Topical compositions and methods for treating alopecia

Abstract

The present invention relates generally to the field of therapeutic compositions and methods for the treatment of inflammatory conditions and disorders of the skin and hair, particularly alopecia.

Description

[Technical Field]

[0001] The present invention relates generally to the field of therapeutic compositions and methods for the treatment of inflammatory conditions and disorders of the skin and hair, particularly alopecia. [Background technology]

[0002] Hair loss is a significant psychological stressor that can cause psychological anxiety, especially for women. In recent years, much research has been conducted into the causes and treatment strategies for hair loss. The underlying causes of hair loss are diverse, among which are hormonal imbalance (androgenetic alopecia, AGA), chronic inflammatory conditions associated with autoimmune diseases (alopecia areata, AA), and chemotherapy-induced alopecia (CIA). Both AGA and AA are caused by activated NKG2D, which produces the Th1 cytokine interferon-γ. + CD8 + This chronic local inflammation around the hair follicle disrupts the balance between immune tolerance and local apoptosis, ultimately leading to significant hair loss in that area.

[0003] The most common type of hair loss is androgen deficiency syndrome (AGA), which is characterized by progressive hair loss due to shrinking hair follicles, and its underlying causes are androgen levels and genetic mutations in the androgen receptor gene. Men with AGA have approximately 50 times higher levels of 5-alpha-reductase, the enzyme responsible for the conversion of testosterone to dihydrotestosterone (DHT), in the scalp compared to men without AGA.

[0004] Despite the existence of several oral or topical treatments for androgenetic alopecia (AGA), such as minoxidil for hair loss in men and women, finasteride for men, and cyclosporine A (CsA) [1-2], none of these have been shown to be reversible. [3] CsA is a known immunosuppressant used in the setting of organ rejection after transplantation and certain autoimmune diseases. It is known to inhibit the activation of helper T cells and suppress interferon-γ production. Its potential applicability to androgenetic alopecia stems from the hirsutism observed in patients treated with oral CsA, which led researchers to propose oral CsA for androgenetic alopecia (MPA, a type of androgenetic alopecia) and androgenetic alopecia (AA). However, subsequent clinical trials of oral CsA alone or in combination with systemic corticosteroids have yielded questionable and often unpredictable results, with success rates ranging from 25 to 77%. [4] This is in addition to the known risks of systemic CsA therapy, such as elevated blood pressure, altered blood glucose levels, gastrointestinal upset, tremors, and numbness.

[0005] AA presents particular challenges. While experts have proposed several systemic and topical treatments for this condition [4], none were approved by the FDA until 2022. These included first-line treatment with potent topical corticosteroids applied daily for at least 6–12 weeks and 3–6 months, and topical scalp treatments such as topical CsA for treating AA of the scalp and eyebrows, but not for AA of the beard. In June 2022, the FDA approved baricitinib, an immunomodulator and Janus kinase (JAK) inhibitor, as a first-line treatment for AA at a daily oral dose of 2 mg or 4 mg for a 36-week period [5–6]. However, several questions remain regarding the efficacy and safety of this treatment for AA in individual and large-scale cases.

[0006] In summary, the current situation is clearly deficient in effective and generally safe treatments for all forms of alopecia, particularly AA, which is further exacerbated by the magnitude of the problem, with a lifetime prevalence of AA of approximately 2% in both genders. Thus, there is an unmet need to find effective and safe topical treatments that can prevent, reduce, and reverse hair loss in AGA, AA, and other forms of alopecia.

[0007] References 1.Gupta AK et al 2022. Comparison of oral minoxidil, finasteride, and dutasteride for treating androgenetic alopecia. J Dermatolog Treat 33:2946-2962. 2.Goldust M et al 2022. Does topical minodixil in concentrations higher than 5% provide additional clinical benefit? Clin Exp Dermatol 47:1951-1955. 3.Santos Z et al 2015. Drug discovery for alopecia: Gone today, hair tomorrow. Expert Opin Drug Discov 10:269-292. 4.Meah N et al 2020. The Alopecia Areata Consensus of Experts (ACE) study: Results of an international expert opinion on treatments for alopecia areaata. JAAD 83:123-130. 5.King B et al 2022. Two Phase 3 trials of baricitinib for alopecia areata. NEJM 386:1687-1699. 6.Lensing M and Jabbari A 2022. An overview of JAK / STAT pathways and JAK inhibition in alopecia areata. Front Immunol 13:955035. Summary of the Invention

[0008] For the treatment of alopecia, local drug delivery would be a preferred option. Oral prescription drugs generally require safety considerations regarding associated adverse effects, which is one of the reasons why few oral drugs have been approved as systemic treatments for alopecia. For example, oral cyclosporine A (CsA) is associated with a relatively high adverse effect profile, including, among others, nephrotoxicity, immunosuppression, hypertension, neuropathy, and a relatively high recurrence rate.

