Topical composition comprising estetrol component and use of said composition for wound healing

Abstract

The present invention relates to topical compositions comprising an estetrol component and the use of said compositions for delivering an effective amount of an estetrol component to the skin, in particular for wound healing. Optionally, the estetrol component may be included in a composition that also includes an ingredient that facilitates wound healing. In particular, the present invention relates to compositions such as gels, in particular hydrogels, creams and ointments, comprising estetrol. The composition of the invention has beneficial effects on wound healing process and patient rehabilitation.

Description

Field of the Invention

[0001] The present invention broadly relates to treating skin wounds using an effective amount of an estetrol component. In addition, the present invention relates to a pharmaceutical composition suitable for treating skin wounds comprising an estetrol component. The pharmaceutical composition and related methods of the present invention have a beneficial effect on wounds, the wound healing process, and ultimately patient recovery. Background Art

[0002] During life, each individual is exposed to adverse stimuli or events that can cause skin wounds many times, such as skin wounds caused by injuries, surgery, burns and pathological factors, and optionally also wounds of subcutaneous tissue. Usually, these wounds heal without causing much burden to the patient after routine and adequate care (including practices such as disinfection and / or temporary covering of the wound site). However, complications may occur if the patient's wound heals poorly, if there is excessive inflammation or infection at the wound site, or if the wound site covers a large area of ​​the individual.

[0003] Although wound healing is often considered a "basic" function of skin tissue, it is the result of a complex combination of molecular mechanisms. In normal skin tissue (i.e., uninjured skin tissue), the epidermis (outermost layer of skin) and dermis (dermis) form a protective layer against external stimuli (Kolarsick et al., JDNA, 2011). When the protective layer is damaged, a repair process is initiated to repair the damage. This repair process has been described in detail in the art and includes subsequent steps such as hemostasis (blood clotting), inflammation, proliferation (new tissue growth), and maturation (tissue remodeling).

[0004] A more detailed study revealed that the above repair process is characterized by a variety of molecular mechanisms, which have also been the subject of research in this field (e.g. Rodrigues et al., Physiol Rev, 2019). In short, during the coagulation phase (usually considered to be part of the inflammatory phase), a fibrin clot is produced to prevent further blood loss. The inflammatory phase is characterized by initial vasoconstriction and subsequent vasodilation, as well as the recruitment of cells such as neutrophils, monocytes and macrophages. The proliferative phase is characterized by angiogenesis, fibroblast differentiation and granulation tissue formation. The formation of granulation tissue enables epithelial cells (keratinocytes) that migrate to cover the wound surface to re-epithelialize. In addition, the late stage of the proliferative phase includes fibroblast-mediated wound contraction. The final stage of wound healing, the maturation stage, is characterized by the rearrangement and cross-linking of collagen fibers formed at the wound site, thereby increasing the tensile strength of the wound and inducing the formation of scar tissue. In individuals with impaired wound healing, one or more of the above stages or their sub-processes are disturbed or essentially absent (Avishai et al., EPMA J, 2017).

[0005] The importance of proper wound care cannot be overstated, especially since the prevalence of chronic wounds is expected to increase in the coming decades along with the prevalence of chronic diseases such as diabetes, cancer and autoimmune diseases. Epidemiological studies have warned of this growing healthcare burden. Based on global, regional and national data from 195 countries and territories, the prevalence has increased significantly over a 10-year period, with 605,036,000 cases in 2015 compared to 492,883,000 in 2005 (reported by the Global Burden of Disease (GBD) 2015 Collaborative Report on Incidence and Prevalence of Disease and Injury, Lancet, 2016). Global estimates show that at least 7 million people suffer complications after surgery each year, of which at least 1 million die. Delayed healing of acute wounds increases the risk of postoperative morbidity and mortality. A good example is surgical site infection, the second leading cause of nosocomial infection. The main complications of acute wounds are related to age-related healing failure and hormone deficiencies. In this regard, the population aged over 65 years (currently 15-28% of the total population) is increasing worldwide.

[0006] Despite the current availability of a variety of wound care strategies (both mechanical and pharmacological), there remains an unmet need for effective wound care strategies and pharmaceutical compositions that aid in wound care. Such compositions ideally aid in wound healing in subjects with impaired wound healing, such as elderly subjects and subjects with chronic wounds due to, for example, lack of motility and / or underlying pathology. Summary of the invention

[0007] As confirmed in detail by the attached examples herein, the inventors unexpectedly found that estrol is particularly suitable for wound healing, including wound healing of patients with impaired wound healing characteristics. Through a large number of experiments, it was found that the estrol component can be safely included in the pharmaceutical composition intended to help wound healing. The composition has no or only limited impact on the uterine weight increase that may occur due to unintentional systemic exposure to the topical use of estrogen in female subjects. More specifically, the inventors found that the estrol component of a specific amount is included in the pharmaceutical composition intended to help wound healing, providing an unprecedented compromise between efficacy and adverse reactions (such as uterine weight increase). Uterine weight does not increase, indicating that the systemic effect of the pharmaceutical composition comprising the estrol component is limited, and even at certain dosages, even without systemic effect after including a penetration enhancer.

[0008] As can be seen from in vitro and in vivo wound healing experiments, estrol plays at least a similar role to estradiol in terms of wound closure, epithelial regeneration and anti-inflammatory effects. Estradiol and estrol treatment can increase fibroblast ER expression, while estrol can also promote fibronectin expression and inhibit MMP activity. Estrol also seems to promote dermal fibroblast migration in the scratch test and promote epidermal keratinocyte migration more effectively than estradiol. In addition, estrol regulates wound-related epidermal gene expression slightly more effectively than estradiol and promotes wound closure more effectively than estradiol. These improvements in wound healing are combined with a reduction in systemic effects (such as increased uterine weight), making estrol compositions an attractive alternative to estradiol and other estrogen-based compositions for wound healing.

[0009] Finally, in some embodiments, the composition is additionally characterized by a gradual release profile, which is another advantage for wound healing. Thus, the present invention makes a significant contribution to new and innovative wound care strategies.

[0010] Therefore, a first aspect of the present invention relates to a pharmaceutical composition comprising 0.02% to 1.5% (w / w) of an estetrol component, preferably 0.05% to 1.2% (w / w) of an estetrol component, about 0.02% to about 1% (w / w) of an estetrol component, about 0.03% to about 1% (w / w) of an estetrol component, preferably about 0.04% to about 1% (w / w) of an estetrol component, more preferably about 0.05% to about 1% (w / w) of an estetrol component, most preferably about 0.06% to about 0.5% (w / w) of an estetrol component, more preferably about 0.09% to about 1.1% (w / w), even more preferably 0.1% to 1% (w / w) of an estetrol component, most preferably 0.3% to 0.7% (w / w) of an estetrol component. Preferably, the composition is for topical use, or for topical application, such as for topical application to the skin.

[0011] Preferably, the composition does not produce significant systemic effects on the subject upon or after topical application.

[0012] In certain embodiments, the pharmaceutical composition comprises about 0.03% to about 0.12% (w / w) of the estetrol component, preferably about 0.04% to about 0.08% (w / w) of the estetrol component, more preferably about 0.05% to about 0.07% (w / w) of the estetrol component, and most preferably about 0.06% (w / w) of the estetrol component.

[0013] In a specific embodiment, the pharmaceutical composition further comprises a penetration enhancer capable of penetrating the stratum corneum and / or penetrating the wound eschar.

[0014] In a specific embodiment, the pharmaceutical composition is a composition for topical wound healing.

[0015] In another aspect, the present invention relates to a (pharmaceutical) composition comprising an estetrol component for use in topical wound healing, wherein the composition optionally further comprises a penetration enhancer capable of penetrating the stratum corneum.

[0016] In a specific embodiment, the (pharmaceutical) composition comprises from about 0.01% to about 5% (w / w) of the estetrol component, preferably from about 0.02% to about 1% (w / w) of the estetrol component, more preferably from about 0.03% to about 0.75% (w / w) of the estetrol component, still more preferably from about 0.04% to about 0.5% (w / w) of the estetrol component, and most preferably about 0.06% (w / w) of the estetrol component.

[0017] In another aspect, the present invention is directed to a hydrogel formulation comprising about 0.02% to about 1.5% (w / w) of an estetrol component, more specifically about 0.05 to 1.2% (w / w), even more specifically about 0.09% to about 1.1% (w / w), or about 0.1% to about 1% (w / w).

[0018] In a specific embodiment, the hydrogel comprises about 0.05 to about 1.3% (w / w), more specifically about 0.08 to about 1.2% (w / w), even more specifically about 0.09% to about 1.1% (w / w), or about 0.1% to about 1% (w / w) of the estetrol component. In an alternative embodiment, the hydrogel comprises about 0.03% to about 0.75% (w / w) of the estetrol component, preferably about 0.04% to about 0.5% (w / w) of the estetrol component, more preferably about 0.05% to about 0.25% (w / w) of the estetrol component, and most preferably about 0.06% (w / w) of the estetrol component.

[0019] As can be seen from the Examples, 0.06% (w / w) estradiol had no effect on the increase in uterine weight in the tested mice, which is consistent with the use of commercially available estradiol containing 0.06% The effects on uterine weight gain were in stark contrast.

[0020] In addition, In contrast, the effect of estetrol at higher concentrations (0.22 and 0.5% (w / w)) on the increase in mouse uterine weight was still significantly lower. This indicates that topical application of a composition containing estetrol is less likely to produce systemic effects on a subject, which is particularly important for female subjects. In a specific embodiment, the hydrogel also includes a penetration enhancer to enable it to penetrate the stratum corneum.

[0021] In specific embodiments, the hydrogel is used for topical wound healing.

[0022] Optionally, the (pharmaceutical) composition of any aspect and embodiment described herein is in the form of a formulation selected from the group consisting of an emulsion, a suspension, an ointment, a paste, a lotion, a gel (including a hydrogel), a foam, a mousse and a cream.

[0023] In embodiments wherein the (drug) composition is a hydrogel, the hydrogel is characterized by a favorable release profile, for example more favorable compared to other formulations such as, but not limited to, creams.

[0024] In a preferred embodiment, the hydrogels described herein are characterized in that the average cumulative amount of estetrol component released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is at least about 2.5 μg / cm 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 about 100 μg / cm 2 , more preferably at least about 150 μg / cm 2 , more preferably at least about 200 μg / cm 2 Depending on the wound surface, it may be necessary to reduce the concentration of the estetrol component to avoid excessive cumulative administration of estetrol. This can be determined by a simple calculation by the physician or pharmacist.

[0025] In a preferred embodiment, the hydrogels described herein are characterized in that the average cumulative amount of estratetol component released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over 8 hours is at least about 50 μg / cm 2 , at least about 100 μg / cm 2 , at least about 150 μg / cm 2 , at least about 200 μg / cm 2 , at least about 250 μg / cm 2 , at least about 300 μg / cm 2 , at least about 350 μg / cm 2 , at least about 400 μg / cm 2 , at least about 450 μg / cm 2 More preferably, the hydrogels described herein are characterized by releasing an average cumulative amount of estetrol component of at least about 80 μg / cm2 .

[0026] Preferably, the hydrogels described herein are characterized in that the average % applied dose of the estratetol component entering the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 1 hour is at least about 15%, preferably at least about 20%, and / or the average % applied dose of the estratetol component entering the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 8 hours is at least about 40%, preferably at least about 50%, more preferably at least about 80%, and most preferably at least 90%.

[0027] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise a penetration enhancer in an amount of about 0.5% to about 60% (w / w), or preferably about 0.05% to about 5% (w / w). Preferably, the penetration enhancer comprises a substance or molecule capable of penetrating the stratum corneum (i.e., a penetration enhancing molecule) and a solvent. Preferably, the penetration enhancing molecule is selected from: ethanol, ethers such as diethylene glycol monoethyl ether Benzyl alcohol, fatty acids and esters thereof or any combination thereof. Preferably, the penetration enhancer comprises a solvent comprising one or more polyethylene glycols (PEG), propylene glycol (PG) or a combination thereof. Preferably, PEG is a PEG having a molecular weight between about 200 g / mol and about 600 g / mol, such as a PEG selected from the following group: PEG200, PEG300, PEG400, PEG500, PEG600 or any combination thereof. More preferably, the penetration enhancer comprises a solvent comprising a PEG having a molecular weight of about 400 g / mol, such as PEG400. In certain aspects, the penetration enhancer comprises up to 50% PEG400 (w / w) and / or about 15% to 45% PEG400 (w / w), and / or about 20% to 40% PEG400 (w / w), and / or about 30% to 35% PEG400 (w / w). Most preferably, the penetration enhancer comprises about 14% to about 21% PEG400 (w / w) and / or about 10% to about 25% PEG (w / w). In an alternative preferred embodiment, the penetration enhancer comprises about 0.5% to about 10% of a penetration enhancer, such as PEG400. In a further alternative preferred embodiment, the penetration enhancer comprises about 0.5% to about 5% of a penetration enhancer, such as PEG400.

[0028] In certain embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel comprises benzyl alcohol, preferably in an amount of about 1% to about 3%.

[0029] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise a thickener. In a specific embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel comprises a thickener in an amount of about 0.3% to about 20% (w / w), or preferably about 0.3% to about 3% (w / w), or more preferably 0.5% to 3% (w / w). Preferably, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel comprises a thickener selected from hydroxyethylcellulose (HEC), carboxymethylcellulose (CMC), a high molecular weight cross-linked acrylic acid-based polymer, a non-ionic triblock copolymer or any combination thereof. Preferably, the high molecular weight cross-linked acrylic acid-based polymer is

[0030] Preferably, the HEC is HEC250 HHX. Preferably, the nonionic triblock copolymer has an approximate molecular weight of about 1800 to about 4000 and a polyoxyethylene content of about 70 to about 80%. Preferably, the nonionic triblock copolymer is selected from poloxamers, such as poloxamer 188, poloxamer 407, or a combination thereof.

[0031] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise a preservative. In a specific embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise a preservative in an amount of about 1% to about 10% (w / w), preferably about 1% to about 3% (w / w). Preferably, 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.

[0032] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise an emollient. In a specific embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise an emollient in an amount of about 2.5% to about 30% (w / w), preferably about 8% to about 12% (w / w), most preferably in an amount of about 10% (w / w). Preferably, the emollient is selected from the group consisting of glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, petrolatum and any combination thereof.

[0033] Therefore, in any aspect and embodiment described herein, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component, a penetration enhancer, a thickener and as optional ingredients a preservative and / or an emollient, each of which is preferably selected from the group described herein. In any aspect and embodiment described herein, the (pharmaceutical) composition may be supplemented to 100% (w / w) by an aqueous solution (e.g. water).

[0034] In a specific embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component (w / w):

[0035] - from about 0.1% to about 60% (w / w) of a penetration enhancer, preferably wherein the penetration enhancer comprises a penetration enhancer molecule and a solvent or solvent system;

[0036] - from about 0.3% to about 20% (w / w) of a thickener;

[0037] -optionally preservatives and / or emollients;

[0038] - Make up to 100% (w / w) with water.

[0039] In a further embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise 0.05% to 0.6% (w / w) estetrol, and further comprises:

[0040] - from about 0.1% to about 5% (w / w) of a penetration enhancer, preferably wherein the penetration enhancer comprises a penetration enhancer molecule and a solvent or solvent system;

[0041] - from about 0.3% to about 3% (w / w) of a thickener;

[0042] -optionally preservatives and / or emollients;

[0043] - Make up to 100% (w / w) with water.

[0044] In a further specific embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component (w / w):

[0045] - about 16% to about 20% (w / w) PEG400;

[0046] - about 18% to about 22% (w / w) PG;

[0047] - from about 8% to about 12% (w / w) glycerol;

[0048] - from about 1% to about 2% (w / w) HEC; and

[0049] - about 1.5% to about 2.5% (w / w) benzyl alcohol.

[0050] In an alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component:

[0051] - about 18% to about 22% (w / w) PEG400;

[0052] - about 0.1% to about 1% (w / w) and

[0053] - about 4% to about 6% (w / w)

[0054] In a further alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component:

[0055] - about 25% to about 55% (w / w) PEG400, preferably about 35% to about 45% (w / w) PEG400; - about 0.1% to about 1% (w / w) Preferably about 0.25% to about 0.75% (w / w) and

[0056] - from about 0.1% to about 5% (w / w) Preferably from about 0.75% to about 3% In a further alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition for use or the hydrogel may comprise, in addition to the estetrol component:

[0057] - about 38% to about 45% (w / w) PEG 400;

[0058] - about 0.1% to about 1% (w / w) and

[0059] - about 0.8% to about 3% (w / w)

[0060] Aspects of the present invention encompass each of the (pharmaceutical) compositions, hydrogels and substitutes described herein as a medicament, i.e. in a therapeutic and / or preventive context. In specific embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used for wound healing. In further embodiments, the use in wound healing corresponds to their use as topical preparations in wound treatment. Similarly, aspects of the present invention relate to the use of a pharmaceutical composition or hydrogel described herein for the manufacture of a medicament for topical treatment of wounds. In connection with the foregoing, aspects of the present invention also encompass methods of topical wound treatment comprising applying any of the pharmaceutical compositions or hydrogels described herein to a wound or wound site of a subject.

[0061] In a specific embodiment, the (drug) composition and hydrogel of the above aspects are used to treat acute wounds. Optionally, the acute wound is a surgical wound or a wound caused by an acute injury. The wound can also be a partial thickness wound (e.g., at a skin transplant donor site). In an alternative embodiment, the pharmaceutical composition and hydrogel of the above aspects are used to treat chronic wounds. Optionally, the chronic wound is a wound caused and / or maintained by diabetes. Other major pathogenic factors of chronic wounds are ischemia, radiation, foreign matter, and prolonged external pressure. Chronic wounds are usually divided into infected wounds or ischemic wounds.

[0062] In a specific embodiment, the medical use or treatment described herein results in improved histological healing parameters compared to untreated wounds. Preferably, the medical use or treatment results in improved incidence of complete wound closure, accelerated wound closure and / or facilitated surgical wound closure. In a preferred embodiment, the medical use or treatment improves the quality of healing (also known as cosmetic). In particular for surgical wounds, fewer scars may be observed.

[0063] In specific embodiments, the pharmaceutical compositions and hydrogels described herein exhibit beneficial effects on inflamed wounds. After use, inflammation itself and / or inflammation progression can be prevented. Wound recurrence can also be prevented.

[0064] In specific embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used to treat wounds in subjects with impaired wound healing, including impaired delayed skin wound healing or bacterial delayed wound healing. In specific embodiments, the impaired wound healing is characterized by reduced wound edge migration. In other embodiments, the impaired wound healing is characterized by increased wound edge hyperplasia.

[0065] In a specific embodiment, the (drug) composition and hydrogel of the above aspects are used to treat infected wound sites, combat wounds, burns and chronic leg ulcers. In some embodiments, the infected wound may be associated with reduced epithelial regeneration, increased proliferation, enhanced inflammatory response and disordered wound matrix deposition. In this regard, a particularly studied pathogen is Klebsiella pneumoniae, and the composition containing the estratetol component disclosed herein has been shown to reduce inflammation in a Klebsiella pneumoniae infected wound model.

[0066] In specific embodiments, the (drug) compositions and hydrogels of the above aspects are used to improve epithelial regeneration at a wound site, increase cell proliferation at a wound site, reduce inflammatory response at a wound site, improve matrix deposition (such as matrix remodeling) and improve angiogenesis at a wound site.

[0067] In a particular embodiment, the medical use or treatment described herein results in improved quality of healing, also referred to as cosmetic. This forms an interesting aspect, particularly in the context of surgical wound healing.

[0068] In specific embodiments, the medical use or treatment described herein results in an improvement in the inflammatory profile of the wound site relative to an untreated wound. Preferably, the medical use or treatment results in an improvement in the macrophage and neutrophil profile, indicating a reduction in local wound inflammation relative to an untreated wound.

[0069] Optionally, the subject is an elderly subject, such as a subject of ≥50 years old or preferably ≥60 years old. Optionally, the (drug) composition or hydrogel described herein is applied to the wound site as part of treatment or medical use in at least two different cases. Optionally, the treatment can last for at least 1 week, even 1 month or longer, such as 12 weeks. Alternatively, the (drug) composition or hydrogel described herein is continuously applied to the wound site over a long period of time. Preferably, the long period of time corresponds to at least 1 day, at least 1 week or at least 1 month. In a specific embodiment, the (drug) composition or hydrogel described herein is contained in a wound dressing, a bandage, a patch or a plaster. Each type of scaffold or matrix can be used. Treatment may also require a longer duration, such as 12 weeks, and the form described herein is particularly suitable for facilitating an extended administration period.

[0070] A further aspect of the invention relates to a packaging unit of a pharmaceutical composition comprising a hydrogel as described in any of the embodiments herein. The packaging unit preferably comprises one or more dosage units of the (pharmaceutical) composition (or optional hydrogel) as described herein. Suitable packaging units include any container capable of enclosing and preserving a liquid. Optionally, the packaging unit is a box, a display unit, an ampoule, a bottle, a vial, a tube, a syringe, a cartridge, a bag, a sachet, a pouch, a film, a laminate, a foil, a can, a cylinder or a pressure vessel.

[0071] The above and further aspects and embodiments of the invention will be described in the following sections and in the appended claims.The subject matter of the appended claims is hereby expressly incorporated into this description. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Mean cumulative amount of estetrol monohydrate released per unit area across the isopore membrane for the 1 to 8 h experimental period (expressed as the square root of time) for the 10 formulations in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution (μg / cm 2 ). Error bars represent standard deviation of the mean (n=6).

