Topical compositions containing an estetrol component and the use of such compositions for wound healing

Abstract

The present invention relates to topical compositions containing an estetrol component and the use of such compositions to deliver an effective amount of the estetrol component to the skin, particularly for wound healing. Optionally, the estetrol component may be included in a composition that further comprises components favorable for wound healing. The present invention particularly relates to compositions such as gels, especially hydrogels, creams, and ointments, that contain estetrol. The compositions of the present invention have beneficial effects on the wound healing process and patient recovery.

Description

[Technical Field]

[0001] The present invention generally relates to treating skin wounds with an effective amount of an estetrol component. Furthermore, the present invention relates to pharmaceutical compositions containing an estetrol component suitable for treating skin wounds. The pharmaceutical compositions and related methods that are the subject of the present invention have a favorable effect on wounds, the wound healing process, and ultimately, patient recovery. [Background technology]

[0002] Throughout life, every individual is repeatedly exposed to harmful stimuli or events that cause skin wounds, such as trauma, surgery, burns, and pathologically induced wounds to the skin and optionally underlying tissue. In many cases, these wounds heal without significant strain on the subject after appropriate routine care, including actions such as disinfecting and / or temporarily covering the wound site. However, complications can arise if the individual has impaired wound healing, if there is excessive inflammation or infection at the wound site, or if the wound site covers a significant area of ​​the individual.

[0003] Wound healing is often perceived as a "basic" function of skin tissue, but it is the culmination of various complex molecular mechanisms. In normal skin tissue (i.e., skin tissue without a wound), the epidermis (the outermost layer of the skin) and the dermis (the corium) form a protective layer against external stimuli (Non-Patent Document 1). When this protective layer is damaged, a repair process is initiated aimed at repairing the damage. This repair process has been well described in the art and includes subsequent steps such as hemostasis (blood clotting), inflammation, proliferation (new tissue growth), and maturation (tissue remodeling).

[0004] Upon closer examination, the repair process is characterized by several molecular mechanisms that have been the subject of research in the art (e.g., Non-Patent Document 2). Briefly, during the coagulation phase (generally considered part of the inflammatory phase), fibrin clots are generated to prevent further blood loss. The inflammatory phase is characterized by initial vasoconstriction followed by vasodilation and the recruitment of cells such as neutrophils, monocytes, and macrophages. The proliferative phase is characterized by angiogenesis, fibroblast differentiation, and granulation tissue formation. Granulation tissue formation allows for re-epithelialization by epithelial cells (keratinocytes) that migrate to cover the wound site. Furthermore, the latter part of the proliferative phase leads to fibroblast-mediated wound contraction. The final wound healing phase, or maturation phase, is characterized by 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 wound healing disorders, one or more of the above phases or their sub-processes are disrupted or essentially absent (Non-Patent Document 3).

[0005] The importance of proper wound care cannot be overstated, as the prevalence of chronic wounds is expected to increase over the coming decades, particularly as a result of the increasing prevalence of chronic diseases such as diabetes, cancer, and autoimmune disorders. Epidemiological studies have already warned of this rapid increase in healthcare burden. Based on global, regional, and national data from over 195 countries and regions, a significant 10-year increase in prevalence has been reported, from 492,883,000 in 2005 to 605,036,000 in 2015 (reported by [unclear text]). Global estimates suggest that at least 7 million people suffer from postoperative complications each year, including at least 1 million deaths. Delayed acute wound healing increases the risk of postoperative morbidity and mortality. A good example is surgical site infection, which is the second leading cause of hospital-acquired infections. Major complications in acute wounds are associated with age-related impaired healing and hormone deficiencies. Here, the population aged 65 and over (currently 15% to 28% of the total) is increasing worldwide.

[0006] Although numerous wound care strategies (both mechanical and pharmaceutical) currently exist, there remains an unmet need for effective wound care strategies and pharmaceutical compositions that aid in such wound care. Such compositions would ideally aid wound healing in elderly subjects and subjects with impaired wound healing, such as, for example, subjects suffering from chronic wounds caused by reduced mobility and / or underlying pathology. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Kolarsick et al., JDNA, 2011 [Non-patent document 2] Rodrigues et al., Physiol Rev, 2019 [Non-patent document 3] Avishai et al., EPMA J, 2017 [Non-patent document 4] Global Burden of Disease (GBD) 2015 Disease and Injury Incidence and Prevalence Collaborators, Lancet, 2016 Summary of the Invention

[0008] As detailed in the examples contained herein, the inventors have surprisingly found that estetrol is particularly suitable for use in wound healing, including wound healing in subjects characterized by impaired wound healing. Extensive experiments have shown that an estetrol component can be safely included in a pharmaceutical composition intended to promote wound healing. The composition has no or limited effect on the increase in uterine weight that may occur due to inadvertent systemic exposure to estrogen when used topically in female subjects. More specifically, the inventors have found that the inclusion of a specific amount of estetrol component in a pharmaceutical composition intended to promote wound healing provides an unprecedented compromise between efficacy and adverse effects, such as increased uterine weight. The lack of increased uterine weight indicates that the systemic effect of a pharmaceutical composition containing an estetrol component is limited, and even with the inclusion of a penetration enhancer, is almost certainly absent at certain dosages.

[0009] In vitro and in vivo wound healing experiments have shown that estetrol exerts effects at least similar to those of estradiol, for example, on wound closure, re-epithelialization, and anti-inflammation. While estradiol and estetrol treatment increase ER expression in fibroblasts, estetrol also promotes fibronectin expression and inhibits MMP activity. Estetrol also promotes dermal fibroblast migration in scratch assays and appears to promote epidermal keratinocyte migration to a greater extent than estradiol. Furthermore, estetrol has been shown to regulate wound-related epidermal gene expression with a slightly greater magnitude of effect than estradiol, promoting wound closure more efficiently than estradiol. These improvements in wound healing, combined with reduced systemic effects (e.g., on uterine weight gain), make estetrol compositions an attractive alternative to estradiol compositions and other estrogen-based compositions used in wound healing.

[0010] Finally, in some embodiments, the compositions additionally feature a gradual release profile, which is an added advantage in the context of wound healing. Thus, the present invention significantly contributes to new and innovative wound care strategies.

[0011] Thus, in a first aspect, the present invention relates to a pharmaceutical composition comprising 0.02% to 1.5% (wt / wt) of an estetrol component, preferably 0.05% to 1.2% (wt / wt) of an estetrol component, about 0.02% to about 1% (wt / wt) of an estetrol component, about 0.03% to about 1% (wt / wt) of an estetrol component, preferably about 0.04% to about 1% (wt / wt) of an estetrol component, more preferably about 0.05% to about 1% (wt / wt) of an estetrol component, most preferably about 0.06% to about 0.5% (wt / wt) of an estetrol component, more preferably about 0.09% to about 1.1% (wt / wt), and even more preferably 0.1% to 1% (wt / wt) of an estetrol component, most preferably 0.3% to 0.7% (wt / wt) of an estetrol component. Preferably, the composition is for topical use or application, for example to the skin.

[0012] Preferably, the composition does not cause significant systemic effects in the subject upon or after topical application.

[0013] In certain embodiments, the pharmaceutical composition comprises about 0.03% to about 0.12% (wt / wt) of the estetrol component, preferably about 0.04% to about 0.08% (wt / wt) of the estetrol component, more preferably about 0.05% to about 0.07% (wt / wt) of the estetrol component, and most preferably about 0.06% (wt / wt) of the estetrol component.

[0014] In certain embodiments, the pharmaceutical composition further comprises a penetration enhancer to enable penetration through the stratum corneum and / or through a wound eschar.

[0015] In certain embodiments, the pharmaceutical composition is a composition for use in topical wound healing.

[0016] In another aspect, the present invention relates to a (pharmaceutical) composition comprising an estetrol component for use in topical wound healing, optionally further comprising a penetration enhancer that allows penetration through the stratum corneum.

[0017] In certain embodiments, 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, even 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.

[0018] In yet another aspect, the present invention relates to a hydrogel formulation comprising about 0.02% to about 1.5% (wt / wt) estetrol component, more particularly about 0.05% to 1.2% (wt / wt), even more particularly about 0.09% to about 1.1% (wt / wt), or about 0.1% to about 1% (wt / wt) estetrol component.

[0019] In certain embodiments, the hydrogel comprises about 0.05% to about 1.3% (wt / wt), more particularly about 0.08% to about 1.2% (wt / wt), even more particularly about 0.09% to about 1.1% (wt / wt), or about 0.1% to about 1% (wt / wt) of the estetrol component. In alternative embodiments, the hydrogel comprises about 0.03% to about 0.75% (wt / wt) of the estetrol component, preferably 0.04% to about 0.5% (wt / wt), more preferably about 0.05% to about 0.25% (wt / wt), and most preferably about 0.06% (wt / wt) of the estetrol component.

[0020] As can be seen from the Examples section, it was found that 0.06% (w / w) of the estetrol component had no effect on uterine weight gain in the tested mice, which is quite different from the effect on uterine weight gain of the commercially available Estrogel™ containing 0.06% estradiol.

[0021] Furthermore, even higher concentrations of estetrol, 0.22% and 0.5% (w / w), still had significantly less effect on uterine weight gain in mice compared to Estrogel™. This indicates that topical application of compositions containing estetrol is less likely to cause systemic effects in subjects, which is particularly important in female subjects. In certain embodiments, the hydrogel further comprises a penetration enhancer that allows penetration through the stratum corneum.

[0022] In certain embodiments, the hydrogels are used in topical wound healing.

[0023] 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.

[0024] In embodiments where the (pharmaceutical) composition is a hydrogel, the hydrogel is characterized by a favorable release profile, for example, a more favorable release profile compared to other formulations such as, but not limited to, creams.

[0025] In preferred embodiments, the hydrogels described herein exhibit a cross-sectional area of ​​at least about 2.5 μg / cm across an Isopore membrane within the square root of one hour in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 , at least about 5 μg / cm 2 , at least about 7 μg / cm 2, at least about 10 μg / cm 2 , at least about 15 μg / cm 2 , at least about 20 μg / cm 2 , at least about 25 μg / cm 2 , preferably at least about 50 μg / cm 2 , more preferably at least 100 μg / cm 2 , more preferably at least about 150 μg / cm 2 , more preferably at least about 200 μg / cm 2 The average cumulative amount of estetrol component released is characterized by: 0.015 mg / mL. Depending on the surface of the wound, the concentration of the estetrol component may need to be reduced to avoid cumulative administration of too much estetrol. This concentration can be easily calculated by a doctor or pharmacist.

[0026] In preferred embodiments, the hydrogels described herein have a cross-linking capacity of at least about 50 μg / cm across an Isopore membrane within 8 hours in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 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 an average cumulative amount of released estetrol component of at least about 80 μg / cm. 2 The average cumulative amount of estetrol component released is characterized by:

[0027] Preferably, the hydrogels described herein are characterized by an average % of the applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of at least about 15%, preferably at least about 20%, after 1 hour, and / or an average % of the applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of at least about 40%, preferably at least about 50%, more preferably at least 80%, and most preferably at least 90% after 8 hours.

[0028] In any of the above embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel may contain 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 (i.e., a penetration enhancer molecule) that enables penetration through the stratum corneum and a solvent. Preferably, the penetration enhancer molecule is selected from the group consisting of ethanol, ethers such as diethylene glycol monoethyl ether (Transcutol™), benzyl alcohol, fatty acids and their esters, or any combination thereof. Preferably, the penetration enhancer comprises a solvent containing one or more polyethylene glycols (PEGs), propylene glycol (PG), or a combination thereof. Preferably, the 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 group consisting of PEG200, PEG300, PEG400, PEG500, PEG600, or any combination thereof. More preferably, the penetration enhancer comprises a solvent containing PEG having a molecular weight of about 400 g / mol, such as PEG 400. In some embodiments, the penetration enhancer comprises up to 50% PEG 400 (wt / wt), and / or about 15%-45% PEG 400 (wt / wt), and / or about 20%-40% PEG 400 (wt / wt), and / or about 30%-35% PEG 400 (wt / wt). Most preferably, the penetration enhancer comprises about 14%-21% PEG 400 (wt / wt), and / or about 10%-25% PEG (wt / wt). In alternative preferred embodiments, the penetration enhancer comprises about 0.5%-10% of a penetration enhancer, such as, for example, PEG 400. In further alternative preferred embodiments, the penetration enhancer comprises about 0.5%-5% of a penetration enhancer, such as, for example, PEG 400.

[0029] In certain embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel comprises benzyl alcohol, preferably in an amount of about 1% to about 3%.

[0030] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel may contain a thickening agent. In certain embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel contains the thickening agent 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 used, or the hydrogel contains a thickening agent selected from the group consisting of hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), a high molecular weight crosslinked acrylic polymer, a nonionic triblock copolymer, or any combination thereof. Preferably, the high molecular weight crosslinked acrylic polymer is Carbopol™.

[0031] Preferably, the HEC is HEC250 HHX. Preferably, the nonionic triblock copolymer has an approximate molecular mass between about 1800 and 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 combinations thereof.

[0032] In any of the above aspects, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel may contain a preservative. In certain embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel may contain 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.

[0033] In any of the above aspects, the pharmaceutical composition, the pharmaceutical composition used, or the hydrogel may contain an emollient. In certain embodiments, the pharmaceutical composition, the pharmaceutical composition used, or the hydrogel may contain the emollient in an amount of about 2.5% to about 30% (w / w), preferably about 8% to about 12% (w / w), and most preferably 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, mineral oil, and any combination thereof.

[0034] Thus, in any aspect and embodiment described herein, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel may comprise, in addition to the estetrol component, a penetration enhancer, a thickener, and optionally 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 with an aqueous solution, such as water, up to 100% (w / w).

[0035] In certain embodiments, the (pharmaceutical) composition, the (pharmaceutical) composition used, or the hydrogel contains, in addition to the estetrol component (w / w): about 0.1% to about 60% (w / w) of a penetration enhancer (preferably, the penetration enhancer comprises a penetration enhancer molecule and a solvent or solvent system); about 0.3% to about 20% (wt / wt) of a thickener, optionally a preservative and / or an emollient, Water (up to 100% (w / w)), may include:

[0036] In a further embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition used or the hydrogel further comprises 0.05% to 0.6% (w / w) of estetrol, about 0.1% to about 5% (w / w) of a penetration enhancer (preferably, the penetration enhancer comprises a penetration enhancer molecule and a solvent or solvent system); about 0.3% to about 3% (wt / wt) of a thickener; optionally a preservative and / or an emollient, Water (up to 100% (w / w)), may include:

[0037] In a further particular embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition used or the hydrogel contains, in addition to the estetrol component (w / w): Approximately 16% to approximately 20% (wt / wt) PEG 400, Approximately 18% to 22% (weight / weight) PG, about 8% to about 12% (wt / wt) glycerol, About 1% to about 2% (wt / wt) HEC, and about 1.5% to about 2.5% (wt / wt) benzyl alcohol; may include:

[0038] In an alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition used or the hydrogel contains, in addition to the estetrol component: Approximately 18% to approximately 22% (wt / wt) PEG 400, about 0.1% to about 1% (w / w) Carbopol™, and about 4% to about 6% (w / w) Transcutol™; may include:

[0039] In yet an alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition used or the hydrogel comprises, in addition to the estetrol component: about 25% to about 55% (wt / wt) PEG 400, preferably about 35% to about 45% (wt / wt) PEG 400; about 0.1% to about 1% (w / w) Carbopol™, preferably about 0.25% to about 0.75% (w / w) Carbopol™, and about 0.1% to about 5% (w / w) Transcutol™, preferably about 0.75% to about 3% (w / w) Transcutol™; may include:

[0040] In yet an alternative embodiment, the (pharmaceutical) composition, the (pharmaceutical) composition used or the hydrogel comprises, in addition to the estetrol component: Approximately 38% to approximately 45% (wt / wt) PEG 400, about 0.1% to about 1% (w / w) Carbopol™, and about 0.8% to about 3% (w / w) Transcutol™; may include:

[0041] Aspects of the present invention encompass each of the (pharmaceutical) compositions, hydrogels, and alternatives described herein as medicaments, i.e., in a therapeutic and / or prophylactic context. In certain embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used in wound healing. In further embodiments, the use in wound healing corresponds to their use as a topical preparation in the treatment of wounds. 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 the topical treatment of wounds. In relation to the above, aspects of the present invention also encompass a method of topical wound treatment, comprising administering any one of the pharmaceutical compositions or hydrogels described herein to a wound or wound site of a subject.

[0042] In certain embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used in the treatment of acute wounds. Optionally, the acute wound is a wound caused by surgery or acute trauma. The wound can also be a partial-thickness wound (e.g., a skin graft donor site). In alternative embodiments, the pharmaceutical compositions and hydrogels of the above aspects are used in the treatment of chronic wounds. Optionally, the chronic wound is a wound caused and / or maintained by diabetic disease. Other major causative factors of chronic wounds are ischemia, radiation, foreign bodies, and prolonged external pressure. Generally, chronic wounds are divided into infected wounds and ischemic wounds.

[0043] In certain embodiments, the medical uses or treatments described herein result in improved histological healing parameters compared to untreated wounds. Preferably, the medical uses or treatments result in an increased incidence of complete wound closure, accelerated wound closure, and / or accelerated surgical wound closure. In preferred embodiments, the medical uses or treatments result in improved quality of healing, also known as cosmesis. Less scarring is observed, particularly in the case of surgical wounds.

[0044] In certain embodiments, the pharmaceutical compositions and hydrogels described herein have beneficial effects on inflamed wounds, preventing inflammation and / or its progression, and may also prevent wound recurrence.

[0045] In certain embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used in treating wounds in subjects with impaired wound healing, including impaired delayed cutaneous wound healing or bacterial delayed wound healing. In certain 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 proliferation.

[0046] In certain embodiments, the pharmaceutical compositions and hydrogels of the above aspects are used in the treatment of infected wound sites, combat wounds, burns, and chronic leg ulcers. In some embodiments, the infected wounds may be associated with decreased re-epithelialization, increased proliferation, enhanced inflammatory responses, and disrupted wound matrix deposition. One pathogen that has been particularly studied in this regard is Klebsiella pneumoniae, and compositions containing an estetrol component disclosed herein have been shown to reduce inflammation in a Klebsiella pneumoniae-infected wound model.

[0047] In certain embodiments, the (pharmaceutical) compositions and hydrogels of the above aspects are used in improving re-epithelialization of a wound site, increasing cell proliferation at the wound site, reducing the inflammatory response at / in the wound site, improving matrix deposition such as matrix remodeling, and improving vascularization at the wound site.

[0048] In certain embodiments, the medical uses or procedures described herein result in improved quality of healing, also called cosmesis, which constitutes an interesting aspect, particularly in the context of surgical wound healing.

[0049] In certain embodiments, the medical uses or treatments described herein result in an improved inflammation profile at the wound site compared to an untreated wound. Preferably, the medical uses or treatments result in an improved macrophage and neutrophil profile indicative of reduced local wound inflammation compared to an untreated wound.

[0050] Optionally, the subject is an elderly subject, such as a subject aged 50 years or older, or preferably 60 years or older. Optionally, the (pharmaceutical) compositions or hydrogels described herein are applied to the wound site at least twice on different occasions as part of a treatment or medical use. Optionally, the treatment may extend for a period of at least one week, or even more than one month, e.g., 12 weeks. Alternatively, the (pharmaceutical) compositions or hydrogels described herein are applied to the wound site continuously for an extended period of time. Preferably, an extended period corresponds to at least one day, at least one week, or at least one month. In certain embodiments, the (pharmaceutical) compositions or hydrogels described herein are included in a wound dressing, bandage, patch, or plaster. Various scaffolds or matrices can be used. Treatment may require an extended period of time, e.g., 12 weeks, and the forms described herein are particularly suitable for facilitating such extended administration periods.

[0051] A further aspect of the present invention relates to a packaging unit containing a pharmaceutical composition of a hydrogel as described in any embodiment herein. The packaging unit preferably contains one or more dosage units of the (pharmaceutical) composition (or optionally, hydrogel) described herein. Suitable packaging units include any container capable of containing and storing a liquid. Optionally, the packaging unit is a box, display unit, ampoule, bottle, vial, tube, syringe, cartridge, bag, sachet, pouch, film, laminate, foil, can, cylinder, or pressurized container.