[0009] In the context of transdermal delivery, the primary goal is to aid in the transdermal permeability of the drug. In this regard, CsA has very poor skin permeability, primarily due to its molecular weight and low water solubility. The penetration of CsA into the skin and scalp can be improved by incorporating permeation enhancers such as ethanol and terpenes, as has been reported for minoxidil and finasteride, and / or by designing specific delivery systems, such as nanoemulsions, liposomes, and nanocapsules (NCs), incorporating the active ingredient. Another advantage of such delivery systems is that they accumulate in the bulge region of the hair follicle, acting as a drug reservoir.

[0010] Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) is a nontoxic synthetic antioxidant known to promote the metabolism of many reactive oxygen species (ROS) and reduce oxidative stress. Its high water solubility and low molecular weight allow it to penetrate biological membranes. Furthermore, it has been demonstrated to reduce inflammation, possibly through reduced leukocyte infiltration and activation of the Nrf2 signaling pathway. Sporadic small studies of whole-brain radiation therapy have suggested that topical application of tempol to the scalp before radiation is relatively safe and well-tolerated and may have a preventive effect against radiation-induced alopecia. However, subsequent attempts to use several formulations of tempol in this setting have been inconsistent and have not produced publishable results.

[0011] The motivation for this technology was to design and develop a safe and effective topical delivery system using active ingredients, such as CsA and tempol, which are potentially effective in the treatment of AGA and AA. Currently, there are no effective topical treatments for AA. CsA and tempol have different physical and chemical properties and mechanisms of action, which may complement each other. Surprisingly, this study demonstrated that the CsA-tempol combination is versatile and synergistic in terms of efficacy, safety, skin penetration, and protective and restorative effects against AGA and AA, and therefore may provide an effective solution not only for alopecia but also for other common skin conditions.

[0012] Essentially, the anti-inflammatory capabilities and topical applicability of the formulations of the present invention, as supported by the examples and discussion provided herein, make them particularly attractive candidates for a wide range of additional common skin conditions involving local inflammation, pigmentation disorders, pimples, and scarring, some examples being acne, atopic dermatitis, epidermolysis bullosa, hidradenitis suppurativa (HS), ichthyosis, pachyonychia congenita, pemphigus, psoriasis, Raynaud's phenomenon, rosacea, scleroderma, and vitiligo.

[0013] In its broadest sense, the present invention can be described as relating to a topical formulation comprising a matrix polymeric emulsifier composed of or formed from a crosslinked copolymer of acrylic acid and an acrylate, and a nanoemulsion comprising a lipophilic immunosuppressant and a cyclic nitroxide (spin label).

[0014] In some embodiments, the topical formulations of the present invention comprise acrylic acid and acrylates, such as C 10 ~C 30 The formulation may be a gel comprising a matrix polymeric emulsifier comprised of a cross-linked copolymer of alkyl acrylate and a nanoemulsion comprising a lipophilic immunosuppressant and a cyclic nitroxide.

[0015] The term "gel formulation" as used herein encompasses aqueous, emulsified, and non-aqueous gel formulations. Typically, gel formulations contain an amount of aqueous medium or water that allows for stability of the nanoemulsion. Gel formulations are typically formulated for topical use and can be characterized by the permeation profile of the active ingredient. Gel formulations typically contain a matrix material, typically in the form of a polymeric emulsifier. The emulsifier is typically a polymeric emulsifier containing acrylic acid and C 10 ~C 30 It may be a high molecular weight copolymer with an acrylate such as an alkyl acrylate.

[0016] In some embodiments, acrylic acid and an acrylate, such as C 10 ~C 30 Cross-linking between alkyl acrylates, such as alkyl acrylates, can be achieved by using allylpentaerythritol.

[0017] In some embodiments, the gel formulation comprises a mixture of acrylic acid and C 10 ~C 30 It comprises a matrix material in the form of a polymeric emulsifier selected from high molecular weight copolymers with an acrylate, such as an alkyl acrylate, where the acrylic acid and the acrylate are crosslinked with allylpentaerythritol.

[0018] In some embodiments, the gel formulation comprises a mixture of acrylic acid and C 10 ~C 30 The matrix material is in the form of a polymeric emulsifier selected from high molecular weight copolymers of acrylates, such as alkyl acrylates, where the acrylic acid and acrylate are crosslinked with allylpentaerythritol. This polymeric emulsifier is a material with CAS number 138789-85-2.

[0019] In some embodiments, the matrix material is Pemulen gel, a type of pharmaceutical excipient with effective emulsifying properties for forming stable oil-in-water emulsions. Pemulen polymer excipients contain both hydrophilic and hydrophobic moieties, which create a network around suspended oil droplets, providing excellent emulsion stability, often without the need for additional surfactants. The term "Pemulen" as used herein refers to a polymer of acrylic acid and C -copolymer crosslinked with allylpentaerythritol. 10 ~C 30 Pemulen refers to a polymeric emulsifier that is a high molecular weight copolymer with alkyl acrylate. Pemulen refers to a polymeric emulsifier with CAS number 138789-85-2.

