Enhanced dermal penetration of lipophilic adapalene esters

Abstract

Methods for enhanced dermal delivery of adapalene esters to treat skin conditions including acne, rosacea, and photoaging are described. The invention is based on the discovery that applying lipophilic adapalene esters to non-disinfected skin and / or with preserved natural microflora results in significantly enhanced penetration and therapeutic and non-therapeutic efficacy compared to conventional protocols involving preliminary skin disinfection. The methods avoid antimicrobial pretreatments, preserve beneficial skin microorganisms, and achieve high absorption and penetration of the adapalene ester while maintaining safety and reducing treatment complexity.

Description

3518179.0020 ENHANCED DERMAL PENETRATION OF LIPOPHILIC ADAPALENE ESTERS FIELD OF THE INVENTION

[0001] The present invention relates to methods and compositions for enhanced dermal delivery of lipophilic naphthoic acid retinoid compounds, specifically adapalene esters, and their use in treating skin conditions including acne, rosacea, and photoaging. BACKGROUND OF THE INVENTION

[0002] Visible skin damage, particularly on the face, is one of the most common targets of dermatological and cosmetic treatments. Untreated facial skin damage may cause anxiety and psycho-emotional trauma. Facial skin damage is often caused by photoaging, and may present itself as actinic keratosis, solar lentigines, loss of underlying skin collagen, loss of elastin, wrinkles and sagging skin. At an early stage, photoaging may present as inflamed erythema as well as enlarged freckles. Other facial skin damage may be caused by acne, resulting in both inflamed pores and post-acne scarring.

[0003] Topical retinoids are commonly used to treat skin damage cause by photoaging and acne in both cosmetic and prescription drug products.

[0004] Retinoids, particularly third-generation retinoids such as adapalene, are widely recognized as first-line treatments for acne vulgaris, rosacea, and photoaging. Adapalene (6- (3-(1-adamantyl)-4-methoxyphenyl)-2-naphthoic acid) belongs to the naphthoic acid class of retinoids and offers both cosmetic and therapeutic benefits with reduced irritation compared to first and second-generation retinoids.

[0005] However, adapalene in its free acid form is a crystalline solid and presents significant formulation challenges due to its poor solubility in both aqueous and lipophilic phases, being insoluble in water and having very limited solubility in standard cosmetic ingredients. Recent developments have addressed this limitation through the creation of lipophilic adapalene esters, as described in US Patent Publication No.2022 / 0211597A1, which discloses C6-C22esters of adapalene that are soluble in common topical oil-based carriers and is incorporated herein in its entirety.

[0006] The human skin microbiome consists of diverse microbial communities of commensal bacteria. Depending on the subject and environment, healthy facial skin can harbor 1 x 104colony-forming units per square centimeter (CFU / cm2) or more. The skin microbiome includes beneficial bacteria such as Staphylococcus epidermidis, CORE / 3518179.0020 / 232318616.1 13518179.0020 Propionibacterium acnes (also called Cutibacterium acnes), and various other commensals that contribute to skin barrier function and homeostasis.

[0007] Current dermatological and cosmetic treatment protocols for acne, rosacea, and related skin conditions typically follow a standardized approach that begins with skin disinfection prior to application of the retinoid compound. This preliminary step involves: 1. Cleansing with surfactants: Application of surface-active agents to remove sebum, debris, and reduce the count of microorganisms; 2. Alcohol or glycol-based toners: Use of these materials reduce the skin’s bacterial load via membrane disruptors and lowering the water-activity of the skin; and / or 3. Antimicrobial treatments: Application of targeted antibiotics or antimicrobials.

[0008] This conventional approach is based on the widely accepted principle that reducing microbial load enhances the efficacy of subsequent treatments. The goal of disinfection is typically to reduce the colony-forming units (CFU) of microorganisms on the skin surface to minimal levels, with often 90% reduction in colony forming units. Furthermore, disinfection of skin disrupts the skin barrier, and it is believed that this disruption allows for greater rates of diffusion of subsequent topically applied retinoids.

[0009] Despite the widespread adoption of disinfection-first protocols, several limitations have been observed, including compromising the skin's natural barrier function, eliminating beneficial bacteria that support skin health and development of bacterial resistance.