[0052] The above and further aspects and embodiments of the present invention are set out in the following and accompanying claims, the subject matter of which is hereby incorporated with particularity into this specification. [Brief explanation of the drawings]

[0053] [Figure 1]Figure 1 shows the average cumulative amount of estetrol monohydrate released per unit area (μg / cm) from 10 formulations across an Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution over experimental periods between 1 and 8 hours (expressed as the square root of time). Error bars represent the standard deviation of the mean (n=6). [Figure 2] Figure 1 shows the average cumulative amount of estetrol monohydrate released per unit area (μg / cm) from seven aqueous gel formulations across an Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution over experimental periods between 1 and 8 hours (expressed as the square root of time). Error bars represent the standard deviation of the mean (n=6). [Figure 3] Figure 1 shows the average cumulative amount of estetrol monohydrate released per unit area (μg / cm2) from three cream formulations across an isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution over experimental periods (expressed as the square root of time) between 1 and 8 hours. Error bars represent the standard deviation of the mean (n=6). [Figure 4] Figure 1 shows the mean percentage (%) of applied dose of estetrol monohydrate released across the isopore membrane into the receptor solution from four aqueous gel formulations in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution over experimental periods between 1 and 8 hours (expressed as the square root of time). Error bars represent the standard deviation of the mean (n=5-6). [Figure 5] Figure 1 shows the average cumulative amount of estetrol monohydrate released per unit area (µg / cm) from four aqueous gel formulations across an isopore membrane in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution over experimental periods (expressed as the square root of time) between 1 and 8 hours. Error bars represent the standard deviation of the mean (n = 5-6). [Figure 6]Figure 1 shows the effect of topical placebo (PBO), EstroGel™ (EG), AG24, AG25, and AG26 application on uterine weight and morphology. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before wounding (6 mice per group). EstroGel™, AG24, AG25, AG26, or placebo was applied in a thin layer over the wound 1 day before wounding, at the time of wounding, and 1, 2, 3, and 4 days after wounding. Changes in uterine morphology over 5 days were assessed by measuring uterine weight (A) and taking uterine photographs (B). Scale bar = 10 mm. Results are expressed as mean ± sem. Differences relative to placebo were determined using a paired t-test. *P value ≤ 0.05, ***P value ≤ 0.001. [Figure 7] This figure shows that topical E4 treatment promotes re-epithelialization in a mouse model of delayed wound healing induced by LPS. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before wounding (6 mice per group). EstroGel™, AG24, AG25, AG26, or placebo was then applied in a thin layer over the top of the wound 1 day before wounding, at the time of wounding, and 1, 2, 3, and 4 days after wounding. Histological images were used to calculate the percentage of wound re-epithelialization on day 5. Tissue sections were subjected to K14 immunohistochemistry to visualize the newly forming epidermis. The degree of re-epithelialization was calculated 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 relative to placebo were determined using a paired t-test. *P value ≤ 0.05, ***P value ≤ 0.001, n = 6 mice per group. Mean + SEM. [Figure 8] Figure 1 shows that topical E4 treatment results in a reduction in wound neutrophil numbers. Immunohistochemistry and quantification of wound neutrophils (A) and representative images for each treatment group (B). **p≦0.01, n=6 mice per group. Mean + SEM. [Figure 9]Topical E4 treatment results in a reduction in wound macrophage numbers. Immunohistochemistry and quantification of wound macrophages (A) and representative images for each treatment group (B). *p≦0.05, **p≦0.01, n=6 mice per group. Mean + SEM. [Figure 10] (Figure 1) Topical E4 treatment promotes a resolved wound phenotype, decreasing M1 and increasing M2 marker expression. RNA from wound tissue was isolated and wound expression of M1 (TNF-α and IL1-β) and M2 (Fizz1 and Ym1) markers was quantified via qPCR. *p≦0.05, **p≦0.01, n=6 mice per group. Mean + SEM. [Figure 11] Figure 1 shows that in vitro polarization of peritoneal macrophages isolated from experimental mice demonstrates that local treatment exerts systemic effects on immune cell phenotype. Peritoneal macrophages were isolated upon completion of in vivo studies. Cells were cultured and polarized in vitro toward M1 or M2 phenotypes, and expression of M1 (TNF-α and iNOS) and M2 (Arg1 and Ym1) markers was quantified via qPCR. *p≦0.05, n=3 replicates for cells pooled from n=6 mice. Mean + SEM. [Figure 12] Figure 1 shows the effect of topical placebo (PBO), EstroGel™ (EG), AG26, and AG28 application on uterine weight and morphology. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before wounding (6 mice per group). EstroGel™, AG26, AG28, or their placebos were applied in a thin layer over the top of the wound on day 0 only (single dose) or on days -1, 0, 1, and 2 (repeated doses). Changes in uterine morphology over 3 days were assessed by measuring uterine weight. Results are expressed as mean ± sem. Differences relative to placebo were determined using a paired t-test. *P value ≤ 0.05, and ***P value ≤ 0.001. [Figure 13]A) Effect of topical placebo (PBO), EstroGel™ (EG), AG26, and AG28 application on re-epithelialization. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before wounding (six mice per group). EstroGel™, AG26, AG28, or their placebos were then applied in a thin layer over the top of the wound on day 0 only (single dose) or on days -1, 0, 1, and 2 (repeated doses). Histological images were used to calculate the percentage of wound re-epithelialization on day 3. Histological sections were subjected to K14 immunohistochemistry to visualize the newly forming epidermis. The degree of re-epithelialization was calculated 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, and ***P value ≦0.001. n = 6 mice per group. Mean + SEM. B) and C) A single topical E4 administration already results in a reduction in wound neutrophil counts in a mouse model of LPS-induced delayed wound healing. Eight-week-old female mice were injected subcutaneously with LPS 24 hours and 2 hours before wounding (6 mice per group). EstroGel™ (EG, 0.06% E2 gel), AG24 (0.5% E4 gel), AG25 (0.22% E4 gel), and AG26 (0.06% E4 gel), or placebo (PBO) was then applied in a thin layer over the top of the wound on day 0 only (single dose) or on days −1, 0, 1, and 2 (repeated doses). Neutrophil cell numbers per cm were quantified on tissue sections on day 3. **p≦0.01, n=6 mice per group. B) Mean+SEM and C) individual data points. [Figure 14] Figure 1 shows that both E2 and E4 promote migration of human dermal fibroblasts. (A) Percentage of closure was calculated 36 hours after treatment with vehicle, E2, or E4 across a range of concentrations. Data from four independent fibroblast donors (n=8). (B) Representative images from the same donor. **p≦0.01, *p≦0.05. Mean + SEM. [Figure 15]Figure 1 shows an evaluation of whether medium / serum composition influences the effects of E2 and E4 on human dermal fibroblast migration. Cells were maintained in DMEM containing 10% charcoal-stripped (CS) FBS, then switched to either 2% CS-FBS, 0% FBS, or 2% FBS, scratched, and treated with vehicle, E2, or E4. Percentage of closure was calculated after 24 hours. Data (A) and representative images (B) from a single donor (n=3). Mean + SEM. [Figure 16] Figure 1 shows that both E2 and E4 promote migration of high-passage mouse dermal fibroblasts. Percentage of closure was calculated 24 hours after treatment with vehicle, E2, or E4 across a range of concentrations. Data from cells isolated from a single mouse (n=4). Representative images from cells isolated from a single mouse. **p≦0.001, *p≦0.05. Mean+SEM. [Figure 17] Figure 1 shows that the effect of E4 on mouse dermal fibroblast migration is greater in high-passage cells. Percentage of closure was calculated 24 hours after treatment with vehicle, E2, or E4. Data from low-passage cells (a, b) and high-passage cells (c, d). Results are shown for cells isolated from three independent mice (n=12). Representative images from cells isolated from the same mouse. **p≦0.001, *p≦0.05. Mean+SEM. [Figure 18] Figure 1 shows that E4 increases the expression of both ERα and ERβ in mouse dermal fibroblasts (MDFs). Expression of ERα (Esr1) and ERβ (Esr2) was measured by qPCR using RNA isolated from MDFs treated with E2, E4, or an ERα (PTT) agonist and an ERβ (DPN) agonist at 10-7 M. Data from cells obtained from n=3 independent mice. **p≦0.01, *p≦0.05. Mean + SEM. [Figure 19]Figure 1 shows that E2 and E4 inhibit MMP2 activity in supernatants from human dermal fibroblasts (HDFs). HDFs from n=3 independent donors were treated with E2 or E4. Zymography was performed including standards for MMP2 and MMP9. Data from n=6 experiments on n=3 independent donors (A). A representative zymogram (B) is shown from a single experiment / donor. **p≦0.01, *p≦0.05. Mean+SEM. [Figure 20] Figure 1 shows that E4 treatment increases the expression of ECM components in dermal fibroblasts from diabetic mice, but not in wild-type mice. Expression of Col1a1 and Fn1 was measured by qPCR using RNA isolated from MDFs obtained from diabetic (db / db) or wild-type mice and treated with E2 and E4. Data from cells obtained from n=3 independent mice. *p≦0.05. Mean + SEM. [Figure 21] Figure 1 shows that both E2 and E4 promote migration of normal human epidermal keratinocytes (NHEK). Percentage of closure was calculated 24 hours after treatment with vehicle, E2, or E4 across a range of concentrations. Data from a single primary NHEK donor (n=9-15) (A: 15% human keratinocyte growth supplement (HKGS), B: 30% HKGS). Representative image field (C) from a single experiment. **p≦0.01, *p≦0.05. Mean + SEM. [Figure 22] This figure shows that E4-treated primary mouse epidermal keratinocytes show increased expression of both ERα and ERβ, and a strong tendency toward altered keratinocyte phenotype markers. Expression of ERα (Esr1), ERβ (Esr2), the epithelial-mesenchymal transition marker Snail, and the keratinocyte differentiation marker Krt1 were measured by qPCR using RNA isolated from MEKs treated for 24 hours with various concentrations of E2 or E4, or E2 / E4 plus the ER antagonist ICI. Data from cells obtained from n=3 independent mice. *p≦0.05. Mean + SEM. [Figure 23]Figure 1 shows that experiments in the human immune THP1 cell line reveal anti-inflammatory activity of both E2 and E4. THP1 cells were differentiated into a macrophage phenotype by PMA treatment and subsequently polarized to an M1 or M2 phenotype. 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 E2 or E4 at various concentrations. n=3. Mean + SEM. [Figure 24] Figure 1 shows that pro-inflammatory markers are reduced in M1-stimulated L929-differentiated murine bone marrow-derived macrophages (BMDMs) after treatment with E2 or E4. BMDMs were differentiated using 20% ​​L929 growth medium and polarized to an M1 phenotype using 20 ng / ml IFN-γ and 10 pg / ml LPS for 6 or 24 hours. Combined treatment with E2 or E4 strongly tended to reduce the expression of M1 markers iNOS, TNF-α, and IL1-β (measured via qPCR) using RNA isolated from treated cells. Data from pooled cells obtained from n=3 independent mice. Mean + SEM. [Figure 25] This figure shows that pro-inflammatory markers are reduced in M1-stimulated MCSF-differentiated mouse bone marrow-derived macrophages (BMDMs) after treatment with E2 or E4. BMDMs were differentiated using 30 ng / ml MCSF and polarized to M1 using 100 ng / ml IFN-γ and 10 pg / ml LPS for 6 hours. Cells were then co-treated with 10-7 M E2 or E4. Expression of M1 markers iNOS, TNF-α, and IL1-β was measured by qPCR using RNA isolated from treated cells. Data from pooled cells obtained from n=3 independent mice. *p≦0.05. Mean + SEM. [Figure 26]Figure 1 shows that proinflammatory markers are reduced in M1-polarized mouse peritoneal macrophages after treatment with E2 or E4. Freshly isolated peritoneal macrophages were immediately pretreated with 10-7 M E2 or E4 and subsequently polarized to the proinflammatory M1 phenotype using 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 treated cells. Data from cells obtained from n=3 independent mice. **p≦0.01, *p≦0.05. Mean + SEM. [Figure 27] Figure 1 shows that combined treatment of HDFs with ER-specific antagonists suggests that both ERα and ERβ are involved in E4-promoted fibroblast migration. The percentage of closure was calculated 24 hours after treatment with vehicle, E2, E4, or E2 / E4 and the ERα-specific antagonist MPP or the ERβ-specific antagonist PHTPP. Data from two independent fibroblast donors (n=6). *p≦0.05 vs. E4. Mean + SEM. [Figure 28] This figure shows that combined treatment of mouse bone marrow-derived macrophages (BMDMs) with ER-specific antagonists suggests that both ERα and ERβ are involved in E4-promoted anti-inflammatory activity. BMDMs were isolated, differentiated using 20% ​​L929 GM, and polarized to a pro-inflammatory M1 phenotype using 100 ng / ml IFN-γ and 10 pg / ml LPS for 6 hours. E2- and E4-stimulated (10-7 M) 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. n = 3 independent mice. **p ≤ 0.01 vs. E4. Mean + SEM. [Figure 29](Figure 1) Combined treatment of BMDMs with ER-specific antagonists suggests that both ERα and ERβ are involved in E4-promoted anti-inflammatory activity. BMDMs were differentiated using 20% ​​L929 GM and polarized to M1 over 6 days using 100 ng / ml IFN-γ and 10 pg / ml LPS. M1-polarized cells were treated with E2, E4, Estrogel™ (EG), AG23 placebo, or AG23 active formulation. iNOS was measured via qPCR. n = 5-6 independent mice. *p < 0.05. **p < 0.01. Mean + SEM. DETAILED DESCRIPTION OF THE INVENTION

[0054] As used herein, the singular forms "a," "an," and "the" include both singular and plural referents unless the context clearly dictates otherwise.

[0055] The terms "comprising," "comprises," and "comprised of," as used herein, are synonymous with "including," "includes," or "containing," and these terms are inclusive or open-ended and do not exclude additional, unrecited components, elements, or method steps. These terms also encompass "consisting of" and "consisting essentially of," which enjoy well-established meanings in patent terminology.

[0056] The recitation of numerical ranges by endpoints includes all numbers and fractions falling within the particular range, as well as the recited endpoints. This applies to numerical ranges whether the numerical range is introduced by the phrase "from to," or "between," or otherwise.

[0057] The terms "about" or "approximately," as used herein, when referring to a measurable value such as a parameter, amount, time interval, etc., are meant to encompass variations from the value relative to the specified value, such as variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less, and even more preferably ±0.1% or less, from the value relative to the specified value, provided that such variations are appropriate for practicing the disclosed invention. It will be understood that values ​​modified by "about" or "approximately" are themselves specifically and preferably disclosed.

[0058] On the other hand, the term "one or more" or "at least one," e.g., one or more members or at least one member of a group of members, is self-explanatory and, by way of further example, encompasses, among other things, reference to any one of said members, or any two or more of said members, e.g., any three or more, four or more, five or more, six or more, seven or more, etc. of said members, up to all of said members. As another example, "one or more" or "at least one" may refer to 1, 2, 3, 4, 5, 6, 7, or more.

[0059] The background discussion to the invention herein is included to explain the context of the invention and is not an admission that any of the material referred to was published, known, or common general knowledge in any country as of the priority date of any of the claims.

[0060] Throughout this disclosure, various publications, patents, and published patent specifications are referenced with an identifying citation. All publications cited herein are incorporated by reference in their entirety. In particular, the teachings or sections of such publications specifically mentioned herein are incorporated by reference.

[0061] Unless otherwise defined, all terms used in disclosing the present invention, including technical and scientific terms, have the meanings commonly understood by those skilled in the art to which this invention belongs. For further guidance, definitions of terms are included so that the teachings of the present invention can be better understood. When a particular term is defined in relation to a particular aspect of the present invention or a particular embodiment of the present invention, such relation or meaning is meant to apply throughout this specification, i.e., in the context of other aspects or embodiments of the present invention, unless otherwise defined. For example, an embodiment relating to a product is also applicable to corresponding features of the method and use.

[0062] In the following sections, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined can be combined with any other aspect(s) or embodiment(s), unless expressly stated to the contrary. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0063] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, even if some embodiments described herein include some features but not other features included in other embodiments, combinations of features from different embodiments are intended to be within the scope of the present invention and form different embodiments, as would be understood by one of ordinary skill in the art. For example, the appended claims encompass alternative combinations of the claimed embodiments, as would be understood by one of ordinary skill in the art.

[0064] The inclusion of estrogen in pharmaceutical compositions suitable for topical application has been described in the art. One example of a commercially available topical formulation is EstroGel™, a gel containing estradiol. EstroGel™ is indicated for the treatment of moderate to severe vasomotor symptoms and moderate to severe atrophy of the vulva and vagina due to menopause. Although EstroGel™ has potential benefits in wound healing, it has not been marketed for this indication itself. Furthermore, conventional estrogen formulations are known to be suboptimal for direct application to individuals and to be characterized by a number of potential adverse effects that may be associated with the use and / or unintended systemic exposure of estradiol. Such adverse effects may include nausea, vomiting, stomach cramps, bloating, swelling, weight gain, breast pain, breast tenderness, headache, vaginal itching, vaginal discharge, menstrual irregularities, spotting, hair loss, etc. (See Mayo Clinic report on transdermal estradiol: Estradiol (Transdermal Route) Side Effects - Mayo Clinic). Thus, known pharmaceutical compositions exert a significant amount of undesirable systemic effects on individuals. As described in the summary of the present disclosure, pharmaceutical compositions containing an estetrol component are provided that have at least similar wound healing properties as pharmaceutical compositions described in the art, but exhibit significantly reduced systemic effects. For example, the known wound healing composition that contains estradiol will cause a clear increase in uterine weight, which indicates the systemic response to estradiol, while the composition that contains estetrol component will only increase uterine weight to a lesser extent, and some compositions may even show virtually no effect on uterine weight, while still being effective in wound healing.This observation also applies when containing penetration enhancer.This allows the formulation of pharmaceutical compositions that contain relatively low estrogen dosage.The wound healing properties of the estetrol component at such low doses are even more remarkable when one considers that estetrol has historically been considered a weak estrogen compared to other estrogens, such as estradiol (Gerard et al., J Endocrinol, 2015). Thus, neither the compositions described herein nor the medical uses of the estetrol component for wound healing can be predicted or anticipated based on what is known in the art.

[0065] With this in mind, a first aspect of the present invention relates to a pharmaceutical composition comprising about 0.02% to about 0.18% (weight / weight) of an estetrol component.

[0066] The term "estetrol component" as used throughout this specification includes substances selected from the group consisting of estetrol, estetrol esters in which at least one hydrogen atom of a hydroxyl group is replaced by an acyl radical of a hydrocarbon carboxylic acid, sulfonic acid, or sulfamic acid having 1 to 25 carbon atoms, estetrol hydrates, such as estetrol monohydrate, and combinations thereof. Reference to "estetrol" throughout any part of this specification is understood to contemplate any estetrol-containing component (i.e., compound) and / or estetrol derivative (such as those described above). More preferably, in the context of this disclosure, a particularly preferred estetrol component suitable for the dosage units or cosmetic or medical uses and treatment methods described herein is estetrol (including its hydrates). Most preferably, the estetrol component is estetrol monohydrate.

[0067] The term "estetrol" as used herein refers to 1,3,5(10)-estratriene-3,15α,16α,17β-tetrol or 15α-hydroxyestriol, as well as estetrol hydrates, such as estetrol monohydrate. "Estetrol" or "E4" for short is an estrogenic steroid produced by fetal human liver (PubChem CID: 27125). Estetrol can be described as a 3-hydroxysteroid corresponding to 17β-estradiol, with two additional hydroxyl groups substituted at the 15α and 16α positions. Estetrol is known to be an estrogen receptor agonist (Coelingh Bennink et al., Estetrol review: profile and potential clinical applications, Climacteric, 2008). When the estetrol component described herein refers to estetrol, the estetrol may be endogenous estetrol. Alternatively, estetrol may be synthesized chemically, by the use of (mutant) recombinant enzymes, or by any combination thereof. Alternatively, estetrol may be known in the art by its molecular formula C 18 H 24 O4, or structural formula (I): [ka] It can be represented by:

[0068] "(wt / wt)", or alternatively designated throughout the art by terms such as "weight per weight" or "weight to weight", refers to the percent contribution of a particular molecule or substance within a composition or mixture measured by its weight (i.e., mass).

[0069] In certain embodiments, the composition comprises about 0.01% to about 0.18% (w / w) of an estetrol component, e.g., estetrol. In preferred embodiments, the composition comprises about 0.02% to about 0.16% (w / w) of an estetrol component, e.g., estetrol, preferably about 0.03% to about 0.14% (w / w) of an estetrol component, e.g., estetrol, more preferably about 0.04% to about 0.12% (w / w) of an estetrol component, e.g., estetrol, even more preferably about 0.05% to about 0.10% (w / w) of an estetrol component, e.g., estetrol, and even more preferably about 0.05% to about 0.08% (w / w) of an estetrol component, e.g., estetrol.