[0020] Acrylic acid and C formed by crosslinking with allylpentaerythritol 10 ~C 30 Acrylate copolymers, also known as alkyl acrylates, are copolymers of acrylic acid and various alkyl acrylate materials. 10 ~C 30 "Alkyl" is intended to encompass not any one alkyl, but rather a mixture of alkyl acrylates that are crosslinked with allylpentaerythritol along with acrylic acid to provide a copolymer.

[0021] In some embodiments, the gel formulations of the present invention may further comprise at least one thickening agent to increase viscosity and topical applicability.

[0022] According to some embodiments, types of applicable thickeners that can be used include: - synthetic raw material components: polymers (polyacrylates and their derivatives, such as carbopol and polycarbophil), poloxamers, polypropylene glycols, and waxes, each of which constitutes a separate and independent embodiment of the present invention; - Natural ingredients: polysaccharides (cellulose, hyaluronic acid, and chitosan, and derivatives thereof, each of which constitutes a separate and independent embodiment of the present invention); - polyproteins: (natural gelatin, collagen, and synthetic polyamino acids such as polyglutamic acid, each constituting a separate and independent embodiment of the present invention); Silicone derivatives: as mineral clays such as magnesium aluminum silicate, and polymeric silicons such as elastomers, each constituting a separate and independent embodiment of the present invention.

[0023] In some embodiments, the thickening agent may be selected from polyacrylates and their derivatives, carbopol, polycarbophil, poloxamer, polypropylene glycol, waxes, polysaccharides, cellulose, hyaluronic acid, chitosan, its derivatives, natural polyproteins, gelatin, collagen, polyamino acids, mineral clays, magnesium aluminum silicate, and polymeric silicon.

[0024] In some embodiments, the gel formulations of the present invention may further comprise an immunosuppressant, such as a lipophilic immunosuppressant, and a nanoemulsion of a cyclic nitroxide. The term "immunosuppressant," as used herein, encompasses any agent capable of reducing or suppressing the activity of at least one biomarker of the immune system or inflammatory marker in vivo or in vitro.

[0025] In some embodiments, the immunosuppressant selected may be a steroid, a cell growth inhibitor, an antibody, an immunophilin drug, mycophenolate, a tumor necrosis factor (TNF-α) inhibitor, etc. Non-limiting examples of immunosuppressants include azathioprine (Imuran), cyclosporine A (CsA), mercaptopurine (Purinethol, 6-MP), rapamycin, fujimycin, and methotrexate.

[0026] In some embodiments, the immunosuppressant may be selected from azathioprine (Imuran), cyclosporine A (CsA), mercaptopurine (Purinethol, 6-MP), rapamycin, fujimycin, and methotrexate.

[0027] In some embodiments, the immunosuppressant can be CsA.

[0028] In some embodiments, the nanoemulsion and matrix material can be mixed into a stable gel, as defined herein.

[0029] The cyclic nitroxides used in the formulations of the present invention are spin-label cyclic compounds having a ring nitrogen atom bonded to an oxygen atom bearing an unpaired electron. Typically, the cyclic nitroxide is a cyclic nitroxide of structure (I) as defined herein. Alternatively, the cyclic nitroxide is a cyclic compound that may or may not have an endocyclic double bond and / or one or more substituted moieties, and a ring oxygen atom bearing an unpaired electron.

[0030] In some embodiments, the gel formulation comprises: (1) Acrylic acid and C 10 ~C 30 a matrix of a crosslinked copolymer with an alkyl acrylate; (2) A nanoemulsion comprising an immunosuppressant (which may be lipophilic) and a cyclic nitroxide, wherein the cyclic nitroxide is a compound of formula I: TIFF2026501730000001.tif43170 formula, A represents a carbon atom or a carbon chain containing up to 3 carbon atoms (e.g., providing a 4-, 5-, or 6-atom ring structure; if A is a 3-carbon atom chain, providing a 6-membered heterocyclic ring structure such as piperidinyl or tempol; if A is a 2-carbon atom chain, providing a 5-membered heterocyclic ring structure such as pyrrolidinyl or proxyl, optionally containing one or more double bonds, i.e., a pyrroline structure), wherein at least one (or only one) of the carbon atoms is optionally substituted with an oxygen atom or an oxygen-containing group (oxazolinyl or doxyl), and / or one or more of the carbon atoms is substituted with one or two bromine atoms; Each of R1, R2, R3, and R4 is independently selected from H and C1-C5 alkyl; or each of R1 and R2 together with the carbon atoms to which they are attached form a 3- to 7-membered ring; and / or R3 and R4, together with the carbon atoms to which they are attached, form a 3- to 7-membered ring; R5 represents a group selected from an aldehyde, a ketone, a carboxylic acid, a carbonyl group, -O-, -S-, -OH, -SH, -COOH, -COONH2, -CN, and a primary amine (e.g., -NH2), a secondary amine (e.g., -NH-R), a tertiary amine (e.g., -NR'R"), or a quaternary amine (e.g., a charged amine); and O represents an oxygen radical, Nanoemulsion and may include:

[0031] Each of R, R', and R" used in reference to the amine group may independently be a C1-C5 alkyl or any other carbon group.