[0010] The therapeutic efficacy of topically-applied active ingredients such as retinoids is dependent on absorption and penetration of the active ingredient into the skin. There remains a need for improved methods of delivering retinoid compounds that maximize therapeutic efficacy while minimizing adverse effects and preserving skin microbiome integrity. SUMMARY OF THE INVENTION

[0011] The present invention is based on the surprising and unexpected discovery that lipophilic adapalene esters achieve superior dermal penetration and bioavailability when applied to non-disinfected skin and / or skin that that otherwise exhibits a higher microbial load of dermal commensal bacteria, compared to disinfected skin and conventional protocols involving preliminary disinfection steps that reduce the concentration of dermal commensal bacteria. CORE / 3518179.0020 / 232318616.1 23518179.0020

[0012] In various aspects, the present invention relates to methods of inhibiting or reducing the signs of acne, rosacea, and photoaging in a subject or inhibiting or reducing wrinkles and sagging facial skin of a subject.

[0013] The present invention is directed to a method of treating the skin of a subject. The method comprises topically applying to the skin of the subject a composition comprising an adapalene ester of Formula I:Formula I wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms.

[0014] In accordance with some embodiments, the skin of the subject to which the composition is applied is non-disinfected skin.

[0015] In accordance with some embodiments, the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2.

[0016] The present is further directed to a composition comprising an adapalene ester of Formula I for use in a method of treating acne or rosacea in a subject in need thereof, the method comprising administering the composition topically to the skin of the subject, and the skin of the subject to which the composition is applied is non-disinfected skin.

[0017] The present is further directed to a composition comprising an adapalene ester of Formula I for use in a method of treating acne or rosacea in a subject in need thereof, the method comprising administering the composition topically to the skin of the subject, and the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2.

[0018] The details of various embodiments of the invention are set forth in the description below. CORE / 3518179.0020 / 232318616.1 33518179.0020

[0019] Other objects and features will be in part apparent and in part pointed out hereinafter. BREIF DESCRIPTION OF THE DRAWINGS

[0020] FIG.1 graphically depicts the in vivo measurements of the total quantity of adapalene ester absorbed topically on undisinfected skin reported in Table 2a of Example 1.

[0021] FIG.2 graphically depicts the in vivo measurements of the total quantity of adapalene ester absorbed topically on disinfected skin reported in Table 2b of Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention relates to the surprising discovery that application of adapalene esters to non-disinfected skin and / or skin that that otherwise exhibits a higher microbial load of commensal bacteria results in enhanced penetration rate, depth of penetration, and overall bioavailability of the adapalene ester as compared to disinfected skin and conventional protocols involving preliminary disinfection steps that reduce the concentration of commensal bacteria. As a result, therapeutic response may be accelerated due to enhanced delivery and bioavailability.

[0023] The present discovery is contrary to the current state-of-art knowledge of transdermal and percutaneous absorption of drug and drug-like compounds. Normally, increased dermal delivery is achieved by disrupting the skin barrier with disinfectant agents like surfactants, alcohols, and glycols.

[0024] In accordance with one embodiment, the adapalene ester is applied to skin surfaces containing at least about 1 x 103or more colony-forming units per square centimeter (CFU / cm2) of naturally occurring commensal bacteria. As used herein, dermal commensal bacteria include the indigenous microbial communities normally present on healthy human skin, including but not limited to Staphylococcus epidermidis, Cutibacterium acnes, Malassezia species, and other commensals. The concentration or loading or commensal bacteria on skin can be readily determined as described in Example 1 as well as other methods known in the art. CORE / 3518179.0020 / 232318616.1 43518179.0020 Adapalene Ester Compounds

[0025] The adapalene esters useful in the present invention are lipophilic derivatives of adapalene. These third generation retinoids include esters of adapalene with C6-C22 alcohols. The esters of adapalene include those of Formula I:wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms. The hydrocarbyl can be branched or linear, saturated or unsaturated. In various embodiments, R1is a saturated or unsaturated, linear or branched, aliphatic hydrocarbyl group. Preferably, R1 is a substituted or unsubstituted branched or linear alkyl or alkenyl having 6 or more carbon atoms, such as C6-C22, C6-C18, C8-C16, or C12-C14carbon chain. When R1 is alkenyl, the substituent has at least one double bond, but can optionally have two or more double bonds. Examples of acceptable hydrocarbyl groups (R1) include C6-C22alkyl or alkenyl groups. Among them C12 and C16 alkyl, C18-1 alkenyl, and C22-1 alkenyl are preferable. Diesters of Formula I are also envisioned in which R1is derived from a diol and is substituted or unsubstituted hydrocarbyl as described above having three, four, five, six or more carbon atoms. The alkyl ester chain or diester chain is preferably one that that is soluble in an oil phase and is mutually soluble with lipophilic emollients typically used in topical skin products. The adapalene esters may be in the form of an amorphous solid (i.e., non-crystalline solid).