[0070] In certain alternative embodiments, the composition contains about 0.18% (w / w) or less of an estetrol component, such as estetrol. In preferred embodiments, the composition contains about 0.16% (w / w) or less of an estetrol component, such as estetrol, preferably about 0.14% (w / w) or less of an estetrol component, such as estetrol, more preferably about 0.12% (w / w) or less of an estetrol component, such as estetrol, even more preferably about 0.10% (w / w) or less of an estetrol component, such as estetrol, and even more preferably about 0.08% (w / w) or less of an estetrol component, such as estetrol.

[0071] 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.

[0072] In a preferred embodiment, estetrol is present or used herein as the monohydrate. Thus, in certain embodiments, the pharmaceutical composition contains about 0.01% to about 0.18% (w / w) estetrol monohydrate. In a preferred embodiment, the composition contains about 0.02% to about 0.16% (w / w) estetrol monohydrate, preferably about 0.03% to about 0.14% (w / w) estetrol monohydrate, more preferably about 0.04% to about 0.12% (w / w) estetrol monohydrate, even more preferably about 0.05% to about 0.10% (w / w) estetrol monohydrate, and even more preferably about 0.05% to about 0.08% (w / w) estetrol monohydrate.

[0073] In another specific embodiment, the pharmaceutical composition comprises about 0.18% (w / w) or less of estetrol monohydrate. In a preferred embodiment, the composition comprises about 0.16% (w / w) or less of estetrol monohydrate, preferably about 0.14% (w / w) or less of estetrol monohydrate, more preferably about 0.12% (w / w) or less of estetrol monohydrate, even more preferably about 0.10% (w / w) or less of estetrol monohydrate, and even more preferably about 0.08% (w / w) or less of estetrol monohydrate.

[0074] The above embodiments do not exclude the presence of non-estetrol estrogen components in the pharmaceutical composition. It is also contemplated that there may be different estetrol components within a single composition. In such embodiments, the composition may, by way of example and not limitation, include estetrol, or more specifically, both estetrol monohydrate and esters of estetrol.

[0075] "Topical application" means application to a specific location on the body or on the body. In particular, application to a body surface such as the skin or mucous membranes is envisaged. When used topically, a local effect is usually sought, in particular a local effect on the skin and mucous membranes. Topical application also includes vaginal application, although topical application is preferably understood in the present context as application to the skin.

[0076] In certain embodiments, the pharmaceutical composition comprises a penetration enhancer that enables penetration through the stratum corneum. In the context of the present invention, the term "penetration enhancer" is used interchangeably with terms including, but not limited to, "penetration enhancer," "penetration enhancer," "penetration inducer," and even "skin enhancer." Those skilled in the art will understand that the term "penetration enhancer" as used throughout this disclosure refers to a penetration enhancer molecule as part of a solvent or solvent system. Various penetration enhancers are well described in the art, including, but not limited to, those listed in the CPE database (Vasyuchenko et al., Pharmaceutics, 2021). Exemplary, non-limiting penetration enhancers are further described herein.

[0077] The composition of skin and its various cellular structural layers are known. Those skilled in the art understand that human skin can generally be considered to comprise three distinct layers: the epidermis, the dermis, and the subcutaneous tissue. The epidermis is the upper layer of skin, primarily composed of keratinocytes, epithelial cells that proliferate and differentiate to ultimately produce the stratum corneum (the outermost layer of dead skin cells). The epidermis forms a barrier against environmental pathogens such as bacteria, controls the amount of water released from the body, and plays an important role in wound healing. The second skin layer, the dermis (alternatively "corium" or "cutaneous connective tissue"), is located between the subcutaneous tissue and the epidermis and primarily comprises (mesenchymal) fibroblasts. The dermis is firmly connected to the epidermis by a basement membrane, a sheet of extracellular matrix. The dermis is also significantly thicker than the epidermis, and fibroblasts within the dermis produce the extracellular matrix (e.g., collagen, glycosaminoglycans including hyaluronic acid, elastic fibers, etc.). The main roles of the dermis are to maintain the thickness and elasticity of the skin, to keep the skin moist (due to the water-holding capacity of glycosaminoglycans, including hyaluronic acid), and to heal wounds, i.e., by reforming and rebuilding the damaged extracellular matrix. The deepest layer of the skin is commonly referred to as the "subcutaneous tissue," which is referred to interchangeably in the art by terms such as "subcutaneous tissue," "hypoderm," "subcutis," and "facial fascia."

[0078] Therefore, those skilled in the art will understand that the "stratum corneum" referred to herein refers to the outer layer of the epidermis, which is composed of many layers of highly differentiated keratinocytes, mainly made of the proteinaceous 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 penetration of pharmaceutically active agents across the skin is rate-limited by the stratum corneum.Therefore, the penetration enhancer envisioned by the present invention helps at least the estetrol component to penetrate across the stratum corneum and / or wound eschar, particularly facilitating penetration in burn wounds.

[0079] The term "active pharmaceutical ingredient", which is used interchangeably with "pharmaceutically active agent" throughout this disclosure, shall be interpreted in accordance with the World Health Organization definition of the term: "A substance used in a finished medicinal product (FPP) that exhibits pharmacological activity or is otherwise intended to have a direct effect in the diagnosis, care, mitigation, treatment or prevention of disease, or in the restoration, correction or alteration of physiological function in humans."

[0080] The compositions described herein are particularly suitable for use in wound healing, and therefore may be referred to indifferently as "pharmaceutical compositions" in any given case. Preferably, the compositions described herein are used for topical wound healing. The phrase "used for topical wound healing" referred to herein relates to the treatment of one or more wounds on a subject, where the area of ​​the wound may be defined by a certain surface area. Furthermore, this phrase refers to the use of the compositions described herein for the treatment of a wound on the skin of a subject. The term "topical wound healing" further refers to the topical administration (i.e., application) of the compositions described herein to the wound area. Topical administration may also include, but is not limited to, the use of transdermal administration means, such as a transdermal patch, as further discussed throughout this disclosure.

[0081] As used herein, the terms "wound area" and "wound size" refer to a physical measure of disruption of the normal continuity of a structure. Wound area or size may be expressed, for example, in square centimeters. Depending on the wound size or area, the concentration of the composition applied to the wound may need to be adjusted to avoid exceeding the maximum tolerated dose. In such cases, the composition may be prepared specifically for the subject, or various grades or concentrates may be pre-prepared. Thus, for larger wounds, lower concentrations of the composition according to the present invention are recommended, while higher concentrations can be used for smaller wounds. Extrapolating the results from mouse studies to human conditions, a concentration of 0.06% (wt / wt) in mice can be calculated to be equivalent to 0.1 mg of the estetrol component in the hydrogel applied to a human subject. Therefore, it can be assumed that topical application of approximately 0.1 mg of the estetrol component to a subject will not have systemic effects, such as uterine thickening, in female subjects. The higher dose of 0.5% (wt / wt) in mice can be extrapolated to 0.9 mg or approximately 1 mg of estetrol, which allows it to be administered locally without significant risk of systemic effects, or at least with less systemic effects than the commercially available Estrogel™ containing 0.06% estradiol, which is regulatory approved for topical application.

[0082] The terms "subject," "individual," or "patient" may be used interchangeably herein and typically and preferably refer to humans, but may also include reference to non-human animals, preferably warm-blooded animals, and even more preferably mammals, such as non-human primates, rodents, canines, felines, equines, ovine, porcines, and the like. The term "non-human animal" includes all vertebrates, e.g., mammals such as non-human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and the like, and non-mammals such as chickens, amphibians, reptiles, and the like. In certain embodiments, the subject is a non-human mammal. Preferred subjects are human subjects, including those of all genders and all ages. The term "subject" is intended to cover both adult subjects, newborn subjects, and fetuses. Thus, both adult and neonatal subjects are intended to be included. Examples of subjects include humans, dogs, cats, cows, goats, and mice. Preferred subjects in the context of the present invention are further defined below.

[0083] In another yet related aspect, the present invention relates to an estetrol component used in wound healing, more particularly in localized or topical wound healing. Accordingly, the present invention contemplates the use of an effective amount of an estetrol component for use in wound healing by applying the estetrol component to the skin or wound area of ​​a subject. The term "effective amount" refers to the amount necessary to achieve a physiological effect. The physiological effect may be achieved by a single dose or multiple doses. In certain embodiments, the present invention relates to an estetrol component used in wound healing in the presence of a penetration enhancer that enables penetration through the stratum corneum. In a further embodiment, the present invention relates to a pharmaceutical composition used in wound healing, comprising an effective amount of the estetrol component and a penetration enhancer. Preferably, the estetrol component used in wound healing is estetrol, most preferably estetrol monohydrate. In an alternative embodiment, the present invention relates to a pharmaceutical composition used in wound healing, comprising an effective amount of the estetrol component combined with a second composition comprising a penetration enhancer. In such embodiments, the particular order of administration of the first and second compositions to the wound area (i.e., wound site) is not particularly limited, and thus the compositions may be applied either sequentially or (nearly) simultaneously.

[0084] In certain embodiments, the pharmaceutical composition is used in wound healing and contains about 0.01% to about 5% (wt / wt), e.g., about 0.08% to about 1.2% (wt / wt), about 0.09% to about 1.1% (wt / wt), or about 0.1% to about 1% (wt / wt) of an estetrol component (such as estetrol, preferably estetrol monohydrate) and a penetration enhancer, preferably about 0.02% to about 2.5% (wt / wt) of an estetrol component and a penetration enhancer, more preferably about 0.02% to about 2% (wt / wt) of an estetrol component. and penetration enhancer, more preferably from about 0.03% to about 1.5% (wt / wt) of estetrol component and penetration enhancer, even more preferably from about 0.03% to about 1% (wt / wt) of estetrol component and penetration enhancer, even more preferably from about 0.03% to about 0.75% (wt / wt) of estetrol component and penetration enhancer, even more preferably from about 0.04% to about 0.5% (wt / wt) of estetrol component and penetration enhancer, and most preferably about 0.06% (wt / wt) of estetrol component and penetration enhancer.

[0085] In an alternative embodiment, the pharmaceutical composition is used in wound healing and contains from about 0.01% to about 5% (w / w), e.g., from about 0.08% to about 1.2% (w / w), from about 0.09% to about 1.1% (w / w), or from about 0.1% to about 1% (w / w) of an estetrol component (such as estetrol, preferably estetrol monohydrate) and a penetration enhancer, preferably about 2.5% (w / w) or less of an estetrol component and a penetration enhancer, more preferably about 2% (w / w) or less of an estetrol component. More preferably, the estetrol component and penetration enhancer are about 1.5% (wt / wt) or less, even more preferably about 1% (wt / wt) or less, even more preferably about 0.75% (wt / wt) or less, still more preferably about 0.5% (wt / wt) or less, and most preferably about 0.1% (wt / wt) or less.

[0086] Preferably, the pharmaceutical compositions referred to herein are hydrogels or are contained within hydrogels. A gel is a semi-solid system in which a liquid is solidified by a gel framework-forming agent. In a hydrogel, the gel-forming liquid is water or an aqueous solution. As used herein, the term "hydrogel" refers to an aqueous solution of an active ingredient solidified primarily by a macromolecular hydrophilic substance to form a gel. Macromolecular hydrophilic substances, and thus polymeric materials, swell upon contact with water and, depending on the concentration, produce solutions that exhibit pseudoplastic flow behavior or plastic structures containing essential aqueous components. Thus, hydrophilic gels consist of water or an aqueous solution gelled, usually with hydrophilic macromolecular compounds. Gels constructed with a hydrophilic macromolecular framework are generally thixotropic. In contrast to creams, gels are referred to as true single-phase systems.

[0087] Hydrogels improve or restore moisture balance to the wound bed by balancing moisture retention with the absorption of excess fluids. Hydrogels contemplated herein may comprise any suitable polymer or combination of polymers, including, but not limited to, hydrophilic polymers, acrylic acid, acrylamide, and 2-hydroxyethyl methacrylate. Hydrogels are particularly preferred forms of the compositions disclosed herein given their ability to provide moisture balance to the wound bed by balancing moisture retention with the absorption of excess fluids. Examples of hydrogels include, but are not limited to, synthetic hydrogels, stimuli-sensitive hydrogels, (poly)peptide-based hydrogels, hybrid hydrogels, and DNA-based hydrogels. Methods for producing each of these hydrogel categories have been described in the art and are therefore known to those skilled in the art.

[0088] By way of illustration and not limitation, examples of synthetic hydrogels include double-network hydrogels. Examples also include nanocomposite hydrogels. Stimuli-sensitive hydrogels are characterized by their ability to undergo changes in swelling that can be mediated by external stimuli (e.g., pH, temperature, ionic strength, solvent type, electric field, magnetic field, light, and chelating species). Examples of stimuli-sensitive hydrogels include, but are not limited to, hydrogels formed from recombinant block copolypeptide segments of natural structural proteins, such as elastin peptide blocks, silk peptide blocks, silk-like peptide blocks, and elastin-like peptide blocks, and recombinant triblock copolymers of one or more polypeptide sequences.

[0089] The term "hybrid hydrogel" is used herein to refer to a hydrogel that contains components from at least two different classes of molecules, e.g., synthetic polymers and biological macromolecules, interconnected by either covalent or non-covalent bonds.

[0090] In the gel formulations described herein, the presence of some additives such as propylene glycol, glycerol, Transcutol®, benzyl alcohol, and PEG400 can increase the solubility of estetrol.The use of these additives in gel formulations makes estetrol soluble and completely available for penetration through the membrane, or through the stratum corneum and / or wound eschar, and / or into the burn wound.This contributes to an increase in the diffusion rate across the membrane, or through the stratum corneum and / or wound eschar, and / or into the burn wound, as predicted by Fick's law of diffusion.This is because the increased solubility of estetrol between the two sides of the membrane creates a higher concentration gradient of estetrol.

[0091] The cream diffusion profile shows that estetrol release is slower in the same time period compared with gel formulation.In cream formulation, estetrol component is likely located in the internal phase of emulsion, and after diffusing to the external phase of emulsion, it must be able to diffuse through the barrier membrane or similarly through the stratum corneum and / or wound eschar, and / or into the burn wound.However, the high partition coefficient of the cream base prevents estetrol from rapidly diffusing along its concentration gradient.The overall process is slower, and the release rate of estetrol is dramatically reduced.

[0092] It is understood that all references to "composition" and "pharmaceutical composition" encompass any hydrogel described herein, and vice versa.

[0093] Preferably, the hydrogels contemplated herein include a penetration enhancer to enable penetration through the stratum corneum. More preferably, the hydrogels referred to herein are hydrogels that include about 0.02% to about 10% (w / w) of an estetrol component, such as estetrol, preferably estetrol monohydrate. Preferably, the hydrogels referred to herein are hydrogels containing about 0.02% to about 5% (wt / wt) of an estetrol component. More preferably, the hydrogels referred to herein are hydrogels containing about 0.02% to about 2.5% (wt / wt) of an estetrol component. More preferably, the hydrogels contain about 0.03% to about 0.75% (wt / wt) of an estetrol component, preferably about 0.04% to about 0.5% (wt / wt) of an estetrol component, more preferably about 0.05% to about 0.25% (wt / wt) of an estetrol component, for example, about 0.08% to about 1.2% (wt / wt), about 0.09% to about 1.1% (wt / wt), or about 0.1% to about 1% (wt / wt) of an estetrol component. Most preferably, the hydrogels contain about 0.06% (wt / wt) of an estetrol component.

[0094] Alternatively, the hydrogel referred to herein is the hydrogel that comprises less than about 10% (wt / wt) estetrol content.Preferably, the hydrogel referred to herein is the hydrogel that comprises less than about 5% (wt / wt), more preferably, the hydrogel referred to herein is the hydrogel that comprises less than about 2.5% (wt / wt), more preferably, the hydrogel that comprises less than about 0.75% (wt / wt), preferably less than about 0.5% (wt / wt), more preferably less than about 0.25% (wt / wt), more preferably less than about 0.1% (wt / wt), most preferably less than about 0.08% (wt / wt).

[0095] In certain embodiments where the (pharmaceutical) composition is a hydrogel, the hydrogel is characterized by a favorable release profile compared to other formulations, such as, but not limited to, creams. In a preferred embodiment, the hydrogels described herein release at least about 2.5 μg / cm across an isoporous membrane within 1 hour in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 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 2In an alternative embodiment, the hydrogels described herein are characterized by an average cumulative amount of released estetrol component of about 25 μg / cm across an isoporous membrane within 1 hour in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 ~about 200μg / cm 2 The hydrogels described herein are characterized by an average cumulative amount of estetrol component released of about 25 μg / cm2 within 1 hour across an Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 ~Approx. 100μg / cm 2 In a further alternative preferred embodiment, the hydrogels described herein are characterized by an average cumulative amount of released estetrol component of about 25 μg / cm across an Isopore membrane within 1 hour in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 ~Approx. 50μg / cm 2 The average cumulative amount of estetrol component released is characterized by:

[0096] Preferably, the hydrogels described herein have a cross-sectional area of ​​at least about 350 μg / cm across an Isopore membrane within 8 hours in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 , preferably at least about 400 μg / cm 2 , more preferably at least about 450 μg / cm 2 The average cumulative amount of estetrol component released is characterized by:

[0097] Preferably, the hydrogels described herein are characterized by an average % applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of at least about 15%, preferably at least about 20%, after 1 hour, and / or an average % applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of at least about 40%, preferably at least about 50%, more preferably at least 80% after 8 hours. In alternative embodiments, the hydrogels described herein are characterized by an average % applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of about 15% to about 30% after 1 hour, and / or an average % applied dose of the estetrol component to a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution of about 45% to about 90% after 8 hours.

[0098] In a highly preferred embodiment, the hydrogels described herein provide a cross-sectional area of ​​approximately 2.5 μg / cm across an isoporous membrane in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 / √hr, at least about 5 μg / cm 2 / √hr, at least about 7 μg / cm 2 / √hr, at least about 10 μg / cm 2 / √hr, at least about 15 μg / cm 2 / √hr, at least about 20 μg / cm 2 / √hr, at least about 25 μg / cm 2 / √ hour ~ approx. 50μg / cm 2 The release rate (i.e., slope) of the estetrol component per square root of √√hr and after 8 hours was approximately 50 μg / cm 2 ~Approx. 100μg / cm 2and optionally a percentage of the released amount of estetrol of about 75% to about 95%. Even more preferably, the hydrogels described herein have a mean cumulative released amount of estetrol of about 30 μg / cm across an isoporous membrane in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 / √ hour ~ approx. 35μg / cm 2 The release rate (i.e., slope) of the estetrol component per square root of √√hr and after 8 hours was approximately 75 μg / cm 2 ~about 90μg / cm 2 and optionally a percentage of the released amount of estetrol of about 85% to about 90%. Most preferably, the hydrogels described herein have a mean cumulative released amount of estetrol of about 33 μg / cm across an isoporous membrane in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 / √ hour ~ approx. 34μg / cm 2 The estetrol component release rate (i.e., slope) was approximately 82 μg / cm after 8 hours. 2 ~Approx. 83μg / cm 2 and optionally a percentage of the amount of estetrol released of about 86% to about 88%.

[0099] In embodiments where the composition is a cream, the cream has a cross-sectional area of ​​about 2.5 μg / cm across the isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 / √hr, at least about 5 μg / cm 2 / √hr, at least about 7 μg / cm 2 / √hr, at least about 10 μg / cm 2 / √hr, at least about 15 μg / cm 2 / √hr, at least about 20 μg / cm 2 / √hr, or 15μg / cm 2 / √ hour ~ approx. 75μg / cm 2The cream may be characterized by an estetrol ingredient release rate (i.e., slope) of about 25 μg / cm across the isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 / √ hour ~ approx. 55μg / cm 2 The estetrol component release rate (i.e., slope) may be characterized by the rate of release per square root of the estetrol component.

[0100] The cream was further purified to approximately 1 μg / cm after 1 hour across the isopore membrane in a 40:30:30 (vol / vol / vol) ethanol:PEG400:water receptor solution. 2 ~Approx. 100μg / cm 2 Preferably, the cream has an average cumulative released amount of estetrol component of about 5 μg / cm after 1 hour across the Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 ~about 80μg / cm 2 The cream may be further characterized by an average cumulative amount of estetrol component released of about 25 μg / cm after 8 hours across an Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 ~Approx. 150μg / cm 2 Preferably, the cream has an average cumulative released amount of estetrol component of about 50 μg / cm after 8 hours across the Isopore membrane in a 40:30:30 (v / v / v) ethanol:PEG400:water receptor solution. 2 ~Approx. 120μg / cm 2 The average cumulative amount of estetrol component released may be characterized as:

[0101] As will be apparent to one skilled in the art, the present disclosure encompasses the use of each of the specific forms of the pharmaceutical compositions disclosed herein for use in wound healing. Accordingly, the present invention also relates to the hydrogels disclosed herein for use in wound healing.