[0032] The A group may also or alternatively be selected from carbon groups that form an endocyclic double bond with an adjacent carbon atom, for example, the A group may be in the form =CR5, where the carbon atom designated =C forms a double bond with the adjacent carbon atom.

[0033] In some embodiments, the cyclic nitroxide compound can be a five-membered heterocyclic ring structure.

[0034] In some embodiments, the cyclic nitroxide compound can be a six-membered heterocyclic ring structure.

[0035] In some embodiments, in the cyclic nitroxide compound of Formula (I), each of R1, R2, R3, and R4 is H or C1-C5 alkyl.

[0036] In some embodiments, in the cyclic nitroxide compound of Formula (I), each of R 1 , R 2 , R 3 , and R 4 is different from H.

[0037] In some embodiments, in the cyclic nitroxide compound of Formula (I), each of R1, R2, R3, and R4 is a C1-C5 alkyl selected from methyl, ethyl, propyl, isopropyl, butyl, and pentyl.

[0038] In some embodiments, in the cyclic nitroxide compounds of Formula (I), each of R1, R2, R3, and R4 is a different C1-C5 alkyl, ie, has a different number of carbon atoms or a different structure.

[0039] In some embodiments, in the cyclic nitroxide compounds of Formula (I), each of R1, R2, R3, and R4 is a linear C1-C5 alkyl.

[0040] In some embodiments, in the cyclic nitroxide compound of Formula (I), each of R 1 , R 2 , R 3 , and R 4 is a methyl group.

[0041] In some embodiments, the cyclic nitroxide compound of Formula (I) can be a compound of Formula (II): TIFF2026501730000002.tif47170In the formula, A and R5 are each as defined above.

[0042] In some embodiments, in the cyclic nitroxide compounds of Formula (I) or Formula (II), A is a carbon group containing 1, 2, or 3 carbon atoms, at least one of which is bonded to an oxygen-containing group. The oxygen-containing group can be a hydroxyl group or an ether group, or the oxygen-containing group is an oxygen-containing group selected from among the groups defining R. In other words, the A-R group can be a group selected from A=O, A-OH, A-COOH, and A-COONH, where A is a carbon atom or a carbon group as defined, as desired, and the carbon atom can be an SP or SP carbon atom.

[0043] When referring to a "carbon group," it should be clear that the group contains one or more carbon atoms and one or more other atoms, e.g., H or heteroatoms, whereby the carbon group is arranged to provide a linear, branched, or interrupted carbon chain. Typically, a carbon group contains 1 to 3 carbon atoms, with each carbon atom further bonded to a hydrogen atom or other atom as specified to provide complete and proper valence. In some embodiments, A is a group having a carbon skeleton selected from -C-, -CC-, and -CCC- and an appropriate number of H atoms, where one of the hydrogen atoms may be replaced with an oxygen-containing group as specified, and all other substituents are hydrogen atoms. In some embodiments, A is a group having a carbon skeleton selected from -C-, -CC-, and -CCC-, where one of the hydrogen atoms is replaced with an oxygen-containing group as specified, and all other substituents are hydrogen atoms, or one or two of the carbon atoms are replaced with one or two Br atoms, and all other substituents are hydrogen atoms.

[0044] In some embodiments, A is a group that has or forms at least one endocyclic double bond.

[0045] In some embodiments, A is a group selected from -CH-, -CH-CH2-, -CH-CH2-CH2-, and -CH2-CH-CH2-, and the A-R5 group is selected from -CHR5-, -CHR5-CH2-, -CHR5-CH2-CH2-, and -CH2-CHR5-CH2-.

[0046] In some embodiments, the A-R5 group is -CHR5-, -CHR5-CH2-CH2-, or -CH2-CHR5-CH2-, where R5 is an oxygen-containing group as defined.

[0047] In some embodiments, the A-R5 group is -CH(OH)-, -CH(OH)-CH2-CH2-, or -CH2-CH(OH)-CH2-.

[0048] In some embodiments, the A-R5 group is -CH2-CH(OH)-CH2-.

[0049] In some embodiments, the cyclic nitroxide compound is a five-membered heterocyclic structure and the A-R5 group is -CH2-CH(OH)-CH2-.