[0026] In one preferred embodiment, the adapalene ester is oleyl adapalenate wherein R1is an oleyl group (also referred to as C18-1), a C18linear alkenyl according to Formula II: CORE / 3518179.0020 / 232318616.1 53518179.0020, to Formula III:

[0028] Thus, in accordance with one embodiment, the composition applied to the skin of the subject comprises oleyl adapalenate. In another embodiment, the composition applied to the skin of the subject comprises erucyl adapalenate.

[0029] Esterification of the adapalene can be accomplished by the following process. The adapalene, which is a carboxylic acid, can be added in stoichiometric quantities to the long chain alcohol in a solvent. The choice of solvent can include using excess of the long chain alcohol. Esterification catalysts, such as sulfonic acids, organo-tin, or organic-titanium catalysts can be used to increase the rate of reaction, while the water of reaction can be removed by a reflux condenser. Downstream purification by column chromatography, distillation, solvent washing, and re-crystallization may be used to further purify the adapalene ester compound.

[0030] One preferred method of producing adapalene esters is via transesterification, with the starting material being methyl adapalenate, the methyl ester of adapalene. This process is similar to the esterification process described above. However, the transesterification releases methanol, as opposed to water, and methanol is removed from reaction by evaporation. The long chain alcohol replaces the methanol in the ester group. CORE / 3518179.0020 / 232318616.1 63518179.0020

[0031] Diesters of adapalene can be similarly prepared by reacting one mole of a diol with two moles of adapalene. Examples of suitable diols include propylene glycol, butylene glycol and neopentyl glycol. Compositions

[0032] The adapalene ester can be formulated in a premix composition with a non-polar lipophilic carrier such as cosmetically or pharmaceutically acceptable oils and emollients. Suitable non-polar lipophilic carriers include but are not limited to emollient esters, triglycerides, vegetable oils, polyesters, polyol esters, wax esters such as jojoba oil, guerbet esters, fatty alcohol, vegetable waxes, hydrocarbon fluids such as mineral oils, petrolatum, alkanes and isoalkanes, silicones, and other topical lipophilic emollients known in the art. Typically, the adapalene ester is soluble in the lipophilic carrier at a minimum of about 5%, about 6%, about 7%, about 8%, about 9%, about 10% or more by weight. Typically, the concentration of the adapalene ester in a premix composition will be at least about 10% by weight, and the weight ratio of adapalene ester to lipophilic carrier is from about 1:9 to about 3:7. The premix composition may be in the form of a homogeneous liquid solution or single phase amorphous solid.

[0033] Topical, external compositions for use in the present invention typically comprise from about 0.01% to about 2% by weight or from about 0.1% to about 2% by weight of one or more adapalene esters of Formula I dissolved or dispersed in oils and emollients, typically referred to as an oil phase, where the oil phase is further dispersed in a physiologically acceptable topical formulation, such as water-in-oil or oil-in-water emulsion, or a suspension of oil droplets within aqueous matrix such as a gel. The topical compositions can be readily formulated from the premix composition described above by mixing the premix composition with other cosmetically or pharmaceutically acceptable ingredients.

[0034] The lipophilic adapalene ester herein may be combined in an external composition with other ingredients intended to inhibit photoaging and acne, or to reduce aging spots, discoloration spots, redness, blemishes, fine lines, and wrinkles. Examples of such ingredients include, but are not restricted to plant-derived exosomes, ascorbic acid and derivatives thereof, ferulic acid, azelaic acid, kojic acid, mandelic acid, alpha-hydroxy acids, beta-hydroxy acids, lipohydroxyacids, fruit acids, gluconolactone, heparan sulfate, arbutin, niacinamide, resveratrol, hydroquinone, exfoliants, keratolytics, plant extracts, marine extracts, ferment extracts, isoflavones, bisabolol, anti-aging peptides, retinol, retinoic acid, retinyl esters, retinoic acid esters, and other retinoids. CORE / 3518179.0020 / 232318616.1 73518179.0020 Methods of Treatment