[0073] Figure 2Average cumulative amount of estetrol monohydrate released per unit area across the isopore membrane for the seven aqueous gel formulations in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over a 1 to 8 hour experimental period (expressed as the square root of time) (μg / cm 2 ). Error bars represent standard deviation of the mean (n=6).

[0074] Figure 3 Mean cumulative amount of estetrol monohydrate released per unit area across the isopore membrane for the three cream formulations in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over a 1 to 8 h experimental period (expressed as the square root of time) (μg / cm 2 ). Error bars represent standard deviation of the mean (n=6).

[0075] Figure 4 Mean percentage (%) of estetrol monohydrate released across the isopore membrane into the receptor solution in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over a 1 to 8 hour experimental period (expressed as the square root of time) for the four aqueous gel formulations applied dose. Error bars represent standard deviation of the mean (n=5-6).

[0076] Figure 5 Average cumulative amount of estetrol monohydrate released per unit area across the isopore membrane for the four aqueous gel formulations in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over a 1 to 8 hour experimental period (expressed as the square root of time) (μg / cm 2 ). Error bars represent standard deviation of the mean (n=5-6).

[0077] Figure 6 .Topical placebo (PBO), Effects of (EG), AG24, AG25, and AG26 application on uterine weight and morphology. Eight-week-old female mice were injected subcutaneously with LPS 24 and 2 hours before injury (6 mice per group). LPS was applied in a thin layer on top of the wound one day before, at the time of injury, and 1, 2, 3, and 4 days after injury. AG24, AG25, AG26, or placebo. Changes in uterine morphology at 5 days were assessed by measuring uterine weight (A) and taking uterine photographs (B). Scale bar = 10 mm. Results are presented as mean ± sem. Differences from placebo were determined using paired t-tests; *P value ≤ 0.05, ***P value ≤ 0.001

[0078] Figure 7Topical E4 treatment promotes epithelial regeneration in a mouse model of LPS-induced delayed wound healing. Eight-week-old female mice were injected subcutaneously with LPS 24 and 2 hours before wounding (6 mice per group). E4 was then applied in a thin layer on top of the wound one day before, at the time of wounding, and 1, 2, 3, and 4 days after wounding. AG24, AG25, AG26, or placebo. The percentage of wound epithelial regeneration on day 5 was calculated using histological images. K14 immunohistochemistry was performed on histological sections to visualize the newly formed epidermis. The extent of epithelial regeneration was defined as the length of the new epidermis divided by the distance between the wound edges, multiplied by 100. Results are presented as mean ± SEM. Differences from placebo were determined using paired t-tests; *P value ≤ 0.05, ***P value ≤ 0.001, n = 6 mice per group. Mean + SEM.

[0079] Figure 8 Topical E4 treatment leads to a decrease in the number of neutrophils in the wound. Immunohistochemistry and quantification of neutrophils in the wound (A), representative images of each treatment group (B). **p≤0.01, n=6 mice per group. Mean + SEM.

[0080] Fig. 9 Topical E4 treatment leads to a decrease in the number of wound macrophages. Immunohistochemistry and quantification of wound macrophages (A), representative images of each treatment group (B). *p≤0.05, **p≤0.01, n=6 mice per group. Mean + SEM.

[0081] Fig.10 Topical E4 treatment promoted a pro-resolution wound phenotype with decreased expression of M1 markers and increased expression of M2 markers. Wound tissue RNA was isolated and wound expression of M1 (TNF-a and IL1-β) and M2 (Fizz1 and Ym1) markers was quantified by qPCR. *p≤0.05, **p≤0.01, n=6 mice per group. Mean + SEM.

[0082] Fig.11 In vitro polarization of peritoneal macrophages isolated from experimental mice demonstrates systemic effects of local treatment on immune cell phenotype. Peritoneal macrophages were isolated after completion of in vivo studies. Cells were cultured and polarized to either M1 or M2 phenotype in vitro, and expression of M1 (Tnf-α and iNOS) and M2 (Arg1 and Ym1) markers was quantified by qPCR. *p≤0.05, n=3 replicates on pooled cells from n=6 mice. Mean + SEM.

[0083] Fig.12 .Topical placebo (PBO), Effects of (EG), AG26, and AG28 application on uterine weight and morphology. Eight-week-old female mice were injected subcutaneously with LPS 24 and 2 hours before wounding (6 mice per group). LPS was applied in a thin layer on top of the wound only on day 0 (single administration) or on days -1, 0, 1, and 2 (repeated administration). AG26, AG28, or their placebo. Uterine morphological changes were assessed on day 3 by measuring uterine weight. Results are presented as mean ± sem. Differences from placebo were determined using paired t-tests; *P value ≤ 0.05, ***P value ≤ 0.001

[0084] Figure 13. A) Local placebo (PBO), Effects of (EG), AG26, and AG28 application on epithelial regeneration. Eight-week-old female mice were injected subcutaneously with LPS 24 and 2 hours before wounding (6 mice per group). LPS was applied in a thin layer on top of the wound only on day 0 (single administration) or on days -1, 0, 1, and 2 (repeated administration). AG26, AG28, or their placebo. Histological images were used to calculate the percentage of wound epithelial regeneration on day 3. Histological sections were subjected to K14 immunohistochemistry analysis to observe the newly formed epidermis. The extent of epithelial regeneration was defined as the length of the new epidermis divided by the distance between the wound edges, multiplied by 100. Results are expressed as mean ± sem. Differences from placebo were determined using a paired t-test; *P value ≤ 0.05, ***P value ≤ 0.001, n = 6 mice per group. Mean + SEM. B) and C) A single topical E4 administration has led to a decrease in the number of wound neutrophils in the LPS-induced delayed wound healing mouse model. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before injury (6 mice per group). Apply in a thin layer on top of the wound only on day 0 (single administration) or on days -1, 0, 1, and 2 (repeated administration). (EG, 0.06% E2 gel), AG24 (0.5% E4 gel), AG25 (0.22% E4 gel) and AG26 (0.06% E4 gel) or placebo (PBO). The number of neutrophils per square centimeter was quantified on tissue sections at day 3. **p≤0.01, n=6 mice per group. B) Mean+SEM and C) individual data points.

[0085] Fig.14 .E2 and E4 both promote migration of human dermal fibroblasts. (A) Percent closure was calculated after 36 h of treatment with vehicle, a range of concentrations of E2 or E4. Data are from four independent fibroblast donors (n=8). (B) Representative images from the same donor. **p≤0.01, *p≤0.05. Mean + SEM.

[0086] Fig.15 . To evaluate whether the medium / serum composition affects the effect of E2 and E4 on the migration of human dermal fibroblasts. Cells were cultured in DMEM containing 10% charcoal-stripped (CS) FBS, then switched to 2% CS-FBS, 0% FBS, or 2% FBS, scraped and treated with vehicle, E2, or E4. Percent closure was calculated after 24 hours. Data (A) and representative images (B) are from a single donor (n=3). Mean + SEM.

[0087] Fig.16 .E2 and E4 both promote migration of high-passage mouse dermal fibroblasts. Percent closure was calculated 24 hours after treatment with vehicle, a range of concentrations of E2 or E4. Data are from cells isolated from a single mouse (n=4). Representative images of cells isolated from a single mouse. **p≤0.001, *p≤0.05. Mean + SEM.

[0088] Fig.17 .The effect of E4 on mouse dermal fibroblast migration is greater in high-passage cells. Percent closure was calculated 24 hours after treatment with vehicle, E2 or E4. Data are from low-passage cells (a, b) and high-passage cells (c, d). Results are shown for cells isolated from 3 independent mice (n=12). Representative images of cells isolated from the same mouse. **p≤0.001, *p≤0.05. Mean + SEM.

[0089] Fig.18 E4 increased the expression of ERα and ERβ in mouse dermal fibroblasts (MDF). -7 M) RNA isolated from treated MDFs, expression of ERα (Esr1), ERβ (Esr2) was measured by qPCR. Data are from cells from n=3 independent mice. **p≤0.01, *p≤0.05. Mean + SEM.

[0090] Fig.19 .E2 and E4 inhibit MMP2 activity in human dermal fibroblast (HDF) supernatants. HDFs from n=3 independent donors were treated with E2 or E4. Zymograms were performed, including standards for MMP2 and MMP9. Data (A) are from n=6 experiments, n=3 independent donors. Representative zymograms from a single experiment / donor are shown (B). **p≤0.01, *p≤0.05. Mean+SEM.

[0091] Fig. 20.E4 treatment increases the expression of ECM components in dermal fibroblasts from diabetic mice but not from wild-type mice. Expression of Col1a1 and Fn1 was measured by qPCR using RNA isolated from MDFs of diabetic (db / db) mice or wild-type mice treated with E2, E4. Data are from cells from n=3 independent mice. *p≤0.05. Mean+SEM.

[0092] Fig.21 .E2 and E4 both promote migration of normal human epidermal keratinocytes (NHEKs). After treatment with vehicle, a range of concentrations of E2 or E4, the percentage of closure was calculated after 24 hours. Data (A: 15% human keratinocyte growth supplement (HKGS), B: 30% HKGS) are from a single primary NHEK donor (n=9-15). Representative image areas (C) are from a single experiment. **p≤0.01, *p≤0.05. Mean + SEM.

[0093] Fig. 22 .E4-treated primary mouse epidermal keratinocytes showed a strong trend toward increased expression of ERα and ERβ, as well as changes in keratinocyte phenotypic markers. Expression of ERα (Esr1), ERβ (Esr2), epithelial-mesenchymal transition marker Snail, and keratinocyte differentiation marker Krt1 were measured by qPCR using RNA isolated from MEK treated with a range of concentrations of E2 or E4 or E2 / E4 + ER antagonist ICI for 24 hours. Data are from cells from n=3 independent mice. *p≤0.05. Mean + SEM.

[0094] Fig.23 Experiments in the human immune THP1 cell line showed that both E2 and E4 have anti-inflammatory activity. THP1 cells were differentiated into a macrophage phenotype by PMA treatment and then polarized to either the M1 or M2 phenotype. The expression of the M1 marker TNF-α and the M2 marker CCL17 was then measured by qPCR using RNA isolated from polarized cells treated with different concentrations of E2 or E4. n=3. Mean + SEM.

[0095] Fig.24.Proinflammatory markers were reduced after treating M1-stimulated L929-differentiated mouse bone marrow-derived macrophages (BMDM) with E2 or E4. MBDM were differentiated using 20% ​​L929 growth medium and polarized to the M1 phenotype using 20ng / ml IFN-γ, 10pg / ml LPS for 6 or 24 hours. Using RNA isolated from treated cells, co-treatment with E2 or E4 resulted in a strong trend of reduced expression (measured by qPCR) of M1 markers iNOS, Tnf-α, and IL1-β. Data are from pooled cells from n=3 independent mice. Mean + SEM.

[0096] Fig.25 Proinflammatory markers were reduced after treatment of M1-stimulated, MCSF-differentiated mouse bone marrow-derived macrophages (BMDM) with E2 or E4. BMDM were differentiated with 30 ng / ml MCSF and polarized to M1 with 100 ng / ml IFN-γ and 10 pg / ml LPS for 6 hours. -7 Cells were co-treated with E2 or E4 of M1. Expression of M1 markers iNOS, Tnf-α, and IL1-β was measured by qPCR using RNA isolated from treated cells. Data are from pooled cells from n=3 independent mice. *p≤0.05. Mean+SEM.

[0097] Fig.26 In M1-polarized mouse peritoneal macrophages, pro-inflammatory markers were reduced after treatment with E2 or E4. Freshly isolated peritoneal macrophages were immediately treated with 10 -7 M cells were pretreated with E2 or E4 and then polarized to a proinflammatory M1 phenotype with 100 ng / ml IFN-γ and 10 pg / ml LPS for 6 hours. Expression of M1 markers iNOS, Tnf-α, and IL1-β was measured by qPCR using RNA isolated from pretreated cells. Data are from cells from n=3 independent mice. **p≤0.01*p≤0.05. Mean + SEM.

[0098] Fig. 27 .Co-treatment of HDFs with ER-specific antagonists showed that both ERα and ERβ were involved in E4-promoted fibroblast migration. Percent closure was calculated 24 hours after treatment with vehicle, E2, E4, or E2 / E4 and ERα-specific antagonist MPP or ERβ-specific antagonist PHTPP. Data are from two independent fibroblast donors (n=6). *p≤0.05vsE4. Mean+SEM.

[0099] Fig.28Mouse bone marrow-derived macrophages (BMDM) co-treated with ER-specific antagonists showed that both ERα and ERβ were involved in the anti-inflammatory activity promoted by E4. BMDM were isolated, differentiated using 20% ​​L929GM, and polarized to the pro-inflammatory M1 phenotype using 100 ng / ml IFN-γ and 10 pg / ml LPS for 6 h. E2 and E4 (10 -7 M) Stimulated M1 polarized cells were co-treated with the ERα-specific antagonist MPP or the ERβ-specific antagonist PHTPP. Expression of the pro-inflammatory marker IL1-β was measured by qPCR using RNA isolated from treated cells. Cells were from n=3 independent mice. **p≤0.01 vs E4. Mean + SEM.

[0100] Fig.29 .BMDM co-treatment with ER-specific antagonists showed that both ERα and ERβ were involved in the anti-inflammatory activity promoted by E4. BMDM were differentiated with 20% L929 GM and polarized to M1 with 100ng / ml IFN-γ and 10pg / ml LPS for 6 hours. M1 polarized cells were treated with (EG), AG23 placebo or AG23 active formulations. iNOS was measured by qPCR. Cells were from n=5-6 independent mice. *p≤0.05. **p≤0.01. Mean+SEM. DETAILED DESCRIPTION OF THE INVENTION

[0102] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0103] As used herein, the terms "comprising, comprises, or comprised of" are synonymous with "including, includes, containing, or contains" and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms also include "consisting of" and "consisting essentially of", which have well-known meanings in patent terms.

[0104] Numerical ranges recited by endpoints include all numbers and fractions subsumed within the corresponding ranges as well as the recited endpoints. This applies to numerical ranges regardless of whether they are introduced by the expression "from ... to ..." or the expression "between ... and ..." or another expression.

[0105] As used herein, the term "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., is meant to include variations of the specific value, such as ±10% or less, preferably ±5% or less, more preferably ±1% or less, and still more preferably ±0.1% or less, of the specific value, as long as these variations are suitable for implementation in the disclosed invention. It should be understood that the value referred to by the modifier "about" or "approximately" itself is also specifically and preferably disclosed.

[0106] Although the term "one or more" or "at least one" (such as one or more members or at least one member of a group of members, etc.) is clear in itself, by way of further example, the term particularly includes reference to any one of the members or any two or more of the members, such as any ≥3, ≥4, ≥5, ≥6 or ≥7 of the members, etc., up to all of the members. In another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0107] The discussion of the background of the invention herein is included for the purpose of explaining the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known or part of the common general knowledge in any country as at the priority date of any claim.

[0108] Throughout this disclosure, various publications, patents, and published patent specifications are cited by explicit reference. All documents cited in this specification are incorporated herein by reference in their entirety. In particular, the teachings or portions of such documents specifically mentioned herein are incorporated herein by reference.

[0109] Unless otherwise defined, all terms, including technical and scientific terms, used in disclosing the invention have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. By way of further guidance, term definitions are included to better understand the teachings of the present invention. When a particular term is defined in conjunction with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is intended to apply throughout the specification, i.e., also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products also apply to corresponding features of methods and uses.

[0110] In the following paragraphs, different aspects or embodiments of the invention are defined in more detail. Unless explicitly indicated to the contrary, each aspect or embodiment so defined can be combined with any other aspect or embodiment. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature indicated as being preferred or advantageous.

[0111] When "one embodiment" is mentioned throughout the specification, "embodiment" means that the specific features, structures or characteristics described in conjunction with the embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily all refer to the same embodiment. In addition, in one or more embodiments, specific features, structures or characteristics can be combined in any suitable manner, which will be apparent to those skilled in the art from this disclosure. In addition, although some embodiments described herein include some of the other embodiments, but do not include other features included in the other embodiments, the combination of features of different embodiments is meant to be within the scope of the present invention and to form different embodiments, as will be understood by those skilled in the art. For example, as will be understood by those skilled in the art, in the appended claims, alternative combinations of the claimed embodiments are included.

[0112] Including estrogens in pharmaceutical compositions suitable for topical application has been described in the art. An example of a commercially available topical formulation is It is a gel that contains estradiol. Indicated for the treatment of moderate to severe vasomotor symptoms and moderate to severe menopausal symptoms of vulvar and vaginal atrophy. In terms of wound healing, it has potential benefits, but it has not yet been listed with this indication. In addition, it is well known that conventional estrogen preparations are not suitable for direct application to individuals, and have the characteristics of a large number of adverse reactions, which may be related to the use of estradiol and / or accidental systemic exposure. Such adverse reactions may include nausea, vomiting, gastrospasm, abdominal distension, swelling, weight gain, breast pain, breast tenderness, headache, vaginal itching, vaginal discharge, menstrual disorders, spotting, alopecia, etc. (see Mayo Clinic report on transdermal estradiol: Estradiol (Transdermal Route) Side Effects-Mayo Clinic). Therefore, known pharmaceutical compositions are accompanied by considerable adverse systemic effects on individuals. As described in the summary of the invention of the present disclosure, a pharmaceutical composition comprising an estradiol component is provided, which has at least the wound healing properties similar to the pharmaceutical composition described in the art, but shows a significantly weakened systemic effect. For example, although the known wound healing compositions comprising estradiol cause a significant increase in uterine weight, indicating a systemic response to estradiol, the compositions comprising the estrrol component increase uterine weight to a lesser extent, and some compositions even have little effect on uterine weight, but are still effective for wound healing. Even after the addition of a penetration enhancer, this observation remains effective. This makes it possible to formulate a pharmaceutical composition comprising a relatively low estrogen dose. In view of the fact that estrrol has always been considered to be a weak estrogen compared to other estrogens (such as estradiol) (Gérard et al., J Endocrinol, 2015), the wound healing properties of such low doses of the estrrol component are also very significant. Therefore, based on knowledge known in the art, it is impossible to anticipate or envision the medical use of the compositions described herein and the estrrol component for wound healing.

[0113] In view of this, a first aspect of the present invention relates to a pharmaceutical composition comprising from about 0.02% to about 0.18 (w / w) of an estetrol component.

[0114] As used throughout this document, the term "estetrol component" encompasses a substance selected from the group consisting of: estetrol, esters of estetrol, esters of estetrol in which at least one hydrogen atom of a hydroxyl group is replaced by an acyl group of a hydrocarbon carboxylic acid, sulfonic acid or sulfamic acid having 1 to 25 carbon atoms, hydrates of estetrol such as monohydrated estetrol; and combinations thereof. It should be understood that when estetrol is mentioned in any part throughout this specification, any estetrol-containing component (i.e., compound) and / or estetrol derivative (such as those mentioned above) is also contemplated. More preferably, in the context of the present disclosure, a particularly preferred estetrol component suitable for use in the dosage units or cosmetic or medical uses and methods of treatment described herein is estetrol (including hydrates thereof). Most preferably, the estetrol component is monohydrated estetrol.

[0115] As used herein, the term "estetrol" refers to 1,3,5 (10) -estraene -3,15α,16α,17β-tetrol or 15α-hydroxy estriol and hydrates of estetrol, such as monohydrated estetrol. "Estetrol", or "E4" for short, is an estrogenic steroid produced by human fetal liver (PubChem CID: 27125). Estetrol can be described as a 3-hydroxy steroid corresponding to 17β-estradiol in which the 15α and 16α positions are substituted by two additional hydroxyl groups. It is known that estetrol is an estrogen receptor agonist (Coelingh Bennink et al., Estetrol review: profile and potential clinical applications, Climacteric, 2008). In the case where the estetrol component described herein represents estetrol, the estetrol may be endogenous estetrol. Alternatively, estetrol may be chemically synthesized, synthesized by using a (mutated) recombinant enzyme, or synthesized by any combination thereof. Alternatively, in the art, estetrol may be represented by its molecular formula: 18 H 24 O4, or represented by structural formula (I):

[0116] Formula (I)

[0117]

[0118] "(w / w)", or as expressed in the art by terms such as "weight ratio" or "weight to weight", refers to the contribution of a particular molecule or substance within a composition or mixture, measured by its weight (ie, mass).

[0119] In a specific embodiment, the composition comprises about 0.01% to about 0.18% (w / w) of the estetrol component (e.g., estetrol). In a preferred embodiment, the composition comprises about 0.02% to about 0.16% (w / w) of the estetrol component (e.g., estetrol), preferably about 0.03% to about 0.14% (w / w) of the estetrol component (e.g., estetrol), more preferably about 0.04% to about 0.12% (w / w) of the estetrol component (e.g., estetrol), still more preferably about 0.05% to about 0.10% (w / w) of the estetrol component (e.g., estetrol), even more preferably about 0.05% to about 0.08% (w / w) of the estetrol component (e.g., estetrol).

[0120] In alternative specific embodiments, the composition comprises about 0.18% (w / w) or less of an estetrol component (e.g., estetrol). In preferred embodiments, the composition comprises about 0.16% (w / w) or less of an estetrol component (e.g., estetrol), preferably about 0.14% (w / w) or less of an estetrol component (e.g., estetrol), more preferably about 0.12% (w / w) or less of an estetrol component (e.g., estetrol), more preferably about 0.10% (w / w) or less of an estetrol component (e.g., estetrol), even more preferably about 0.08% (w / w) or less of an estetrol component (e.g., estetrol).

[0121] Optionally, the composition comprises about 0.04% to 1% (w / w) estetrol. In certain embodiments, the composition comprises about 0.05% to 0.5% (w / w) estetrol. In further embodiments, the composition comprises about 0.06% to 0.5% (w / w) estetrol.