[0050] In some embodiments, the cyclic nitroxide compound is a six-membered heterocyclic structure and the A-R5 group is -CH2-CH(OH)-CH2-.

[0051] In some embodiments, the cyclic nitroxide can be 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, tempol. TIFF2026501730000003.tif56170

[0052] In some embodiments, the cyclic nitroxide compound can be a compound of the following formula: TIFF2026501730000004.tif51170

[0053] In some embodiments, the cyclic nitroxide compound can be a compound of the following formula: TIFF2026501730000005.tif51170

[0054] In some embodiments, the cyclic nitroxide can be chosen from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

[0055] In some embodiments, the nanoemulsion may comprise a lipophilic immunosuppressant, such as CsA, and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

[0056] In some embodiments, the gel formulation comprises: (1) Acrylic acid and C 10 ~C 30 a matrix of a crosslinked copolymer with an alkyl acrylate, and (2) A nanoemulsion comprising a lipophilic immunosuppressant, such as CsA, and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl; may include at least one of:

[0057] In some embodiments, the formulation may include CsA and tempol.

[0058] In some embodiments, the formulation may contain CsA at a concentration in the range of about 0.01% to about 0.5% (wt / wt), or more specifically, at a concentration in the range of about 0.01-0.05%, 0.05-0.1%, 0.1-0.2%, 0.2-0.3%, 0.3-0.4%, 0.4-0.5% (wt / wt), or up to 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (wt / wt).

[0059] In some embodiments, the formulation may contain tempol at a concentration in the range of about 0.1% to about 5% (w / w), or more specifically, at a concentration in the range of about 0.1-0.5%, 0.5-1%, 1-1.5%, 1.5-2%, 2-2.5%, 2.5-3%, 3-3.5%, 3.5-4%, 4-4.5%, or 4.5-5% (w / w), or at concentrations in the ranges of up to 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5% (w / w).

[0060] In some embodiments, the formulation may include CsA at a concentration of about 0.1% and tempol at a concentration of about 0.5% (w / w).

[0061] In some embodiments, the gel formulation may further comprise one or more additional active ingredients or inert additives. Depending on the type of formulation desired and various other factors, the hair growth preparation may comprise a variety of ingredients that can be mixed and dissolved, as known in the pharmaceutical and / or cosmetic arts. In general, the formulations of the present invention may comprise diluents, buffers, fragrances, binders, surfactants, thickeners, lubricants, preservatives, pH adjusters, fungicides, antioxidants, emulsifiers, stabilizers, flavors, and colorants.

[0062] One of the unique features of the formulation of the present invention is manifested in the particle size of the encapsulated nanoemulsion.

[0063] In some embodiments, the nanoemulsion can have a particle size in the range of about 200 nm to about 300 nm, or more specifically, the particle size can be in the range of 200-210 nm, 210-220 nm, 220-230 nm, 230-240 nm, 240-250 nm, 250-260 nm, 260-270 nm, 270-280 nm, 280-290 nm, or 290-300 nm.

[0064] In some embodiments, the particle size is 100-110 nm, 110-120 nm, 120-130 nm, 130-140 nm, 140-150 nm, 150-160 nm, 160-170 nm, 170-180 nm, 180-190 nm, 190-200 nm, 200-210 nm, 210-220 nm, 220-230 nm, 230-240 nm, 240-250 nm, 250-260 nm, 260-270 nm, 270-280 nm, 280-290 nm, 290-300 nm, 300-310 nm, 310-320 nm, 320-330 nm, 330-340 nm, 340-350 nm, 350-360 nm, 360-370 nm, 370-380 nm, 380-390 nm, 390-400 nm, 400-410 nm, 420-420 nm, 430-440 nm, 440-450 nm, 450-460 nm, 460-470 nm, 470-480 nm, 480-490 nm, 490-500 nm, 500-510 nm, 510-520 nm, 520-530 nm, 530-540 nm, 540-550 nm, 550-560 nm, 560-570 nm, 570-580 nm, 580-590 nm, 590-600 nm, 600-610 nm, 61 nm, 300-310 nm, 310-320 nm, 320-330 nm, 330-340 nm, 340-350 nm, 350-360 nm, 360-370 nm, 370-380 nm, 380-390 nm, 390-400 nm, 400-410 nm, 410-420 nm, 420-430 nm, 430-440 nm, 440-450 nm, 450-460 nm, 460-470 nm, 470-480 nm, 480-490 nm, or 490-500 nm.

[0065] In some embodiments, the nanoemulsion comprises acrylic acid and C 10 ~C 30 The nanoemulsion can be encapsulated in a matrix of a cross-linked copolymer with alkyl acrylate in the range of 60% to 100% or more specifically, the nanoemulsion can be encapsulated in a matrix of a cross-linked copolymer with acrylic acid and C 10 ~C 30 It may be up to 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% encapsulated in a matrix of crosslinked copolymer with alkyl acrylate, or within the range of 60-65%, 70-75%, 80-85%, 90-95%, or 95-100% encapsulated in the matrix.