[0102] Hydrogels are a preferred form of pharmaceutical composition disclosed throughout this disclosure, but this does not exclude other topical formulations known in the art. Accordingly, suitable formulations include, but are not limited to, emulsions, suspensions, ointments, pastes, lotions, gels (including hydrogels), foams, mousses, sprays, and creams. All of these terms are intended to correspond to their generally accepted meanings. Similarly, these topical formulations can be applied directly to the skin or in combination with dressings, patches, bandages, band-aids, tampons, plasters, and the like to prevent the formulation from peeling off the skin and, in some embodiments, to shield the wound from external influences such as dirt and microorganisms.

[0103] "Emulsion" refers to any mixture of at least two liquids that are not mixable (i.e., immiscible, unblendable), whereby the first liquid is distributed in small droplets (dispersed phase) throughout the second liquid (dispersion medium). Thus, in certain embodiments, the pharmaceutical compositions described herein are oil-in-water emulsions or water-in-oil emulsions. Emulsions are widely used in skin care formulations and can be classified as creams and lotions. In this context, "suspension" broadly refers to a heterogeneous mixture that contains solids dispersed in a liquid phase, where the solids are not dissolved and have a size large enough to allow sedimentation.

[0104] "Cream" generally refers to a water-in-oil emulsion in which an aqueous phase is dispersed in an oily phase, but can equally well be an oil-in-water emulsion in which an oil is dispersed within an aqueous base. It is generally recognized that creams differ from emulsions in that instead of emulsions being stable suspensions of small immiscible droplets of a fluid that is immiscible with another fluid portion of the emulsion, creams refer to a specific subset of emulsions that are more viscous and usually contain more lipophilic and / or surfactant components.

[0105] A "lotion" is a liquid composition of low to medium viscosity. Generally, lotions have a lower viscosity than creams, although in some cases the viscosity of both may be similar. Lotions may contain insoluble finely divided powder materials in a dispersion medium through the use of suspending and dispersing agents. Alternatively, lotions may have a liquid material as the dispersed phase that is immiscible with the vehicle, usually 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 lotions allows for quick and uniform application over large surface areas. Lotions are typically intended to dry on the skin, leaving a thin coating of their active ingredients on the surface of the skin.

[0106] "Ointment" generally refers to a thicker oil-in-water cream, i.e., a semi-solid substance containing an ointment base and, optionally, one or more pharmaceutically active ingredients (in the context of the present invention, the estetrol component). Examples of suitable ointment bases include hydrocarbon bases, absorption bases, water-removable bases, and water-soluble bases. "Paste" generally differs in that ointments contain a higher percentage of solids. Overall, pastes are more absorbent and less greasy than ointments based on the same set of ingredients / additives.

[0107] As used herein, "foam" refers to a dispersion of gas particles in a liquid medium. Examples of liquid media in foams include oil-in-water emulsions, water-in-oil emulsions, ethanol, water, solvents, liquid oils, propylene glycol, and glycerin. Those skilled in the art will appreciate that foams can be generated by reducing the surface tension of a liquid mixed with a gaseous substance(s), causing bubble formation. Foams are popular because they are easily applied to large areas of skin, do not leave an oily or greasy film, and are rapidly absorbed into the skin. "Mousse" refers to a substance that is similar to foam, but is generally less watery. "Spray" refers to a drug-containing solution that is filled into a device suitable for atomizing the drug-containing solution and released in a mist under pressure.

[0108] As noted above, any of the pharmaceutical compositions disclosed herein, including but not limited to, hydrogels, may include a penetration enhancer. Preferably, the penetration enhancer comprises a molecule that enables penetration through the stratum corneum (i.e., a penetration-enhancing molecule) and a solvent or solvent system. Optionally, the penetration enhancer is present in the compositions disclosed herein, including but not limited to, hydrogels, in an amount of about 0.5% to about 60% (wt / w), preferably about 1% to about 50% (wt / w), more preferably about 2.5% to about 45% (wt / w), more preferably about 5% to about 40% (wt / w), more preferably about 10% to about 30% (wt / w), or alternatively, about 0.1% to about 5% (wt / w). Optionally, the penetration enhancer molecule may be present in the hydrogel or other compositions disclosed herein in an amount of about 0.1% to about 25% (w / w), preferably about 0.5% to about 15% (w / w), more preferably about 1% to about 10% (w / w), more preferably about 2.5% to about 7.5% (w / w), and more preferably about 3.5% to about 5% (w / w). Optionally, the solvent (system) may be present in the hydrogel or other compositions disclosed herein in an amount of about 1% to about 60% (w / w), preferably about 5% to about 50% (w / w), more preferably about 10% to about 40% (w / w), more preferably about 15% to about 30% (w / w), and more preferably about 18% to about 25% (w / w).

[0109] In the present invention, the penetration enhancer described herein is not particularly limited and may therefore comprise or consist of a molecule selected from the group consisting of suberin, lignin, and cutin, including dimethyl sulfoxide, ethanol, propylene glycol, glycerin, propyl ethylene glycol, urea, dimethylacetamide, sodium lauryl sulfate, poloxamer, span, tween, lecithin, terpene, and combinations thereof. Preferred penetration enhancer molecules in the context of the present invention include ethanol, ethers, benzyl alcohol, fatty acids and their esters, or any combination thereof. Particularly preferred penetration enhancer molecules in the context of the present invention include Transcutol™, benzyl alcohol, and any combination thereof. Benzyl alcohol (CHCHOH) may be referred to interchangeably in the art as "phenylmethanol," "phenylcarbinol," and "benzenemethanol." Transcutol™ is the commonly accepted trade name for 2-(2-ethoxyethoxy)ethanol, which is known in the art as diethylene glycol monoethyl ether (CH). 14 Optionally, the pharmaceutical compositions described herein contain about 1% to about 20% (w / w) Transcutol™ as a penetration enhancer, preferably about 2% to about 10% (w / w) Transcutol™, more preferably about 3% to about 8% (w / w) Transcutol™, and most preferably about 4% to about 6% (w / w) Transcutol™.

[0110] The penetration enhancers of the present invention comprise a penetration enhancer molecule and a solvent or solvent system. Preferred solvents for penetration enhancers include, but are not limited to, polyethylene glycol (PEG), propylene glycol (PG), and combinations thereof. "Polyethylene glycol" may be interchangeably referred to by terms such as, but not limited to, polyethylene oxide or poly(oxyethylene), poly(ethylene oxide), and polyoxyethylene, which are well described in the art, and thus polyethylene glycol has the chemical formula H-(O-CH-CH). n Those skilled in the art will understand that polyethylene glycol is characterized by a -OH group (where n is an integer). Polyethylene glycol is a petroleum-derived polyether compound. Preferred PEGs are those characterized by a molecular weight of about 150 g / mol to about 5000 g / mol, more preferably about 200 g / mol to about 2500 g / mol, even more preferably about 250 g / mol to about 1000 g / mol, and most preferably about 300 g / mol to about 600 g / mol. Thus, the PEG referred to herein may be selected from the group consisting of PEG200, PEG300, PEG400, PEG500, PEG600, and any combination thereof. Most preferably, the penetration enhancer comprises, as a solvent or part of a solvent, a PEG having a molecular weight of about 400 g / mol, such as, but not limited to, PEG400. Propylene glycol is commonly referred to in the art as propane-1,2-diol, α-propylene glycol, 1,2-propanediol, and 1,2-dihydroxypropane. Propylene glycol is characterized by the chemical formula CH3CH(OH)CH2OH.

[0111] Preferably, the penetration enhancer comprises about 5% to about 50% (wt / wt), preferably about 5% to about 35% (wt / wt), or about 10% to about 45% (wt / wt) PEG, preferably PEG400, and / or about 10% to about 35% (wt / wt) PG. More preferably, the penetration enhancer comprises about 10% to about 30% (wt / wt) PEG, preferably PEG400, and / or about 12% to about 30% (wt / wt) PG. More preferably, the penetration enhancer comprises about 12% to about 25% (wt / wt) PEG, preferably PEG400, and / or about 15% to about 25% (wt / wt) PG. More preferably, the penetration enhancer comprises about 14% to about 23% (wt / wt) PEG, preferably PEG400, and / or about 16% to about 23% (wt / wt) PG. More preferably, the penetration enhancer comprises about 16% to about 22% (wt / wt) PEG, preferably PEG 400, e.g., about 18% to about 22% (wt / wt) PEG or about 16% to about 20% (wt / wt) PEG, and / or about 18% to about 22% (wt / wt) PG.

[0112] Optionally, the pharmaceutical composition (optionally a hydrogel) contains benzyl alcohol. The term "benzyl alcohol" should be interpreted according to its general interpretation in the art and, therefore, refers to an aromatic alcohol characterized by the chemical formula CHCHOH. When present, the amount of benzyl alcohol in the composition is not particularly limited. However, preferably, the amount of benzyl alcohol is about 0.1% to about 10% (wt / wt), more preferably about 0.5% to about 5% (wt / wt), more preferably about 1% to about 3% (wt / wt), and even more preferably about 1.5% to about 2.5% (wt / wt).

[0113] Optionally, the pharmaceutical composition (which may be a hydrogel) contains a thickening agent. "Thickening agent" may alternatively be referred to by terms such as, but not limited to, "thickening agent" and "viscosifying agent." As used in the context of the present invention, "thickening agent" refers to any substance or molecule that, when added to a liquid or semi-solid composition, increases the viscosity and / or texture of the composition. Thus, when present, the thickening agent may be selected from the group consisting of carboxylic acid polymers, crosslinked polyacrylate polymers, polyacrylamide polymers, polysaccharides, diblock polymers, triblock polymers, gums, and any combination thereof. In embodiments in which the thickening agent is or comprises a polysaccharide, the polysaccharide may be selected from the group consisting of cellulose, cellulose derivatives, carboxymethylcellulose, cellulose acetate propionate carboxylate, hydroxyethylcellulose, hydroxyethylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylhydroxyethylcellulose, hydroxyalkylated cellulose, lignin, cutin, suberin, microcrystalline cellulose, sodium cellulose sulfate, scleroglucan, and any combination thereof. Suitable gums that may function as thickening agents include acacia, agar, algin, alginic acid, cetyl alcohol, ammonium alginate, amylopectin, calcium alginate, calcium carrageenan, carnitine, carrageenan, dextrin, gelatin, gellan gum, guar gum, guar hydroxypropyltrimonium chloride, hectorite, hyaluronic acid, hydrated silica, hydroxypropyl chitosan, hydroxypropyl guar, karaya gum, kelp, locust bean gum, natto gum, potassium alginate, potassium carrageenan, propylene glycol alginate, sclerotium gum, sodium carboxymethyl dextran, sodium carrageenan, tragacanth gum, xanthan gum, and any combination thereof.

[0114] In certain embodiments, the pharmaceutical compositions described herein (which are optionally hydrogels) contain a viscosity enhancing agent described herein in an amount of about 0.1% to about 25% (w / w), preferably about 0.3% to about 20% (w / w), more preferably about 0.4% to about 15% (w / w), more preferably about 0.5% to about 10% (w / w), more preferably about 0.75% to about 5% (w / w), or about 0.3% to about 3% (w / w). Preferred viscosity enhancing agents in the context of the present invention include, but are not limited to, viscosity enhancing agents selected from the group consisting of hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), high molecular weight crosslinked acrylic polymers, nonionic triblock copolymers, or any combination thereof. More preferred viscosity enhancing agents in the context of the present invention include, but are not limited to, hydroxyethyl cellulose, high molecular weight crosslinked acrylic polymers, nonionic triblock copolymers, and any combination thereof. Preferred high molecular weight crosslinked acrylic polymers are the carbomers, which are referred to interchangeably throughout the art by the trade name Carbopol™. Carbopol™ homopolymers (i.e., acrylic acid crosslinked with allyl sucrose or allyl pentaerythritol), Carbopol™ copolymers (acrylic acid and C crosslinked with allyl pentaerythritol), 10 ~C 30Both alkyl acrylates) and Carbopol™ interpolymers (carbomer homopolymers or copolymers comprising block copolymers of polyethylene glycol and long-chain alkyl acid esters) are contemplated. A particularly preferred Carbopol™ in the context of the present invention is Carbopol™ 980, alternatively referred to throughout the art as "Carbomer Homopolymer Type C USP NF," which is a homopolymer of acrylic acid crosslinked with allyl sucrose or allyl pentaerythritol in a cosolvent of cyclohexane and ethyl acetate having a viscosity of 40,000 cP to 60,000 cP. A preferred hydroxyethyl cellulose is HEC250 HHX. A preferred nonionic triblock copolymer is one having a molecular weight of about 1,800 g / mol to about 4,000 g / mol and a polyoxyethylene content of about 70% to about 80%. Highly preferred non-ionic triblock copolymers include poloxamer 188, poloxamer 407, or a combination thereof.

[0115] In certain embodiments, the thickening agent is or comprises HEC and is present in the pharmaceutical composition in an amount of about 0.1% to about 10% (w / w), preferably 0.2% to about 7.5% (w / w), more preferably about 0.5% to about 5% (w / w), more preferably about 0.75% to about 2.5% (w / w), and most preferably about 1% to about 2% (w / w). In certain embodiments, the thickening agent is or comprises Carbopol™ and is present in the composition in an amount of about 0.1% to about 10% (w / w), preferably 0.2% to about 7.5% (w / w), more preferably about 0.5% to about 5% (w / w), more preferably about 0.75% to about 2.5% (w / w), and most preferably about 0.75% to about 1.5% (w / w). In alternative embodiments, Carbopol™ is present in the composition in an amount of about 0.1% to about 1% (w / w). In certain embodiments, the thickening agent is or comprises Poloxamer 188 and is present in the composition in an amount of about 0.1% to about 30% (w / w), preferably about 1% to about 15% (w / w), and more preferably about 2.5% to about 10% (w / w). In certain embodiments, the thickening agent is or comprises Poloxamer 407 and is present in the composition in an amount of about 0.1% to about 30% (w / w), preferably about 5% to about 25% (w / w), and more preferably about 10% to about 20% (w / w).

[0116] In certain embodiments, the viscosity enhancing agent is or comprises CMC and is present in the pharmaceutical composition in an amount of about 0.1% to about 10% (wt / wt), preferably in an amount of 0.2% to about 7.5% (wt / wt), more preferably in an amount of about 0.5% to about 5% (wt / wt), more preferably in an amount of about 0.75% to about 2.5% (wt / wt), and most preferably in an amount of about 1% to about 2% (wt / wt) or about 1.5% (wt / wt).

[0117] Optionally, the pharmaceutical composition (optionally a hydrogel) includes a preservative. In certain embodiments, the composition includes a preservative in an amount of 0.5% to 20% (w / w), preferably 1% to about 10% (w / w), and more preferably about 1% to about 3% (w / w). The specific preservative is not particularly limited in the present invention and may therefore be selected from the group consisting of lysozyme, nisin, quaternary ammonium preservatives, parabens, phenoxyethanol, benzyl alcohol, chlorobutanol, phenol, sorbic acid, thimerosal, natural preservatives, and any combination thereof. A preferred preservative in the context of the present invention is benzyl alcohol.

[0118] Optionally, the pharmaceutical composition (which may be a hydrogel) includes an emollient. As used throughout this disclosure, the term "emollient" refers to a substance useful not only for preventing and / or treating dry skin, but also for providing extra protection to the skin. In the present invention, the specific emollient is not particularly limited and may therefore be selected from the group consisting of glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, mineral oil, and any combination thereof. A particularly preferred emollient in the context of the present invention is glycerol. The emollient may be 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), and more preferably about 8% to about 12% (w / w). In a further embodiment, the emollient may be glycerol, which is present in the composition in an amount of from about 2.5% to about 30% (wt / wt), preferably from about 5% to about 25% (wt / wt), more preferably from 7.5% to about 20% (wt / wt), more preferably from about 8% to about 12% (wt / wt), and most preferably in an amount of about 10% (wt / wt).

[0119] As described in detail throughout this disclosure, any of the pharmaceutical compositions described herein may contain a certain amount of ingredients (i.e., pharmaceutically active agents and / or additives) in addition to the estetrol component. In any of the embodiments of the present invention, a solvent can be added to reach a certain concentration of the ingredient. In a further embodiment, an aqueous solution is used to supplement the composition. The term "aqueous solution" refers to any solution containing water or any solution in which the solvent is water. Furthermore, "aqueous solution" is used to describe a solution that exhibits similarities with water or an aqueous solution, including but not limited to properties such as appearance, odor, color, taste, viscosity, pH, absorbance, or physical state at a particular temperature. In such an embodiment, the aqueous solution may be water. In a further alternative embodiment, a non-aqueous solution is used to supplement the composition. In yet another alternative embodiment, a mixture of a non-aqueous solution and an aqueous solution is used to supplement the composition.

[0120] As defined herein, the pH of a composition, solution, or formulation may be measured using a variety of methods known to those skilled in the art. + pH indicators may be used that change color by absorbing or releasing ions, where the resulting color is indicative of a particular pH value. Alternatively, pH meters may be used that measure the potential difference between a pH electrode and a reference electrode. The potential difference is related to the acidity or pH of the solution.

[0121] In view of the above, exemplary pharmaceutical compositions according to the present invention comprise, in addition to the estetrol component at any one of the concentrations recited herein: about 0.1% to about 60% (wt / wt) of a penetration enhancer; about 0.3% to about 20% (wt / wt) of a thickener, optionally a preservative and / or an emollient, Water (up to 100% (w / w)), Includes:

[0122] In certain embodiments, the pharmaceutical composition consists essentially of or consists of (in addition to the estetrol component) a penetration enhancer, a thickener, a preservative, and an emollient. In a preferred embodiment, the composition according to the present invention comprises, in addition to the estetrol component: about 0.1% to about 10% (wt / wt) of penetration enhancer molecules, preferably about 0.25% to about 8% (wt / wt) of penetration enhancer molecules; about 10% to about 60% (wt / wt) of a penetration enhancer solvent; about 0.3% to about 20% (wt / wt) thickener, preferably about 0.3% to about 10% (wt / wt) thickener, more preferably about 0.3% to about 5% (wt / wt) thickener, and most preferably about 0.3% to about 3% (wt / wt) thickener; optionally a preservative and / or an emollient, Water (up to 100% (w / w)), Includes:

[0123] In still further preferred embodiments, exemplary pharmaceutical compositions according to the present invention comprise, in addition to the estetrol component at any one of the concentrations recited herein: about 1% to about 7.5% (wt / wt) of penetration enhancer molecules, preferably about 0.5% to about 5% (wt / wt) of penetration enhancer molecules; about 15% to about 45% (wt / wt) of a penetration enhancer solvent; about 0.3% to about 20% (wt / wt) thickener, preferably about 0.3% to about 10% (wt / wt) thickener, more preferably about 0.3% to about 5% (wt / wt) thickener, and most preferably about 0.3% to about 3% (wt / wt) thickener; optionally a preservative and / or an emollient, Water (up to 100% (w / w)), Includes:

[0124] In certain embodiments, the pharmaceutical composition (optionally a hydrogel) contains, in addition to the estetrol component at any one of the concentrations recited herein: about 8% to about 40%, preferably about 16% to about 20%, most preferably about 18% (wt / wt) PEG 400; about 9% to about 44%, preferably about 18% to about 22%, most preferably about 20% (wt / wt) PG; about 4% to about 24%, preferably about 8% to about 12%, and most preferably about 10% (wt / wt) glycerol; about 0.5% to about 4%, preferably about 1% to about 2%, most preferably about 1.5% (w / w) HEC; and about 0.75% to about 1.25%, preferably about 1.5% to about 2.5%, most preferably about 2% (w / w) benzyl alcohol; Comprise, consist essentially of, or consist of.

[0125] In certain embodiments, the pharmaceutical composition (optionally a hydrogel) contains, in addition to the estetrol component at any one of the concentrations recited herein: about 9% to about 44%, preferably about 18% to about 22%, and most preferably about 20% (wt / wt) PEG 400; about 0.01% to about 2%, preferably about 0.1% to about 1%, and most preferably about 0.5% (w / w) Carbopol™; and about 0.1% to about 15%, 2% to about 12%, preferably about 4% to about 6%, most preferably about 5% (w / w) Transcutol™; Comprise, consist essentially of, or consist of.