[0122] In a preferred embodiment, estetrol is present or used herein in the form of a monohydrate. Therefore, in a specific embodiment, the pharmaceutical composition comprises about 0.01% to about 0.18% (w / w) of estetrol monohydrate. In a preferred embodiment, the composition comprises about 0.02% to about 0.16% (w / w) of estetrol monohydrate, preferably about 0.03% to about 0.14% (w / w) of estetrol monohydrate, more preferably about 0.04% to about 0.12% (w / w) of estetrol monohydrate, more preferably about 0.05% to about 0.10% (w / w) of estetrol monohydrate, even more preferably about 0.05% to about 0.08% (w / w) of estetrol monohydrate.

[0123] In alternative specific embodiments, the pharmaceutical composition comprises about 0.18% (w / w) or less estetrol monohydrate. In a preferred embodiment, the composition comprises about 0.16% (w / w) or less estetrol monohydrate, preferably about 0.14% (w / w) or less estetrol monohydrate, more preferably about 0.12% (w / w) or less estetrol monohydrate, more preferably about 0.10% (w / w) or less estetrol monohydrate, even more preferably about 0.08% (w / w) or less estetrol monohydrate.

[0124] The above embodiments do not exclude the presence of non-estrrol estrogen components in the pharmaceutical composition. It is also envisioned that different estrrol components are present in a single composition. In such embodiments, the composition may include estrrol, or more specifically, estrrol monohydrate and estrrol esters, by way of example and without limitation.

[0125] "Topical application" means application to a specific location on or in the body. In particular, application to a body surface, such as the skin or mucous membranes, is contemplated. When used topically, a local effect is generally sought, in particular a local effect on the skin and mucous membranes. Although topical application also includes application to the vagina, in this context topical application is preferably understood as application to the skin.

[0126] In a specific embodiment, the pharmaceutical composition comprises a penetration enhancer capable of penetrating the stratum corneum. In the context of the present invention, the term "penetration enhancer" can be used interchangeably with terms including but not limited to "permeability enhancer", "permeability increaser", "permeability inducer" and even "skin enhancer". It will be appreciated by those skilled in the art that the term "penetration enhancer" is used throughout this disclosure to represent a penetration enhancer molecule that is part of a solvent or solvent system. Different penetration enhancers have been described in detail in the art, including but not limited to the penetration enhancers listed in the CPE database (Vasyuchenko et al., Pharmaceutics, 2021). Exemplary and non-limiting penetration enhancers are further described herein.

[0127] The composition and different cellular structural layers of the skin are known. As will be appreciated by those skilled in the art, human skin can generally be considered to comprise three different layers: the epidermis, the dermis and the subcutaneous layer. The epidermis is the upper layer of the skin and is mainly composed of keratinocytes, i.e. epithelial cells that proliferate and differentiate to ultimately produce the stratum corneum (the outermost layer of dead skin cells). The epidermis forms a barrier to environmental pathogens such as bacteria, regulates the amount of water released by the body, and plays a major role in wound healing. The second layer of skin, the dermis (or "dermis" or "skin connective tissue"), is located between the subcutaneous layer and the epidermis and is mainly composed of (mesenchymal) fibroblasts. The dermis is tightly connected to the epidermis by a basement membrane (i.e. a sheet-like extracellular matrix). The dermis is also much thicker than the epidermis, and the fibroblasts in the dermis produce the extracellular matrix (collagen, glycosaminoglycans including hyaluronic acid, elastic fibers...). The main functions of the dermis are to maintain skin thickness and elasticity, maintain skin moisture (through the water-retaining capacity of glycosaminoglycans including hyaluronic acid), and wound healing, i.e., by reforming and remodeling the damaged extracellular matrix. The deepest layer of the skin is usually referred to as the "subcutaneous layer (layer)", which is annotated interchangeably in the art with terms such as "subcutis" and "hypoderm", "subcutis" and "superfacial fascia".

[0128] Therefore, it will be understood by those skilled in the art that the "stratum corneum" referred to herein refers to the outermost epidermis composed of multiple layers of terminally differentiated keratinocytes (mainly composed of the protein substance keratin). The composition, function and details of the stratum corneum have been described in detail in the art (e.g., Matsui and Amagai, Int Immunol, 2015). It is generally believed that the rate at which a pharmaceutically active agent penetrates into the skin is limited by the stratum corneum. Therefore, the penetration enhancer contemplated by the present invention helps at least the estetrol component penetrate into the stratum corneum and / or the wound eschar. Penetration is particularly achievable in burn wounds.

[0129] The term "pharmaceutically active ingredient" is used interchangeably with "pharmaceutically active agent" in this disclosure and should be interpreted in accordance with the World Health Organization's definition of that term: "a substance used in a finished pharmaceutical product (FPP) that is intended to exhibit pharmacological activity or otherwise have a direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or in restoring, correcting or modifying a physiological function in humans."

[0130] The compositions described herein are particularly suitable for wound healing and are therefore interchangeably referred to as "pharmaceutical compositions" in any given case. Preferably, the compositions described herein are used for topical wound healing. The expression "for topical wound healing" referred to herein relates to treating one or more wounds of a subject, wherein the area of ​​the wound can be defined by a certain surface area. In addition, the expression refers to treating a skin wound of a subject using the composition described herein. The term "topical wound healing" further refers to topically administering (i.e., applying) the composition described herein to the wound area. Topical administration may also involve the use of transdermal administration methods, such as, but not limited to, transdermal patches, as further discussed in this disclosure.

[0131] The terms "wound area" and "wound size" as used herein refer to a physical measure of the disruption of the normal continuity of the structure. The wound area or wound size can be expressed, for example, in square centimeters. Depending on the wound size or area, it may be necessary to adjust the concentration of the composition to be applied to the wound so as not to exceed the maximum tolerated dose. In this case, the composition can be prepared specifically for the subject, or different grades or concentrations of the composition can be prepared in advance. Therefore, for larger wounds, it is recommended to use a lower concentration of the composition of the present invention, while for smaller wounds, a higher concentration can be used. If the results of the mouse study are generalized to the human case, it can be calculated that a concentration of 0.06% (w / w) in mice is equivalent to 0.1 mg of the estratetol component in the hydrogel applied to human subjects. Therefore, it can be assumed that topical application of about 0.1 mg of the estratetol component to the subject will not produce systemic effects, such as thickening of the uterus in female subjects. The higher dose of 0.5% (w / w) in mice can be extended to 0.9 mg or about 1 mg of estratetol, which can be applied topically without significant risk of systemic effects, or at least more than the commercially available hydrogel containing 0.06% estradiol. The systemic effects are smaller and the It has received regulatory approval for topical application.

[0132] The terms "subject", "individual" or "patient" are used interchangeably herein and generally and preferably refer to humans, but non-human animals, preferably warm-blooded animals, and even more preferably mammals, such as non-human primates, rodents, canines, felines, equines, sheep, pigs, etc., may also be encompassed. The term "non-human animal" includes all vertebrates, such as mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and non-mammals, such as chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a non-human mammal. Preferred subjects are human subjects, including all genders and all age categories. The term "subject" is intended to cover adult subjects, newborn subjects, and fetuses. Therefore, both adult and newborn subjects should be covered. Examples of subjects include humans, dogs, cats, cattle, goats, and mice. Preferred subjects in the context of the present invention are further defined below.

[0133] In another related aspect, the present invention relates to an estrol component for wound healing, more specifically local or topical wound healing. Therefore, the present invention contemplates using an effective amount of an estrol component for wound healing by applying the estrol component to the skin or wound area of ​​a subject. The term "effective amount" refers to the amount required to obtain a physiological effect. The physiological effect can be achieved by a single dose or repeated doses. In certain embodiments, the present invention relates to an estrol component that can penetrate the stratum corneum in the presence of a penetration enhancer for wound healing. In a further embodiment, the present invention relates to a pharmaceutical composition for wound healing comprising an effective amount of an estrol component and a penetration enhancer. Preferably, the estrol component for wound healing is estrol, most preferably estrol monohydrate. In an alternative embodiment, the present invention relates to a pharmaceutical composition for wound healing, comprising an effective amount of an estrol component and a second composition comprising a penetration enhancer. In such embodiments, the specific order in which the first and second compositions are applied to the wound area (i.e., wound site) is not particularly limited. Therefore, these compositions can be applied sequentially or (close to) simultaneously.

[0134] In certain embodiments, the pharmaceutical composition is for wound healing and comprises about 0.01% to about 5% (w / w), for example, about 0.08 to about 1.2% (w / w), about 0.09% to about 1.1% (w / w), or about 0.1% to about 1% (w / w) of an estetrol component (e.g., estetrol, preferably estetrol monohydrate) and a penetration enhancer, preferably about 0.02% to about 2.5% (w / w) of an estetrol component and a penetration enhancer, more preferably about 0.02% to about 2% (w / w) of The estetrol component and the permeation enhancer, more preferably about 0.03% to about 1.5% (w / w) of the estetrol component and the permeation enhancer, still more preferably about 0.03% to about 1% (w / w) of the estetrol component and the permeation enhancer, still more preferably about 0.03% to about 0.75% (w / w) of the estetrol component and the permeation enhancer, even more preferably about 0.04% to about 0.5% (w / w) of the estetrol component and the permeation enhancer, and most preferably about 0.06% (w / w) of the estetrol component and the permeation enhancer.

[0135] In alternative embodiments, the pharmaceutical composition is for wound healing and comprises about 0.01% to about 5% or less (w / w), such as about 0.08 to about 1.2% (w / w), about 0.09% to about 1.1% (w / w), or about 0.1% to about 1% (w / w) of an estetrol component (e.g., estetrol, preferably estetrol monohydrate) and a penetration enhancer, preferably about 2.5% (w / w) or less of the estetrol component and the penetration enhancer, more preferably about 2% (w / w) or less. Less estetrol component and permeation enhancer, more preferably about 1.5% (w / w) or less estetrol component and permeation enhancer, even more preferably about 1% (w / w) or less estetrol component and permeation enhancer, even more preferably about 0.75% (w / w) or less estetrol component and permeation enhancer, even more preferably about 0.5% (w / w) or less estetrol component and permeation enhancer, most preferably about 0.1% (w / w) or less estetrol component and permeation enhancer.

[0136] Preferably, the pharmaceutical composition described herein is a hydrogel, or is contained in a hydrogel. A gel is a semisolid system in which a liquid is solidified by a gel skeleton forming agent. In a hydrogel, the liquid that forms the gel is water or an aqueous solution. The term "hydrogel" as used herein refers to an aqueous solution of an active ingredient, which is primarily solidified with a macromolecular hydrophilic substance to form a gel. Macromolecular hydrophilic substances and polymer materials swell when in contact with water, and depending on the concentration, a solution having a pseudoplastic flow behavior or a plastic structure containing an essential aqueous component is produced. Therefore, a hydrophilic gel is composed of water or an aqueous solution that is usually gelled with a hydrophilic macromolecular compound. Gels constructed with a hydrophilic macromolecular scaffold are generally thixotropic. In contrast to creams, gels are referred to as true single-phase systems.

[0137] The hydrogel improves or restores the moisture balance of the wound bed by absorbing excess liquid to balance hydration. The hydrogel contemplated herein may include any suitable polymer or combination of polymers, such as but not limited to hydrophilic polymers, acrylic acid, acrylamide and 2-hydroxyethyl methacrylate. Hydrogels are particularly preferred forms of the compositions disclosed herein because they can provide moisture balance for the wound bed by absorbing excess liquid to balance hydration. Examples of hydrogels include but are not limited to synthetic hydrogels, irritation-sensitive hydrogels, (poly)peptide-based hydrogels, hybrid hydrogels and DNA-based hydrogels. The production methods of each of these hydrogel classes have been described in the art, and are therefore known to those skilled in the art.

[0138] By way of example and not limitation, examples of synthetic hydrogels include double network hydrogels. Examples also include nanocomposite hydrogels. Stimuli-sensitive hydrogels are characterized in that they can undergo swelling changes mediated by external stimuli (e.g., pH, temperature, ionic strength, solvent type, electric field, magnetic field, light, and chelates). Examples of stimulus-sensitive hydrogels include, but are not limited to, hydrogels formed by block co-polypeptide recombinant fragments of natural structural proteins (e.g., elastin, silk, filamentous, and elastin-like peptide blocks) and recombinant triblock copolymers of one or more polypeptide sequences.

[0139] The term "hybrid hydrogel" herein refers to a hydrogel comprising components derived from at least two different molecular classes (eg, synthetic polymers and biomacromolecules) that are interconnected in a covalent or non-covalent manner.

[0140] In the gel formulations described herein, some excipients (e.g., propylene glycol, glycerol, The presence of benzyl alcohol and PEG400 can increase the solubility of estetrol. The use of these excipients in a gel formulation can allow estetrol to dissolve and completely penetrate through the membrane, or similarly, penetrate through the stratum corneum and / or wound eschar and / or enter the burn wound. This helps to increase the rate of diffusion through the membrane or similarly, penetrate through the stratum corneum and / or wound eschar and / or enter the burn wound, as predicted by Fick's law of diffusion, because a higher estetrol concentration gradient can be generated by increasing the solubility of estetrol between the two sides of the membrane.

[0141] Compared with the gel formulation, the cream diffusion curve shows that the release rate of estetrol is slower in the same time. In the cream formulation, the estetrol component may be located in the internal phase of the emulsion and needs to diffuse to the external phase of the emulsion before it can diffuse through the barrier membrane, or similarly, diffuse through the stratum corneum and / or wound eschar and / or into the burn wound. However, the high partition coefficient of the cream matrix prevents the rapid diffusion of estetrol along its concentration gradient. The whole process is slow and the release rate of estetrol drops sharply.

[0142] It should be understood that any reference to a "composition" and "pharmaceutical composition" encompasses any hydrogel described herein, and vice versa.

[0143] Preferably, the hydrogels contemplated herein comprise a penetration enhancer capable of penetrating the stratum corneum. More preferably, the hydrogels referred to herein are hydrogels comprising from about 0.02% to about 10% (w / w) of an estetrol component (eg estetrol, preferably estetrol monohydrate). Preferably, the hydrogel referred to herein is a hydrogel comprising about 0.02% to about 5% (w / w) estetrol component, more preferably, the hydrogel referred to herein is a hydrogel comprising about 0.02% to about 2.5% (w / w) estetrol component, more preferably, the hydrogel comprises about 0.03% to about 0.75% (w / w) estetrol component, preferably about 0.04% to about 0.5% (w / w) estetrol component, more preferably about 0.05% to about 0.25% (w / w) estetrol component, for example, about 0.08% to about 1.2% (w / w), about 0.09% to about 1.1% (w / w) or about 0.1% to about 1% (w / w), most preferably the hydrogel comprises about 0.06% (w / w) estetrol component.

[0144] Alternatively, the hydrogel described herein is a hydrogel comprising about 10% (w / w) or less of the estetrol component. Preferably, the hydrogel described herein is a hydrogel comprising about 5% (w / w) or less of the estetrol component, more preferably, the hydrogel described herein is a hydrogel comprising about 2.5% (w / w) or less of the estetrol component, more preferably, the hydrogel comprises about 0.75% (w / w) or less of the estetrol component, preferably, about 0.5% (w / w) or less of the estetrol component, more preferably, about 0.25% (w / w) or less of the estetrol component, more preferably, about 0.1% (w / w) or less of the estetrol component, most preferably, about 0.08% (w / w) or less of the estetrol component.

[0145] In certain embodiments where the (drug) composition is a hydrogel, the hydrogel is characterized by a favorable release profile, e.g. when compared to other formulations such as, but not limited to, creams. In preferred embodiments, the hydrogels described herein are characterized by an average cumulative amount of estratetol component released across an isopore membrane in a 40:30:30 v / v / v receptor solution of ethanol:PEG400:water of at least about 2.5 μg / cm 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 about 100 μg / cm2 , more preferably at least about 150 μg / cm 2 , more preferably at least about 200 μg / cm 2 In an alternative embodiment, the hydrogels described herein are characterized in that the average cumulative amount of estratetol component released across the isopore membrane in 1 hour in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 25 μg / cm 2 About 200 μg / cm 2 Depending on the wound surface, it may be necessary to reduce the concentration of the estratetol component to avoid excessive cumulative administration of estratetol. This can be easily calculated by a physician or pharmacist. In a preferred alternative embodiment, the hydrogel described herein is characterized in that the average cumulative amount of estratetol component released across the isopore membrane in 1 hour in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 25 μg / cm 2 About 100 μg / cm 2 In a further preferred alternative embodiment, the hydrogel described herein is characterized in that the average cumulative amount of estratetol component released across the isopore membrane in 1 hour in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 25 μg / cm 2 About 50 μg / cm 2 .

[0146] Preferably, the hydrogels described herein are characterized in that the average cumulative amount of estratetol component released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution over 8 hours is at least about 350 μg / cm 2 , preferably at least about 400 μg / cm 2 , more preferably at least about 450 μg / cm 2 .

[0147] Preferably, the hydrogels described herein are characterized in that the average % applied dose of the estratetol component in the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 1 hour is at least about 15%, preferably at least about 20%, and / or the average % applied dose of the estratetol component in the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 8 hours is at least about 40%, preferably at least about 50%, more preferably at least 80%. In alternative embodiments, the hydrogels described herein are characterized in that the average % applied dose of the estratetol component in the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 1 hour is about 15% to about 30%, and / or the average % applied dose of the estratetol component in the receptor solution of 40:30:30 v / v / v ethanol:PEG400:water after 8 hours is about 45% to about 90%.

[0148] In a highly preferred embodiment, the hydrogels described herein are characterized in that the release rate (i.e., slope) of the estetrol component is about 2.5 μg / cm 2 / √h, at least about 5μg / cm 2 / √h, at least about 7μg / cm 2 / √h, at least about 10μg / cm 2 / √h, at least about 15μg / cm 2 / √h, at least about 20μg / cm 2 / √h, at least about 25μg / cm 2 / √h to about 50μg / cm 2 / √h, and the average cumulative amount of estratetol component released after 8 hours is about 50 μg / cm 2 About 100 μg / cm 2 , and optionally, the percentage of the amount of estetrol released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 75% to about 95%. More preferably, the hydrogel described herein is characterized in that the estetrol component release rate (i.e., slope) is about 30 μg / cm 2 / √h to about 35μg / cm 2 / √h, the average cumulative amount of estratetol released after 8 hours is about 75μg / cm 2 About 90 μg / cm 2 , and optionally the percentage of the amount of estetrol released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 85% to about 90%. Most preferably, the hydrogel described herein is characterized in that the estetrol component release rate (i.e., slope) is about 33 μg / cm2 / √h to about 34μg / cm 2 / √h, the average cumulative amount of estratetol released after 8 hours is about 82μg / cm 2 About 83 μg / cm 2 , and optionally the percentage of estetrol released across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 86% to about 88%.

[0149] In embodiments where the composition is a cream, the cream may be characterized by a release rate (i.e., slope) of the estetrol component across an isopore membrane of about 2.5 μg / cm in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution. 2 / √h, at least about 5μg / cm 2 / √h, at least about 7μg / cm 2 / √h, at least about 10μg / cm 2 / √h, at least about 15μg / cm 2 / √h, at least about 20μg / cm 2 / √h, or 15μg / cm 2 / √h to about 75μg / cm 2 Preferably, the cream is characterized in that the release rate (i.e., slope) of the estratetrol component across the isopore membrane in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution is about 25 μg / cm 2 / √h to about 55μg / cm 2 / √h.

[0150] The cream is also characterized in that the average cumulative amount of the estratetol component released across the isopore membrane after 1 hour in a receptor solution of 40:30:30 v / v / v ethanol:PEG400:water is about 1 μg / cm 2 About 100 μg / cm 2 Preferably, the cream is characterized in that the average cumulative amount of the estratetol component released through the isopore membrane after 1 hour in a receptor solution of 40:30:30 v / v / v ethanol:PEG400:water is about 5 μg / cm 2 About 80 μg / cm 2 The cream is also characterized in that the average cumulative amount of the estratetol component released across the isopore membrane after 8 hours in a receptor solution of 40:30:30 v / v / v ethanol:PEG400:water is about 25 μg / cm 2 About 150 μg / cm 2Preferably, the cream is characterized in that, in a receptor solution of 40:30:30 v / v / v ethanol:PEG400:water, the average cumulative amount of the estratetol component released across the isopore membrane after 8 hours is about 50 μg / cm 2 About 120 μg / cm 2 .

[0151] It is obvious to those skilled in the art that the present disclosure encompasses the use of each specific form of the pharmaceutical composition disclosed herein for wound healing. Therefore, the present invention also relates to the hydrogel disclosed herein for wound healing.

[0152] Although hydrogel is the preferred form of the pharmaceutical composition disclosed in the present disclosure, this does not exclude other topical preparations known in the art. Therefore, suitable preparations include, but are not limited to, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, sprays and creams. Each of these terms is intended to correspond to its generally accepted meaning. Similarly, these topical preparations can be directly applied to the skin, or can be used in combination with dressings, patches, bandages, band-aids, tampons, plasters, etc., to prevent the preparation from falling off the skin, and in some embodiments protect the wound from external influences, such as dirt and microorganisms.

[0153] "Emulsion" refers broadly to any mixture of at least two immiscible (i.e., immiscible, non-mixable) liquids, whereby the first liquid is distributed in small droplets (dispersed phase) in the second liquid (dispersion medium). Thus, in certain embodiments, the pharmaceutical compositions described herein are oil-in-water or water-in-oil emulsions. Emulsions are widely used in skin care formulations and can be divided into creams and lotions. "Suspensions" in this context broadly refer to heterogeneous mixtures containing solids dispersed in a liquid phase, which solids are insoluble and large enough to allow precipitation.