[0066] In some embodiments, the gel formulations may remain stable at 37° C. for at least 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 weeks or more, or for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more, or for at least 1, 2, 3, 4, 5, 6 years or more. Stability may be measured and verified in terms of retention of active ingredient concentration over time or retention of a physical property of the formulation, such as viscosity, both of which measurements are exemplified herein.

[0067] For example, the viscosity of the gel formulation may remain relatively constant or stable within ±20% at 37°C for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months.

[0068] In some embodiments, the gel formulation may preferentially distribute to deeper skin layers, such as the epidermis and / or dermis. The term "preferentially," as used herein, means that the distribution, amount, or concentration of at least one active ingredient in the epidermis and / or dermis is increased compared to its distribution, amount, or concentration in other tissues, particularly the stratum corneum.

[0069] In some embodiments, the gel formulation may have increased penetration into the epidermal and dermal layers of the skin compared to the penetration of the formulation into the stratum corneum. The term "penetration," as used herein, means an increased amount or concentration of at least one active ingredient in the epidermis and / or dermis compared to its amount or concentration in the stratum corneum.

[0070] The degree of increase in the amount or concentration of the active ingredient in the epidermis and / or dermis can be in the range of up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more compared to the amount or concentration in the stratum corneum, or up to 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, and more compared to the amount or concentration in the stratum corneum. Such measurements are exemplified in the present invention.

[0071] Another aspect of the present invention is to provide clinical and cosmetic methods and uses of gel formulations and their permutations according to the provisions of the present invention for promoting hair growth and / or skin histomorphological remodeling on the human skin or scalp. These effects can be assessed by known qualitative and quantitative methods, such as visual or quantitative assessment of hair density and histological assessment of skin morphology. Such measurements are exemplified in the present invention.

[0072] The term "hair growth" as used herein includes the maintenance, induction, stimulation, promotion, and regeneration of hair development in a human or animal subject, as well as the growth of defective hair, the extension of the anagen phase in the pilar cycle, and the conversion of vellus hair to terminal hair.

[0073] In some embodiments, particularly when using formulations comprising CsA and tempol, the effects of CsA and tempol on treating or promoting hair growth on the human skin or scalp and / or on the histomorphological remodeling of the skin may be synergistic.

[0074] The term "synergistic" means that the effect of the combination of active ingredients (CsA and Tempol) or gel formulation is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold greater than the effect of a gel formulation of a single active ingredient (CsA or Tempol).

[0075] In yet another aspect, the present invention provides methods and uses of the above gel formulations in the treatment of alopecia in various clinical conditions.

[0076] The term "alopecia", as used herein, encompasses all types of hair growth failure and partial or total hair loss, including, but not limited to, androgenic alopecia, androgenetic alopecia (AGA), toxic alopecia, alopecia areata (AA), telogen effluvium, alopecia due to endocrine, metabolic, and nutritional disorders, drug-induced alopecia, mechanical alopecia, alopecia due to skin diseases, cicatricial alopecia, congenital alopecia, and trichotillomania.

[0077] In some embodiments, the alopecia is androgenetic alopecia (AGA) or alopecia areata (AA).

[0078] One aspect to be considered in the context of clinical methods and uses is the effective amount, concentration, or dose of an active ingredient applied to a subject to be treated, as indicated by the term "therapeutically effective" amount, concentration, or dose, which typically refers to an amount, concentration, or dose, or active ingredient, associated with a therapeutic effect that can be recognized or measured by recognizable clinical criteria, such as alleviation of clinical symptoms of a disease or condition, a decrease in one or more recognized biomarkers thereof, or patient report of improvement in clinical symptoms.

[0079] In some embodiments, improvement or reduction of such clinical symptoms or biomarkers may involve daily, weekly, or monthly topical administration of a therapeutically effective amount of a gel formulation of the present invention.

[0080] In some embodiments, improvement or reduction of such clinical symptoms or biomarkers may involve topical administration of a therapeutically effective amount of the formulation one or more times daily.

[0081] In yet another aspect, the present invention provides the use of the gel formulations of the present invention, and permutations thereof, in the manufacture of a topical medicament for promoting hair growth on the skin or scalp of a human and / or for the histomorphological remodeling of the skin.

[0082] In some embodiments, the present invention provides the use of a gel formulation of the present invention in the manufacture of a topical medicament for various symptoms of alopecia.

[0083] The topical formulations of the present invention may be developed into multiple forms, including liquid or semi-liquid preparations such as lotions, emulsions, creams, ointments, liniments, sprays, aerosols, oils, pastes, gels, tonics, solutions, or suspensions, and may be adapted for application to the scalp and / or skin.