[0035] The present invention provides methods of treating the skin of a subject comprising applying to the skin of the subject a composition comprising an adapalene ester of Formula I. In some embodiments, the method is a therapeutic method and involves treating a skin condition in a subject in need thereof. The terms “treat”, “treating” and “treatment” in the context of the present invention refer to methods taken to prevent, cure, delay, reduce the severity of or ameliorate one or more symptoms of the skin condition, and encompass inhibiting or reducing signs of the skin condition. In some embodiments, the skin condition is acne, for example, acne vulgaris. The treatment of acne with the composition of the invention is considered to be therapeutic because it may reduce of the risk of long-term complications of acne, such as scarring, hyperpigmentation and post-inflammatory erythema. In some embodiments, the skin condition is rosacea. Rosacea is a common, chronic skin condition that mainly affects the face, causing redness, flushing, visible blood vessels, and sometimes pimples or bumps. The treatment of rosacea with the composition of the invention is considered to be therapeutic because it may reduce the risk of skin thickening and persistent inflammation. Without being bound by any such theory, the adapalene structure is believed to inhibit lipoxygenase enzymes inherent on skin. These enzymes convert facial oils to inflammatory cytokines which begins a cascade of inflammatory response. This lipoxygenase inhibition by third generation retinoids is useful for ameliorating the redness of inflammatory acne and rosacea.

[0036] In other embodiments, the method is a non-therapeutic method and involves treating the skin of a healthy subject. For example, in some embodiments, the composition of the invention also provides a cosmetically useful, non-therapeutic reduction in redness in those without acne, rosacea or other skin disease condition. In some embodiments, the method comprises reducing wrinkled or sagging facial skin. In other such embodiments, the method comprises inhibiting or reducing the signs of photoaging. Such treatments are considered non-therapeutic (cosmetic) because they are not the result of an underlying pathology (i.e. they occur in healthy subjects) and the subject is not at risk of developing a pathological state in the absence of treatment.

[0037] Wrinkled and sagging facial skin often occurs as a natural result of aging. Moreover, glucagon-like peptide-1 (GLP-1) receptor agonists such as semaglutide are used extensively in weight loss treatment. In addition to weight loss, subjects may also experience an undesirable increase in wrinkles and sagging skin, particularly in facial areas. In CORE / 3518179.0020 / 232318616.1 83518179.0020 accordance with one embodiment, the non-therapeutic method described herein is employed for inhibiting or reducing wrinkles and sagging facial skin of a subject.

[0038] Typically, the skin of the subject (in both therapeutic and non-therapeutic methods) is treated in accordance with the present invention once to twice daily or more, preferably in the evening. Treatment areas include any area of the skin exhibiting the signs of the condition to be treated, including but not limited to face, neck, chest, back, or other affected areas.

[0039] In accordance with one embodiment of the present invention, the skin of the subject to which the composition is applied has not been subjected to pretreatment disinfection. Pretreatment disinfection includes antimicrobial treatments, alcohol-based toners, soaps, antibacterial cleansers, or any other microorganism-reducing treatments. Following any such microorganism-reducing treatments, and in the absence of maintaining the skin in a sterile environment, the population or microbial load of commensal bacteria on human skin will recover relatively quickly in the natural environment such that the skin is no longer disinfected. Accordingly, in accordance with these and other embodiments, pretreatment disinfection to reduce the microbial load on the skin to be treated is not conducted at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours or longer prior to application of the composition comprising an adapalene ester of Formula I to the skin of the subject.

[0040] As used herein, “non-disinfected skin” refers to skin that has not been subjected to such pretreatment disinfection within the time periods as described above.

[0041] The microbial load of commensal bacteria on non-disinfected skin can vary significantly depending on various factors including the subject’s environment and activities, but typically is at least about 1 x 103CFU / cm2. Furthermore, depending on a variety of factors, the concentration of commensal bacteria on human skin can vary substantially from one subject to another and may remain elevated even immediately after disinfection treatment. Accordingly, in accordance with another embodiment of the of the present invention, the therapeutic and non-therapeutic methods described herein comprises application of a composition comprising an adapalene ester of Formula I to skin of the subject having a concentration of dermal commensal bacteria of at least about 1 x 103CFU / cm2, at least about 2 x 103CFU / cm2, at least about 3 x 103CFU / cm2, at least about 4 x 103CFU / cm2, at least about 5 x 103CFU / cm2, at least about 6 x 103CFU / cm2, at least about 7 x 103CFU / cm2, at least about 8 x 103CFU / cm2, at least about 9 x 103CFU / cm2, at least about 1 x 104CFU / cm2, at least about 1.3 x 104CFU / cm2, at least about 1.5 x 104CORE / 3518179.0020 / 232318616.1 93518179.0020 CFU / cm2, at least about 1.8 x 104CFU / cm2, at least about 2 x 104CFU / cm2, at least about 2.5 x 104CFU / cm2, at least about 3 x 104CFU / cm2, at least about 3.5 x 104CFU / cm2, at least about 4 x 104CFU / cm2, at least about 4.5 x 104CFU / cm2, at least about 5 x 104CFU / cm2, at least about 5.5 x 104CFU / cm2, at least about 6 x 104CFU / cm2, at least about 6.5 x 104CFU / cm2, at least about 7 x 104CFU / cm2, at least about 7.5 x 104CFU / cm2, at least about 8 x 104CFU / cm2, at least about 8.5 x 104CFU / cm2, at least about 9 x 104CFU / cm2, at least about 9.5 x 104, at least about 1 x 105CFU / cm2or higher. In accordance with these and other embodiments, the skin to which the composition comprising the adapalene ester is applied may be disinfected or non-disinfected.