[0154] "Cream" usually refers to a water-in-oil emulsion, where the aqueous phase is dispersed in the oil phase, but can also refer to an oil-in-water emulsion, where the oil is dispersed in an aqueous base. It is generally accepted that creams differ from lotions in that emulsions are stable suspensions of small droplets of an immiscible fluid in the emulsion, whereas creams refer to a specific class of emulsions that are more viscous and typically contain more lipophilic and / or surfactant components.

[0155] "Lotion" is a liquid composition of low to medium viscosity. Typically, lotions have a lower viscosity than creams, but in some cases, the viscosities of the two may be similar. Lotions may contain finely powdered materials that are insoluble in the dispersion medium through the use of suspending agents and dispersants. Alternatively, lotions may have a dispersed phase liquid material that is immiscible with the vehicle and is typically dispersed by an emulsifier or other suitable stabilizer. In one embodiment, the lotion is in the form of an emulsion with a viscosity between 100 and 1000 centistokes. The fluidity of the lotion allows for rapid and uniform application over a wide surface area. Lotions are typically intended to dry on the skin, leaving a thin layer of the medicinal ingredient on the surface of the skin.

[0156] "Ointment" refers broadly to a higher viscosity oil-in-water cream, i.e., a semisolid material containing an ointment base and one or more pharmaceutically active ingredients (in the present invention, an estetrol component). Examples of suitable ointment bases include hydrocarbon bases, absorbent bases, water-removable bases, and water-soluble bases. "Pastes" are generally different from ointments because they contain a greater proportion of solids. In general, pastes are more absorbent and less greasy than ointments based on the same ingredients / excipients.

[0157] As used herein, "foam" refers to a dispersion of gas particles in a liquid medium. Oil-in-water emulsions, water-in-oil emulsions, ethanol, water, solvents, liquid oils, propylene glycol and glycerol can be used as examples of liquid media in foams. It will be appreciated by those skilled in the art that foams can be produced by reducing the surface tension of a liquid, mixing in gaseous substances, and forming bubbles. The acceptability of foams is that they are easy to apply to large areas of skin, do not leave a greasy film, and can be quickly absorbed by the skin. "Mousse" refers to a substance that is very similar to foam, but is generally used to refer to a substance with a lower water content. "Spray" refers to a medicated solution that is loaded into a device suitable for spraying the medicated solution and released in a mist with the help of pressure.

[0158] As described above, any pharmaceutical composition (such as but not limited to the hydrogel disclosed herein) may include a penetration enhancer. Preferably, the penetration enhancer includes a molecule (i.e., a penetration enhancing molecule) that can penetrate the stratum corneum and a solvent or solvent system. Optionally, the penetration enhancer is present in the composition (such as but not limited to the hydrogel disclosed herein) in an amount of about 0.5% to about 60% (w / w), preferably in an amount of about 1% to about 50% (w / w), more preferably in an amount of about 2.5% to about 45% (w / w), more preferably in an amount of about 5% to about 40% (w / w), more preferably in an amount of about 10% to about 30% (w / w) or alternatively in an amount of about 0.1% to about 5% (w / w). Optionally, the permeation enhancer molecule is present in the composition, such as but not limited to the hydrogel disclosed herein, in an amount of about 0.1% to about 25% (w / w), preferably in an amount of about 0.5% to about 15% (w / w), more preferably in an amount of about 1% to about 10% (w / w), more preferably in an amount of about 2.5% to about 7.5% (w / w), more preferably in an amount of 3.5% to about 5% (w / w). Optionally, the solvent (system) is present in the composition, such as but not limited to the hydrogel disclosed herein, in an amount of about 1% to about 60% (w / w), preferably in an amount of about 5% to about 50% (w / w), more preferably in an amount of about 10% to about 40% (w / w), more preferably in an amount of about 15% to about 30% (w / w), more preferably in an amount of 18% to about 25% (w / w).

[0159] The penetration enhancers described herein are not particularly limited to the present invention and thus may include or consist of molecules selected from the group consisting of: suberin, lignin, keratin, including dimethyl sulfoxide, ethanol, propylene glycol, glycerol, propyl glycol, urea, dimethylacetamide, sodium lauryl sulfate, poloxamer, span, tween, lecithin, terpenes and combinations thereof. Preferred penetration enhancing molecules in the context of the present invention include ethanol, ether, benzyl alcohol, fatty acids and their esters or any combination thereof. Particularly preferred penetration enhancer molecules in the context of the present invention include Benzyl alcohol and any combination thereof. Benzyl alcohol (C6H5CH2OH) is interchangeably referred to in the art as "phenylmethanol", "phenylmethyl alcohol" and "benzyl alcohol". is the generally accepted trade name for 2-(2-ethoxyethoxy)ethanol, which is also commonly identified in the art with diethylene glycol monoethyl ether (C6H 14 Optionally, the pharmaceutical compositions described herein comprise about 1% to about 20% (w / w), preferably about 2% to about 10% (w / w), more preferably about 3% to about 8% (w / w), most preferably from about 4% to about 6% (w / w) as a penetration enhancer.

[0160] The penetration enhancers of the present invention include a penetration enhancer molecule and a solvent or solvent system. Preferred solvents for the penetration enhancers include, but are not limited to, polyethylene glycol (PEG), propylene glycol (PG), and combinations thereof. "Polyethylene glycol" may be interchangeably referred to as, but not limited to, polyethylene oxide or poly(ethylene oxide), poly(ethylene oxide), and polyoxyethylene, which terms are well described in the art and are therefore known to those skilled in the art, who understand that polyethylene glycol is characterized by a chemical formula of H-(O-CH2-CH2) n -OH, wherein n is an integer. Polyethylene glycol is a polyether compound derived from petroleum. Preferred PEG is a PEG having a molecular weight of about 150 g / mol to about 5000 g / mol, more preferably about 200 g / mol to about 2500 g / mol, more preferably about 250 g / mol to about 1000 g / mol, and most preferably about 300 g / mol to about 600 g / mol. Therefore, the PEG mentioned herein can be selected from: PEG200, PEG300, PEG400, PEG500, PEG600 and any combination thereof. Most preferably, the penetration enhancer comprises a PEG having a molecular weight of about 400 g / mol (such as, but not limited to, PEG400) as a solvent or part of a solvent. Propylene glycol, commonly referred to in the art as propane-1,2-diol, α-propylene glycol, 1,2-propylene glycol, 1,2-dihydroxypropane. Propylene glycol is characterized in that the chemical formula is CH3CH(OH)CH2OH.

[0161] Preferably, the penetration enhancer comprises about 5% to about 50% (w / w), preferably about 5% to about 35% (w / w) or about 10% to about 45% (w / w) PEG, preferably PEG400, and / or about 10% to about 35% (w / w) PG. More preferably, the penetration enhancer comprises about 10% to about 30% (w / w) PEG, preferably PEG400, and / or about 12% to about 30% (w / w) PG. More preferably, the penetration enhancer comprises about 12% to about 25% (w / w) PEG, preferably PEG400, and / or about 15% to about 25% (w / w) PG. More preferably, the penetration enhancer comprises about 14% to about 23% (w / w) PEG, preferably PEG400, and / or about 16% to about 23% (w / w) PG. More preferably, the penetration enhancer comprises about 16% to about 22% (w / w) PEG, preferably PEG400, such as about 18% to about 22% (w / w) PEG or about 16% to about 20% (w / w) PEG, and / or about 18% to about 22% (w / w) PG.

[0162] Optionally, the pharmaceutical composition (optionally a hydrogel) comprises benzyl alcohol. The term "benzyl alcohol" should be interpreted according to its usual interpretation in the art and thus refers to an aromatic alcohol characterized by the chemical formula C6H5CH2OH. If present, the amount of benzyl alcohol in the composition is not particularly limited. However, the amount of benzyl alcohol is preferably from about 0.1% to about 10% (w / w), more preferably from about 0.5% to about 5% (w / w), more preferably from about 1% to about 3% (w / w), and more preferably from about 1.5% to about 2.5% (w / w).

[0163] Optionally, the pharmaceutical composition (which may be a hydrogel) comprises a thickener. "Thickener" may also be represented by terms such as, but not limited to, "thickener" and "viscosity agent". "Thickener" used in the context of the present invention represents any substance or molecule that increases the viscosity and / or texture of the composition when added to a liquid or semisolid composition. Therefore, if there is a thickener, the thickener may be selected from the following groups: carboxylic acid polymers, cross-linked polyacrylate polymers, polyacrylamide polymers, polysaccharides, diblock polymers, triblock polymers, gums, and any combination thereof. In embodiments where the thickener is a polysaccharide or comprises a polysaccharide, the polysaccharide may be selected from the following groups: cellulose, cellulose derivatives, carboxymethyl cellulose, cellulose acetate propionate carboxylate, hydroxyethyl cellulose, hydroxyethyl ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, methyl hydroxyethyl cellulose, hydroxyalkylated cellulose, lignin, cutin, 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, pullulan, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar gum, 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.

[0164] In certain embodiments, the pharmaceutical compositions described herein (which are optionally hydrogels) comprise a thickening agent as described herein in an amount of about 0.1% to about 25% (w / w), preferably in an amount of about 0.3% to about 20% (w / w), more preferably in an amount of about 0.4% to about 15%, more preferably in an amount of about 0.5% to about 10% (w / w), more preferably in an amount of about 0.75% to about 5% (w / w), or in an amount of about 0.3% to about 3% (w / w). In the context of the present invention, preferred thickening agents include, but are not limited to, thickening agents selected from the following groups: hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), high molecular weight cross-linked acrylic acid-based polymers, non-ionic triblock copolymers, or any combination thereof. In the context of the present invention, more preferred thickening agents include, but are not limited to, hydroxyethyl cellulose, high molecular weight cross-linked acrylic acid-based polymers, non-ionic triblock copolymers, and any combination thereof. A preferred high molecular weight cross-linked acrylic acid-based polymer is carbomer, which is interchangeably used under the trade name throughout the art. express. Homopolymer (i.e. acrylic acid cross-linked with allyl sucrose or allyl pentaerythritol), Copolymer (acrylic acid and C10-C30 alkyl acrylate crosslinked with allyl pentaerythritol), Interpolymers (carbomer homopolymers or copolymers, which contain block copolymers of polyethylene glycol and long chain alkyl acid esters) are all contemplated. Particularly preferred in the present invention are for 980, also known in the art as "Carbomer Homopolymer Type C USP NF", is a homopolymer of acrylic acid crosslinked with allyl sucrose or allyl pentaerythritol, with a viscosity of 40,000 to 60,000 cP in a co-solvent of cyclohexane and ethyl acetate. The preferred hydroxyethyl cellulose is HEC250 HHX. The preferred nonionic triblock copolymer is a nonionic triblock copolymer having a molecular weight of about 1800 g / mol and 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 a combination thereof.

[0165] In certain embodiments, the thickening agent is or includes HEC and is present in the pharmaceutical composition in an amount of about 0.1% to about 10% (w / w), preferably in an amount of 0.2% to about 7.5% (w / w), more preferably in an amount of about 0.5% to about 5% (w / w), more preferably in an amount of about 0.75% to about 2.5% (w / w), and most preferably in an amount of about 1% to about 2% (w / w). In certain embodiments, the thickening agent is or includes and is present in the composition in an amount of about 0.1% to about 10% (w / w), preferably in an amount of about 0.2% to about 7.5% (w / w), more preferably in an amount of about 0.5% to about 5% (w / w), more preferably in an amount of about 0.75% to about 2.5% (w / w), and most preferably in an amount of about 0.75% to about 1.5% (w / w). In alternative embodiments, In certain embodiments, the thickening agent is or includes poloxamer 188 and is present in the composition in an amount of about 0.1% to about 30% (w / w), preferably in an amount of about 1% to about 15% (w / w), and more preferably in an amount of about 2.5% to about 10% (w / w). In certain embodiments, the thickening agent is or includes poloxamer 407 and is present in the composition in an amount of about 0.1% to about 30% (w / w), preferably in an amount of about 5% to about 25% (w / w), and more preferably in an amount of about 10% to about 20% (w / w).

[0166] In certain embodiments, the thickening agent is or includes CMC and is present in the pharmaceutical composition in an amount of about 0.1% to about 10% (w / w), preferably in an amount of 0.2% to about 7.5% (w / w), more preferably in an amount of about 0.5% to about 5% (w / w), more preferably in an amount of about 0.75% to about 2.5% (w / w), and most preferably in an amount of about 1% to about 2% (w / w) or about 1.5% (w / w).

[0167] Optionally, the pharmaceutical composition (which is optionally a hydrogel) comprises a preservative. In a specific embodiment, the composition comprises a preservative in an amount of 0.5% to 20% (w / w), preferably in an amount of 1% to about 10% (w / w), more preferably in an amount of about 1% to about 3% (w / w). The exact preservative is not particularly limited for the present invention and can therefore be selected from the following group: lysozyme, nisin, quaternary ammonium preservatives, parabens, phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, natural preservatives and any combination thereof. In the context of the present invention, a preferred preservative is benzyl alcohol.

[0168] Optionally, the pharmaceutical composition (which can be a hydrogel) comprises an emollient. "Emollient" used in the present disclosure refers to a material that can be used to prevent and / or treat dry skin and provide additional protection for the skin. Specific emollients are not particularly limited for the present invention and can therefore be selected from the following group: glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerol, vaseline, petroleum and any combination thereof. An emollient particularly preferred in the context of the present invention is glycerol. The amount of emollient in the composition can be from about 2.5% to about 30% (w / w), preferably from about 5% to about 25% (w / w), more preferably from about 7.5% to about 20% (w / w), more preferably from about 8% to about 12% (w / w). In a further embodiment, the emollient may be glycerin, which is present in the composition in an amount of about 2.5% to about 30% (w / w), preferably about 5% to about 25% (w / w), more preferably about 7.5% to about 20% (w / w), more preferably about 8% to about 12% (w / w), and most preferably about 10% (w / w).

[0169] As described in detail in the present disclosure, any pharmaceutical composition described herein may also include a certain amount of components (i.e., pharmaceutically active agents and / or excipients) in addition to the estetrol component. In any embodiment of the present invention, a solvent may be added to reach a certain concentration of the components. In a further embodiment, an aqueous solution is used to supplement the composition. The term "aqueous solution" refers to any solution containing water or a solvent that is water. In addition, "aqueous solution" is used to describe a solution that has something in common with water or an aqueous solution, and is not limited to features such as appearance, odor, color, taste, viscosity, pH, absorbance, or physical state at a specific temperature. In such an embodiment, the aqueous solution may be water. In an alternative further embodiment, a non-aqueous solution is used to supplement the composition. In an alternative embodiment, a mixture of a non-aqueous solution and an aqueous solution is used to supplement the composition.

[0170] As defined herein, the pH of a composition, solution or formulation can be measured using various methods known to those skilled in the art. pH indicators that change color by absorbing or releasing H+ ions can be used, wherein the color they produce indicates a specific pH value. Alternatively, a pH meter can be used to measure the potential difference between a pH electrode and a reference electrode. The potential difference is related to the acidity or pH value of the solution.

[0171] In view of the above, the exemplary pharmaceutical composition of the present invention comprises, in addition to any concentration of estetrol component listed herein, further comprising:

[0172] - from about 0.1% to about 60% (w / w) of a penetration enhancer;

[0173] - from about 0.3% to about 20% (w / w) of a thickener;

[0174] -optionally preservatives and / or emollients;

[0175] - Make up to 100% (w / w) with water.

[0176] In certain embodiments, the pharmaceutical composition consists essentially of a penetration enhancer, a thickener, a preservative and an emollient (except the estetrol component), or consists of a penetration enhancer, a thickener, a preservative and an emollient (except the estetrol component). In a preferred embodiment, the composition of the present invention further comprises, in addition to the estetrol component:

[0177] - from about 0.1% to about 10% (w / w) of penetration enhancer molecules, preferably from about 0.25% to about 8% (w / w) of penetration enhancer molecules;

[0178] - from about 10% to about 60% (w / w) of a penetration enhancer solvent;

[0179] - from about 0.3% to about 20% (w / w) thickener, preferably from about 0.3% to about 10% (w / w) thickener, more preferably from about 0.3% to about 5% (w / w) thickener, most preferably from about 0.3% to about 3% (w / w) thickener;

[0180] -optionally preservatives and / or emollients;

[0181] - Make up to 100% (w / w) with water.

[0182] In a further preferred embodiment, the pharmaceutical composition of the present invention comprises, in addition to any concentration of the estetrol component listed herein, further comprising:

[0183] - from about 1% to about 7.5% (w / w) of permeation enhancer molecules, preferably from about 0.5% to about 5% (w / w) of permeation enhancer molecules;

[0184] - from about 15% to about 45% (w / w) of a penetration enhancer solvent;

[0185] - from about 0.3% to about 20% (w / w) thickener, preferably from about 0.3% to about 10% (w / w) thickener, more preferably from about 0.3% to about 5% (w / w) thickener, most preferably from about 0.3% to about 3% (w / w) thickener;

[0186] -optionally preservatives and / or emollients;

[0187] - Make up to 100% (w / w) with water.

[0188] In a specific embodiment, the pharmaceutical composition (optionally a hydrogel) comprises, consists essentially of, or consists of the following ingredients in addition to the estetrol component at any concentration listed herein:

[0189] - about 8% to about 40%, preferably about 16% to about 20%, most preferably about 18% (w / w) PEG400;

[0190] - about 9% to about 44%, preferably about 18% to about 22%, most preferably about 20% (w / w) PG;

[0191] - from about 4% to about 24%, preferably from about 8% to about 12%, most preferably about 10% (w / w) glycerol;

[0192] - from about 0.5% to about 4%, preferably from about 1% to about 2%, most preferably about 1.5% (w / w) HEC; and

[0193] - about 0.75% to about 1.25%, preferably about 1.5% to about 2.5%, most preferably about 2% (w / w) benzyl alcohol.

[0194] In a specific embodiment, the pharmaceutical composition (optionally a hydrogel) comprises, consists essentially of, or consists of the following ingredients in addition to the estetrol component at any concentration listed herein:

[0195] - from about 9% to about 44%, preferably from about 18% to about 22%, most preferably about 20% (w / w) PEG400;

[0196] - from about 0.01% to about 2%, preferably from about 0.1% to about 1%, most preferably about 0.5% (w / w) and

[0197] - from about 0.1% to about 15%, 2% to about 12%, preferably from about 4% to about 6%, most preferably about 5% (w / w)

[0198] In a specific embodiment, the pharmaceutical composition (optionally a hydrogel) comprises, consists essentially of, or consists of the following ingredients in addition to the estetrol component at any concentration listed herein:

[0199] - from about 10% to about 55%, preferably from about 30% to about 50%, most preferably about 40% to 45% (w / w) PEG400;

[0200] - from about 0.01% to about 2%, preferably from about 0.1% to about 1%, most preferably about 0.5% (w / w) and

[0201] - from about 0.1% to about 15%, from 0.2% to about 10%, preferably from about 0.5% to about 5%, most preferably from about 1% to about 2.5% (w / w)

[0202] The pharmaceutical compositions described herein, such as but not limited to the hydrogels described herein, are expected to be used as therapeutic and preventive drugs. More specifically, the compositions described herein, such as but not limited to the hydrogels described herein, are expected to be used as medical uses for wound healing. In other words, the present invention relates to the use of the compositions described herein (e.g., hydrogels) to manufacture drugs for wound healing (preferably local wound healing). In other words, the present invention relates to a method for treating a wound, preferably a local wound treatment method, comprising applying any one of the compositions or hydrogels described herein to a wound (site) of a subject. In the context of the present invention, the compositions described herein (e.g., hydrogels) are used as topical preparations, i.e., local wound treatment methods applied to a wound site.

[0203] Throughout this disclosure, the term "therapy" or "treatment" refers to one or more symptoms or measurable markers that alleviate or measurably alleviate a pathological condition, in the context of the present invention, one or more wounds. These terms include both therapeutic treatments of wounds that have already developed (i.e., wounds that have already formed) and preventive or prophylactic measures, wherein the purpose of treatment is to prevent the occurrence and / or recurrence, development and progression of wounds in a subject (i.e., the subject's skin). By way of illustration and limitation, the prophylactic use of the pharmaceutical composition described herein may be applied to a skin site that appears fragile in an elderly subject to prevent bedsores. Another example may be a skin site applied to a subject where a surgical insertion site will be formed in the foreseeable future. Measurable reduction includes any statistically significant decrease in measurable inflammatory markers, wound area and / or wound depth and / or wound width. Statistical significance as used herein refers to a p value below 0.05, which is a generally accepted critical value in statistical analysis as understood by those skilled in the art. More specific indications of healing wound sites will be described in detail below. Beneficial or desired clinical outcomes of medical use (i.e., treatment) may include, but are not limited to, relief of pain and / or discomfort, improvement of one or more biomarkers, reduction of wound extent, stabilization of wound status (i.e., no worsening), acceleration of wound healing process, improvement of patient quality of life, etc.

[0204] Those skilled in the art know that in order to achieve effective therapeutic treatment, it is necessary to apply a therapeutically effective dose to the subject. Therefore, in the context of the present disclosure, "effective amount" refers to the amount required for obtaining a physiological effect. The physiological effect can be achieved by a single dose or multiple doses. "Therapeutic effective amount" or "therapeutic effective dose" represents the amount of the estrol component that produces a clinically positive response to the subject suffering from one or more wounds when applied. Similarly, "preventive effective amount" or "preventive effective dose" refers to the amount of the estrol component that inhibits or delays wound occurrence or wound progression. Those skilled in the art know that terms such as "quantity", "amount" and "level" are synonyms and have clear meanings in the art, and understand that these terms refer to the relative quantification of the estrol component part of a pharmaceutical composition (such as, but not limited to, a hydrogel) in the context of the present application, or refer to the absolute quantification of the estrol component when indicated, which is considered to be an effective amount for the application described herein when applied to the skin of the subject.