[0084] More broadly, due to its anti-inflammatory effect and topical applicability, the formulations of the present invention may potentially be used for a wide variety of additional common skin conditions, particularly those commonly referred to as inflammatory, immune, and / or autoimmune skin conditions. The most common examples are widespread localized skin inflammation, pigmentation disorders, pimples, and scars. Further clinically relevant examples include acne, atopic dermatitis, epidermolysis bullosa, hidradenitis suppurativa (HS), ichthyosis, pachyonychia congenita, pemphigus, psoriasis, Raynaud's phenomenon, rosacea, scleroderma, and vitiligo. Research into the applicability of the formulations to such conditions is currently underway.

[0085] The term "about" generally means at least ±5%, ±10%, ±15%, ±20%, ±25% deviation from the respective value.

[0086] The present invention further comprises: A topical formulation comprising a matrix polymer emulsifier composed of a crosslinked copolymer of acrylic acid and acrylate, and a nanoemulsion comprising an immunosuppressant and a cyclic nitroxide; to provide.

[0087] In any configuration of the formulations disclosed herein, the formulation may be a gel formulation selected from an aqueous gel formulation, an emulsion gel formulation, and a non-aqueous gel formulation.

[0088] In any configuration of the formulations disclosed herein, the matrix polymer emulsifier is a high molecular weight copolymer of acrylic acid and an acrylate.

[0089] In any configuration of the formulations disclosed herein, the acrylate may be C 10 ~C 30 It may be an alkyl acrylate.

[0090] In any configuration of the formulations disclosed herein, the matrix polymer emulsifier may be selected from high molecular weight copolymers of acrylic acid and acrylates, where the acrylic acid and acrylates are crosslinked with allylpentaerythritol.

[0091] In any configuration of the formulations disclosed herein, the formulation includes at least one thickening agent.

[0092] In any configuration of the formulations disclosed herein, the thickening agent is selected from polyacrylates and their derivatives, carbopol, polycarbophil, poloxamer, polypropylene glycol, waxes, polysaccharides, cellulose, hyaluronic acid, chitosan and its derivatives, natural polyproteins, gelatin, collagen, polyamino acids, mineral clays, magnesium aluminum silicate, and polymeric silicon.

[0093] In any configuration of the formulations disclosed herein, the nanoemulsion and matrix polymer emulsifier are provided as a stable gel.

[0094] In any configuration of the formulations disclosed herein, the immunosuppressant is selected from among agents that reduce or suppress the activity of the immune system in vivo, and optionally is selected from steroids, cell growth inhibitors, antibodies, immunophilins, mycophenolates, and tumor necrosis factor (TNF-α) inhibitors.

[0095] In any configuration of the formulations disclosed herein, the immunosuppressant is selected from azathioprine (Imuran), cyclosporine A (CsA), mercaptopurine (Purinethol, 6-MP), rapamycin, fujimycin, and methotrexate.

[0096] In any configuration of the formulations disclosed herein, the immunosuppressant is CsA.

[0097] In any configuration of the formulations disclosed herein, the formulation may comprise: - Acrylic acid and C 10 ~C 30 a matrix of a crosslinked copolymer with an alkyl acrylate; a nanoemulsion comprising a lipophilic immunosuppressant and a cyclic nitroxide, wherein the cyclic nitroxide is a compound of formula I: TIFF2026501730000006.tif43170 formula, A represents a carbon atom or a carbon chain containing up to three carbon atoms, one of which is substituted with an oxygen atom or an oxygen-containing group, or one or more of which is substituted with one or two bromine atoms; Each of R1, R2, R3, and R4 is independently selected from H and C1-C5 alkyl; or each of R1 and R2 together with the carbon atoms to which they are attached form a 3- to 7-membered ring, and / or each of R3 and R4 together with the carbon atoms to which they are attached form a 3- to 7-membered ring; R5 represents a group selected from an aldehyde, a ketone, a carboxylic acid, a carbonyl group, -O-, -S-, -OH, -SH, -COOH, -COONH2, -CN, and a primary amine, a secondary amine, a tertiary amine, or a quaternary amine; and O represents an oxygen radical, Nanoemulsion and Includes.

[0098] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a five-membered heterocyclic ring structure.

[0099] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a six-membered heterocyclic ring structure.

[0100] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a five-membered heterocyclic ring structure containing an endocyclic double bond.

[0101] In any configuration of the formulations disclosed herein, each of R1, R2, R3, and R4 of the cyclic nitroxide compound of Formula (I) is independently C1-C5 alkyl.

[0102] In any configuration of the formulations disclosed herein, each of R1, R2, R3, and R4 of the cyclic nitroxide compound of Formula (I) is independently selected from methyl, ethyl, propyl, isopropyl, butyl, and pentyl.