[0126] In certain embodiments, the pharmaceutical composition (optionally a hydrogel) contains, in addition to the estetrol component at any one of the concentrations recited herein: about 10% to about 55%, preferably about 30% to about 50%, and most preferably about 40% to 45% (weight / weight) of PEG 400; about 0.01% to about 2%, preferably about 0.1% to about 1%, and most preferably about 0.5% (w / w) Carbopol™; and about 0.1% to about 15%, 0.2% to about 10%, preferably about 0.5% to about 5%, and most preferably about 1% to about 2.5% (w / w) of Transcutol™; Comprise, consist essentially of, or consist of.

[0127] The pharmaceutical compositions described herein, including but not limited to the hydrogels described herein, are contemplated for use as medicaments in both therapeutic and prophylactic contexts. More particularly, the compositions described herein, including but not limited to the hydrogels described herein, are contemplated for medical use in wound healing. In other words, the present invention relates to the use of the compositions, such as the hydrogels described herein, for the manufacture of a medicament for wound healing, preferably topical wound healing. In yet another way, the present invention relates to a method for treating a wound, preferably a topical wound treatment method, comprising administering to a subject the wound (site) any one of the compositions or hydrogels described herein. In the context of the present invention, the compositions, such as the hydrogels described herein, are used as topical preparations, i.e., topical wound treatment means applied to the wound site.

[0128] As used throughout this disclosure, the terms "therapy" or "treatment" refer to the alleviation or measurable reduction of one or more symptoms or measurable markers of a medical condition, in the context of the present invention, of one or more wounds. These terms encompass both therapeutic treatment of existing wounds (i.e., established wounds) and prophylactic or preventative measures in which the goal of treatment is to prevent the occurrence and / or recurrence, development, and progression of wounds on a subject, i.e., the subject's skin. By way of example and not limitation, a preventative use of the pharmaceutical compositions described herein could be to administer to potentially vulnerable skin sites in elderly subjects to prevent the development of pressure ulcers. An alternative example could be application to a skin site of a subject scheduled for the creation of a surgical insertion site in the near future. A 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 of less than 0.05, which is a commonly accepted cut-off score in statistical analysis, as will be understood by those skilled in the art.More specific indications for wound healing sites will be described in more detail below.Beneficial or desirable clinical results of medical use (i.e., treatment) can include, but are not limited to, alleviating pain and / or discomfort, improving one or more biological markers, reducing wound area, stabilizing (i.e., not worsening) wound condition, accelerating wound healing progress, improving patient quality of life, etc.

[0129] Those skilled in the art will recognize that to achieve effective therapeutic treatment, a therapeutically effective dose must be administered to the subject. Accordingly, in the context of the present disclosure, "effective amount" refers to the amount necessary to achieve a physiological effect. The physiological effect may be achieved by a single or multiple administrations. A "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of an estetrol component that, when administered, results in a clinically positive response in treating a subject suffering from one or more wounds. Similarly, a "prophylactically effective amount" or "prophylactically effective dose" refers to an amount of an estetrol component that prevents or slows the occurrence or progression of wounds. Those skilled in the art will recognize that terms such as "amount," "quantity," and "level" are synonymous and have clearly defined meanings in the art, and will understand that in the context of the present application, they refer to the relative quantification of the estetrol component portion of a pharmaceutical composition, such as a hydrogel, or, where indicated, the absolute quantification of the estetrol component that, when applied to the skin of a subject, would be considered an effective amount for the uses described herein.

[0130] Optionally, the pharmaceutical compositions and hydrogels described herein are used in the treatment of acute wounds. In the present invention, the cause of acute wounds is not particularly limited, and thus includes both wounds caused by trauma and wounds induced by surgery. In the present invention, the cause of trauma is not limited, and thus includes both accidental trauma and malicious trauma (i.e., combat wounds). Non-limiting examples of acute wounds include abrasions (i.e., skin scraped or scraped), incisions (i.e., cleanly cut wounds), lacerations (i.e., torn and / or ripped wounds), puncture wounds (i.e., wounds with a relatively small wound opening caused by a relatively tapered object), and avulsion wounds (skin wounds pulled or torn).

[0131] Optionally, the pharmaceutical compositions and hydrogels described herein are used in the treatment of burn wounds. Those skilled in the art will understand that "burn wound" refers to a specific type of tissue trauma caused by contact with heat, flame, chemicals, electricity, or radiation. First-degree burns are characterized primarily by redness, second-degree burns are characterized by the presence of one or more vesicular patches (i.e., blister formation), and third-degree burns are characterized by the presence of necrosis. In the art, first-degree and second-degree burns generally refer to partial-thickness burns (i.e., tissue destruction extending from the epidermis to the dermis but not penetrating the dermis), while third-degree burns generally refer to full-thickness burns (i.e., destruction characterized by complete penetration of the dermis).

[0132] Optionally, the pharmaceutical compositions and hydrogels described herein are used in the treatment of chronic wounds. The term "chronic wound" as used herein refers to any wound that has not undergone or has not undergone the standard wound healing process. Therefore, wounds can be clinically classified as acute or chronic based on their healing time. In particular, surgical wounds can become chronic wounds, which are referred to as surgical wounds that cannot heal by secondary healing. The term "chronic wound" can be used interchangeably with synonyms such as, but not limited to, "recalcitrant wound," "difficult-to-heal wound," "non-healing wound," and "complex wound." Chronic wounds have been extensively characterized in the art (e.g., Vanwijck, Bull Mem Acad R Med Belg, 2001). The healing process of chronic wounds can be dysregulated by many factors that prolong the wound healing phase by one or more stages. 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 a prolonged or uncontrolled inflammatory phase, persistent infection, the formation of drug-resistant microbial biofilms, and the inability of dermal and / or epidermal cells to respond to repair stimuli.

[0133] As used herein, "inflammation" broadly refers to the physiological process by which vascular tissue responds to trauma. Similarly, the term "inflammatory process" refers to the process by which soluble inflammatory mediators cooperate with cellular components to contain and eliminate any factors that cause distress. The term "inflammatory mediator" broadly refers to any molecular mediator of the inflammatory process. Inflammatory mediators can act locally at the site of tissue injury and / or infection or at more distant sites. Certain inflammatory mediators are activated by the inflammatory process, while others are produced and / or released from cellular sources in response to inflammation or upon activation by other inflammatory mediators. Examples of inflammatory mediators of the inflammatory response include, but are not limited to, plasma proteases, complement, kinins, coagulation proteins, fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, platelet-activating factors, peptides, amines, and proinflammatory cytokines.

[0134] Terms such as "skin inflammation" or "skin inflammation" used herein should be interpreted according to the meaning generally accepted in the prior art, and therefore refer to any local immune response of the skin. The cause of skin inflammation is generally the occurrence of trauma such as wounds. Therefore, skin inflammation may be considered to be the result of cellular interactions in the skin of a subject, where immune cells remain the most important cell type. The skin inflammation referred to herein refers to the "standard" inflammation observed in wounds, but also refers to excessive inflammation that exceeds the normal limit of inflammation, which may be the result of bacterial or fungal infection at the wound site or a defective host response (for example, in the case of diabetes).Non-limiting examples of bacteria that may be involved in wound infection include, but are not limited to, Staphylococcus aureus, coagulase-negative staphylococci, Corynebacteria, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Acinetobacter baumanii, Serratia marcescens, Stenotrophonas maltophilia, Streptococcus agalactiae, Enterobacter cloacae, and the like. cloacae, Enterococci, Klebsiella pneumoniae, Morganella morganii, Providencia stuarii, Alcaligenes faecalis, Citrobacter amalonaticus, Citrobacter koseri, Klebsiella oxytoca, Kocuria kristinae, 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.

[0135] 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, trauma, and combat wounds), burns, and chronic leg ulcers (Crompton et al., Lab Invest, 2016). Klebsiella pneumoniae has been associated in the art with decreased re-epithelialization, increased proliferation, enhanced inflammatory responses, and disrupted wound matrix deposition. The inventors have found that the topical compositions described herein are particularly suitable for use in reducing inflammation in wounds infected with Klebsiella pneumoniae and wounds considered at risk for developing infection with Klebsiella pneumoniae.

[0136] Non-limiting examples of chronic wounds include vascular ulcers, pressure ulcers, and diabetic ulcers. Vascular ulcers include arterial ulcers and venous ulcers. Thus, in certain embodiments, the chronic wound is a wound selected from the group consisting of arterial ulcers, venous ulcers, pressure ulcers, diabetic ulcers, and combinations thereof. As used herein, "venous ulcers" are caused by elevated venous pressure caused by venous valve insufficiency. Pressure-induced changes in the permeability of the blood vessel wall result in leakage of fibrin and other plasma components into perivascular spaces, and the accumulation of this fibrin has a negative impact on wound healing. Collagen synthesis is downregulated by fibrin, leading to the formation of fibrin cuffs around capillaries, which create a barrier to normal vascular function and trap blood-derived growth factors. The term "arterial ulcer" refers to a chronic wound resulting from arterial insufficiency, which may be caused by atherosclerosis or embolism, leading to narrowing and ischemia of the arterial lumen and preventing timely healing of minor trauma. "Pressure ulcers" develop as a result of prolonged, unchanging pressure and shear forces on the skin and underlying muscle tissue, causing reduced oxygen tension, ischemia-reperfusion injury, and tissue necrosis. Finally, "diabetic ulcers" develop as a result of aging and diabetes. Furthermore, diabetes can exacerbate vascular pathology, which in turn can worsen arterial insufficiency, venous insufficiency, and / or pressure ulcers. Additional abnormalities that contribute to the development of diabetic ulcers in diabetic patients include neuropathy (often associated with vasculopathy), impaired muscle metabolism, and certain microvascular pathologies caused by hyperglycemia. Macroscopic pathologies observed in chronic wounds, particularly diabetic wounds, generally include, but are not limited to, abnormalities in cellular phenotypes, such as low mitogenic capacity, low motility, and an inability to respond to environmental factors.

[0137] In certain embodiments, the pharmaceutical compositions described herein (which may be hydrogels) are used in wound healing in subjects characterized by impaired wound healing. Impaired wound healing can be caused by underlying pathologies and / or wound infection, as described throughout this disclosure. Infection is a common cause of delayed wound healing. Live bacteria (and the bacterial toxins subsequently produced) cause an excessive inflammatory response and tissue damage. Possible consequences of bacterial wound infection include the development of abscesses, cellulitis, osteomyelitis, and limb loss (e.g., in diabetic patients). Furthermore, inflammatory cells recruited to the wound site by infection produce proteases that can degrade the extracellular matrix and growth factors present at the wound site. A significant portion of bacteria colonizing a wound can form biofilms, resulting in increased bacterial survival and increased production of virulence factors. Thus, in certain embodiments, the subject is characterized by a wound containing a biofilm.

[0138] In certain embodiments, the pharmaceutical composition is used to treat a wound in a subject considered to be at risk of developing a chronic wound or who has previously developed at least one chronic wound.

[0139] In embodiments in which the subject is 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).

[0140] The present invention is not particularly limited to the specific manifestations and causes of impaired wound healing. Thus, in certain embodiments, the impaired wound healing process may be selected from the group of wound healing processes consisting of hemostasis (blood clotting), inflammation, proliferation (new tissue growth), and maturation (tissue remodeling), or any combination thereof. Furthermore, the impaired wound healing process may be characterized by the occurrence of infection, hypoxia, necrotic tissue, exudate (cells and fluids seeping out of the wound), excessive levels of inflammatory cytokines, and any combination thereof.

[0141] Optionally, the subject is characterized by impaired wound healing due to insufficient or absent blood coagulation. Immediately after trauma in a healthy subject, platelets adhere to damaged blood vessels, initiate a release response, and initiate a hemostatic response. This results in a blood coagulation cascade, which prevents excessive bleeding and provides temporary protection for the wound area. Platelets have been described to release numerous growth factors, cytokines, and other survival or apoptosis-inducing factors. Important components of the platelet release response include platelet-derived growth factor (PDGF) and transforming growth factor A1 and transforming growth factor 2 (TGF-A1 and TGF-2), which attract inflammatory cells (e.g., leukocytes, neutrophils, and macrophages).

[0142] Optionally, the subject is characterized by impaired wound healing due to a defect in the inflammatory wound healing phase. In healthy subjects, the inflammatory phase is initiated in response to capillary injury, which leads to the formation of a temporary clot matrix containing fibrin and fibronectin, among other components. This provisional matrix fills the wound area and induces the influx of effector cells. Platelets present within the clot release multiple cytokines that recruit inflammatory cells (e.g., neutrophils, monocytes, and macrophages, among others), fibroblasts, and endothelial cells.

[0143] Optionally, the subject is characterized by impaired wound healing due to defects in the proliferative phase. In healthy subjects, the proliferative phase is characterized by active angiogenesis, which generates new capillaries, which provide nutrients to the wound site and support the proliferation of fibroblasts. These fibroblasts synthesize and deposit extracellular matrix (ECM) components, thereby replacing the provisional matrix. The fibroblasts are further characterized by contractile properties mediated by smooth muscle actin organized into microfilament bundles or stress fibers.

[0144] Optionally, the subject is characterized by impaired wound healing due to a lack of a remodeling phase. In healthy subjects, the final healing phase involves gradual remodeling and re-epithelialization of granulation tissue. Proteolytic enzymes such as matrix metalloproteinases (MMPs) and their inhibitors (TIMPs, tissue inhibitors of metalloproteinases) play an important role in the remodeling phase. During re-epithelialization, fibronectin and type III collagen, the main components of granulation tissue (i.e., new stromal tissue), are gradually replaced by type I collagen, and elastin is replenished. Elastin contributes to skin elasticity and is initially absent from granulation tissue. Eventually, cell density within the wound normalizes due to apoptosis of vascular cells and fibroblasts.

[0145] Wound edge proliferation is part of the wound re-epithelialization process, which represents the process of covering (i.e., resurfacing, providing) new epithelium. It has been documented that in skin wounds, re-epithelialization progresses from the peripheral wound margin (i.e., wound edge) toward the center of the wound. Re-epithelialization is part of the proliferative phase and typically begins approximately 16 to 24 hours after trauma, with the activation of keratinocytes through the recruitment of neutrophils, monocytes, and macrophages to the wound site, as detailed above. Activated keratinocytes are characterized by changes in the cell cytoskeleton and cell surface receptors. Furthermore, activated keratinocytes are hyperproliferative and produce components of the dermal-epidermal junction. For example, activated keratinocytes produce matrix metalloproteinase 9 (MMP-9), which causes degradation of the dermal-epidermal junction and allows keratinocytes to migrate across the wound. Keratinocyte migration is an early event in wound re-epithelialization. Further studies of activated keratinocytes in wounds have led to the observation that a migrating, adherent epithelial cell sheet at the wound edge migrates toward the center of the wound. Various mechanisms have been proposed for the process of keratinocyte migration across the wound bed, all of which are envisioned and recognized in the context of the present invention.

[0146] Optionally, impaired wound healing may be observed and / or manifested through reduced wound edge movement. As used herein, "wound edge" refers to the periphery of the wound area, i.e., the portion of the wound area adjacent to the untraumatized tissue area of ​​the subject's skin. One skilled in the art can observe and / or measure the wound area, and thus the wound edge. Such observations and / or measurements can be performed at multiple time points to determine whether the subject's wound is healing at a "normal" rate (i.e., within the range considered typical in healthy subjects) or whether any delay in the initiation or slowdown of wound healing has occurred. Optionally, in the absence of any treatment, impaired wound healing in a subject with impaired wound healing is manifested by a reduction in wound edge movement of at least 25%, preferably at least 50%, more preferably at least 75%, or most preferably at least 100%, compared to a healthy subject.

[0147] In certain embodiments, the compositions and hydrogels of the above aspects are used to improve re-epithelialization at a wound site. In this context, improved re-epithelialization may refer to the overall re-epithelialization process, but may also refer to a specific improvement in a specific aspect thereof. Thus, in certain embodiments, improved wound healing when treated with the compositions described herein may improve a process selected from the group consisting of improved re-epithelialization at the wound site, increased cell proliferation at the wound site, reduced inflammatory response at or in the wound site, improved matrix deposition at the wound site, hair follicle-mediated re-epithelialization, bottom-up re-epithelialization in partial-thickness wounds, and any combination thereof.

[0148] Improved re-epithelialization may be characterized by increased and / or accelerated production of provisional matrix. In such embodiments, the amount of provisional matrix produced may be increased by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, and more preferably at least about 100%, and / or the production of said provisional matrix may be accelerated by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, and more preferably at least about 100%, compared to a subject not treated with a composition (optionally a hydrogel) described herein.

[0149] Alternatively or in addition to increased and / or promoted provisional matrix production, improved re-epithelialization may be characterized by increased and / or promoted keratinocyte activation (i.e., proliferation) and / or migration. 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%, and more preferably at least about 100%, and / or keratinocyte migration may be increased by at least about 10%, preferably at least about 25%, preferably at least about 50%, preferably at least about 75%, and more preferably at least about 100%, compared to a subject not treated with a composition (optionally a hydrogel) described herein.

[0150] In certain embodiments, the compositions and hydrogels of the above aspects are used to increase cell proliferation at a wound site in a subject. Cell proliferation includes, but is not limited to, proliferation of endothelial cells, fibroblasts, and / or keratinocytes. In certain embodiments, proliferation of cells selected from the group consisting of 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 in a subject not treated with a composition described herein.

[0151] In certain embodiments, the compositions described herein are used to reduce an inflammatory response at a wound site in a subject. In such embodiments, the compositions described herein are 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 a wound site in a subject. 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, kinins, coagulation proteins, fibrinolytic proteins, lipid mediators, prostaglandins, leukotrienes, and platelet-activating factor (PAF). In certain embodiments, the compositions (optionally hydrogels) described herein are used to reduce the amount of one or more pro-inflammatory molecules selected from the group consisting of interleukin-1β (IL1-β), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor alpha (TNF-α), interferon gamma (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) produced at a wound site by at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 75%, and most preferably about 100%, compared to the level of the one or more pro-inflammatory molecules present at a wound site in a subject not treated with a composition described herein. Preferably, the medical use or treatment results in an improved macrophage and neutrophil profile indicative of reduced local wound inflammation compared to an untreated wound, hi certain embodiments, treatment according to the present invention reduces the number of inflammatory cells, including but not limited to macrophages and neutrophils, within the wound.Thus, in certain embodiments, topical administration of a composition according to the present invention promotes a resolving wound phenotype, in which M1 marker expression is decreased and M2 marker expression is increased. In some embodiments, administration of a composition of the present invention reduces the number of both innate and adaptive immune cells. Typically, in further embodiments, administration of the compositions described herein can be observed to have beneficial effects on the function of other immune cells, such as dendritic cells, Langerhans cells, and mast cells.

[0152] In a preferred embodiment, the use of a composition (e.g., a hydrogel) containing an estetrol component results in improved histological healing parameters compared to wounds not treated with any wound healing composition. More preferably, the use of a composition containing an estetrol component results in improved histological healing parameters compared to wounds not treated with a wound healing composition not containing an estetrol component. Optionally, the histological healing parameter is a histological skin parameter selected from the group consisting of 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 assessed histologically by the presence of newly formed epidermis, spinous and / or granular epidermal differentiation markers, migrating cells, proliferating cells, collagen fiber deposition, immune cell markers, wound protease levels, and matrix composition, respectively. All of these parameters have been described in detail in the art (e.g., Gupta and Kumar, Plast Aesthet Res, 2015). Alternative histological parameters that can be obtained from one or more observations include, but are not limited to, the length of the re-epithelialized area, the distance between wound borders, the wound depth, the wound width, the connective tissue thickness, and the thickness of the native dermis at the wound edge, the orientation of the dermal matrix, the wound cellularity, and the wound vascularization.

[0153] In a preferred embodiment, the compositions described herein can be used to improve the incidence of complete wound closure in subjects, which are optionally subjects characterized by wound healing disorders.Complete wound closure refers to the complete closure of the skin surface, i.e., the complete resurfacing with new epithelium.In this embodiment, the incidence of complete wound closure by using a composition comprising estetrol components is increased by at least 25%, preferably at least 50%, more preferably at least 75%, and most preferably at least 100%, compared with wounds not treated with the composition, or based on the wound healing history of the subject.

[0154] In a preferred embodiment, the compositions described herein can be used to accelerate the time to achieve wound closure in a subject, optionally a subject characterized by impaired wound healing. In such an embodiment, the time to achieve wound closure in 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 subject's wound healing history. Those skilled in the art will understand that certain parameters detailed throughout this disclosure, such as, but not limited to, the certain time required to achieve complete wound closure, will depend, inter alia, on the size of the wound.