[0205] Optionally, the pharmaceutical compositions and hydrogels described herein are used to treat acute wounds. The causes of acute wounds are not particularly limited for the present invention, and therefore include wounds caused by injury and wounds caused by surgery. The causes of injury are not limited for the present invention, and therefore include accidental injuries and malicious injuries (i.e., combat wounds). Non-limiting examples of acute wounds include abrasions (i.e., scrapes or scratches on the skin), incisions (i.e., clean incisional wounds), lacerations (i.e., torn and / or rough wounds), punctures (i.e., wounds with relatively small wound openings caused by relatively sharp objects), and avulsions (stretched or torn skin wounds).

[0206] Optionally, the pharmaceutical compositions and hydrogels described herein are used to treat burn wounds. It is understood by those skilled in the art that "burn wound" refers to a specific type of tissue damage caused by contact with heat, flames, chemicals, electricity or radiation. First-degree burns are mainly characterized by redness; second-degree burns are characterized by the presence of one or more blister points (i.e., blistering); and third-degree burns are characterized by the presence of necrosis. First-degree and second-degree burns are generally referred to in the art as partial thickness burns (i.e., tissue destruction that extends through the epidermis but does not pass through the dermis), while third-degree burns are generally referred to as full-thickness burns (i.e., the destruction is characterized by extending completely to the dermis).

[0207] Optionally, the pharmaceutical compositions and hydrogels described herein are used to treat chronic wounds. "Chronic wounds" as referred to herein refer to any wound that has failed or has not yet recovered according to the standard wound healing process. Therefore, wounds can be clinically classified as acute or chronic according to the time frame of wound healing. It is worth noting that surgical wounds can become chronic wounds and are referred to as surgical wounds that fail to heal through secondary healing (secondary intention). The term "chronic wound" can be used interchangeably with synonyms, such as but not limited to "difficult wounds", "difficult to heal wounds", "unhealing wounds" and "complex wounds". Chronic wounds have been described in detail in the art (e.g., Vanwijck, Bull Mem Acad R Med Belg, 2001). The healing process of chronic wounds may be disordered due to a variety of factors, thereby prolonging one or more stages of wound healing. Non-limiting examples include, but are not limited to, infection, tissue hypoxia, necrosis, exudate and excessive levels of inflammatory cytokines. Common features of chronic wounds include prolonged or uncontrolled inflammatory periods, persistent infections, the formation of resistant microbial biofilms, and the inability of dermal and / or epidermal cells to respond to repair stimuli.

[0208] "Inflammation" used herein refers extensively to the physiological process that vascular tissue responds to injury.Similarly, the term "inflammatory process" refers to the process that soluble inflammatory mediators and cellular components work together to limit and remove any painful material.The term "inflammatory mediators" refers extensively to any molecular mediator of inflammatory process.Inflammatory mediators can work locally at the position of tissue damage and / or infection, and can work at more distant positions.Some inflammatory mediators are activated by inflammatory process, while other inflammatory mediators produce and / or release from cell sources after responding to inflammation or being activated by other inflammatory mediators.The example of the inflammatory mediator of inflammatory response includes but is not limited to plasma protease, complement, kinin, coagulation protein, fibrinolytic protein, lipid mediator, prostaglandin, leukotrienes, platelet activating factor, peptide, amine and proinflammatory cytokine.

[0209] Terms such as "inflammation of the skin" or "skin inflammation" as used herein should be interpreted according to the generally accepted meaning in the current art and therefore refer to any local immune response of the skin. The cause of skin inflammation is usually the occurrence of an injury, such as a wound. Therefore, skin inflammation can be considered to be the result of cellular interactions in the subject's skin, of which immune cells remain the most important cell type. The skin inflammation mentioned herein refers to both "standard" inflammation observed in wounds and excessive inflammation that exceeds the limits of normal inflammation and may be the result of bacterial or fungal infection at the wound site or a defective host response (e.g. diabetes). Non-limiting examples of bacteria that may be involved in wound infection include, but are not limited to, Staphylococcus aureus, coagulase-negative staphylococci, Corynebacterium, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Acinetobacter baumannii, Serratia marcescens, Stenotrophomonas maltophilia, Streptococcus agalactiae, Enterobacter cloacae, Enterococcus, Klebsiella pneumoniae, Morganella morganii, Providencia stuartii, Alcaligenes faecalis, Citrobacter amalonicidus, Citrobacter kluyveri, Klebsiella oxytoca, Kochella kluyveri, and Pseudomonas stutzeri. Non-limiting examples of fungi that may be involved in wound infection include, but are not limited to, Candida albicans, Candida parapsilosis, and Aspergillus niger.

[0210] A bacterium of particular interest in the context of the present invention is Klebsiella pneumoniae, which is known to act as a wound pathogen in infected wound sites, such as, but not limited to, acute wounds (including surgical, injury and combat wounds), burns and chronic leg ulcers (Crompton et al., Lab Invest, 2016). In the art, Klebsiella pneumoniae is associated with reduced epithelial regeneration, increased proliferation, enhanced inflammatory response and disordered wound matrix deposition. The inventors have found that the topical compositions described herein are particularly suitable for reducing inflammation in wounds infected with Klebsiella pneumoniae, as well as wounds that are considered to be at risk of infection by Klebsiella pneumoniae.

[0211] Non-limiting examples of chronic wounds include vascular ulcers, pressure sores, and diabetic ulcers. Vascular ulcers include arterial ulcers and venous ulcers. Therefore, in certain embodiments, chronic wounds are wounds selected from the following groups: arterial ulcers, venous ulcers, pressure sores, diabetic ulcers, and combinations thereof. "Venous ulcers" as used herein are caused by elevated venous pressure caused by venous valve defects. Changes in the permeability of the vascular wall caused by pressure lead to leakage of fibrin and other plasma components into perivascular locations, and the accumulation of fibrin has a negative impact on wound healing. Collagen synthesis is downregulated by fibrin, resulting in the formation of fibrin cuffs around capillaries, thereby creating a barrier for normal vascular function and limiting blood-derived growth factors. "Arterial ulcers" refer to chronic wounds caused by insufficient blood supply to the arteries due to atherosclerosis or embolism, resulting in narrowing and ischemia of the arterial lumen, which prevents minor injuries from healing in time. "Pressure sores" are formed due to the long-term lack of pressure and shear stress relief on the skin and underlying muscle tissue, resulting in reduced oxygen tension, ischemia-reperfusion injury, and tissue necrosis. Finally, “diabetic ulcers” are a consequence of aging and diabetes. Diabetes may also exacerbate vasculopathy, which in turn exacerbates arterial insufficiency, venous insufficiency, and / or pressure ulcers. Other abnormalities that contribute to the development of diabetic ulcers in patients with diabetes include neuropathy (often associated with vascular damage), defects in muscle metabolism, and certain microangiopathy caused by hyperglycemia. The macroscopic pathology seen in chronic (especially diabetic) wounds often includes abnormalities in cellular phenotypes such as, but not limited to, low mitogenicity, low motility potential, and an inability to respond to environmental factors.

[0212] In a specific embodiment, the pharmaceutical composition described herein (which may be a hydrogel) is used for wound healing of a subject characterized by impaired wound healing. Impaired wound healing may be caused by potential pathology and / or wound infection as described in the present disclosure. Infection is a common cause of delayed wound healing. Live bacteria (and the bacterial toxins produced subsequently) induce excessive inflammatory responses and tissue damage. Potential consequences of bacterial infection of wounds include the occurrence of abscesses, cellulitis, osteomyelitis, or limb loss (e.g., in diabetic patients). In addition, inflammatory cells recruited to the wound site produce proteases that can degrade the extracellular matrix and growth factors present in the wound site after infection. A considerable portion of wound colonization bacteria can form biofilms, thereby causing an increase in bacterial survival and increasing the production of virulence factors. Therefore, in certain embodiments, the subject is a subject characterized by wounds comprising biofilms.

[0213] In certain embodiments, the pharmaceutical composition is used to treat a wound in a subject considered to be at risk for developing a chronic wound or who has developed at least one chronic wound at an earlier point in time.

[0214] In embodiments where the subject is a subject characterized by impaired wound healing, the impaired wound healing occurs at least about 10% slower, preferably at least about 20% slower, preferably at least about 30% slower, preferably at least about 40% slower, preferably at least 50% slower, preferably at least 60% slower, preferably at least 70% slower, preferably at least 80% slower, preferably at least 90% slower, compared to wound healing in subjects not considered or suspected of having impaired wound healing (i.e., subjects considered healthy or generally healthy).

[0215] The specific manifestations of impaired wound healing and their causes are not particularly limited to the present invention. Therefore, in certain embodiments, the stage at which the wound healing of the subject may be impaired is selected from the following wound repair stages: hemostasis (blood clotting), inflammation, proliferation (new tissue growth) and maturation (tissue remodeling) or any combination thereof. The process of impaired wound healing may also be characterized by the occurrence of infection, hypoxia, necrotic tissue, exudate (cells and fluids seeping out of the wound), excessive inflammatory cytokines and any combination thereof.

[0216] Optionally, the subject is characterized by impaired wound healing due to insufficient or absent coagulation function. Immediately after injury to a healthy subject, platelets attach to the damaged blood vessel, initiate a release response, and initiate a hemostatic response. This results in a blood coagulation cascade that prevents excessive bleeding and provides temporary protection to the injured area. Platelets have been described to release a variety of growth factors, cytokines, and other survival or apoptosis inducers. Key components of the platelet release response include platelet-derived growth factor (PDGF) and transforming growth factors Al and 2 (TGF-A1 and TGF-2), which attract inflammatory cells (e.g., leukocytes, neutrophils, and macrophages).

[0217] Optionally, the subject is characterized by impaired wound healing caused by insufficient inflammatory wound healing phase. In healthy subjects, the inflammatory phase is initiated in response to capillary damage, which results in the formation of a temporary blood clot matrix, which includes components such as fibrin and fibronectin. The temporary matrix fills the wound area and triggers the influx of effector cells. Platelets in the blood clot release a variety of cytokines, which recruit inflammatory cells (e.g., neutrophils, monocytes, macrophages, etc.), fibroblasts, and endothelial cells.

[0218] Optionally, the subject is characterized by impaired wound healing caused by a defect in the proliferation phase. In healthy subjects, the proliferation phase is characterized by active angiogenesis, thereby generating new capillaries, allowing nutrients to be delivered to the wound site and supporting fibroblast proliferation. These fibroblasts synthesize and deposit extracellular matrix (ECM) components to replace the temporary matrix. The fibroblasts also have contractile properties, mediated by smooth muscle actin organized in microfilament bundles or stress fibers.

[0219] Optionally, the subject is characterized by impaired wound healing due to an insufficient remodeling phase. In healthy subjects, the final healing phase involves gradual remodeling of granulation tissue and re-epithelialization. Proteolytic enzymes, such as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs, tissue inhibitors of metalloproteinases) play a key role in the remodeling phase. During the re-epithelialization process, fibronectin and type III collagen, the main components of granulation tissue (i.e., new matrix tissue), are gradually replaced by type I collagen and supplemented by elastin. Elastin contributes to skin elasticity and is initially absent in granulation tissue. Finally, the cell density in the wound returns to normal through apoptosis of vascular cells and fibroblasts.

[0220] Wound edge proliferation is a part of the wound epithelial regeneration process, and the latter represents the process of covering (i.e. resurfacing, providing) with new epithelium. In skin wounds, it has been confirmed that epithelial regeneration develops from the wound edge (i.e. wound edge) around to the wound center. Epithelial regeneration is a part of the proliferation stage, and usually about 16 to about 24 hours after injury by activating keratinocytes as described above due to neutrophils, monocytes and macrophages recruited to the wound site. The feature of activated keratinocytes is that cytoskeleton and cell surface receptors change. In addition, activated keratinocytes have excessive proliferation and produce the components of dermis-epidermis junction. For example, activated keratinocytes produce matrix metalloproteinase 9 (MMP-9), which can cause dermis-epidermis junction degradation, and allow the keratinocytes to migrate on the wound. Keratinocyte migration is an early event of wound epithelial regeneration. Further research on activated keratinocytes in wounds leads to the observation that the wound edge moves the sticky epithelial sheet to migrate to the wound center. Different mechanisms have been proposed for the process by which keratinocytes migrate across a wound bed, each of which is envisioned and understood in the context of the present invention.

[0221] Optionally, impaired wound healing can be observed and / or manifested by reduced migration of the wound edge of the wound. "Wound edge" as used herein refers to the periphery of the wound area, i.e. the portion of the wound area adjacent to the uninjured tissue area of ​​the subject's skin. Those skilled in the art can observe and / or measure the wound area, and the wound edge as a result. Such observations and / or measurements can be carried out at multiple time points to determine whether the subject's wound heals at a "normal" rate (i.e., a rate within the range of rates considered to be common in healthy subjects) or whether any wound healing delay occurs or a rate reduction occurs. Optionally, in the absence of any treatment, the wound healing of a subject with impaired wound healing is manifested as a reduction of at least 25%, preferably at least 50%, more preferably at least 75%, or most preferably at least 100% in wound edge migration compared to healthy subjects.

[0222] In a specific embodiment, the composition and hydrogel of the above aspects are used to improve the epithelial regeneration of the wound site. In this context, improved epithelial regeneration can refer to the entire process of epithelial regeneration, but also refers to a certain improvement of its specific aspects. Therefore, in certain embodiments, the wound healing improvement after treatment with the composition described herein can improve the process selected from the following group: improvement of epithelial regeneration of the wound site, increase of cell proliferation at the wound site, reduction of inflammatory response at or within the wound site, improvement of matrix deposition at the wound site, epithelial regeneration mediated by hair follicles, epithelial regeneration of partial thickness wounds from bottom to top, and any combination thereof.

[0223] Improved epidermal regeneration is characterized by increased and / or accelerated production of provisional matrix. In such embodiments, the amount of provisional matrix produced can be increased by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, more preferably at least about 100%, and / or the production of the provisional matrix can be accelerated by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, more preferably at least about 100%, compared to a subject not treated with a composition described herein (optionally a hydrogel).

[0224] Alternatively or in addition to increased and / or accelerated production of provisional matrix, improved epidermal regeneration may also be characterized by increased and / or accelerated activation (i.e., proliferation) and / or migration of keratinocytes. In such embodiments, the amount of activated keratinocytes may be increased by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, more preferably at least about 100%, and / or the migration of keratinocytes may be increased by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, more preferably at least about 100%, compared to a subject not treated with a composition described herein (optionally a hydrogel).

[0225] In a specific embodiment, the composition and hydrogel of the above aspects are used to increase cell proliferation at a wound site of a subject. Cell proliferation may include, but is not limited to, proliferation of endothelial cells, fibroblasts and / or keratinocytes. In certain embodiments, cell proliferation of a cell group selected from endothelial cells, fibroblasts, keratinocytes and any combination thereof is increased by at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 75%, and most preferably at least 100%, compared to cell proliferation at a wound site of a subject not treated with the composition described herein.

[0226] In a specific embodiment, the composition described herein is used to reduce the inflammatory response of the subject's wound site. In such embodiments, the composition described herein is used to reduce the amount of one or more pro-inflammatory molecules and / or increase the amount of one or more anti-inflammatory molecules at the subject's wound site. Non-limiting examples of pro-inflammatory molecules contemplated herein include but are not limited to interleukin-1β (IL-1β), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α (TNF-α), interferon γ (IF-γ), interleukin 12 (IL-12), histamine, serotonin, neuropeptides, plasma proteases, complement, kinin, coagulation proteins, fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, and platelet activating factor (PAF). In certain embodiments, the compositions described herein (optionally hydrogels) are used to reduce the amount of one or more pro-inflammatory molecules produced at a wound site by at least 10%, preferably at least 25%, more preferably at least 50%, still more preferably at least 75%, and most preferably about 100%, compared to the level of one or more pro-inflammatory molecules present at a wound site of a subject not treated with the compositions described herein, wherein the pro-inflammatory molecules are selected from interleukin-1β (IL1-β), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α (TNF-α), interferon γ (IF-γ), interleukin 12 (IL-12), histamine, serotonin, neuropeptides, plasma proteases, complement, kinins, coagulation proteins, fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, and platelet activating factor (PAF). Preferably, the medical use or treatment results in an improvement in the macrophage and neutrophil profiles, indicating a reduction in local wound inflammation compared to an untreated wound. In a specific embodiment, the treatment according to the present invention reduces the number of inflammatory cells (such as but not limited to macrophages and neutrophils) in the wound. Therefore, in a specific embodiment, topical administration of a composition according to the present invention promotes a pro-healing wound phenotype, wherein M1 marker expression is reduced and M2 marker expression is increased. In some embodiments, after administration of the composition of the present invention, the number of both innate and acquired immune cells is reduced. Generally, in further embodiments, when the composition described herein is administered, beneficial effects on the function of other immune cells (such as dendritic cells, Langerhans cells and mast cells) can be observed.

[0227] In a preferred embodiment, compared with a wound not treated with any wound healing composition, a composition (e.g., hydrogel) comprising an estrrol component is used to improve histological healing parameters. More preferably, compared with a wound not treated with a wound healing composition not containing an estrrol component, a composition comprising an estrrol component is used to improve histological healing parameters. Optionally, histological healing parameters are histological skin parameters selected from the following groups: epidermal closure, epidermal differentiation, epidermal migration, granulation tissue formation and epidermal hyperplasia, granulation tissue and matrix formation, inflammation and late matrix remodeling, which can be performed histologically by the presence of newly formed epidermis, spinous and / or granular epidermal differentiation markers, migratory cells, proliferating cells, collagen fiber deposition, immune cell markers, wound protease levels and matrix composition. Each of these parameters has been described in detail in the art (e.g., Gupta and Kumar, Plast Aesthet Res, 2015). Surrogate histological parameters that may be derived from one or more observations include, but are not limited to, length of the epithelial regeneration zone, distance between wound borders, depth of the wound, width of the wound, thickness of connective tissue, thickness of the native dermis at the wound edge, orientation of the dermal matrix, wound cell properties, wound vascularization.

[0228] In preferred embodiments, the compositions described herein can be used to improve the incidence of complete wound closure in a subject, optionally a subject characterized by impaired wound healing. Complete wound closure means that the skin surface is completely closed, i.e., completely re-covered by new epithelium. In such embodiments, the incidence of complete wound closure using a composition comprising an estetrol component is increased by at least 25%, preferably at least 50%, more preferably at least 75%, and most preferably at least 100%, compared to wounds not treated with the composition, or based on the wound healing history of the subject.

[0229] In preferred embodiments, the compositions described herein can be used to accelerate the time to achieve wound closure in a wound of a subject, the subject being optionally a subject with impaired wound healing. In such embodiments, the time to achieve wound closure in a wound of a subject is reduced by at least 25%, preferably at least 50%, more preferably at least 75%, and most preferably more than 80%, compared to a wound not treated with the composition, or based on the wound healing history of the subject. The skilled artisan recognizes that certain parameters described in detail in the present disclosure, such as, but not limited to, the specific time required to achieve complete wound closure, depend on the size of the wound, etc.

[0230] In preferred embodiments, the compositions described herein can be used to promote surgical wound closure in a subject, the subject being optionally a subject characterized by impaired wound healing. In such embodiments, the compositions described herein can increase the speed of surgical wound closure and / or increase the chances of a subject achieving wound closure of a wound whose area and / or depth exceeds the area and / or depth of a wound that can be healed by the subject without the use of a composition comprising an estetrol component. In some embodiments, the quality of healing is improved, particularly in infected wounds.

[0231] The terms "formulation" or "composition" are used interchangeably herein. In any embodiment of the composition described herein, it is apparent that the composition may include one or more pharmaceutically or cosmetically acceptable carriers (i.e., excipients) that are not described in detail in the present disclosure. The term "pharmaceutically acceptable" as used herein is consistent with the art and refers to being compatible with the other ingredients of the pharmaceutical or cosmetic composition and harmless to the recipient thereof. In a particularly preferred embodiment of the present invention, the pharmaceutical composition according to the present invention is designed for daily administration, i.e., it represents a daily dosage unit. The excipients that can be used in the pharmaceutical composition are not particularly limited and can therefore be one or more excipients selected from the following: active pharmaceutical ingredient excipients, adhesive excipients, carrier excipients, co-processing excipients, coating system excipients, controlled release excipients, diluent excipients, disintegrant excipients, dry powder inhalation excipients, effervescent system excipients, emulsifier excipients, lipid excipients, lubricant excipients, sustained release excipients, penetration enhancer excipients, penetration enhancer excipients, pH adjuster excipients, plasticizer excipients. In some embodiments, the invention relates to a pharmaceutical composition comprising a preservative adjuvant, a preservative excipient, a solubilizing agent excipient, a solvent excipient, a sustained-release excipient, a sweetener excipient, a flavoring excipient, a thickener excipient, a viscosity modifier excipient, a filler excipient, a compacting excipient, a dry granulation excipient, a hot melt extrusion excipient, a wet granulation excipient, a quick-release excipient, an excipient that increases bioavailability, a dispersing excipient, a solubility enhancing excipient, a stabilizer excipient, a capsule filling excipient or any combination thereof. The technician knows that it is common practice to use such media and reagents for pharmaceutically active substances, so the incorporation of these excipients is well known in the art. Obviously, all the ingredients used should be nontoxic at the concentration contained in the final pharmaceutical composition, and should not negatively interfere with the activity of the estrone component, which is preferably present in the pharmaceutical composition as the main pharmaceutically active ingredient. In some embodiments, the technician is classified as more than one excipient belonging to the same excipient group and added to the pharmaceutical composition. In further embodiments, more than one excipient, wherein different excipients belong to different groups, is added to the pharmaceutical composition.In certain embodiments, an excipient may fulfill more than one function and / or be classified by a skilled person as belonging to different groups or classes of excipients.