[0042] The present invention is further directed to treatment methods in which the microbial load of commensal bacteria on the skin of the subject to be treated is purposely substantially maintained or even increased prior to application of the of the composition comprising an adapalene ester of Formula I.

[0043] Make-up (foundation, mascara, etc.) and other cosmetics (e.g., moisturizers, perfumes, etc.) previously applied to a subject’s skin may interfere with absorption and penetration of the adapalene ester of Formula I into the skin to be treated in accordance with the present invention. In order to enhance the effectiveness of the treatment methods disclosed herein, the method may further comprise preparing the skin of the subject for application of the composition comprising an adapalene ester of Formula I in a manner that maintains or even increases the microbial load of commensal bacteria on the skin of the subject.

[0044] For example, in one embodiment, the skin of the subject is prepared by wiping the skin of the subject to be treated with a non-aseptic wipe (e.g., a towel or washcloth) that may be dry or wetted with water to remove cosmetics and other substances that might interfere with absorption and penetration of the adapalene ester into the skin to be treated. By using a non-aseptic wipe to prepare the skin of the subject, the microbial load of commensal bacteria is substantially maintained or even increased.

[0045] In another embodiment, the skin of the subject is prepared for application of the composition comprising an adapalene ester of Formula I by contacting the skin of the subject to be treated with water to remove cosmetics and other substances that might interfere with absorption and penetration of the adapalene ester into the skin to be treated. The skin is then dried in a non-sterile environment, for example, in ambient air and / or by wiping the skin of the subject to be treated with a dry, non-aseptic wipe. CORE / 3518179.0020 / 232318616.1 103518179.0020

[0046] By preparing the skin of the subject to be treated in a non-sterile environment and or by using a non-aseptic wipe, and not subjecting the skin of the subject to which the composition is applied to pretreatment disinfection, the microbial load of commensal bacteria is advantageously substantially maintained or even increased.

[0047] The treatment methods described herein provide several advantages over known therapeutic and non-therapeutic methods employing adapalene and similar naphthoic acid class of retinoids, including: enhanced penetration of the adapalene ester active and improved bioavailability in target tissue layers; more rapid therapeutic response due to enhanced delivery; microbiome preservation and improved maintenance of beneficial skin microorganisms that support barrier function; and simplified treatment protocols and fewer treatment steps resulting in less cumulative skin irritation and reduced overall treatment costs. Embodiments of the Invention

[0048] Embodiment 1 is a method of treating the skin of a subject, the method comprising topically applying to the skin of the subject a composition comprising an adapalene ester of Formula I:wherein R1 is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the skin of the subject to which the composition is applied is non-disinfected skin.

[0049] Embodiment 2 is the method according to Embodiment 1, wherein pretreatment disinfection to reduce the microbial load on the skin to be treated is not conducted at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours or longer prior to application of the composition comprising an adapalene ester of Formula I to the skin of the subject.

[0050] Embodiment 3 is a method of treating the skin of a subject, the method comprising applying to the skin of the subject a composition comprising an adapalene ester of Formula I: CORE / 3518179.0020 / 232318616.1 113518179.0020 wherein R1isor two carbon atoms and wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2.