[0155] In preferred embodiments, the compositions described herein can be used to promote surgical wound closure in subjects, which are optionally subjects characterized by wound healing disorders.In such embodiments, the compositions described herein can increase the speed of surgical wound closure and / or increase the likelihood that a subject will achieve wound closure for wounds with area and / or depth that exceed the area and / or depth of the wound that can be healed by a subject without using a composition that contains estetrol components.In some embodiments, the quality of healing is improved, especially for infected wounds.

[0156] The terms "formulation" and "composition" may be used interchangeably herein. It is clear that in any embodiment of a composition described herein, the composition may comprise one or more pharmaceutically or cosmetically acceptable carriers (i.e., additives) not described in detail throughout this disclosure. The term "pharmaceutically acceptable" as used herein is consistent with the art and means compatible with other ingredients of a pharmaceutical or cosmetic composition and not harmful to its recipient. 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 corresponds to a daily dosage unit. The additives that can be used in the pharmaceutical composition are not particularly limited, and therefore may be one or more additives selected from the group consisting of active pharmaceutical ingredient additives, binder additives, carrier additives, co-processed additives, coating system additives, controlled release additives, diluent additives, disintegrant additives, dry powder inhalation additives, effervescent system additives, emulsifier additives, lipid additives, lubricant additives, modified release additives, permeation enhancer additives, penetration enhancer additives, pH adjuster additives, plasticizer additives, preservative additives, solubilizer additives, solvent additives, sustained release additives, sweetener additives, taste making additives, thickener additives, viscosity modifier additives, filler additives, compaction additives, dry granulation additives, hot melt extrusion additives, wet granulation additives, immediate release additives, bioavailability enhancing additives, dispersion additives, solubility enhancing additives, stabilizer additives, capsule filling additives, or any combination thereof. Those skilled in the art will recognize that the use of such media and agents for pharmaceutically active substances is common practice, and therefore the inclusion of these additives is well known in the art. It is clear that all ingredients used should be non-toxic at the concentrations contained in the final pharmaceutical composition and should not negatively interfere with the activity of the estetrol component, which is preferably present as the main pharmaceutically active ingredient in the pharmaceutical composition.In certain embodiments, two or more additives that one skilled in the art would classify as belonging to the same group of additives are added to the pharmaceutical composition. In further embodiments, two or more additives are added to the pharmaceutical composition, where different additives belong to different groups. In certain embodiments, an additive may fulfill more than one function and / or may be classified by one skilled in the art as belonging to different groups or classes of additives.

[0157] As mentioned above, in the context of the present invention, the details regarding the subject suffering from the wound are not particularly limited. A preferred subject is an elderly subject. "Elderly subject" refers to an elderly subject, i.e., a subject whose age is approaching or exceeding the subject's life expectancy. An elderly subject is defined by an age of 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 group consisting of a young child (i.e., a juvenile subject), an adolescent subject, and an adult subject. In certain embodiments described herein, the subject is diagnosed as or considered to be palliative.

[0158] Optionally, the pharmaceutical compositions contemplated by the present invention (which may optionally be hydrogels) may contain additional skin active ingredients capable of providing skin care benefits. Skin care benefits may include, but are not limited to, benefits related to the cosmetic appearance of the skin. The additional skin active ingredients may provide immediate and short-term (i.e., acute) benefits and / or long-term and sustained (i.e., chronic) benefits.

[0159] Optionally, the pharmaceutical compositions contemplated by the present invention (which may optionally be hydrogels) may contain at least one additional pharmaceutically active ingredient in addition to the estetrol component. In certain embodiments, the at least one additional pharmaceutically active ingredient is selected from the group consisting of anti-inflammatory agents, analgesics, and anti-infective agents. The anti-inflammatory component may be a steroidal anti-inflammatory component, a 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.

[0160] In certain embodiments, the pharmaceutical compositions contemplated by the present invention (which may optionally be hydrogels) are used in wound healing in the context of skin grafting. In such embodiments, the compositions described herein can be combined with skin grafting techniques, such as, but not limited to, those that rely on stem cell therapy, bioengineered skin, skin equivalents, skin substitutes, synthetic skin, or combinations thereof. In certain embodiments, the compositions used herein are used in wound healing after skin grafting where extensive trauma to a significant area of ​​a subject's skin is deemed necessary, such as, but not limited to, in the context of burn wounds. Healing of wounds from skin graft donor sites is also contemplated herein.

[0161] The pharmaceutical composition described herein (optionally a hydrogel) is administered (i.e., applied) to the wound site. The composition may be applied by pouring, dripping, spraying, rubbing, or any other suitable means. Optionally, the composition is administered once to the wound site. Alternatively, the composition is administered to the wound site multiple times, preferably at substantially regular intervals. In certain embodiments, the composition is administered daily to the wound site. Optionally, the composition is administered to the wound site and maintained at the wound site (i.e., not removed from the wound site) for an extended 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, and more preferably at least 1 month. In such embodiments, it is understood that the wound is continuously exposed to the composition for the indicated amount of time.

[0162] The term "continuous" / "continuously" as used herein means that the components are administered at relatively regular intervals without significant (therapeutic) interruptions. Of course, minor interruptions that do not affect the overall efficacy of the method may be made, and indeed such deviations are encompassed by the present invention.

[0163] The pharmaceutical compositions described herein (optionally hydrogels) can be part of, or contained within, any suitable device for application to the skin and / or wound site of a subject, preferably on or near the wound site, to deliver a pharmaceutically active ingredient to the skin (and / or wound site). Alternatively, the compositions described herein can be applied to the subject's skin and / or wound site by the subject, another subject, or a skilled medical professional, and then applied to any suitable device for applying the composition to the subject's skin or wound site (i.e., the composition is used in conjunction with a device for application to the skin).

[0164] Preferably, the pharmaceutical composition is applied to the subject's skin using a dressing. Numerous 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 scaffold dressings, hydrocolloid dressings, occlusive dressings, or combinations thereof. Dressings may be adhesive or non-adhesive. The term "occlusive dressing" as used herein refers to a dressing that prevents contact with air and / or bacteria and retains one or more of moisture, heat, body fluids, and medications. Those skilled in the art can select an appropriate wound healing dressing for use with a particular wound, and such selection can be made depending on parameters such as, but not limited to, the type of wound, the size of the wound, and the progress of wound healing.

[0165] Optionally, the dressing is a hydrogel dressing. Hydrogel dressings are composed mostly of water within a network of fibers that maintain the integrity of the polymer gel. Water is released from the dressing to maintain an appropriate moisture level in the wound. Examples of hydrogel dressings include, but are not limited to, Tegagel™ and Intrasite™.

[0166] Methods and protocols for producing any of the above coatings have been described in the art. The estetrol component may be included in / on the coating during its manufacture, but may also be applied to a pre-fabricated coating. By way of example and not limitation, the estetrol component may be impregnated into a pre-fabricated coating or a portion thereof. Alternatively, the estetrol component may be coated onto a pre-fabricated coating or a portion thereof.

[0167] In certain embodiments, the pharmaceutical composition (optionally a hydrogel) is contained in a skin substitute (i.e., a skin substitute or dermal substitute). The skin substitute provides a three-dimensional biomatrix that fulfills the function of the dermal layer of skin, capable of covering open skin wounds either temporarily or permanently. The material of the skin substitute is not particularly limited and may therefore include biological materials, synthetic materials, or combinations thereof. Non-limiting examples of biological materials include, but are not limited to, human or porcine skin and human or porcine intestinal submucosa. The biological skin substitute may include various components, including, but not limited to, collagen, glycosaminoglycans, fibronectin, hyaluronic acid, elastin, and any combination thereof.

[0168] 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, band-aids, patches, and plasters.

[0169] While the present invention has been described in conjunction with specific embodiments thereof, it is evident from the foregoing description that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims. Aspects and embodiments of the invention disclosed herein are further supported by the following non-limiting examples. [Example]

[0170] Example 1. Manufacturing process Aqueous gel formulation Batch AG23, Batch AG24, Batch AG25, Batch AG26 i. Water (1st addition) was weighed into Duran (Container 1). ii. The sample from step (i) was placed on a hotplate stirrer at 800 rpm to create a vortex. iii. The polymer (Carbopol™) was weighed into the contents of step (ii) via a weigh boat and dispersed while maintaining a vortex. The sample was left stirring for at least 1 hour to allow the polymer to disperse. iv. The weight of the polymer dispersion in step (iii) was recorded. v. The sample from step (iv) is then cooled to room temperature under standard conditions (121°C ± 2°C, 2 x 10 5 The mixture was autoclaved at 400 KPa for 15 min. vi. Once the autoclaved sample had cooled, the sample from step (v) was reweighed and additional water was added to compensate for the loss of volatiles due to evaporation. vii. Solvent (PEG 400, Transcutol™ P (diethylene glycol monoethyl ether)) was weighed into a separate container. viii. Estetrol monohydrate was weighed into the contents of step (vii) and stirred (hot plate magnetic stirrer at lab room temperature) at 500 rpm for 2.5 hours to dissolve the drug. ix. In a biosafety laminar flow hood, the contents of step (viii) were filtered through multiple Spartan (regenerated cellulose) (0.2 μm) sterile filters into multiple 20 mL autoclaved vials to avoid bulk contamination in case of filter breakage. x. The contents of step (ix) were combined and weighed into a pre-autoclaved Duran container. xi. The contents of step (x) were poured into the contents of step (vi) and mixed using an overhead stirrer and spatula. The mixing speed and time were recorded in a laboratory notebook. xii. Water (approximately 4% of the total volume) was used to rinse the container from step (x) and poured into container 1. xiii. The formulation was allowed to stand at least overnight. xiv. The pH of the sample was adjusted to a target pH of between 6 and 6.5 using sodium hydroxide solution and weighed with water. xv. Please note that manufacturing steps (ix) to (xiv) were carried out under sterile conditions in a laminar flow hood.

[0171] Batch AG30, Batch AG22 (Carbopol™ 980) i. Water (1st addition) was measured into 150 mL of Duran (Container 1). ii. The sample from step (i) was placed on a hotplate stirrer at 800 rpm to create a vortex. iii. The polymer (Carbopol™ 980) was weighed into the contents of step (ii) via a weigh boat and dispersed while maintaining a vortex. The sample was left stirring for at least 1 hour to allow the polymer to disperse. iv. The weight of the polymer dispersion in step (iii) was recorded. v. PEG 400 and glycerol were weighed and added to the contents of step (iv). vi. The sample from step (v) is then cooled to standard conditions (121°C ± 2°C, 2 x 10 5 The mixture was autoclaved at 400 KPa for 15 min. vii. Once the autoclaved sample had cooled, the sample from step (v) was re-weighed and additional water was added to compensate for the loss of volatiles due to evaporation. viii. Solvent (propylene glycol (PG), Transcutol™ P) was weighed into 100 mL of Duran. ix. Estetrol monohydrate was weighed into the contents of step (viii) and stirred at 500 rpm (hot plate magnetic stirrer at lab room temperature). x. In a biosafety laminar flow hood, the contents of step (ix) were filtered through multiple Spartan (0.2 μm) sterile filters into multiple 20 mL autoclaved vials to avoid bulk contamination if the filters broke. xi. The contents of step (x) were combined and weighed into 100 mL of pre-autoclaved Duran. xii. Sodium hydroxide (18%) (pre-filtered through a 0.2 μm sterile Spartan filter) was poured into the contents of step (xi) and mixed. xiii. The contents of step (xii) were quickly poured into the contents of step (vii) and mixed using a spatula. xiv. Water (approximately 4% of the total volume) was used to rinse the container from step (x) and poured into container 1. xv. The thickened sample was allowed to stand at least overnight. xvi. The pH of the sample was measured and adjusted to pH 7-7.5, along with the remaining sterile water, by adding sodium hydroxide solution (also pre-sterilized through a 0.2 μm Spartan filter) as needed. Please note that manufacturing steps (x) to (xvi) were carried out under sterile conditions in a laminar flow hood.

[0172] Batch AG27 (Poloxamer) i. Water (1st addition) was weighed into 150 mL of Duran and chilled in a refrigerator maintained at 2°C to 8°C for 20 minutes. ii. The sample from step (i) was placed on a hotplate stirrer and the poloxamers (P407 followed by P188) were weighed and added while vortexing at 800 rpm. iii. The sample from step (ii) was stirred at 200 rpm using an overhead stirrer in a water bath maintained at 2°C to 8°C for 6 hours to dissolve the poloxamer. iv. In a separate container, PEG 400, benzyl alcohol (BA), and PG were weighed together. v. Estetrol monohydrate was weighed and added to the contents of step (iv) and left stirring overnight on a hot plate stirrer at 500 rpm. Once the drug was dissolved, the required amount of solution from step (v) was weighed into the original container containing the poloxamer solution. The sample was left on a magnetic stirrer at 300 rpm overnight. vii. The pH of the sample from step (vi) was adjusted to 7-7.5.

[0173] Batches AG28 (hydroxyethyl cellulose (HEC)), AG29 (carboxymethyl cellulose (CMC)) i. The solvent system for the formulation was prepared by weighing out all solvents (except water / buffer for the active system only). ii. The drug was added to the solvent system for the active formulation and stirred at 500 rpm overnight. iii. After the drug was dissolved, the water / pH 7.0 buffer solution was added to the solvent system while stirring the Duran. iv. The appropriate amount of premix was weighed into a separate 150 mL of Duran. v. The polymer was dispersed by stirring at 800 rpm to form a vortex and the formulation was left stirring overnight at 500 rpm. For AG28, the pH was adjusted to pH 7-7.5. For AG29, no pH adjustment was required because it contained phosphate-phosphate buffer (pH 7.0).

[0174] Batch AG15 (Poloxamer) i. Water (1st addition) was weighed into 150 mL of Duran and chilled in a refrigerator maintained at 2°C to 8°C for 20 minutes. ii. The sample from step (i) was placed on a hotplate stirrer and the poloxamers (P407 followed by P188) were weighed and added while vortexing at 800 rpm. iii. The sample from step (ii) was stirred at 200 rpm using an overhead stirrer in a water bath maintained at 2°C to 8°C for 6 hours to dissolve the poloxamer. iv. In a separate container, the PEG400, BA, and PG were weighed together. v. Estetrol was weighed and added to the contents of step (iv) and left to stir overnight on a hot plate stirrer at 500 rpm. This step was omitted when producing placebo. Once the drug was dissolved, the required amount of solution from step (v) was weighed into the original container containing the poloxamer solution. The sample was left on a magnetic stirrer at 300 rpm overnight. vii. The pH of the sample from step (vi) was adjusted to 7-7.5.

[0175] Batch AG17 i. Water (1st addition) was measured into 150 mL of Duran (Container 1). ii. The sample from step (i) was placed on a hotplate stirrer at 800 rpm to create a vortex. iii. The polymer (Carbopol™) was weighed into the contents of step (ii) via a weigh boat and dispersed while maintaining a vortex. The sample was left stirring for at least 1 hour to allow the polymer to disperse. iv. The weight of the polymer dispersion in step (iii) was recorded. v. The sample from step (iv) is then cooled to room temperature under standard conditions (121°C ± 2°C, 2 x 10 5 The mixture was autoclaved at 400 KPa for 15 min. vi. Once the autoclaved sample had cooled, the sample from step (v) was reweighed and additional water was added to compensate for the loss of volatiles due to evaporation. vii. Benzyl alcohol, PEG 400, and PG were weighed into separate 100 mL Duran. viii. Estetrol monohydrate was weighed into the contents of step (vii) and stirred at 500 rpm overnight (hot plate magnetic stirrer at laboratory room temperature) to dissolve the drug. This step was omitted for the placebo formulation. ix. Sodium hydroxide (18%) (1.38 g) was poured into the contents of step (vi) and mixed. x. The contents of step (ix) were quickly poured into the contents of step (vi) and mixed using a spatula. xi. Water (4.5g) was used to rinse the container from step (ix) and poured into container 1. xii. The thickened sample was allowed to stand at least overnight. xiii. The pH of the sample was measured and the remaining water was added slowly while continuing to measure the pH.

[0176] Batch AG18 (HEC), Batch AG19 (CMC) i. Vehicle systems for the placebo and active formulations were prepared by weighing out all solvents (except water / buffer for the active system only). ii. The drug was added to the solvent system for the active formulation and stirred at 500 rpm overnight. iii. After the drug was dissolved, the water / pH 7.0 buffer solution was added to the solvent system while stirring the Duran. iv. The appropriate amount of premix was weighed into a separate 150 mL of Duran. The polymer was dispersed by stirring at 800 rpm to form a vortex, and the formulation was left stirring overnight at 500 rpm. For v.AG18, the pH was adjusted to pH 7-7.5. For AG19, no pH adjustment was required because it contained a phosphate-phosphate buffer (pH 7.0).

[0177] Batch AG20, Batch AG23 i. Steps (i) to (vi) from AG17 were followed. ii. Solvent (PEG 400, Transcutol™ P) was weighed into a separate container (100 mL). iii. Estetrol monohydrate was weighed into the contents of step (ii) and stirred (hot plate magnetic stirrer at lab room temperature) at 500 rpm for 2.5 hours to dissolve the drug. This step was omitted for the placebo formulation. iv. In a biosafety laminar flow hood, the contents of step (iii) were filtered through multiple Spartan (regenerated cellulose) (0.2 μm) sterile filters into multiple 20 mL autoclaved vials to avoid bulk contamination in case of filter breakage. Initially, the sample was planned to be filtered through a Nalgene Bottle System filter (PES) using a vacuum pump, but the filter broke, so Spartan syringe filters were used. v. The contents of step (iv) were combined and weighed into 100 mL of pre-autoclaved Duran. vi. Pour the contents of step (v) into the contents of step (i) and mix using a spatula. vii. Water (4.5g) was used to rinse the container from step (v) and poured into container 1. viii. The formulation did not thicken so it was left to stand at least overnight. ix. The pH of the sample was adjusted to between 6 and 6.5 using sodium hydroxide solution and weighed with water. Note that the process was initially performed using sodium hydroxide (18%), but the Carbopol™ flocculated, i.e., failed to hydrate. Furthermore, while the initial target pH was 7-7.5, polymer precipitation was observed above pH 6.5; to avoid this issue, the sample was repeated and placed between pH 6 and 6.5. Please note that manufacturing steps (iv) to (ix) were carried out under sterile conditions in a laminar flow hood.

[0178] Batch AG21, Batch AG22 i. Steps (i) to (vi) from AG17 were followed. ii. Solvents (PEG 400, PG, Trans P) were weighed into 100 mL of Duran. iii. Estetrol monohydrate was weighed into the contents of step (i) and stirred at 500 rpm (hot plate magnetic stirrer at laboratory room temperature). The drug dissolved within 2.5 hours of stirring for AG22 and overnight for AG21. This step was omitted for the placebo formulation. iv. In a biosafety laminar flow hood, the contents of step (viii) were filtered through multiple Spartan (0.2 μm) sterile filters into multiple 20 mL autoclaved vials to avoid bulk contamination if the filters broke. v. The contents of step (ix) were combined and weighed into 100 mL of pre-autoclaved Duran. vi. Sodium hydroxide (18%) (1.38 g) (pre-filtered through a 0.2 μm sterile Spartan filter) was poured into the contents of step (x) and mixed. vii. The contents of step (xi) were quickly poured into the contents of step (vi) and mixed using a spatula. viii. Water (4.5g) was used to rinse the container from step (x) and poured into container 1. ix. The sample thickened and the sample was allowed to stand at least overnight. x. The pH of the sample was measured and adjusted to pH 7-7.5, along with the remaining sterile water, by adding sodium hydroxide solution (also pre-sterilized through a 0.2 μm Spartan filter) as needed. Please note that manufacturing steps (ix) to (xv) were carried out under sterile conditions in a laminar flow hood.

[0179] [Table 1] TIFF2025534465000003.tif254170

[0180] cream Batch CR01, Batch CR05, Batch CR12 i. Placebo and active drug vehicle systems were prepared by weighing out all vehicles (except water / buffer for the active drug system only). ii. The drug was added to the solvent system for the active cream and stirred at 500 rpm overnight. iii. After the drug was completely dissolved, the water / pH 7.0 buffer solution was added to the solvent system while stirring the container (Duran). iv. The oil phase was then prepared. For CR01, the oil phase was placed in a 75°C water bath to melt it. For v.CR05 and CR12, the oil phase was placed in an oven at 160°C for 2 hours (simulating a sterilization process). Once the oil phase had cooled to room temperature and re-solidified, it was placed in a water bath (75°C) to re-melt it. vi. Prior to processing the active and placebo creams, the required amount of solvent-based premix was weighed into separate Duran containers. vii. The Duran with the premix was placed in a water bath with the homogenizer head for 5 minutes. viii. For CR01 and CR12, the cream was processed through an Ultra-turrax equipped with a 25G dispersing head at 10000 rpm for 2 minutes. ix. For CR05, during homogenization, the cream was homogenized instead at 5000 rpm for 5 minutes to avoid overflowing. x. After processing, the cream was stirred by hand with a metal spatula until it reached room temperature. xi. These were left to harden overnight. xii. The pH of the formulation was adjusted to pH 7-7.5.