[0232] As described above, in the context of the present invention, there are no particular limitations on the details of the subject affected by the wound. Preferred subjects are elderly subjects. "Elderly subjects" refer to subjects who are old, i.e., close to or exceeding the age of the subject's life expectancy. Elderly subjects are defined as being at least 60 years old, preferably at least 70 years old, at least 75 years old, at least 80 years old, at least 85 years old, and most preferably at least 85 years old. In alternative embodiments, the subject is selected from the following groups: infants (i.e., juvenile subjects), adolescent subjects, and adult subjects. In certain embodiments described herein, the subject is diagnosed with palliative care or is considered for palliative care.

[0233] Optionally, the pharmaceutical compositions contemplated by the present invention (which may optionally be hydrogels) may include additional skin active ingredients that can provide skin care benefits. Skin care benefits may include, but are not limited to, benefits associated with the cosmetic appearance of the skin. Additional skin active ingredients may provide immediate and short-term (i.e., acute) benefits and / or long-term and long-lasting (i.e., chronic) benefits.

[0234] Optionally, the pharmaceutical composition contemplated by the present invention (which may optionally be a hydrogel) may also contain at least one other pharmaceutically active ingredient in addition to the estetrol component. In a specific embodiment, at least one other pharmaceutically active ingredient is selected from: an anti-inflammatory agent, an analgesic agent, and an anti-infective agent. The anti-inflammatory component may be a steroidal anti-inflammatory component, a non-steroidal anti-inflammatory component, or a combination thereof. The analgesic component (i.e., a component capable of inducing pain relief) may be a non-opioid analgesic or an opioid analgesic. Suitable anti-infective agents include, but are not limited to, antibiotics.

[0235] In a specific embodiment, the pharmaceutical compositions contemplated by the present invention (which may optionally be hydrogels) are used for wound healing in the context of skin transplantation. In such embodiments, the compositions described herein may be combined with skin transplantation techniques, such as, but not limited to, skin transplantation techniques that rely on stem cell therapy, bioengineered skin, skin equivalents, skin substitutes, synthetic skin, or combinations thereof. In a specific embodiment, the compositions used herein are used for wound healing after skin transplantation that is considered necessary due to extensive damage to a considerable area of ​​the subject's skin, such as, but not limited to, skin transplantation in burn wounds. The healing of skin transplant donor site wounds is also contemplated herein.

[0236] Pharmaceutical composition as described herein (which is optionally a hydrogel) is applied (i.e., applied) to the wound site. Compositions can be applied by pouring, dripping, spraying, rubbing or any other appropriate manner. Optionally, the composition is applied to the wound site once. Alternatively, the composition is applied to the wound site at multiple time points, preferably at the time points of the substantially regular dispersion. In certain embodiments, the composition is applied to the wound site daily. Optionally, the composition is applied to the wound site and maintained (i.e., not removed from the wound site) for a long period of time, 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 should be understood that the wound is continuously exposed to the composition within the indicated time amount.

[0237] The term "continuously" as used herein refers to the administration of the ingredients at relatively regular intervals without (therapeutically) significant interruptions. Of course, minor interruptions may occur, but do not affect the overall effectiveness of the method, and in fact, the present invention covers such exceptions.

[0238] The pharmaceutical compositions described herein (optionally hydrogels) can be part of, i.e., contained in, any device suitable for application to the skin and / or wound site of a subject, preferably on or near the wound site, to deliver the pharmaceutically active ingredient to the skin (and / or wound site). Alternatively, the compositions described herein can be applied to any device suitable for application to the skin and / or wound site of a subject by the subject, another subject, or a skilled medical practitioner prior to application to the skin or wound site of a subject (i.e., the composition is used in conjunction with a device for application to the skin).

[0239] Preferably, the pharmaceutical composition is applied to the skin of the experimenter by a dressing. Various types of dressings have been described in the art, including but not limited to gauze dressings, tulle dressings, alginate dressings, polyurethane dressings, film dressings, polysaccharide paste dressings, granular dressings, foam dressings, silicone dressings, synthetic polymer stent dressings, hydrocolloid dressings, occlusive dressings or combinations thereof. Dressings can be viscous or non-viscous. The term "occlusive dressing" used herein refers to a dressing that prevents air and / or bacteria from contacting and retains one or more of the following materials: moisture, heat, body fluids and medicines. The technician can select suitable wound healing dressings for specific wounds, and the selection can be carried out according to parameters such as but not limited to wound type, wound size and wound healing progress.

[0240] Optionally, the dressing is a hydrogel dressing. Hydrogel dressings are largely composed of water in a network of fibers that maintain the integrity of the polymer gel. The water in the dressing is released to maintain adequate moisture levels in the wound. Examples of hydrogel dressings include, but are not limited to and

[0241]

[0242] Methods and protocols for producing any of the above dressings have been described in the art. The estetrol component may be added to the dressing during the manufacture of the dressing, but may also be applied to pre-made dressings. For example (but not limited to), the pre-made dressing or a portion thereof may be impregnated with the estetrol component. Alternatively, the pre-made dressing or a portion thereof may be coated with the estetrol component.

[0243] In a specific embodiment, the pharmaceutical composition (which is optionally a hydrogel) is included in a skin substitute (i.e., a skin substitute or a dermal substitute). The skin substitute provides a three-dimensional biomatrix that implements the function of the skin dermis, which can temporarily or permanently cover an open skin wound. The material of the skin substitute is not particularly limited, and therefore may include a biomaterial, a synthetic material, or a combination thereof. Non-limiting examples of biomaterials include, but are not limited to, human or pig skin and human or pig intestinal submucosa. Biological skin substitutes may include different ingredients, including, but not limited to, collagen, glycosaminoglycans, fibronectin, hyaluronic acid, elastin, and any combination thereof.

[0244] Alternatively, the pharmaceutical composition may be contained in any other device suitable for application to the skin tissue of a subject, and thus may be contained in devices such as, but not limited to, bandages, adhesive bandages, patches, and plasters.

[0245] Although the present invention has been described in conjunction with specific embodiments of the present invention, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art based on the foregoing description. Therefore, it is intended that all such substitutions, modifications and variations as follows be included within the spirit and broad scope of the appended claims. Aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting examples. Example

[0246] Example 1. Manufacturing process

[0247] Aqueous gel preparation

[0248] Batch AG23, AG24, AG25, AG26

[0249] i. Weigh water (first addition) into Duran (container 1).

[0250] ii. Place the sample from step (i) on a hot plate stirrer at 800 rpm to create a vortex.

[0251] iii. Weighing polymer by weighing boat Disperse it in the contents of step (ii) and keep vortexing. Stir the sample for at least one hour to allow the polymer to disperse.

[0252] iv. Record the weight of the polymer dispersion in step (iii).

[0253] v. The sample in step (iv) was sterilized under high pressure under standard conditions (121°C ± 2°C, 2x10 5 Pa, 15 minutes).

[0254] vi. After the autoclaved sample has cooled, reweigh the sample from step (v) and add additional water to compensate for volatile losses due to evaporation.

[0255] vii. The solvent (PEG 400, P (diethylene glycol monoethyl ether)) was weighed into a separate container.

[0256] viii. Estetrol monohydrate was weighed into the contents of step (vii) and stirred at 500 rpm for 2.5 hours (hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug.

[0257] ix. In a biosafety laminar flow hood, filter the contents of step (viii) through multiple Spartan (regenerated cellulose) 0.2 μm sterile filters into multiple 20 mL autoclave bottles to avoid bulk contamination if the filters break.

[0258] x. Combine and weigh the contents of step (ix) into a pre-autoclaved Duran container.

[0259] xi. Pour the contents of step (x) into the contents of step (vi) and mix using an overhead stirrer and spatula; record the mixing speed and time in a laboratory notebook.

[0260] xii. Rinse the container in step (x) with water (about 4% of the total volume) and pour it into container 1.

[0261] xiii. Let the preparation stand for at least one night

[0262] xiv. Adjust the pH of the sample to a target pH between 6-6.5 using sodium hydroxide solution and adjust the weight with water.

[0263] xv. It is worth noting that manufacturing steps (ix) to (xiv) are all performed under sterile conditions in a laminar flow hood.

[0264] Batch AG30, AG22( 980)

[0265] i. Weigh water (first addition) into 150 mL Duran (Container 1).

[0266] ii. Place the sample from step (i) on a hot plate stirrer at 800 rpm to create a vortex.

[0267] iii. Weigh the polymer by weighing boat ( 980) and disperse it in the contents of step (ii), keeping vortexing. Stir the sample for at least one hour to allow the polymer to disperse.

[0268] iv. Record the weight of the polymer dispersion in step (iii).

[0269] v. Weigh PEG 400 and glycerol and add to the contents of step (iv).

[0270] vi. The sample in step (v) was sterilized under high pressure under standard conditions (121°C ± 2°C, 2x10 5 Pa, 15 minutes).

[0271] vii. After the autoclaved sample has cooled, reweigh the sample from step (v) and add additional water to compensate for volatile losses due to evaporation.

[0272] viii. The solvent (propylene glycol (PG), P) Weigh into 100 mL Duran.

[0273] ix. Estetrol monohydrate was weighed into the contents of step (viii) and stirred at 500 rpm (hot plate magnetic stirrer at laboratory room temperature).

[0274] x. In a biosafety laminar flow hood, filter the contents of step (ix) through multiple Spartan 0.2 μm sterile filters into multiple 20 mL autoclave bottles to avoid bulk contamination if the filters break.

[0275] xi. Combine the contents of step (x) and weigh into pre-autoclaved 100 mL Duran.

[0276] xii. Pour 18% sodium hydroxide (previously filtered through a 0.2 μm sterile Spartan filter) into the contents of step (xi) and mix.

[0277] xiii. Quickly pour the contents of step (xii) into the contents of step (vii) and mix with a spatula.

[0278] xiv. Rinse the container in step (x) with water (about 4% of the total volume) and pour it into container 1.

[0279] xv. Allow the thickened sample to sit at least overnight.

[0280] xvi. Measure the pH of the sample and add the remaining sterile water and sodium hydroxide solution (also previously sterilized through a 0.2 μm Spartan filter) to adjust the pH to 7-7.5 as needed.

[0281] It is worth noting that manufacturing steps (x) to (xvi) were all performed under sterile conditions in a laminar flow hood.

[0282] Batch AG27 (Poloxamer)

[0283] i. Weigh water (first addition) into a 150 mL Duran container and cool in a refrigerator at 2-8°C for 20 minutes.

[0284] ii. Place the sample from step (i) on a hot plate stirrer, weigh the poloxamer (P407, then P188), and add while vortexing at 800 rpm.

[0285] iii. The sample from step (ii) was stirred with an overhead stirrer at 200 rpm for 6 hours until the poloxamer was dissolved in a water bath at 2-8°C.

[0286] iv. In a separate container, weigh PEG 400, benzyl alcohol (BA) and PG together.

[0287] v. Weigh estetrol monohydrate and add it to the contents of step (iv) and stir on a hot plate stirrer at 500 rpm overnight.

[0288] vi. After the drug is dissolved, weigh the required amount of solution from step (v) and add it to the first container containing the poloxamer solution. Place the sample on a magnetic stirrer and stir at 300 rpm overnight.

[0289] vii. Adjust the pH of the sample in step (vi) to 7-7.5.

[0290] Batch AG28 (HEC), AG29 (CMC)

[0291] i. Prepare the solvent system for the formulation by weighing all solvents except water / buffer specific to the active system.

[0292] ii. Add the drug to the solvent system of the active formulation and stir at 500 rpm overnight.

[0293] iii. After the drug is dissolved, add the water / pH 7.0 buffer solution to the solvent system while stirring the Duran.

[0294] iv. Weigh an appropriate amount of premix into a separate 150 mL Duran.

[0295] v. The polymer was dispersed while stirring at 800 rpm to form a vortex, and the formulation was stirred at 500 rpm overnight.

[0296] vi. For AG28, the pH is adjusted to pH 7-7.5, whereas for AG29, no pH adjustment is required as it contains buffered phosphate-phosphate pH 7.0.

[0297] Batch AG15 (Poloxamer)

[0298] i. Water (first addition) was weighed into 150 mL Duran and cooled in a refrigerator maintained at 2-8°C for 20 minutes.

[0299] ii. Place the sample from step (i) on a hot plate stirrer, weigh the poloxamer (P407, then P188), and add while vortexing at 800 rpm.

[0300] iii. The sample from step (ii) was stirred with an overhead stirrer at 200 rpm for 6 hours until the poloxamer was dissolved in a water bath maintained at 2-8°C.

[0301] iv. In a separate container, weigh PEG400, BA and PG together.

[0302] v. Weigh estetrol and add it to the contents of step (iv), then stir overnight on a hot plate stirrer at 500 rpm. This step was omitted when making the placebo.

[0303] vi. After the drug is dissolved, weigh the required amount of solution from step (v) and add it to the first container containing the poloxamer solution. Place the sample on a magnetic stirrer and stir at 300 rpm overnight.

[0304] vii. Adjust the pH of the sample in step (vi) to 7-7.5.

[0305] Batch AG17

[0306] i. Weigh water (first addition) into 150 mL Duran (Container 1).

[0307] ii. Place the sample from step (i) on a hot plate stirrer at 800 rpm to create a vortex.

[0308] iii. Weighing polymer by weighing boat Disperse it in the contents of step (ii) and keep vortexing. Stir the sample for at least one hour to allow the polymer to disperse.

[0309] iv. Record the weight of the polymer dispersion in step (iii).

[0310] v. The sample in step (iv) was sterilized under high pressure under standard conditions (121°C ± 2°C, 2x10 5 Pa, 15 minutes).

[0311] vi. After the autoclaved sample has cooled, reweigh the sample from step (v) and add additional water to compensate for volatile losses due to evaporation.

[0312] vii. Weigh benzyl alcohol, PEG 400, and PG into separate 100 mL Duran.

[0313] viii. Weigh estetrol monohydrate into the contents of step (vii) and stir overnight at 500 rpm (hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug. This step was omitted for the placebo preparation

[0314] ix. Pour 18% sodium hydroxide (1.38 g) into the contents of step (vi) and mix.

[0315] x. Quickly pour the contents of step (ix) into the contents of step (vi) and mix with a spatula.

[0316] xi. Rinse the container from step (ix) with water (4.5 g) and pour it into container 1.

[0317] xii. Allow the thickened sample to sit for at least overnight.

[0318] xiii. Measure the pH of the sample and gradually add the remaining water while continuing to measure the pH.

[0319] Batch AG18(HEC), AG19(CMC)

[0320] i. Prepare the solvent systems for placebo and active formulations by weighing all solvents except water / buffer specific to the active system.

[0321] ii. Add the drug to the solvent system of the active formulation and stir at 500 rpm overnight.

[0322] iii. After the drug is dissolved, add the water / pH 7.0 buffer solution to the solvent system while stirring the Duran.

[0323] iv. Weigh an appropriate amount of premix into a separate 150 mL Duran.

[0324] The polymer was dispersed while stirring at 800 rpm to form a vortex, and the formulation was then stirred at 500 rpm overnight.

[0325] v. For AG18, the pH is adjusted to pH 7-7.5, while AG19 does not require pH adjustment as it contains buffered phosphate-phosphate pH 7.0.

[0326] Batch AG20, AG23

[0327] i. Follow steps (i)-(vi) in AG17.

[0328] ii. Solvent (PEG 400, P) were weighed into a separate container (100 mL).

[0329] iii. Weigh estetrol monohydrate into the contents of step (ii) and stir at 500 rpm for 2.5 hours (hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug. This step was omitted for the placebo formulation.

[0330] iv. In a biosafety laminar flow hood, filter the contents of step (iii) through multiple Spartan (regenerated cellulose) 0.2 μm sterile filters into multiple 20 mL autoclave bottles. This is to avoid contamination of the entire sample if the filter breaks. It should be noted that initially the sample will be filtered through a Nalgene bottle system filter (PES) with the aid of a vacuum pump, but the filter broke, so a Spartan syringe filter was used.

[0331] v. Combine the contents of step (iv) and weigh into pre-autoclaved 100 mL Duran.

[0332] vi. Pour the contents of step (v) into the contents of step (i) and mix with a spatula.

[0333] vii. Rinse the container from step (v) with water (4.5 g) and pour it into container 1.

[0334] viii. Since the preparation did not thicken, it was left to stand at least overnight.

[0335] ix. Adjust the pH of the sample to between 6-6.5 with sodium hydroxide solution and weigh with water. Note that the process was initially performed using 18% sodium hydroxide, but Also, the initial target pH was 7-7.5, however, polymer precipitation was observed above pH 6.5, so repeated sampling was performed and maintained between pH 6-6.5 to avoid this problem.

[0336] It is worth noting that manufacturing steps (iv) to (ix) were performed under sterile conditions in a laminar flow hood.

[0337] Batch AG21, AG22

[0338] i. Follow steps (i) to (vi) in AG17.

[0339] ii. Weigh the solvent (PEG 400, PG, Trans P) into 100 ml Duran.

[0340] iii. Weigh estratetrol monohydrate into the contents of step (i) and stir at 500 rpm (hot plate magnetic stirrer at laboratory room temperature). The drug for AG22 dissolved within 2.5 hours of stirring, and the drug for AG21 dissolved after stirring overnight. This step was omitted for the placebo preparation.

[0341] iv. In a biosafety laminar flow hood, filter the contents of step (viii) through multiple Spartan 0.2 μm sterile filters into multiple 20 mL autoclave bottles to avoid bulk contamination if the filters break.

[0342] v. Combine the contents of step (ix) and weigh into pre-autoclaved 100 mL Duran.

[0343] vi. Pour 18% sodium hydroxide (1.38 g) (previously filtered through a 0.2 μm sterile Spartan filter) into the contents of step (x) and mix.

[0344] vii. Quickly pour the contents of step (xi) into the contents of step (vi) and mix with a spatula.

[0345] viii. Rinse the container in step (x) with water (4.5 g) and pour it into container 1.

[0346] ix. The sample thickened and was allowed to sit for at least overnight.

[0347] x. Measure the pH of the sample and add the remaining sterile water and sodium hydroxide solution (also sterilized beforehand through a 0.2 μm Spartan filter) to adjust the pH to 7-7.5 as needed.

[0348] It is worth noting that manufacturing steps (ix) to (xv) were performed under sterile conditions in a laminar flow hood.

[0349] Table 1: Composition (% w / w) of estetrol monohydrate (E4) hydrogel formulations.

[0350]

[0351] Cream batches CR01, CR05, CR12

[0352] i. Prepare the placebo and active solvent systems by weighing out all solvents except the water / buffer specific to the active system.

[0353] ii. Add the drug to the solvent system of the active cream and stir at 500 rpm overnight.

[0354] iii. After the drug was completely dissolved, water / pH 7.0 buffer solution was added to the solvent system while stirring the vessel (Duran).

[0355] iv. Then prepare the oil phase. For CR01, place the oil phase in a 75°C water bath until melted.

[0356] v. For CR05 and CR12, place the oil phase in an oven at 160°C for 2 hours (to simulate the sterilization process). After the oil phase cools to room temperature and resolidifies, place it in a water bath (75°C) until the oil phase melts again.

[0357] vi. Prior to processing the active and placebo creams, the required amount of the solvent system premix was weighed into separate Durans.

[0358] vii. Place the Duran with premix in a water bath with homogenizer for 5 minutes.

[0359] viii. For CR01 and CR12, the creams were processed using an Ultra-turrax equipped with a 25G disperser head at 10,000 rpm for 2 minutes.

[0360] ix. For CR05, during homogenization, the cream was homogenized at 5000 rpm for 5 minutes to avoid overflow.

[0361] x. After processing, stir the cream manually with a metal spatula until the cream reaches room temperature.

[0362] xi. Leave them overnight to cure.

[0363] xii. Adjust the pH of the formulation to pH 7-7.5.

[0364] Batch CR10, CR13, CR14, CR15, CR16

[0365] i. Prepare the placebo and active solvent systems by weighing out all solvents except the water / buffer specific to the active system.

[0366] ii. Add the drug to the solvent system of the active cream and stir at 500 rpm overnight.

[0367] iii. After the drug was completely dissolved, water / pH 7.0 buffer solution was added to the solvent system while stirring Duran.

[0368] The oil phases of CR13, CR14 and CR15 were placed in an oven at 160°C for 2 hours (sterilization) and then left at room temperature to resolidify.

[0369] v. Sterilize the aqueous phase by filtering through a 0.2 μm sterile PES syringe filter.

[0370] vi. Weigh the required amount of water into 250 mL of Duran, which has been pre-sterilized by autoclave.

[0371] vii. Place the oil phase in a 75°C water bath until melted.

[0372] viii. Place the aqueous phase together with the homogenizer head in a water bath and equilibrate for 5 minutes.

[0373] ix. For each cream, the oil phase was added to the corresponding water phase and homogenized using an Ultra-turrax equipped with a 25G disperser head at 10,000 rpm for 2 minutes.

[0374] x. After processing, stir the cream manually with a metal spatula until the cream reaches room temperature.

[0375] xi. Leave them overnight to cure.