[0051] Embodiment 4 is the method according to any one of Embodiments 1 to 3, wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 2 x 103CFU / cm2, at least about 3 x 103CFU / cm2, at least about 4 x 103CFU / cm2, at least about 5 x 103CFU / cm2, at least about 6 x 103CFU / cm2, at least about 7 x 103CFU / cm2, at least about 8 x 103CFU / cm2, at least about 9 x 103CFU / cm2, at least about 1 x 104CFU / cm2, at least about 1.3 x 104CFU / cm2, at least about 1.5 x 104CFU / cm2, at least about 1.8 x 104CFU / cm2, at least about 2 x 104CFU / cm2, at least about 2.5 x 104CFU / cm2, at least about 3 x 104CFU / cm2, at least about 3.5 x 104CFU / cm2, at least about 4 x 104CFU / cm2, at least about 4.5 x 104CFU / cm2, at least about 5 x 104CFU / cm2, at least about 5.5 x 104CFU / cm2, at least about 6 x 104CFU / cm2, at least about 6.5 x 104CFU / cm2, at least about 7 x 104CFU / cm2, at least about 7.5 x 104CFU / cm2, at least about 8 x 104CFU / cm2, at least about 8.5 x 104CFU / cm2, at least about 9 x 104CFU / cm2, at least about 9.5 x 104, at least about 1 x 105CFU / cm2or higher.

[0052] Embodiment 5 is the method according to any one of Embodiments 1 to 4, wherein R1is a saturated or unsaturated, linear or branched, aliphatic hydrocarbyl group.

[0053] Embodiment 6 is the method according to any one of Embodiments 1 to 5, wherein R1is unsubstituted linear or branched alkyl or alkenyl.

[0054] Embodiment 7 is the method according to any one of Embodiments 1 to 6, wherein R1is C8, C12, or C16alkyl.

[0055] Embodiment 8 is the method according to any one of Embodiments 1 to 6, wherein R1is a linear eighteen or twenty-two carbon alkenyl.

[0056] Embodiment 9 is the method according to any one of Embodiments 1 to 6, wherein R1is an oleyl group according to Formula II: CORE / 3518179.0020 / 232318616.1 123518179.0020to 9, wherein the composition comprises a lipophilic carrier.

[0058] Embodiment 11 is the method according to any one of Embodiments 1 to 10, wherein the composition comprises one or more additional ingredients selected from the group consisting of plant-derived exosomes, ascorbic acid and derivatives thereof, ferulic acid, azelaic acid, kojic acid, mandelic acid, alpha-hydroxy acids, beta-hydroxy acids, lipohydroxyacids, fruit acids, gluconolactone, heparan sulfate, arbutin, niacinamide, resveratrol, hydroquinone, exfoliants, keratolytics, plant extracts, marine extracts, ferment extracts, isoflavones, bisabolol, anti-aging peptides, retinol, retinoic acid, retinyl esters, retinoic acid esters, and other retinoids.

[0059] Embodiment 12 is the method according to any one of Embodiments 1 to 11, wherein the method is a therapeutic method comprising treating a skin condition in a subject in need thereof.

[0060] Embodiment 13 is the method according to Embodiment 12, wherein the skin condition is acne or rosacea.

[0061] Embodiment 14 is the method according to any one of Embodiments 1 to 11, wherein the method is a non-therapeutic method and the subject is a healthy subject.

[0062] Embodiment 15 is the method according to Embodiment 14, wherein the method comprises treating photoaging or wherein the method comprises treating wrinkled or sagging facial skin and the composition is topically applied to the facial skin of the subject.

[0063] Embodiment 16 is the method according to any one of Embodiments 1 to 15, wherein the skin of the subject is prepared for application of the composition comprising an adapalene ester of Formula I while maintaining or increasing the microbial load of commensal bacteria on the skin of the subject. CORE / 3518179.0020 / 232318616.1 133518179.0020

[0064] Embodiment 17 is the method according to Embodiment 16, wherein preparing the skin of the subject for application of the composition comprising an adapalene ester of Formula I comprises wiping the skin of the subject to be treated with a non-aseptic wipe.

[0065] Embodiment 18 is the method according to Embodiment 16, wherein preparing the skin of the subject for application of the composition comprising an adapalene ester of Formula I comprises: contacting the skin of the subject to be treated with water; and drying the skin of the subject in a non-sterile environment.

[0066] Embodiment 19 is the method according to Embodiment 18, wherein drying the skin of the subject comprises wiping the skin of the subject to be treated with a non-aseptic wipe.

[0067] The following non-limiting example is provided to further illustrate the present invention. EXAMPLE 1: Comparative Treatment Study

[0068] A model study was performed on human skin treated with a composition in the form of an emulsion containing oleyl adapalenate lotion, 0.5 % wt / wt, and a lipophilic carrier. Disinfection Step

[0069] Ten live human subjects had one of their forearms pretreated with ethanol for disinfection, and their other forearms were not disinfected. Each forearm had a treatment site of 4 cm2. The site was swabbed and microbial load was measured via quantitative polymerase chain reaction measurement (qPCR) which detects the quantity of microbial DNA present. The observed qPCR signal was converted to concentration units of CFU / cm2using the established calibration curve of the instrument. The interpolated microbial counts for the disinfected and undisinfected sites are reported in Table 1.