[0181] Batch CR10, Batch CR13, Batch CR14, Batch CR15, Batch CR16 i. Placebo and active drug vehicle systems were prepared by weighing out all vehicles (except water / buffer for the active drug system only). ii. The drug was added to the solvent system for the active cream and stirred at 500 rpm overnight. iii. After the drug was completely dissolved, the water / pH 7.0 buffer solution was added to the solvent system while stirring the Duran. The oil phases of CR13, CR14, and CR15 were placed in an oven (sterilized) at 160°C for 2 hours and allowed to resolidify at room temperature. v. The aqueous phase was sterilized by filtration using a sterile PES syringe filter (0.2 μm). vi. The required amount of aqueous phase was weighed into 250 mL of Duran that had been pre-sterilized by autoclaving. vii. The oil phase was placed in a 75°C water bath to melt. viii. The aqueous phase was placed in a water bath with the homogenizer head and allowed to equilibrate for 5 minutes. ix. For each cream, the oil phase was added to the respective water phase and homogenized using an Ultra-turrax equipped with a 25G dispersing head at 10000 rpm for 2 minutes. x. After processing, the cream was stirred by hand with a metal spatula until it reached room temperature. xi. These were left to harden overnight. xii. The pH of the formulation was adjusted to pH 7-7.5. It should be noted that manufacturing steps (v) to (xii) were performed under sterile conditions in a laminar flow hood and the sterile container was only opened in the sterile laminar flow hood.

[0182] [Table 2] TIFF2025534465000005.tif254170

[0183] Example 2. In vitro release experiment 1 Following 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 utilized are shown in Table 3.

[0184] [Table 3]

[0185] Full-scale in vitro release experiments were performed using the experimental conditions identified during method development and feasibility studies. Results for all gel and cream formulations tested are shown in Figure 1, and tabular results are shown in Table 4.

[0186] Aqueous gel formulation The release rates of the aqueous gel formulations are shown in Figure 2 and Table 4. Formulations AG18, AG19, and AG23 had the highest release rate (161 μg / cm 2 / √hr~221μg / cm 2 / √hr), followed by AG21 (51 μg / cm 2 / √hr), with AG22 (sterilized), AG22 (autoclave), and AG15 having the lowest release rate (3 μg / cm 2 / √hr~10μg / cm 2 / √hr). As a general trend, the observed release rates correlated with 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).

[0187] The aqueous gels with the lowest release rates (AG21, AG22 (sterilized), AG22 (autoclaved), and AG15) were found to immediately release a large amount of the dosed API into the receptor solution (approximately 50% or more release at t = 0.5 h). As a result of the large release of API, linear steady-state drug release was not achieved in many formulations (r 2 <0.9). Therefore, no inferences or statistical comparisons were made using the aqueous gel formulation.

[0188] Cream formulation The release rates of the cream formulations are shown in Figure 3 and supported by Table 4. CR16 (0.50% w / w) had the highest release rate, followed by CR14 (0.35% w / w) and finally CR01 (0.24% w / w), where the average release rate was 32 μg / cm 2 / √ hour~53μg / cm 2 / √hr. Following the same trend as the aqueous gels, the release rate correlated with the concentration of API present in the formulation, with the highest release rate occurring in the formulation with the highest drug loading (CR16, containing 0.50% API, 52.20 μg / cm 2 The average drug release (μg / cm) of the cream formulation was observed at a release rate of 1 / √hr. 2 One-way statistical analysis (p / √hr) was performed using the Tukey-Kramer method (n = 6). There was no statistical difference between the release rates of CR14 and CR16 (p > 0.05), but the release rate of CR01 was observed to have a significantly lower release rate (p < 0.05).

[0189] [Table 4]

[0190] Example 3. In vitro release experiment 2 Following the results of the first in vitro release experiment (Example 2), a second IVRT experiment was performed using the four formulations and experimental parameters outlined in Table 5.

[0191] [Table 5]

[0192] A second full-scale in vitro release experiment was conducted using the experimental parameters and four aqueous gel formulations detailed in Table 5. Results for all formulations tested are shown in Figures 4 and 5, and tabular results are shown in Tables 6 and 7.

[0193] The objective of this part of the study was to determine the effect of thermodynamic activity on the release rate of estetrol monohydrate from the formulation by comparing the non-optimized formulation (AG23 0.5% wt / wt API) with various concentrations of optimized formulations (AG24 0.5% wt / wt, AG25 0.22% wt / wt, and AG26 0.06% wt / wt API).

[0194] Considering these four formulations in terms of percentage release of the applied dose of API (Table 17), after 8 hours, 87.95±10.88% of API was released from the AG26 formulation (0.06% wt / wt API), which was significantly higher (p, 0.05) than the other three formulations (AG25 61.51±4.91% (0.22% wt / wt API), AG24 60.67±4.86% (0.5% wt / wt API), and AG23 56.88±5.90% (0.5% wt / wt API)), which were not statistically different from each other (p>0.05).

[0195] Considering the percentage release rate of the applied dose over time (slope), there was a significant difference between formulation AG26 and the other three formulations (AG23, AG24, and AG25) (p<0.05). No statistically significant differences were found between AG23, AG24, and AG25. Statistical analysis was performed by Tukey-Kramer test (n=5-6).

[0196] For this study, formulation AG23 was used in the first full-scale IVRT study at 195 ± 20 μg / cm 2 / hour, compared with approximately 167±13μg / cm 2 In this study, formulation AG24 showed the highest release rate (approximately 182±8 μg / cm 2 / hour), followed by AG23, AG25 (approximately 81 ± 5 μg / cm 2 / h), and finally AG26 (approximately 34 ± 3 μg / cm 2 / hr). The release rate of each formulation is significantly different from the neighboring formulation (p<0.05). In the case of AG23 and AG24, both of which contain 0.5% (wt / wt) estetrol monohydrate, this may be due to problems encountered with the dosing of AG23, or batch-to-batch variability compared to the batch of AG23 used in the previous experiment (which had a release rate very similar to that seen here for AG24), or simply differences in the formulation of AG23 and AG24. For the other two formulations, the difference in release rate is as expected due to the lower API content in these formulations (0.5% (wt / wt) for AG23 and AG24, 0.22% (wt / wt) for AG25, and 0.06% (wt / wt) for AG26).

[0197] To assess differences between the aqueous gel formulations reported above, one-way statistical analysis was performed using the Tukey-Kramer method (n = 5–6).

[0198] [Table 6]

[0199] [Table 7]

[0200] Example 4. In vivo LPS treatment protocol (delayed wound healing model) An in vivo LPS-induced delayed wound healing model was reproduced as previously described (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.).

[0201] Female wild-type (C57BL / 6J) 8-week-old mice were divided into six groups (six animals per experimental group): double placebo control (no LPS, placebo treatment), LPS control (LPS, placebo treatment), LPS and topical EstroGel (estradiol gel, containing 0.06% 17β-estradiol as hemihydrate in a hydroalcoholic gel); LPS and topical AG24 (0.5% E4), LPS and topical AG25 (0.22% E4); LPS and topical AG26 (0.06% E4).

[0202] The day before wounding (day -1), all animals were weighed and anesthetized using oxygen and isoflurane (2%–2.5% isoflurane at a flow rate of 1.25–2 L depending on clinical signs). The animals' hair was shaved, and the dorsal area was prepared. The wound locations (two wounds) were marked on each animal's back. Except for the duplicate placebo group, which received DPBS only, animals received a first subcutaneous injection of 2 μg of K. pneumoniae-derived LPS diluted in DPBS (1 μg per wound, Sigma-Aldrich, UK: L4268) at the wound site. A thin layer of EstroGel, AG24, AG25, AG26, or placebo, totaling 60 μl, was then applied to each animal's back (30 μl per wound). After recovering in a heated cabinet, animals were housed singly in new cages containing Alpha Pad, RO water, food, mash, and a house. On day 0 (24 hours after the initial anesthesia), mice were re-anesthetized, and the dorsal skin was cleaned with chlorhexidine wipes. Two 6-mm dorsal incisions were made. Except for the double placebo mice, which were injected with DPBS, 2 μg of LPS (1 μg per wound) was injected subcutaneously into the wound site as described above. EstroGel, AG24, AG25, AG26, or placebo AG23 was then applied in a thin layer over the top of the wound (60 μl total, 30 μl per wound), taking care not to damage the "LPS blister." The analgesic buprenorphine (0.1 mg / kg) was administered via subcutaneous injection in the nape of the neck postoperatively, and each animal was imaged. Mice were allowed to recover in a heated cabinet and returned to single-housed care. Postoperative observations were performed.

[0203] For treatment groups, topical EstroGel, AG24, AG25, AG26, or placebo was reapplied in a thin layer over the top of the wound during observation on days 1, 2, 3, and 4 (60 μl total, 30 μl per wound).

[0204] On day 5, mice were humanely sacrificed via elevated CO2 concentration and cervical dislocation.

[0205] The uteri were removed and weighed.

[0206] Non-wounded skin (NS) from the treatment site was harvested at the time of wounding (day 0), processed for histological analysis, and flash-frozen. Wound tissue was harvested 5 days after wounding (day 5). Wounds were bisected, and the bottom half of each wound was fixed for histological analysis, while the top half was flash-frozen.

[0207] For immunohistochemistry (IHC) and histological analysis, tissue samples were fixed in 10% buffered formalin, embedded in paraffin wax, and sectioned. Tissue sections were dewaxed in xylene and rehydrated through an ethanol gradient before hematoxylin and eosin staining and IHC for immune cells.

[0208] To isolate and culture mouse peritoneal macrophages, the peritoneal cavity of a euthanized mouse was filled with 5 ml of ice-cold PBS supplemented with 3% FBS and 1% antibiotic-antimycotic. The cell-containing fluid was removed using a needle and syringe and cultured at 1 × 10 cells per ml in RPMI growth medium supplemented with 10% FBS and 1% penicillin / streptomycin. 6 Cells were seeded into 12-well plates at 100 μg / ml of IFN-γ and 1 μg / ml of LPS. For M2 macrophages, 20 μg / ml of anti-IFN-γ and 10 ng / ml of IL-4 were used. Cells were left overnight to allow macrophages to adhere and then washed twice to remove any non-adherent cells. Cells were cultured for an additional 24 hours before polarization into either the M1 or M2 state. M1 macrophages were induced using 100 μg / ml of IFN-γ and 1 μg / ml of LPS. For M2 macrophages, 20 μg / ml of anti-IFN-γ and 10 ng / ml of IL-4 were used.

[0209] For RNA isolation and quantitative real-time PCR, mouse macrophages were collected in Trizol and RNA was isolated using the Trizol Plus RNA Isolation Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using GoScript reverse transcriptase (Promega). Quantitative real-time PCR was performed using 2x Takyon SYBR Green master mix and a CFX Connect thermocycler. Primers for the mouse genes Il-1β, Tnf-α, iNos, Arg1, Fizz1, and Ym1 were used to assess the degree of M1 and M2 polarization of peritoneal macrophages from different treatment groups. Data were normalized using primers for GAPDH. Unless otherwise specified, relative gene expression was determined relative to the M0 PBS + PBO control group.

[0210] Statistical significance was assessed using one-way ANOVA with Tukey post-hoc analysis or paired t-tests, as appropriate.

[0211] Figure 6 clearly shows that application of EstroGel™ promoted uterine weight gain and hypertrophic changes. Topical application of E4 dose-dependently affected uterine weight, with AG26 having no effect and AG24 causing uterine weight gain. The data confirm that, in contrast to EstroGel™ (E2) and high-level E4 formulations (AG24, AG25), topical application of the low-concentration E4 formulation (AG26) to open wounds for 6 days did not result in systemic side effects, manifested as increased uterine weight. Wound closure was then assessed from histological samples. Tissue sections were subjected to K14 immunohistochemistry to visualize the newly forming epidermis. The degree of re-epithelialization was calculated as the length of the neoepidermis divided by the distance between the wound edges, multiplied by 100. As expected, wounds treated with LPS and placebo showed a delay of more than 30% in wound re-epithelialization compared to non-LPS-treated wounds (Figure 7). All four active treatments (EstroGel™, AG24, AG25, and AG26) promoted re-epithelialization. Interestingly, a greater magnitude of promotion and statistical significance was observed in wounds treated with either EstroGel™ or AG26, where re-epithelialization approached that observed in the non-LPS-treated control group. The AG25-treated group failed to achieve any statistical significance relative to the LPS / PBO group.

[0212] Based on the data shown in Figures 6 and 7, it can be concluded that topically applied AG26 formulations can significantly improve wound healing without causing systemic effects such as increased uterine weight. This provides an indication of a preferred dosage range if systemic effects are to be avoided entirely. However, in some cases, higher dosages may be envisioned based on the risk / benefit favorable to the subject at hand.

[0213] Immunohistochemistry was performed to assess the effect of active treatments (EstroGel™, AG24, AG25, and AG26) on local wound immune cell counts. Wound neutrophil levels were significantly increased in LPS / PBO-treated wounds compared with non-LPS / PBO-treated wounds (Figure 8). Treatment with AG24 and AG26 significantly reduced wound neutrophil counts to a similar extent as EstroGel™. AG25 was slightly less effective, but still highly significant, in reducing wound inflammation. Very similar effects of gels containing E2 and E4 on wound macrophage counts were observed (Figure 9). Again, AG25 was slightly less effective. Interestingly, EstroGel™ and AG26 completely reversed the effect of LPS on macrophages, restoring wound macrophage levels to levels comparable to those of the non-LPS / PBO group (Figure 9).

[0214] To further complement the immunohistochemical analysis, wound tissue RNA was isolated and qPCR was performed to evaluate specific markers of M1 and M2 polarization phenotypes (Figure 10). All treatments resulted in a trend toward decreased expression of M1 markers compared to the LPS / PBO control. Interestingly, the magnitude of the effect was greatest in the Estrogel™ and AG26 groups (reaching statistical significance for IL1-β and AG26 treatments). In contrast, the M2 markers Fizz1 and Ym1 were increased by all treatment groups, although in this case the effect of AG26 was the least pronounced of the three AG formulations tested. These data indicate that E4 reduces inflammation and promotes an M2 (pro-healing) wound environment.

[0215] Finally, to further explore the potential systemic effects of local E2 or E4 treatment, the phenotype of peritoneal macrophages isolated from each experimental mouse group at study completion was assessed. Specifically, macrophages from each group were separately isolated by peritoneal lavage, cultured, and polarized toward either the M1 or M2 phenotype (see Example 7). RNA was isolated, and qPCR was performed to quantify the relative levels of Tnf-α and iNOS (M1 markers), and Arg1 and Ym1 (M2 markers, Figure 11).

[0216] Macrophages isolated from the LPS / PBO group were found to exhibit increased expression of M1 markers when polarized to either the M1 or M2 phenotype in vitro. In contrast, the LPS / PBO group showed decreased induction of M2 markers in response to M2 stimulation in vitro. Notably, the M0 LPS / PBO group showed no changes in the expression of M1 or M2 markers compared with controls. In conclusion, topical LPS induces peritoneal macrophages to undergo an exaggerated proinflammatory response.

[0217] These effects were reversed in LPS-treated mice treated locally with EstroGel™ or E4 formulations (AG24, AG25, AG26) (Figure 11). For example, treatment with E4 formulations completely reversed the LPS-induced increase in TNF-α expression in both M1 and M2 in vitro polarized cells. Statistically significant decreases in expression were observed after treatment with AG24, AG25, and AG26. This anti-inflammatory effect was slightly greater with AG24 (the formulation with the highest E4 concentration), especially when considered in conjunction with iNOS expression (Figure 11).

[0218] All four formulations increased the expression of the M2 markers Arg1 and Ym1 in M2-polarized cells compared to cells from LPS / PBO-treated mice (Figure 11). This effect was most pronounced in macrophages isolated from the EstroGel™-treated group, where M2 marker levels in M2-polarized cells exceeded those from control (non-LPS-treated) mice. Interestingly, of the three E4 formulations tested, AG26 (the lowest E4 concentration) produced the greatest increase in M2 marker expression, although AG25 and AG24 were also effective.

[0219] Example 5. In vivo LPS treatment protocol (delayed wound healing model): Comparison of different formulations and variation of treatment duration The in vivo LPS treatment protocol (delayed wound healing model) described in Example 4 was repeated using the following groups of animals: a double placebo group (non-treated control), LPS control (LPS, placebo treatment), LPS and topical EstroGel (estradiol gel, containing 0.06% 17β-estradiol as hemihydrate in a hydroalcoholic gel), repeated administration for 4 days; LPS and topical AG26 (0.06% E4), single dose; LPS and topical AG26 (0.06% E4), repeated administration for 4 days; LPS and topical AG28 (0.06% E4), single dose; LPS and topical AG28 (0.06% E4), repeated administration for 4 days.

[0220] On day 0 only (single dose) or on days -1, 0, 1, and 2 (repeated doses), topical EstroGel™, AG26, AG28, or matching placebo was applied in a thin layer over the top of the wounds during observation (60 μl total, 30 μl per wound).

[0221] On day 3, mice were humanely sacrificed via elevated CO2 concentration and cervical dislocation. Uteri and unwounded and wounded tissue samples were treated as described in Example 4. In contrast to EstroGel, topical application of AG26 and AG28 did not affect uterine weight (Figure 12).

[0222] Wound closure was then assessed from histological samples (FIG. 13). Tissue sections were subjected to K14 immunohistochemistry to visualize the newly forming epidermis. All three active treatments (EstroGel™, AG26, and AG28) promoted re-epithelialization when administered repeatedly for 4 days. AG28 failed to achieve any statistical significance compared with the AG28 PBO group. Interestingly, a greater magnitude of promotion and statistical significance was observed in wounds treated with either AG26 compared with Estrogel™, where re-epithelialization achieved that observed in the LPS-untreated control group.

[0223] Both AG26 and AG28 (four applications) accelerated wound healing compared to placebo in a delayed wound healing mouse model by increasing re-epithelialization (FIG. 13).

[0224] AG26 and AG28 are topical formulations that can improve wound healing after just one application without exerting systemic effects.

[0225] Example 6. Wound healing in db / db mice Eight-week-old female diabetic db / db mice are weighed and the animals are divided into six groups (six animals per experimental group): topical placebo (control), Topical EstroGel™ (estradiol gel, containing 0.06% 17β-estradiol as hemihydrate in a hydroalcoholic gel), topical AG24 (0.5% E4); topical AG25 (0.22% E4); topical AG26 (0.06% E4); Topical AG28 (0.06% E4).

[0226] Mice were anesthetized using oxygen and isoflurane (2%-2.5% isoflurane at a flow rate of 1.25-2 L depending on clinical signs), shaved, and the dorsal skin was cleaned with chlorhexidine wipes. Two 6 mm dorsal incisions were made. EstroGel™, AG24, AG25, AG26, AG28, or placebo was then applied in a thin layer over the top of the wound (60 μL total, 30 μL per wound). Buprenorphine (0.1 mg / kg) was administered via subcutaneous injection in the nape of the neck postoperatively, and each animal was imaged. Mice were allowed to recover in a warming cabinet and returned to single-housed care. Postoperative observations were performed.

[0227] Topical EstroGel™, AG24, AG25, AG26, AG28, or placebo is reapplied in a thin layer over the top of the wound each day during observation (60 μL total, 30 μL per wound). Wounds are imaged for planimetric analysis on days 1, 3, 5, 7, 9, 11, 13, and 14.

[0228] On day 14, mice were sacrificed and the wounds were bisected at their midpoints. The lower half of each wound was then processed for wax histology (placed in a cassette containing fixative). The uteri were carefully removed so that uterine weights could be documented.

[0229] It is expected that application of EstroGel™ will promote uterine weight gain and hypertrophic changes. Topical application of E4 will affect uterine weight in a dose-dependent manner, with AG26 and AG28 having no effect and AG24 causing an increase in uterine weight.

[0230] EstroGel™, AG24, AG25, AG26, and AG28 are expected to accelerate wound healing compared to placebo by increasing re-epithelialization in a mouse model of diabetic wound healing.