[0376] xii. Adjust the pH of the formulation to pH 7-7.5

[0377] It is worth noting that manufacturing steps (v) to (xii) are all performed under sterile conditions in a laminar flow hood, and the sterile container is only opened in the sterile laminar flow hood.

[0378] Table 2: Composition (% w / w) of estetrol monohydrate (E4) cream formulation.

[0379]

[0380]

[0381] Example 2. In vitro release experiment 1

[0382] After method development and small-scale preliminary in vitro release testing (IVRT) experiments, full-scale IVRT experiments were performed using the 10 test formulations described in Example 1. The experimental conditions used are shown in Table 3.

[0383] Table 3: Experimental conditions for full-scale IVRT.

[0384]

[0385] Full-scale in vitro release experiments were performed using the experimental conditions developed during method development and feasibility studies. Results for all tested gel and cream formulations are shown in Table 1. Figure 1 The tabular results are shown in Table 4.

[0386] Aqueous gel preparation

[0387] Figure 2 Table 4 shows the release rates of the aqueous gel formulations. AG18, AG19, and AG23 formulations resulted in the highest release rates (161-221 μg / cm 2 / √hr), followed by AG21 (51μg / cm 2 / √hr), AG22 (sterile), AG22 (autoclaved), and AG15 resulted in the lowest release rates (3-10 μg / cm 2 The overall trend was that the observed release rate was related to the concentration of API, with the formulations with the highest release rates (AG18, AG19, and AG23) also containing the highest concentration of estetrol monohydrate (0.50% w / w).

[0388] The hydrogels with the lowest release rates (AG21, AG22 (sterile), AG22 (autoclaved), and AG15) were found to release a significant amount of the administered API (approximately 50% or more of the amount released at t = 0.5 hours) immediately into the receptor solution. Due to the large amounts of API released, many formulations did not achieve linear steady-state drug release (r2 < 0.9). Therefore, no conclusions or statistical comparisons were made using the hydrogel formulations.

[0389] Cream preparations

[0390] The release rate of the cream formulation is shown in Figure 3and Table 4. CR16 (0.50% w / w) resulted in the highest release rate, followed by CR14 (0.35% w / w), and finally CR01 (0.24% w / w), with an average release rate ranging from 32 to 53 μg / cm 2 Similar to the trend for the hydrogels, the release rate was correlated with the concentration of API present in the formulation, with the highest release rate observed in the formulation with the highest drug loading (CR16, containing 0.50% API, resulting in a release rate of 52.20 μg / cm 2 Tukey-Kramer analysis (n=6) was used to analyze the average drug release (μg / cm 2 There was no statistical difference between the release rates of CR14 and CR16 (p>0.05), while a significantly lower release rate was observed for CR01 (p<0.05).

[0391] Table 4. Ten formulations in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution, across the 1 and 8 hour experimental time points The average release rate of estetrol monohydrate from the membrane (μg / cm 2 / √hr).

[0392]

[0393] Example 3. In vitro release experiment 2

[0394] Based on the results of the first in vitro release experiment (Example 2), a second IVRT experiment was conducted using the four formulations and experimental parameters listed in Table 5.

[0395] Table 5: IVRT study parameters and formulations used in in vitro drug release experiment 2.

[0396]

[0397] A second full-scale in vitro release experiment was conducted using the experimental parameters detailed in Table 5 and four aqueous gel formulations. Results for all tested formulations are shown in Table 5. Figure 4 and Figure 5 The tabular results are shown in Tables 6 and 7.

[0398] The objective of this part of the study was to determine the effect of thermodynamic activity on the release rate of estetrol monohydrate from a formulation by comparing a non-optimized formulation (AG23 0.5% (w / w) API) with optimized formulations of various strengths (AG24 0.5% (w / w), AG25 0.22% (w / w), and AG26 0.06% (w / w) API).

[0399] When considering the percentage release of the API dose applied by these four formulations (Table 17), formulation AG26 (0.06% (w / w) API) released 87.95±10.88% of the API after 8 hours, which was significantly higher (p, 0.05) than the other three formulations, while there was no statistical difference (p>0.05) among the other three formulations (61.51±4.91% AG25 (0.22% (w / w) API), 60.67±4.86% AG24 (0.5% (w / w) API) and 56.88±5.90% AG23 (0.5% (w / w) API)).

[0400] Considering the percentage of the applied dose release rate over time (slope), there was a significant difference (p<0.05) between formulation AG26 and the other three formulations (AG23, AG24 and AG25). No statistically significant differences were found between AG23, AG24 and AG25. Statistical analysis was performed using the Tukey-Kramer test (n=5-6).

[0401] In the current study, formulation AG23 showed a release rate of approximately 167 ± 13 μg / cm 2 / hr, compared with a release rate of 195 ± 20 μg / cm in the first IVRT full-scale experiment. 2 / In the current study, formulation AG24 had the highest release rate (approximately 182 ± 8 μg / cm 2 / hr), followed by AG23 and AG25 (about 81±5μg / cm 2 / hr), and finally AG26 (about 34±3μg / cm 2 Each formulation had a release rate that was significantly different from the next (p<0.05), which in the case of AG23 and AG24 (which both contained 0.5% (w / w) estetrol monohydrate) could be due to issues encountered when dosing AG23 or batch-to-batch differences compared to the AG23 batch used in the previous experiments (which had a release rate closer to that seen here for AG24), or simply formulation differences between AG23 and AG24. For the other two formulations, the differences in release rate were expected given the lower levels of API in these formulations (AG23 and AG24 0.5% w / w, AG25 0.22% (w / w), and AG26 0.06% w / w).

[0402] To evaluate the differences among the above aqueous gel formulations, univariate statistical analysis was performed using Tukey-Kramer (n=5-6).

[0403] Table 6: Mean release rates (μg) of estetrol monohydrate from aqueous gel formulations across isopore membranes in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution between the 1 hour and 8 hour experimental time points / cm 2 / √hr).

[0404]

[0405] *Removed a single duplicate value because it was identified as an outlier by Dixon’s Q test

[0406] **Data from full-scale IVRT experiment 1

[0407] Table 7: Cumulative ester alcohol monohydrate released across isopore membranes from aqueous gel formulations at the 8 hour experimental time point in a 40:30:30 v / v / v ethanol:PEG400:water receptor solution (μg / cm 2 ) and percentage of applied dose.

[0408]

[0409]

[0410] *Removed a single duplicate value because it was identified as an outlier by Dixon’s Q test

[0411] Example 4. In vivo LPS treatment regimen (delayed wound healing model)

[0412] The in vivo LPS-induced delayed wound healing model was reconstructed as described previously (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.).

[0413] Eight-week-old female wild-type (C57BL / 6J) mice were divided into six groups (six animals per experimental group):

[0414] - Double placebo-controlled (no LPS, placebo-treated),

[0415] - LPS control (LPS, placebo treatment),

[0416] -LPS with topical EstroGel (estradiol gel containing 0.06% in a hydroalcoholic gel

[0417] 17β-estradiol hemihydrate)

[0418] -LPS with topical AG24 (0.5% in E4)

[0419] -LPS with topical AG25 (0.22% for E4)

[0420] -LPS with topical AG26 (0.06% in E4).

[0421] One day before injury (day -1), all animals were weighed and anesthetized with oxygen and isoflurane (flow rate 1.25-2L, 2-2.5% isoflurane, depending on clinical symptoms). The animals were shaved and the dorsal area was prepared. The wound location was marked on the back of each animal (2 wounds). The animals received a first subcutaneous injection of 2 μg of Klebsiella pneumoniae-derived LPS diluted in DPBS (1 μg per wound; Sigma Aldrich, UK: L4268) at the wound site, except for the double placebo group, which received DPBS only. A thin layer of EstroGel, AG24, AG25, AG26 or placebo was then applied to the back of each animal, totaling 60 μl (30 μl / wound). The animals recovered in a warm box and were then individually housed in fresh cages equipped with Alpha pads, RO water, food, feed, and housing. On day 0 (24 hours after the first anesthesia), mice were re-anesthetized and the dorsal skin was cleaned with chlorhexidine wipes. Two 6 mm dorsal excisions were performed. 2 μg LPS (1 μg / wound) was injected subcutaneously at the wound site as described above, except for double placebo mice injected with DPBS. EstroGel, AG24, AG25, AG26 or AG23 was then applied as a placebo in a thin layer on top of the wound (60 μl in total; 30 μl / wound), taking care not to damage the "LPS blister". Buprenorphine (0.1 mg / Kg), an analgesic, was injected subcutaneously at the nape of the neck postoperatively, and each animal was imaged. The mice were placed in an incubator and returned to individual breeding boxes. Observations were performed postoperatively.

[0422] On days 1, 2, 3 and 4, for the treatment groups, topical EstroGel, AG24, AG25, AG26 or placebo was reapplied in a thin layer on top of the wound for the duration of the observation period (60 μl total; 30 μl / wound).

[0423] On the fifth day, mice were humanely killed by elevated carbon dioxide concentration and cervical dislocation.

[0424] The uterus was removed and weighed.

[0425] Unwounded skin (NS) was collected from the treated site at the time of wounding (day 0), subjected to histological analysis and snap-frozen. Wound tissue was collected five days after wounding (day 5). The wounds were divided into two halves, the lower half of each wound was fixed for histological analysis, and the upper half was snap-frozen.

[0426] For immunohistochemistry (IHC) and histological analysis, tissue samples were fixed in 10% buffered formalin and embedded in paraffin for sectioning. Tissue sections were dewaxed in xylene and rehydrated through an ethanol gradient before hematoxylin and eosin staining and IHC for immune cells.

[0427] To isolate and culture mouse peritoneal macrophages, fill the peritoneal cavity of euthanized mice with 5 ml of ice-cold PBS supplemented with 3% FBS and 1% antibiotic-antimycotic. Remove the cell-containing fluid using a needle and syringe and culture the cells at 1x10 6 Cells / ml were seeded into 12-well plates in RPMI growth medium supplemented with 10% FBS and 1% penicillin / streptomycin. The cells were left overnight to allow macrophages to adhere and then washed twice to remove any non-adherent cells. The cells were cultured for an additional 24 hours and then polarized to either the M1 or M2 state. M1 macrophages were induced using 100ng / ml IFN-γ and 1μg / ml LPS. For M2 macrophages, 20μg / ml anti-IFN-γ and 10ng / ml IL4 were used.

[0428] To isolate RNA and perform quantitative real-time PCR, mouse macrophages were collected in Trizol and RNA was isolated using the Trizol plus RNA isolation kit (Invitrogen, Thermo Fisher) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using GoScript reverse transcriptase (Promega). Quantitative real-time PCR was performed using 2× Takyon SYBR Green mastermix and a CFX Connect thermal cycler. To assess the extent of M1 and M2 polarization of peritoneal macrophages in different treatment groups, primers for mouse genes Il-1β, Tnf-α, iNos, Arg1, Fizz1, and Ym1 were used. Data were normalized using GAPDH primers. Unless otherwise stated, relative gene expression was set to the M0 PBS+PBO control group.

[0429] Statistical significance was assessed using one-way ANOVA with Tukey post hoc analysis or paired t-test.

[0430] from Figure 6 It can be clearly seen that Application promoted an increase in uterine weight and hypertrophic changes. Topical application of E4 affected uterine weight in a dose-dependent manner, AG26 showed no effect, and AG24 induced an increase in uterine weight. In contrast to the (E2) and high-level E4 formulations (AG24, AG25), topical application of a low-concentration E4 formulation (AG26) to open wounds for 6 days did not result in systemic side effects. These side effects were manifested as increased uterine weight. Next, we assessed wound closure on histological samples. K14 immunohistochemistry was performed on histological sections to visualize the newly formed epidermis. The extent of epithelial regeneration was determined by dividing the length of the new epidermis by the distance between the wound edges and multiplying by 100. As expected, LPS- and placebo-treated wounds showed a >30% delay in epithelial regeneration compared to wounds that did not receive LPS treatment ( Figure 7 All four active treatments ( AG24, AG25, and AG26) all promoted epithelial regeneration. Higher promotion and statistical significance were observed in wounds treated with AG25 or AG26, where epithelial regeneration approached the levels observed in controls not receiving LPS treatment. AG25-treated groups failed to reach statistical significance compared with the LPS / PBO group.

[0431] according to Figure 6 and Figure 7 Based on the data presented, we conclude that topical application of the AG26 formulation improves wound healing particularly well without causing systemic effects such as increased uterine weight. This suggests a preferred dose range if systemic effects are to be avoided completely. However, in some cases, higher doses can be envisioned based on a risk / benefit profile that is favorable to the subject.

[0432] Immunohistochemistry analysis was performed to evaluate the active agent treatment ( The effects of AG24, AG25, and AG26 on the number of local wound immune cells. Compared with non-LPS / PBO treated wounds, the level of neutrophils in LPS / PBO treated wounds was significantly increased ( Figure 8 Treatment with AG24 and AG26 significantly reduced the number of neutrophils in the wound, with the same degree of reduction as AG25 was slightly less effective in reducing wound inflammation, but still very significant. In terms of wound macrophage numbers, very similar effects were observed for gels containing E2 and E4 ( Fig. 9 ). Again, the AG25 was slightly less effective. Interestingly, and AG26 completely reversed the effects of LPS on macrophages and restored wound macrophage levels comparable to those of the non-LPS / PBO group ( Fig. 9 ).

[0433] To further complement the immunohistochemical analysis, wound tissue RNA was isolated and qPCR was performed to assess specific markers of M1 and M2 polarization phenotypes ( Fig.10 ). All treatments resulted in a trend toward decreased expression of M1 markers compared to LPS / PBO controls. Interestingly, The magnitude of the effect was greatest in the IL1-β and AG26 groups (statistically significant with IL1-β and AG26 treatments). In contrast, the M2 markers Fizz1 and Ym1 were increased in all treatment groups, but in this case, the effect of AG26 was the least pronounced of the three AG preparations tested. These data suggest that E4 can both suppress inflammation and promote an M2 (pro-healing) wound environment.

[0434] Finally, to further explore the potential systemic effects of topical E2 or E4 treatment, the phenotype of peritoneal macrophages isolated from each experimental mouse group after completion of the study was evaluated. Specifically, macrophages from each group were isolated by peritoneal lavage, cultured, and polarized toward either the M1 or M2 phenotype (as described in Example 7). RNA was isolated and qPCR was performed to quantify Tnf-α and iNOS (M1 markers) and Arg1 and Ym1 (M2 markers; Fig.11 ) relative level.

[0435] It was found that macrophages isolated from the LPS / PBO group exhibited increased expression of M1 markers when polarized to either M1 or M2 phenotypes in vitro. In contrast, the LPS / PBO group exhibited decreased induction of M2 markers in response to in vitro M2 stimulation. Notably, the M0 LPS / PBO group did not show altered expression of M1 or M2 markers compared to the control group. In conclusion, topical LPS can induce an excessive proinflammatory response in peritoneal macrophages.

[0436] In use These effects were reversed in LPS-treated mice treated topically with either AG24, AG25, or E4 formulations (AG26). Fig.11 ). For example, treatment with the E4 formulation completely reversed the LPS-induced increase in Tnf-α expression in M1 and M2 polarized cells in vitro. Statistically significant reductions in expression were observed after treatment with AG24, AG25, and AG26. The anti-inflammatory effect of AG24 (the formulation with the highest E4 concentration) was slightly stronger, especially when considered together with iNOS expression ( Fig.11 ).

[0437] All four preparations increased the expression of M2 markers Arg1 and Ym1 in M2 polarized cells compared with cells from LPS / PBO treated mice ( Fig.11 ). This effect is seen in This was most evident in isolated macrophages from the treatment groups, where levels of M2 markers in M2-polarized cells exceeded those in control (not treated with LPS) mice. Interestingly, of the three E4 preparations tested, AG26 (the lowest E4 concentration) produced the highest increase in M2 marker expression, although AG25 and AG24 were also effective.

[0438] Example 5. In vivo LPS treatment regimen (delayed wound healing model): comparison of different formulations and duration of treatment changes between.

[0439] The in vivo LPS treatment protocol presented in Example 4 (delayed wound healing model) has been repeated on the following groups of animals:

[0440] - Double placebo group (untreated control),

[0441] - LPS control (LPS, placebo treatment),

[0442] -LPS with topical EstroGel (estradiol gel containing 0.06% in a hydroalcoholic gel

[0443] 17β-estradiol hemihydrate), repeated for 4 days,

[0444] -LPS with topical AG26 (0.06% in E4), single dose,

[0445] -LPS and topical AG26 (0.06% in E4), repeated for 4 days,

[0446] -LPS with topical AG28 (0.06% in E4), single dose,

[0447] -LPS and topical AG28 (0.06% in E4), repeated for 4 days.

[0448] Topical EstroGel, AG26, AG28 or corresponding placebo was applied in a thin layer on top of the wound (60 μl in total; 30 μl / wound) during the observation period only on day 0 (single dose) or days -1, 0, 1 and 2 (repeated doses).

[0449] On day 3, mice were humanely killed by increasing CO2 concentration and cervical dislocation. Uteri and uninjured and injured tissue samples were processed as described in Example 4. In contrast to EstroGel, topical application of AG26 and AG28 did not affect uterine weight ( Fig.12 ).

[0450] Next, we assessed wound closure in histological samples (Figure 13). K14 immunohistochemistry was performed on histological sections to visualize the newly formed epidermis. All three active treatments ( Both AG26 and AG28 promoted epithelial regeneration when administered for 4 consecutive days. AG28 failed to reach statistical significance compared with the AG28PBO group. In contrast, the promotion was greater and statistically significant in wounds treated with AG26, with epithelial regeneration including that observed in controls not treated with LPS.

[0451] Compared to placebo, both AG26 and AG28 (4 applications) accelerated wound healing by increasing epithelial regeneration in a delayed wound healing mouse model ( FIG. 13 ).

[0452] AG26 and AG 28 are topical formulations that improve wound healing with just one application without causing systemic effects.

[0453] Example 6. Wound healing in db / db mice

[0454] Eight-week-old diabetic female db / db mice were weighed and the animals were divided into six groups (6 animals per experimental group):

[0455] - topical placebo (control),

[0456] -Partial (estradiol gel containing 0.06% 17β-estradiol hemihydrate in a hydroalcoholic gel),

[0457] - Topical AG24 (0.5% of E4),

[0458] - Topical AG25 (0.22% for E4),

[0459] - Topical AG26 (0.06% for E4),

[0460] - Topical AG28 (0.06% in E4).

[0461] Mice were anesthetized with oxygen and isoflurane (flow rate 1.25-2 L, 2-2.5% isoflurane, depending on clinical symptoms), shaved and the dorsal skin cleaned with chlorhexidine wipes. Two 6 mm dorsal incisions were performed. AG24, AG25, AG26, AG28 or placebo will be applied as a thin layer on top of the wound (60 μL total; 30 μL per wound). Buprenorphine (0.1 mg / Kg) will be injected subcutaneously via the scruff of the neck postoperatively and each animal will be imaged. Mice will be placed in an incubator and returned to individual vivariums. Observation will be performed postoperatively.

[0462] Topicals will be reapplied in a thin layer over the top of the wound each day during the observation period. AG24, AG25, AG26, AG28 or placebo (60 μL total; 30 μL per wound). Wound images will be taken on days 1, 3, 5, 7, 9, 11, 13 and 14 for planimetric analysis.

[0463] On day 14, mice were sacrificed and wound tissue was cut in half at the midpoint, and the lower half of each wound was processed for wax histology (placed in a box with fixative). The uterus was carefully removed so that uterine weight could be recorded.

[0464] Application is expected to promote increased uterine weight and hypertrophic changes. Topical application of E4 is expected to affect uterine weight in a dose-dependent manner, with AG26 and AG28 showing no effect, while AG24 induces an increase in uterine weight.

[0465] AG24, AG25, AG26, and AG28 are expected to accelerate wound healing by increasing epithelial regeneration in a diabetic wound healing mouse model.

[0466] We can conclude that AG26 and AG28 are topical agents that can improve wound healing without causing systemic effects.

[0467] Example 7. Effect of estetrol in wound-related in vitro assays

[0468] In vitro assays were performed to determine the effective dose of E4 in wound-associated cell types (fibroblasts, keratinocytes, and immune cells) to provide information for E4 dose selection for clinical formulation studies. Second, a mechanistic understanding of the effects of E4 on wound-associated cell function was established.

[0469] 1.0. Methods

[0470] 1.1. Human and mouse cells

[0471] Primary human dermal fibroblasts (HDF) were isolated from abdominal skin or leg skin. Primary neonatal normal human epidermal keratinocytes (NHEK) were purchased (Lonza). Mouse dermal fibroblasts (MDF), mouse epidermal keratinocytes (MEK), mouse peritoneal macrophages, and mouse bone marrow were isolated from C57 / Bl6 (wt), NDb (Lepr+ / -), or Db (Lepr- / -) mice.

[0472] 1.2. Cultivation of dermal fibroblasts and epidermal keratinocytes

[0473] Fibroblasts were isolated and cultured in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 1% amphotericin B. At least 4 days prior to assay, cells were switched to DMEM supplemented with 5% charcoal-stripped FBS.

[0474] Neonatal HEKs were cultured in EpiLife supplemented with 15% human keratinocyte growth supplement (HKGS) and 1% penicillin / streptomycin. MEKs were cultured in CnT basal medium supplemented with 1% penicillin / streptomycin and 1% amphotericin B.

