reduction of inflammation and / or oxidative stress in the hair follicle.

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