[0070] Table 1. Microbial Load of Disinfected and Undisinfected Skin Subject Undisinfected Skin Disinfected SkinCORE / 3518179.0020 / 232318616.1 143518179.0020 6 10970.2 3039.8 7 6993.2 3774.7

[0071] 85%. This is consistent with the scientific and clinical literature that demonstrates standard consumer hygiene practices such as washing with soap (or surfactants) and water, or application of toners containing glycols, alcohols or antimicrobial compounds results in a microbial reduction of 80-90%. Also, typical of literature reports is a microbial load of 1 x 104CFU / cm2on unwashed skin, and this is consistent with the mean load of 3.7 x 104CFU / cm2on the undisinfected sites. Lipophilic Adapalene Ester Penetration

[0072] A fixed quantity of lotion containing the representative adapalene ester, oleyl adapalenate, at 0.5% wt / wt was applied to the treatment areas of both the undisinfected sites and the disinfected sites within about one minute following disinfection. The representative adapalene ester was quantitatively measured at four skin depths (0, 5, 10, 15 µm) and three time points (1, 10 and 20 minutes following application of the oleyl adapalenate lotion) using in vivo Raman Spectroscopy techniques as described in the art. (Reference 1 below).

[0073] 1. Nico C, Bakker Schut TC, Caspers PJ, Puppels GJ. Limits of Detection of Topically Applied Products in the Skin Using In Vivo Raman Spectroscopy. Pharmaceutics. 2024 Feb 22;16(3):304. doi: 10.3390 / pharmaceutics16030304. PMID: 38543198; PMCID: PMC10975793.

[0074] The spectroscopy probe was calibrated to track the amount of adapalene ester and its metabolite in the skin. The sum of adapalene ester and its metabolite in the skin represent the total quantity of adapalene ester absorbed topically. The results of these in vivo measurements are found in Tables 2a and 2b, and shown graphically in FIG.1 and FIG.2, for undisinfected skin and disinfected skin, respectively. The integrating under the curve of the concentration gradients, reported in Table 3, yields the total quantity of active agent absorbed and bioavailable. Application to undisinfected skin resulted in a surprising and dramatic increase in bioavailability of between 47-61.5% as compared to the disinfected skin. CORE / 3518179.0020 / 232318616.1 153518179.0020

[0075] Table 2a. Undisinfected Skin: Mean Concentration (µg / cm3) of Oleyl Adapalenate Absorbed Skin Depth (µm) 1 min 10 min 20 min 0 19.91 12.37 10.27 [007alenate Absorbed Skin Depth (µm) 1 min 10 min 20 min [007. g y µg y p lenate Absorbed Disinfected Skin Undisinfected Skin Increase Bioavailability Achieved by Non-Disinfection

[0078] When introducing elements of the present invention or the preferred embodiments(s) thereof, the articles "a", "an" and "the" are intended to mean that there are one or more of the elements. The terms "comprising", "including" and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0079] In view of the above, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[0080] As various changes could be made in the above methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. CORE / 3518179.0020 / 232318616.1 16

Claims

3518179.0020 CLAIMS:

1. A method of treating the skin of a subject, the method comprising topically applying to the skin of the subject a composition comprising an adapalene ester of Formula I:Formula I wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the skin of the subject to which the composition is applied is non- disinfected skin.

2. The method according to claim 1, wherein pretreatment disinfection to reduce the microbial load on the skin to be treated is not conducted at least 1 hour, at least 2 hours, at least 4 hours, at least 6 hours or longer prior to application of the composition comprising an adapalene ester of Formula I to the skin of the subject.