[0475] 1.3. Scratch damage measurement

[0476] Cells were seeded into 24-well plates and cultured to form confluent monolayers, then scraped with 1 ml pipette tips. Holes were treated with E2, E4 and / or various estrogen receptor agonists and antagonists. The stock solutions of E2, E4 and PHTPP were dissolved in ethanol (EtOH), so that the final concentration of EtOH in the growth medium (GM) did not exceed 0.1%. ICI, PHPTT, MPP, PPT and DPN were dissolved in DMSO, so that the final concentration of DMSO in GM did not exceed 0.05%. In addition to the untreated negative control, a vehicle control containing an equivalent concentration of EtOH and / or DMSO was included. All treatments were diluted to working concentrations in a culture medium supplemented with 2% activated carbon stripped FBS. In order to visualize the scratch at the defined endpoint, cells were stained with crystal violet and imaged on a Nikon E400 bright field microscope. The scratch closure was determined by multiple independent measurements of each well.

[0477] 1.4. Cultivation of THP1 cells

[0478] THP1 cells were maintained in RPMI growth medium supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin. Cells were plated at 2x10 5 Cells / ml were seeded into 12-well or 6-well plates and treated with phorbol 12-myristate 13-acetate (PMA) to induce differentiation into macrophages. Cells were cultured in complete growth medium without PMA for 24 hours and then serum starved for 6 hours before polarization. Cells were polarized into M1 macrophages using 20ng / ml IFN-γ and 10pg / ml LPS (from E. coli) for 6 or 24 hours and collected for RNA isolation or flow cytometry.

[0479] 1.5. Isolation and culture of mouse bone marrow-derived macrophages

[0480] Rinse the bones with DMEM supplemented with 1% penicillin / streptomycin and 1% amphotericin B. Isolate bone marrow cells at 1x106 Cells / ml were seeded into 12-well plates or 6-well plates in DMEM supplemented with 10% FBS and 10% L929 conditioned medium to induce differentiation. After 7-10 days, differentiated macrophages were cultured in serum-free growth medium with 10 -7 The cells were treated with ME2 or E4 for 16 h and polarized to M1 type using 100 ng / ml IFN-γ and 1 μg / ml LPS. Polarized cells were collected 6 h or 24 h later for RNA isolation or flow cytometry.

[0481] 1.6. Isolation and culture of mouse peritoneal macrophages

[0482] Peritoneal macrophages were isolated from C57 / Bl6 mice by peritoneal lavage. The peritoneal cavity of euthanized mice was filled with 5 ml of ice-cold PBS supplemented with 3% FBS. The lavage fluid containing cells was removed using a needle and syringe and the cells were plated at 1x10 6 Cells / ml were seeded into 12-well or 6-well plates in RPMI growth medium supplemented with 10% charcoal-stripped FBS and 1% penicillin / streptomycin. -7 M E2 or E4 were treated for 16 hours and polarized to M1 type cells using 100 ng / ml IFN-γ and 1 μg / ml LPS. After 6 hours or 24 hours, polarized cells were collected for RNA isolation or flow cytometry.

[0483] RNA Isolation and Quantitative Real-Time PCR

[0484] After treatment, human and murine macrophages were collected in Trizol, and RNA was isolated using the Trizolplus RNA isolation kit (Invitrogen, Thermo Fisher) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using GoScript reverse transcriptase (Promega). Quantitative real-time PCR was performed using 2× Takyon SYBR Green mastermix and a CFX Connect thermal cycler. ERα and ERβ primers were used to explore changes in ER expression after treatment of MEK and MDF with E2 and E4. To investigate the effects of E2 and E4 on MEK differentiation and expression of ECM proteins by MDF, primers for Snai1, keratin-1 and fibronectin, collagen I, MMP-2, and MMP-9 were used. Primers for iNOS, IL-1β, and Tnf-α or Ccl17 were used to assess the M1 / M2 polarization state. GAPDH primers were used to normalize the data. Unless otherwise stated, relative gene expression was set to vehicle control or M0 expression.

[0485] Statistical analysis

[0486] Statistical significance was assessed using one-way ANOVA with Tukey post hoc analysis or paired t-test as appropriate.

[0487] 2.0. Keratinocytes and fibroblasts

[0488] 2.1. Both E2 and E4 promote the migration of human dermal fibroblasts (HDF).

[0489] Scratch assays were performed to quantify the effects of different concentrations of E2 and E4 on HDF migration ( Fig.14 ). Compared with vehicle control, 10 -8 M and 10 -7 ME2 treatment significantly accelerated wound closure (157% and 141%, respectively). -6 M) and two minimum concentrations of 10 -8 M and 10 -9 No differences in closure were observed for E2. E4 treatment compared to vehicle control at 10 -8 M, 10 -7 M and 10 -6 M also significantly accelerated scratch closure (152%, 147%, and 142%, respectively). -8 M optimally stimulated fibroblast scratch wound closure, with higher amplitudes of stimulation observed after E2 treatment.

[0490] Next, an additional experiment was performed using HDFs to a) further confirm the effects of E2 and E4 on fibroblasts from the fifth donor and b) explore the relative effects of including CS-FBS in the cell culture medium. Three identical sets of scratch assays (comparing vehicle with 10 -7 Interestingly, a trend was observed in all three cases, where both E2 and E4 promoted scratch closure, with the greatest promotion after E4 treatment ( Fig.15 ). As expected, the FBS (non-CS) group showed faster closure in all treatments, however, the relative effects of E2 and E4 were less pronounced in this group.

[0491] 2.2. Both E2 and E4 promoted the migration of mouse dermal fibroblasts (MDF).

[0492] To further demonstrate the beneficial effects of E4 and to support dosing considerations for future in vivo mouse model studies, we next performed an in vitro scratch assay in mouse dermal fibroblasts (MDF). The biological effects of estrogenic compounds have been extensively demonstrated in mouse models, and the switch to cells isolated from inbred mouse strains is expected to reduce model variability compared to using cells isolated from human donors.

[0493] As with HDFs, treatment of MDFs with E2 or E4 significantly enhanced scratch closure compared with vehicle control treatment ( Fig.16 and 17 In cells from a single mouse, the magnitude of the effect of E4 treatment was smaller than that of E2 treatment, but the range of efficacy appeared to be wider (significance at 3 concentrations of E4 vs 2 concentrations of E2; Fig.16 Adding data from 2 additional mice and comparing low-passage and high-passage cells showed that the magnitude of the effect of E4 was slightly greater than that of E2 ( Fig.17 ). In addition, the beneficial effects of E2 and E4 were greatest in high-passage cells, which is consistent with clinical expectations.

[0494] 2.3. E2 and E4 treatment increased the expression of ER in fibroblasts, while E4 promoted the expression of fibronectin and inhibited the activity of MMP.

[0495] Mouse fibroblasts cultured in vitro were treated with 10 -7 E2, E4, DPN, or PPT treatment of M. As mentioned above, E2 treatment upregulated the expression of ERα and ERβ, whereas treatment with ER agonists preferentially increased the expression of their respective receptors (e.g., PPT increased ERα and DPN increased ERβ expression; Fig.18 All treatments also showed a strong trend towards increased expression of MMP2 and MMP9 ( Fig.18 ). Next, zymography was performed on the cell supernatants of HDFs treated with E2 or E4 to evaluate the effects of E2 and E4 on cell-derived MMP activity. In cells from three independent donors, both E2 and E4 significantly reduced MMP2 activity compared to the control group ( Fig.19 ). The magnitude of the effect (and statistical significance) was greater in cells treated with E4. MMP9 activity was not detected in any of the treatment groups. Finally, we explored the effects of E2 and E4 on the expression of extracellular matrix genes in mouse fibroblasts, specifically collagen 1 (Cola1) and fibronectin (Fn1) ( Fig. 20 In cells derived from wild-type mice, no effect was observed after treatment. However, when cells derived from diabetic (db / db) mice were treated with E2 or E4, a trend toward increased expression of both Co1a1 and Fn1 was observed ( Fig. 20). In db / db-derived cells treated with E4 alone, the expression of fibronectin (Fn1) was increased to statistical significance.

[0496] 2.4E2 and E4 promote epidermal keratinocyte migration and regulate wound-related epidermal gene expression

[0497] The effects of E2 and E4 on keratinocyte migration were evaluated using primary normal human epidermal keratinocytes (NHEK). Experiments were performed in a range of growth media containing different concentrations of human keratinocyte growth supplement (HKGS). Data show that cells cultured in 15% HKGS and 30% HKGS supplements ( Fig.21 ). Under both conditions, 10 -7 / 10 -8 Both E2 and E4 in the M range showed a strong trend towards faster wound closure. Interestingly, this was only true for the 10 -7 ME2 and 10 -7 ME4 reached statistical significance, and the effect size of E4 was slightly higher and more statistically significant.

[0498] We next turned to primary mouse epidermal keratinocytes (MEK) to explore the effects of E2 and E4 on MEK wound-related gene expression. Although not statistically significant, we observed a strong trend toward induction of both ERα and ERβ in cells treated with E2 and E4 (particularly at 10 -7 M; Fig. 22 ). Consistent with the documented beneficial effects of E2, we observed that -7 There was a statistically significant induction of the EMT marker Snail after E2 or E4 treatment of M. In contrast, the differentiation marker keratin 1 (Krt1) showed a down-regulation trend after E2 and E4 treatment. Overall, these data suggest a shift to a less differentiated, pro-healing phenotype after treatment with E2 or E4. In general, the observed effects of E2 and E4 on gene expression were reversed by co-treatment with the ER antagonist ICI.

[0499] 2.5. E2 and E4 exhibited anti-inflammatory activity in vitro.

[0500] The relative anti-inflammatory effects of E2 and E4 were evaluated in vitro. First, several concentrations of E2 and E4 were screened using the human monocytic THP1 cell line. THP1 cells were differentiated into a macrophage phenotype by treatment with PMA and then polarized into either an M1 or M2 phenotype ( Fig.23Successful polarization was confirmed by analyzing the expression of M1 markers (TNF-α) and M2 markers (CCL17). The effect of co-treatment with a range of E2 or E4 concentrations was evaluated. Here, treatment with E2 or E4 resulted in a strong trend toward decreased expression of TNF-α in M1 polarized cells and increased expression of CCL17 in M2 polarized cells ( Fig.23 ).

[0501] For subsequent experiments, mouse bone marrow-derived monocytes were isolated, differentiated (L929 medium) and polarized to a pro-inflammatory M1 phenotype (IFN-γ and LPS; 6 or 24 h), and treated with the optimal concentrations (10 -7 ; Fig.24 ) were co-treated with either E2 or E4. Compared with vehicle treatment, the expression of a series of M1 markers (iNOS, IL1-β, and Tnf-α) decreased in the presence of E2 and E4. Similar effects were observed in cells polarized for 6 or 24 hours ( Fig.24 ). The experiment was then conducted by differentiating mouse bone marrow-derived monocytes into macrophages using 30 ng / ml MCSF instead of L929 conditioned medium. When polarized to M1 (relative to M0), MCSF-stimulated macrophages showed higher levels of M1 marker expression. Similarly, a range of M1 markers (iNOS, IL1-β, and Tnf-α) showed a strong trend toward decreased expression in the presence of both E2 and E4 compared to vehicle treatment ( Fig.25 ). Note that both E2 and E4 resulted in a statistically significant reduction in the M1 marker iNOS in MSCF-differentiated BMDM. In general, the observed anti-inflammatory effects of E4 were slightly greater than those of E2.

[0502] Finally, the anti-inflammatory effects of E2 and E4 were evaluated in freshly isolated mouse peritoneal macrophages. Unlike the BMDM protocol, differentiated peritoneal macrophages were immediately treated with E2 (10 -7 M) or E4(10 -7 M) pretreatment, followed by M1 polarization with IFN-γ and LPS for 6 hours. In peritoneal macrophages, M1 markers (iNOS, IL1-β, and Tnf-α) also showed a strong trend of decreased expression in the presence of both E2 and E4 compared to vehicle treatment ( Fig.26 ). Note the overall higher levels of marker expression in these cells. The E4-mediated reduction in IL1-β reached statistical significance. The observed anti-inflammatory effects of E4 were broadly similar to those of E2.

[0503] 2.6. Preliminary evaluation of ER-specific effects on fibroblasts and immune cells.

[0504] The relative ER-specific effects of E4 were assessed in vitro using co-treatment with highly specific antagonists of ERα (MPP) or ERβ (PHTPP). Preliminary studies showed that E4 had an effect on HDF migration ( Fig. 27 ) and BMDM polarization ( Fig.28 ) may be mediated by ERα. Specifically, co-treatment with the ERα antagonist MPP appears to block E4-induced scratch closure and E4-mediated reduction of IL1-β, whereas the same ERα antagonist has limited effect on the E2 effect. Given the relatively high variability, these studies should be repeated with a larger number of replicates to confirm the observed effects.

[0505] 2.7. Conduct a preliminary assessment of the in vitro activity of the preparation.

[0506] To initially determine the wound healing potential of the formulated gels, we performed AG23 active (ACT) and placebo (PBO) gel formulations as well as (EG; Fig.29 Again, E2 and E4 treatment significantly suppressed the M1 cell phenotype, and E2-containing The same was true for treatment. AG23 placebo had no effect on relative iNOS expression, whereas AG23 ACT (containing E4) significantly reduced iNOS expression compared to AG23 PBO. These data support further evaluation of the E4 gel formulation in ex vivo human wounds.

[0507] 3.0. Summary of results

[0508] Experiments were designed to explore the relative impact of E4 on specific cellular aspects of wound healing. A series of in vitro studies were conducted using fibroblasts, keratinocytes, and immune cells. Crucially, these in vitro studies were conducted in both human and mouse cells, combining the clinical relevance of human cells and cross-species validation of mouse cells to support subsequent in vivo studies.

[0509] Fibroblasts. E4 has been shown to promote migration of human (HDF) and mouse (MDF) dermal fibroblasts. In both cases, the optimal concentration of E4 was 10 -7 and 10 -8 M, with the optimal E2 concentration being broadly similar. Note that in each case, the relative efficacy of E2 and E4 was also similar. In MDS, we report that the effects on migration are greater in high-passage cells (which may mimic the chronic wound environment). In MDFs, treatment with E2 or E4 directly increased cellular expression of ERα and ERβ. In HDSFs, E4 treatment inhibited MMP2 activity in the cell supernatant and appeared to do so to a greater extent than E2. Preliminary assessments suggest that ERα is important in mediating the effects of E4 on HDF migration.

[0510] Keratinocytes. Scratch wounds also healed faster when mouse epidermal keratinocytes (MEK) were treated with E4 or E2. Again, the optimal concentrations of E2 and E4 were the same (10 -7 M), the observed effects depended on the composition of the cell growth medium. As with fibroblasts, both E2 and E4 treatment induced MEK expression of ERα and ERβ (although the effect did not reach statistical significance). E2 and E4 treatment were shown to induce expression of the EMT marker Snail and inhibit differentiation marker keratin 1, both of which are hallmarks of cellular pro-healing responses.

[0511] Anti-inflammatory activity. E4 and E2 have been shown to have anti-inflammatory effects that promote healing in mouse bone marrow-derived macrophages (BMDM), mouse peritoneal macrophages, and human THP-1 cells. In THP-1 cells, E4 reduced the expression of TNF-α (M1 marker) in M1-polarized cells and increased the expression of CCL17 (M2 marker) in M2-polarized cells. In mouse BMDM and peritoneal macrophages, E4 consistently reduced the expression of a panel of M1 markers in M1-polarized cells. This effect was demonstrated at 6 and 24 hours after polarization in cells obtained using three independent differentiation methods: L929 GM or MCSF (BMDM) or in vivo (peritoneal isolated macrophages). In each case, the magnitude of the effect observed with E4 roughly matched that observed with E2. Preliminary evaluations suggest that ERα is important for mediating the anti-inflammatory effects of E4 on BMDM. Pilot studies have shown a reduction in M1 marker expression following treatment with the E4 preparation AG23.

[0512] 4.0. References

[0513] Campbell L,Emmerson E,Davies F,et al.Estrogen promotes cutaneouswound healing via estrogen receptor beta independent of its antiinflammatoryactivities.J Exp Med.2010;207(9):1825-1833.doi:10.1084 / jem.20100500

[0514] Collaborative Group on Hormonal Factors in Breast Cancer.Type andtiming of menopausal hormone therapy and breast cancer risk:individualparticipant meta-analysis of the worldwide epidemiologicalevidence.Lancet.2019;394(10204):1159-1168.doi:10.1016 / S0140-6736(19)31709-X

[0515] Roth GS, Harman SM, Lamberg SI. Altered Ovarian Regulation of WoundHealing during Aging. Proceedings of the Society for Experimental Biology and Medicine. 1981; 166(1):17-23.doi:10.3181 / 00379727-166-41017

[0516] Thornton MJ.Estrogens and aging skin.Dermatoendocrinol.2013;5(2):264-270.doi:10.4161 / derm.23872

[0517] Example 8. Preparation of aqueous gel formulations for stability studies

[0518] Table 8 lists the aqueous gels prepared for stability studies. The preparation method was consistent with the description of AG24 in Example 1.

[0519] Table 8: Composition (% w / w) of estetrol (E4) aqueous gel formulations prepared for stability studies.

[0520]

Claims

1. A composition for topical application, comprising from about 0.01% to about 5%, preferably from 0.02% to 1.5% (w / w) of an estetrol component, or from about 0.02% to about 1% (w / w) of an estetrol component, or from about 0.05% to about 1.2% (w / w) of an estetrol component, more preferably from about 0.09% to about 1.1% (w / w), even more preferably from 0.1% to 1% (w / w) of an estetrol component, and most preferably from 0.3% to 0.7% (w / w) of an estetrol component.

2. The composition of claim 1, further comprising a penetration enhancer capable of penetrating the stratum corneum of the subject's skin.

3. A hydrogel preparation comprising the composition according to claim 1 or 2.

4. A composition or hydrogel according to any one of claims 1 to 3 for use in topical wound healing.

5. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 4, which is a formulation selected from the group consisting of an emulsion, a suspension, an ointment, a paste, a lotion, a gel, a foam, a mousse and a cream.

6. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 5, comprising a penetration enhancer in an amount of 0.05% to 60% (w / w), preferably in an amount of 0.1% to 5% (w / w).

7. The composition, composition for use, hydrogel or hydrogel for use according to claim 6, wherein the penetration enhancer comprises a substance or molecule capable of penetrating the stratum corneum and comprises a solvent, preferably wherein the molecule promoting penetration is selected from the group consisting of ethanol, ethers such as diethylene glycol monoethyl ether Benzyl alcohol, fatty acids and their esters, or any combination thereof; or The penetration enhancer comprises a solvent, and the solvent includes one or more polyethylene glycol (PEG), propylene glycol (PG) or a combination thereof.

8. The composition, the composition for use, the hydrogel or the hydrogel for use according to claim 7, wherein the PEG is a PEG with a molecular weight between about 200 g / mol and about 600 g / mol, such as a PEG selected from the following group: PEG200, PEG300, PEG400, PEG500, PEG600, or any combination thereof.

9. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 8, further comprising benzyl alcohol, preferably in an amount of 1% to 3%, and / or further comprising a thickener in an amount of 0.3% to 20% (w / w), preferably in an amount of 0.5% to 3% (w / w).

10. The composition, composition for use, hydrogel or hydrogel for use according to any one of claims 1 to 9, wherein the thickener is selected from hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), a high molecular weight cross-linked acrylic acid-based polymer, a nonionic triblock copolymer or any combination thereof, preferably wherein the high molecular weight cross-linked acrylic acid-based polymer is Or wherein the HEC is HEC250 HHX, or wherein the nonionic triblock copolymer has an approximate molecular weight between about 1800 and about 4000 and a polyoxyethylene content of about 70% to about 80%.

11. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 10, further comprising a preservative in an amount of 1% to 10% (w / w), preferably 1% to 3% (w / w), preferably, wherein the preservative is selected from: lysozyme, nisin, quaternary ammonium preservatives, parabens, phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, natural preservatives and any combination thereof.

12. A composition, a composition for use, a hydrogel or a hydrogel for use according to any one of claims 1 to 11, further comprising an emollient in an amount of 2.5% to 30% (w / w), preferably 8% to 12% (w / w), preferably, wherein the emollient is selected from glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, petrolatum and any combination thereof.

13. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 12, wherein the composition or formulation is supplemented to 100% (w / w) by an aqueous solution, such as water.

14. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 4, comprising: - from about 0.1% to about 5% (w / w) of a penetration enhancer, preferably from about 1% to about 2.5% (w / w) of a penetration enhancer, more preferably wherein the penetration enhancer comprises a penetration enhancer molecule and a solvent or solvent system; - from about 0.3% to about 3% (w / w) of a thickener; -optionally preservatives and / or emollients; - water supplemented to 100% (w / w); or or contain: - about 38% to about 45% (w / w) PEG 400; - about 0.1% to about 1% (w / w) and - about 0.8% to about 3% (w / w) 15. A composition, a composition for use, a hydrogel or a hydrogel for use according to any one of claims 1 to 14 for use in treating acute wounds, surgical wounds, wounds caused by acute injury, chronic wounds, wounds caused and / or maintained by diabetes, wounds in subjects with impaired wound healing including impaired delayed cutaneous wound healing or bacterial delayed wound healing, wound healing characterized by reduced wound edge migration, infected wound sites, combat wounds, burns and chronic leg ulcers.

16. The composition, the composition for use, the hydrogel or the hydrogel for use according to any one of claims 1 to 14, which is contained in a wound dressing, a bandage, a plaster, a patch or a plaster.

17. A package comprising one or more dosage units of the composition hydrogel according to any one of claims 1 to 14, preferably wherein the packaging unit is a box, a display unit, an ampoule, a bottle, a vial, a tube, a syringe, a cartridge, a bag, a sachet, a pouch, a film, a sheet, a foil, a can, a cylinder or a pressure vessel.

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