[0231] It can be concluded that AG26 and AG28 are topical formulations that can improve wound healing without exerting systemic effects.

[0232] Example 7. Effect of estetrol in wound-related in vitro assays In vitro assays were performed to determine the effective dose of E4 in wound-associated cell types (fibroblasts, keratinocytes, and immune cells), which will inform E4 dose selection for clinical formulation studies. Next, a mechanistic understanding of the effects of E4 on wound-associated cell function was established.

[0233] 1.0. Method 1.1. Human and mouse cells Primary human dermal fibroblasts (HDFs) were isolated from abdominal or leg skin. Primary neonatal normal human epidermal keratinocytes (NHEKs) were purchased from Lonza. Mouse dermal fibroblasts (MDFs), mouse epidermal keratinocytes (MEKs), mouse peritoneal macrophages, and mouse bone marrow were isolated from C57 / Bl6 (wt), NDb (Lepr+ / -), or Db (Lepr- / -) mice.

[0234] 1.2. Cultivation of dermal fibroblasts and epidermal keratinocytes Fibroblasts were dissociated and cultured in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin / streptomycin, and 1% amphotericin B. At least 4 days before performing the assay, cells were switched to DMEM supplemented with 5% charcoal-stripped FBS.

[0235] 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.

[0236] 1.3. Scratch Wound Assay Cells were seeded into 24-well plates and cultured to form confluent monolayers, after which a scratch was made with a 1 ml pipette tip. Wells were treated with E2, E4, and / or various estrogen receptor agonists and antagonists. Stock solutions of E2, E4, and PHTPP were dissolved in ethanol (EtOH) so that the final concentration of EtOH in 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 an untreated negative control, vehicle controls containing equivalent concentrations of EtOH and / or DMSO were included. All treatments were diluted to working concentrations in medium supplemented with 2% charcoal-stripped FBS. To visualize the scratch at a defined endpoint, cells were stained with crystal violet and imaged under a Nikon E400 bright-field microscope. Scratch closure was determined from multiple independent measurements per well.

[0237] 1.4. Cultivation of THP1 cells THP1 cells were maintained in RPMI growth medium supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin. Cells were cultured at 2 x 10 cells per ml. 5 Cells were seeded into 12- or 6-well plates at 1000 x g 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 20 ng / ml IFN-γ and 10 pg / ml LPS (E. coli) for 6 or 24 hours and collected for RNA isolation or flow cytometry.

[0238] 1.5. Isolation and culture of mouse bone marrow-derived macrophages The bones were washed with DMEM supplemented with 1% penicillin / streptomycin and 1% amphotericin B. Bone marrow cells were cultured at 1 × 10 per ml in DMEM supplemented with 10% FBS and 10% L929 culture supernatant.6 Cells were seeded into 12-well or 6-well plates at 10 cells per well to induce differentiation. After 7-10 days, differentiated macrophages were cultured in serum-free growth medium at 10 -7 M cells were treated with E2 or E4 for 16 hours and polarized to M1 type cells using 100 ng / ml IFN-γ and 1 μg / ml LPS. After 6 or 24 hours of polarization, cells were harvested for RNA isolation or flow cytometry.

[0239] 1.6. Isolation and culture of mouse peritoneal macrophages Peritoneal macrophages from C57 / B16 mice were isolated 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 the cells was removed using a needle and syringe and cultured at 1 × 10 per ml in RPMI growth medium supplemented with 10% charcoal-stripped FBS and 1% penicillin / streptomycin. 6 Cells were seeded in 12-well or 6-well plates at 10 cells / well in serum-free growth medium. -7 M cells were treated with E2 or E4 for 16 hours and polarized to M1 type cells using 100 ng / ml IFN-γ and 1 μg / ml LPS. After 6 or 24 hours of polarization, cells were harvested for RNA isolation or flow cytometry.

[0240] 1.7. RNA isolation and quantitative real-time PCR After treatment, human and mouse macrophages were collected in Trizol, and RNA was isolated using the Trizol Plus RNA Isolation Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using GoScript reverse transcriptase (Promega). Quantitative real-time PCR was performed using 2x Takyon SYBR Green master mix and a CFX Connect thermocycler. Primers for ERα and ERβ were used to examine changes in ER expression after E2 and E4 treatment of MEK and MDF. Primers for Snail, keratin 1 and fibronectin, collagen I, MMP-2, and MMP-9 were used to examine the effects of E2 and E4 on MEK differentiation and ECM protein expression by MDF. Primers for iNOS, IL-1β, and TNF-α, or Ccl17 were used to assess M1 / M2 polarization. Data were normalized using primers for GAPDH. Unless otherwise specified, relative gene expression was determined relative to the expression of the vehicle control, i.e., M0.

[0241] 1.8.Statistical analysis Statistical significance was assessed using one-way ANOVA with Tukey post-hoc analysis or paired t-tests, as appropriate.

[0242] 2.0. Keratinocytes and fibroblasts 2.1. Both E2 and E4 promote migration of human dermal fibroblasts (HDFs) A scratch assay was performed to quantify the effects of various concentrations of E2 and E4 on HDF migration (Figure 14). -8 M and 10 -7 Treatment of M with E2 resulted in significantly faster scratch closure compared to vehicle control (157% and 141%, respectively). -6 M) and two minimum concentrations of 10 -8 M and 10 -9No difference in closure was observed with E2 in M. E4 treatment also reduced the closure rate by 10 -8 M, 10 -7 M, and 10 -6 E2 and E4 both resulted in significantly faster scratch closure compared to vehicle controls (152%, 147%, and 142%, respectively). -8 M optimally stimulated scratch wound closure in fibroblasts, where a higher magnitude of stimulation was observed after E2 treatment.

[0243] Additional experiments were then performed using HDFs to a) further confirm the effects of E2 and E4 in fibroblasts from a fifth donor, and b) investigate the relative effect of including CS-FBS in the cell culture medium. Three identical sets of scratch assays (vehicle-treated and E2- or E4-treated for 10 min) were performed in DMEM containing either 2% CS-FBS, 0% FBS, or 2% FBS (no CS). -7 A comparison of E2 and E4 (compared with M) was performed. Interestingly, in all three cases, both E2 and E4 tended to promote scratch closure, with the greatest promotion observed after E4 treatment (Figure 15). As expected, the FBS (no CS) group showed faster closure across all treatments, but the relative effects of E2 and E4 were less pronounced in this group.

[0244] 2.2. Both E2 and E4 promote the migration of mouse dermal fibroblasts (MDFs) To further demonstrate the beneficial effects of E4 and support dosing considerations for future in vivo mouse model studies, we next performed an in vitro scratch assay in mouse dermal fibroblasts (MDFs). The biological effects of estrogenic compounds have been extensively demonstrated in mouse models, and switching to cells isolated from an inbred mouse strain was predicted to reduce the variability of the model compared to using cells isolated from human donors.

[0245] Similar to HDFs, treatment of MDFs with E2 or E4 significantly increased scratch closure compared with vehicle control treatment (Figures 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 broader (significant at three concentrations for E4, compared with two concentrations for E2, Figure 16). Adding data from two additional mice and comparing low-passage and high-passage cells revealed a slightly larger magnitude effect for E4 than E2 (Figure 17). Furthermore, the beneficial effects of both E2 and E4 were greatest in high-passage cells, consistent with clinical expectations.

[0246] 2.3. E2 and E4 treatments increase ER expression in fibroblasts, while E4 promotes fibronectin expression and inhibits MMP activity 10 in vitro cultured mouse fibroblasts -7HDFs were treated with either E2, E4, DPN, or PPT. As previously reported, E2 treatment upregulated the expression of both ERα and ERβ, whereas treatment with ER agonists preferentially increased the expression of these respective receptors (e.g., PPT increased ERα expression, and DPN increased ERβ expression; Figure 18). All treatments also showed a strong tendency toward increased MMP2 and MMP9 expression (Figure 18). Next, zymography was performed on cell supernatants from HDFs treated with either E2 or E4 to assess the effects of E2 and E4 on cell-derived MMP activity. Across cells from three independent donors, both E2 and E4 significantly reduced MMP2 activity compared with controls (Figure 19). The magnitude (and statistical significance) of the effect was greater in cells treated with E4. MMP9 activity was not detected in any of the treatment groups. Finally, we investigated the effects of E2 and E4 on the expression of extracellular matrix genes, specifically collagen 1 (Cola1) and fibronectin (Fn1), in mouse fibroblasts (Figure 20). No effect was observed in cells derived from wild-type mice after treatment. However, a trend toward increased expression of both Colal and Fn1 was observed in cells derived from diabetic (db / db) mice treated with either E2 or E4 (Figure 20). This increase in expression reached statistical significance for fibronectin (Fn1) only in E4-treated db / db-derived cells.

[0247] 2.4. E2 and E4 promote epidermal keratinocyte migration and regulate wound-associated epidermal gene expression The effects of E2 and E4 on keratinocyte migration were evaluated using primary normal human epidermal keratinocytes (NHEK). Experiments were performed in various growth media (with various concentrations of human keratinocyte growth supplement (HKGS)). Data are shown for cells cultured in both 15% and 30% HKGS supplements (Figure 21). In both conditions, 10 -7 M / 10 -8The M ranges E2 and E4 showed a strong tendency for faster wound closure. Interestingly, this was not the case for the 10% HKGS in 15% HKGS. -7 M E2 and 10 -7 Statistical significance was reached only for E4 of M, where E4 showed a slightly larger effect and higher statistical significance.

[0248] Next, we switched to primary mouse epidermal keratinocytes (MEK) and investigated the effects of E2 and E4 on MEK wound-related gene expression. Although not statistically significant, both E2 and E4 (especially 10 -7 A strong tendency for induction of both ERα and ERβ was also observed in cells treated with E2 (Figure 22). Consistent with the documented beneficial effects of E2, -7 A statistically significant induction of the EMT marker Snail was observed after treatment of M with either E2 or E4. In contrast, the differentiation marker keratin 1 (Krt1) showed a tendency to be downregulated by E2 and E4 treatment. Collectively, these data suggest a switch to a less differentiated, prohealing phenotype after treatment with either E2 or E4. Overall, the observed effects of E2 and E4 on gene expression were reversed by combined treatment with the ER antagonist ICI.

[0249] 2.5. E2 and E4 exhibit anti-inflammatory activity in vitro The relative anti-inflammatory effects of both E2 and E4 were evaluated in vitro. Initially, 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 subsequently polarized to either an M1 or M2 phenotype (Figure 23). Successful polarization was confirmed by profiling the expression of M1 markers (TNF-α) and M2 markers (CCL17). The effect of combined treatment with various concentrations of E2 or E4 was evaluated. Here, treatment with either E2 or E4 strongly tended to decrease TNF-α expression in M1-polarized cells and increase CCL17 expression in M2-polarized cells (Figure 23).

[0250] In follow-up 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 hours) and treated with the optimal concentration (10 -7 M1 cells were co-treated with either E2 or E4 (Figure 24). In the presence of both E2 and E4, there was a trend toward decreased expression of various M1 markers (iNOS, IL1-β, and TNF-α) compared with vehicle treatment. Similar effects were observed in cells polarized for either 6 or 24 hours (Figure 24). Next, we undertook experiments using mouse bone marrow-derived monocytes differentiated into macrophages with 30 ng / ml MCSF rather than L929 culture supernatant. MCSF-stimulated macrophages showed much higher levels of M1 marker expression when polarized to M1 (compared to M0). Again, various M1 markers (iNOS, IL1-β, and TNF-α) showed a strong trend toward decreased expression in the presence of both E2 and E4 compared with vehicle treatment (Figure 25). Of note, both E2 and E4 resulted in a statistically significant decrease in the M1 marker iNOS in MSCF-differentiated BMDMs. In general, the observed anti-inflammatory effect of E4 was slightly greater than that of E2.

[0251] Finally, the anti-inflammatory effects of E2 and E4 were evaluated in freshly isolated mouse peritoneal macrophages. Unlike the BMDM protocol, already differentiated peritoneal macrophages were immediately treated with E2 (10 -7 M) or E4(10 -7 M1 cells were pretreated with E2 and E4, followed by M1 polarization using IFN-γ and LPS for 6 hours. Peritoneal macrophages also showed a strong tendency toward decreased expression of M1 markers (iNOS, IL1-β, and TNF-α) in the presence of both E2 and E4 compared with vehicle treatment (Figure 26). Of note, the overall level of marker expression was higher in these cells. The E4-mediated reduction in IL1-β reached statistical significance. The observed anti-inflammatory effect of E4 was similar to that observed with E2.

[0252] 2.6. Preliminary evaluation of ER-specific effects on fibroblasts and immune cells The relative ER-specific effects of E4 were assessed in vitro using combination treatment with highly specific antagonists of ERα (MPP) or ERβ (PHTPP). Preliminary studies suggest that the effects of E4 on both HDF migration (Figure 27) and BMDM polarization (Figure 28) may be mediated by ERα. Specifically, combination treatment with the ERα antagonist MPP appeared to prevent E4-induced scratch closure and E4-mediated IL1-β reduction, whereas the same ERα antagonist had limited impact on the effects of E2. Given the relatively high variability, these studies should be repeated with increased numbers of replicates to confirm the observed effects.

[0253] 2.7. Preliminary evaluation of the activity of the formulation in vitro To provide initial confidence in the wound healing capabilities of the formulated gel, in vitro evaluation of EstroGel™ (EG, Figure 29) was performed alongside AG23 active (ACT) and placebo (PBO) gel formulations. Again, E2 and E4 treatment significantly suppressed the M1 cell phenotype, as did treatment with Estrogel™ containing E2. 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.

[0254] 3.0. Summary of Results Experiments were designed to investigate the relative effects of E4 on specific cellular aspects of wound healing. A series of in vitro studies were undertaken using fibroblasts, keratinocytes, and immune cells. Importantly, these in vitro studies were performed in both human and mouse cells, combining clinical relevance in human cells with cross-species validation in mouse cells to support subsequent in vivo studies.

[0255] Fibroblasts. E4 was found to promote the migration of both human (HDF) and mouse (MDF) dermal fibroblasts. In both cases, the optimal concentration of E4 was 10 -7 M to 10 -8 The optimal concentration of E2 was approximately the same as that of E2. It is noteworthy that the relative efficacy of E2 and E4 was similar in both cases. In MDS, the effect on migration was reported to be greater in high-passage cells, which may mimic the chronic wound environment. In MDF, treatment with either E2 or E4 directly increased cellular expression of ERα and ERβ. In HDSF, E4 treatment inhibited MMP2 activity in the cell supernatant, and appeared to inhibit it to a greater extent than E2. Preliminary evaluation suggests that ERα is important in mediating the effect of E4 on HDF migration.

[0256] Keratinocytes. Mouse epidermal keratinocytes (MEK) also closed scratch wounds more quickly when treated with either E4 or E2. Again, the optimal concentrations for both E2 and E4 were similar (10 -7 M), where the observed effects were dependent on the composition of the cell growth medium. Similar to 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 found to induce expression of the EMT marker Snail and inhibit the differentiation marker keratin 1, both of which are hallmarks of a pro-healing cellular response.

[0257] Anti-inflammatory effects. E4 and E2 were found to exert pro-healing anti-inflammatory effects on mouse bone marrow-derived macrophages (BMDMs), mouse peritoneal macrophages, and human THP-1 cells. In THP-1 cells, E4 reduced the expression of TNF-α (an M1 marker) in M1-polarized cells and increased the expression of CCL17 (an M2 marker) in M2-polarized cells. In both mouse BMDMs and peritoneal macrophages, E4 consistently reduced the expression of a panel of M1 markers in M1-polarized cells. This effect was demonstrated both 6 and 24 hours after polarization and using cells derived from three independent differentiation methods: L929 GM or MCSF (BMDMs), or in vivo (isolated peritoneal macrophages). In all cases, the magnitude of the effect observed with E4 was roughly consistent with that observed with E2. Preliminary evaluation suggests that ERα is important in mediating the anti-inflammatory effects of E4 on BMDMs. A pilot study revealed that the expression of M1 markers was reduced after treatment with the E4 formulation AG23.

[0258] 4.0.References Campbell L, Emmerson E, Davies F, et al. Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities. J Exp Med. 2010;207(9):1825-1833. doi:10.1084 / jem.20100500 Collaborative Group on Hormonal Factors in Breast Cancer. Type and timing of menopausal hormone therapy and breast cancer risk: individual participant meta-analysis of the worldwide epidemiological evidence. Lancet. 2019;394(10204):1159-1168. doi:10.1016 / S0140-6736(19)31709-X Roth GS, Harman SM, Lamberg SI. Altered Ovarian Regulation of Wound Healing during Aging. Proceedings of the Society for Experimental Biology and Medicine. 1981;166(1):17-23. doi:10.3181 / 00379727-166-41017 Thornton MJ. Estrogens and aging skin. Dermatoendocrinol. 2013;5(2):264-270. doi:10.4161 / derm.23872

[0259] Example 8. Preparation of aqueous gel formulations for stability studies Table 8 lists the hydrogels prepared for the stability study. Preparation was similar to that described for AG24 in Example 1.

[0260]

Table 8

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), and most preferably from 0.3% to 0.7% (w / w) of an estetrol component.

2. 10. The composition of claim 1, further comprising a penetration enhancer that enables penetration through the stratum corneum of the subject's skin.

3. A hydrogel formulation comprising the composition of 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, composition used, hydrogel or hydrogel used according to any one of claims 1 to 4, which is a formulation selected from the group consisting of emulsions, suspensions, ointments, pastes, lotions, gels, foams, mousses and creams.

6. 6. The composition, composition used, hydrogel or hydrogel used 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 penetration enhancer comprises a solvent and a substance or molecule that allows penetration through the stratum corneum, preferably the penetration enhancer molecule is selected from the group consisting of ethanol, ethers such as diethylene glycol monoethyl ether (Transcutol™), benzyl alcohol, fatty acids and their esters, or any combination thereof, or 7. The composition, composition used, hydrogel, or hydrogel used of claim 6, wherein the penetration enhancer comprises a solvent comprising one or more of polyethylene glycol (PEG), propylene glycol (PG), or a combination thereof.

8. 8. The composition, composition used, hydrogel, or hydrogel used of claim 7, wherein the 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 group consisting of PEG200, PEG300, PEG400, PEG500, PEG600, or any combination thereof.

9. 9. The composition, composition used, hydrogel or hydrogel used 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 comprising a thickener in an amount of 0.5% to 3% (w / w).

10. The composition, composition used, hydrogel or hydrogel used according to any one of claims 1 to 9, wherein the thickening agent is selected from the group consisting of hydroxyethyl cellulose (HEC), carboxymethyl cellulose (CMC), a high molecular weight cross-linked acrylic polymer, a non-ionic triblock copolymer, or any combination thereof, and preferably the high molecular weight cross-linked acrylic polymer is Carbopol™, or the HEC is HEC250 HHX, or the non-ionic triblock copolymer has an approximate molecular weight between about 1800 and about 4000 and a polyoxyethylene content of about 70% to about 80%.

11. 11. The composition, composition used, hydrogel or hydrogel used 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 said preservative being 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.

12. 12. The composition, composition used, hydrogel or hydrogel used 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 said emollient being selected from the group consisting of glycerol, acetyl alcohol, stearyl alcohol, stearic acid, isopropyl palmitate, squalene, lanolin, glycerin, petrolatum, mineral oil, and any combination thereof.

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

14. 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 comprising a penetration enhancer molecule and a solvent or solvent system; about 0.3% to about 3% (w / w) of a thickening agent; optionally containing a preservative and / or an emollient; containing up to 100% (w / w) water, or about 38% to about 45% (wt / wt) PEG 400; about 0.1% to about 1% (w / w) Carbopol™; and The composition, composition used, hydrogel or hydrogel used according to any one of claims 1 to 4, comprising from about 0.8% to about 3% (w / w) of Transcutol™.

15. 15. The composition, composition used, hydrogel or hydrogel used according to any one of claims 1 to 14 for use in the treatment of acute wounds, surgical wounds, wounds caused by acute trauma, chronic wounds, wounds caused and / or maintained by diabetic disease, wounds in subjects with impaired wound healing including delayed cutaneous wound healing disorders 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, composition for use, hydrogel or hydrogel for use according to any one of claims 1 to 14, which is contained in a wound dressing, bandage, band-aid, patch or plaster.

17. 15. A package containing one or more dosage units of the composition hydrogel according to any one of claims 1 to 14, preferably said packaging unit being a box, display unit, ampoule, bottle, vial, tube, syringe, cartridge, bag, sachet, pouch, film, laminate, foil, can, cylinder or pressurized container.

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