3. A method of treating the skin of a subject, the method comprising applying to the skin of the subject a composition comprising an adapalene ester of Formula I:wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2. CORE / 3518179.0020 / 232318616.1 173518179.0020 4. The method according to claim 1 or 3, wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 2 x 103CFU / cm2, at least about 3 x 103CFU / cm2, at least about 4 x 103CFU / cm2, at least about 5 x 103CFU / cm2, at least about 6 x 103CFU / cm2, at least about 7 x 103CFU / cm2, at least about 8 x 103CFU / cm2, at least about 9 x 103CFU / cm2, at least about 1 x 104CFU / cm2, at least about 1.3 x 104CFU / cm2, at least about 1.5 x 104CFU / cm2, at least about 1.8 x 104CFU / cm2, at least about 2 x 104CFU / cm2, at least about 2.5 x 104CFU / cm2, at least about 3 x 104CFU / cm2, at least about 3.5 x 104CFU / cm2, at least about 4 x 104CFU / cm2, at least about 4.5 x 104CFU / cm2, at least about 5 x 104CFU / cm2, at least about 5.5 x 104CFU / cm2, at least about 6 x 104CFU / cm2, at least about 6.5 x 104CFU / cm2, at least about 7 x 104CFU / cm2, at least about 7.5 x 104CFU / cm2, at least about 8 x 104CFU / cm2, at least about 8.5 x 104CFU / cm2, at least about 9 x 104CFU / cm2, at least about 9.5 x 104, at least about 1 x 105CFU / cm2or higher.

5. The method according to claim 1 or 3, wherein R1is a saturated or unsaturated, linear or branched, aliphatic hydrocarbyl group.

6. The method according to claim 1 or 3, wherein R1 is unsubstituted linear or branched alkyl or alkenyl.

7. The method according to claim 1 or 3, wherein R1 is C8, C12, or C16 alkyl.

8. The method according to claim 1 or 3, wherein R1is a linear eighteen or twenty-two carbon alkenyl.

9. The method according to claim 1 or 3, wherein R1is an oleyl group according to Formula II:CORE / 3518179.0020 / 232318616.1 183518179.0020 11. The method according to claim 1 or 3, wherein the composition comprises one or more additional ingredients selected from the group consisting of plant-derived exosomes, ascorbic acid and derivatives thereof, ferulic acid, azelaic acid, kojic acid, mandelic acid, alpha-hydroxy acids, beta-hydroxy acids, lipohydroxyacids, fruit acids, gluconolactone, heparan sulfate, arbutin, niacinamide, resveratrol, hydroquinone, exfoliants, keratolytics, plant extracts, marine extracts, ferment extracts, isoflavones, bisabolol, anti-aging peptides, retinol, retinoic acid, retinyl esters, retinoic acid esters, and other retinoids.

12. The method according to claim 1 or 3, wherein the method is a therapeutic method comprising treating a skin condition in a subject in need thereof.

13. The method according to claim 12, wherein the skin condition is acne or rosacea.

14. The method according to claim 1 or 3, wherein the method is a non-therapeutic method and the subject is a healthy subject.

15. The method according to claim 14, wherein the method comprises treating photoaging or wherein the method comprises treating wrinkled or sagging facial skin and the composition is topically applied to the facial skin of the subject.

16. The method according to claim 1 or 3, wherein the skin of the subject is prepared for application of the composition comprising an adapalene ester of Formula I while maintaining or increasing the microbial load of commensal bacteria on the skin of the subject.

17. The method according to claim 16, wherein preparing the skin of the subject for application of the composition comprising an adapalene ester of Formula I comprises wiping the skin of the subject to be treated with a non-aseptic wipe.

18. The method according to claim 16, wherein preparing the skin of the subject for application of the composition comprising an adapalene ester of Formula I comprises: contacting the skin of the subject to be treated with water; and drying the skin of the subject in a non-sterile environment.

19. The method according to claim 18, wherein drying the skin of the subject comprises wiping the skin of the subject to be treated with a non-aseptic wipe.

20. A composition comprising an adapalene ester of Formula I: CORE / 3518179.0020 / 232318616.1 193518179.0020 for use in aacne or rosacea a the method comprising administering the composition topically to the skin of the subject, wherein R1 is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the skin of the subject to which the composition is applied is non-disinfected skin.

21. A composition comprising an adapalene ester of Formula I: for use in athe method comprising administering the composition topically to the skin of the subject, wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2.

22. Use of a composition comprising an adapalene ester of Formula I: CORE / 3518179.0020 / 232318616.1 203518179.0020 for thea acne or rosacea a subject in need thereof, the treatment comprising topically applying the medicament to the skin of the subject, wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the skin of the subject to which the medicament is applied is non- disinfected skin.

23. Use of a composition comprising an adapalene ester of Formula I:for the manufacture of a medicament for the treatment of acne or rosacea in a subject in need thereof, the treatment comprising topically applying the medicament to the skin of the subject, wherein R1is substituted or unsubstituted hydrocarbyl having six to twenty-two carbon atoms and wherein the concentration of dermal commensal bacteria on the skin of the subject to which the composition is applied as at least about 1 x 103CFU / cm2. CORE / 3518179.0020 / 232318616.1 21

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