[0103] In any configuration of the formulations disclosed herein, each of R1, R2, R3, and R4 of the cyclic nitroxide compound of formula (I) is a methyl group.

[0104] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound of formula (I) is a compound of formula (II): TIFF2026501730000007.tif39170 wherein A and R5 are as defined herein.

[0105] In any configuration of the formulations disclosed herein, A of the cyclic nitroxide compound of Formula (I) or Formula (II) is a carbon group containing 1, 2, or 3 carbon atoms, at least one of which is bonded to an oxygen-containing group.

[0106] In any configuration of the formulations disclosed herein, the oxygen-containing group is a hydroxyl group or an ether group, or the oxygen-containing group is an oxygen-containing group selected from R5.

[0107] In any configuration of the formulations disclosed herein, the A-R5 group is a group selected from A=O, A-OH, A-COOH, and A-COONH2.

[0108] In any configuration of the formulations disclosed herein, A-R5 is -CHR5-, -CHR5-CH2-, -CHR5-CH2-CH2-, or -CH2-CHR5-CH2-.

[0109] In any configuration of the formulations disclosed herein, the A-R5 group is -CHR5-, -CHR5-CH2-CH2-, or -CH2-CHR5-CH2-, where R5 is an oxygen-containing group.

[0110] In any configuration of the formulations disclosed herein, the A-R5 group is -CH(OH)-, -CH(OH)-CH2-CH2-, or -CH2-CH(OH)-CH2-.

[0111] In any configuration of the formulations disclosed herein, the A-R5 group is -CH2-CH(OH)-CH2-.

[0112] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a five-membered heterocyclic ring structure and the A-R5 group is -CH2-CH(OH)-.

[0113] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a six-membered heterocyclic ring structure and the A-R5 group is -CH2-CH(OH)-CH2-.

[0114] In any configuration of the formulations disclosed herein, the cyclic nitroxide is tempol. TIFF2026501730000008.tif51170

[0115] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a compound of the following formula: TIFF2026501730000009.tif56170

[0116] In any configuration of the formulations disclosed herein, the cyclic nitroxide compound is a compound of the following formula: TIFF2026501730000010.tif56170

[0117] In any configuration of the formulations disclosed herein, the cyclic nitroxide is selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

[0118] In any configuration of the formulations disclosed herein, the nanoemulsion comprises CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

[0119] In any configuration of the formulations disclosed herein, the formulation may comprise: a matrix of a crosslinked copolymer of acrylic acid and a C10 to C30 alkyl acrylate, and - a nanoemulsion comprising CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, It includes at least one of the following:

[0120] In any configuration of the formulations disclosed herein, the nanoemulsion has a particle size in the range of about 200 nm to about 300 nm.

[0121] In any configuration of the formulations disclosed herein, the nanoemulsion may comprise a mixture of acrylic acid and C 10 ~C 30 It is encapsulated in a matrix of a cross-linked copolymer with alkyl acrylate to a degree ranging from 60% to 100%.

[0122] In any configuration of the formulations disclosed herein, the formulations are stable at 37° C. for a period of at least 3 months.

[0123] In any configuration of the formulations disclosed herein, the formulation distributes preferentially to the deeper skin layers.

[0124] In any configuration of the formulations disclosed herein, the formulations have increased penetration into the epidermal and dermal layers of the skin compared to the penetration of the formulation into the stratum corneum.

[0125] In any configuration of the formulations disclosed herein, the formulation comprises CsA and tempol.

[0126] In any configuration of the formulations disclosed herein, CsA is at a concentration ranging from about 0.01% to about 0.5% and Tempol is at a concentration ranging from about 0.1% to about 5% (wt / wt).

[0127] In any configuration of the formulations disclosed herein, the concentration of CsA is about 0.1% and the concentration of Tempol is about 0.5% (w / w).

[0128] In any configuration of the formulations disclosed herein, the formulations are for use in treating or promoting hair growth on the human skin or scalp and / or for the histomorphological remodeling of the skin.

[0129] In any configuration of the formulations disclosed herein, the formulations are for use in treating or promoting hair growth on the skin or scalp of a human and / or in the histomorphological remodeling of the skin, and the effects of CsA and tempol on treating or promoting hair growth on the skin or scalp of a human and / or in the histomorphological remodeling of the skin are synergistic.

[0130] Also provided is the use of the formulations disclosed herein in a method for preventing or treating alopecia.

[0131] In any configuration of the formulations disclosed herein, the alopecia is selected from androgenic alopecia, androgenetic alopecia (AGA), toxic alopecia, alopecia areata (AA), telogen effluvium, alopecia caused by endocrine, metabolic, and nutritional disorders, drug-induced alopecia, mechanical alopecia, alopecia caused by skin diseases, cicatricial alopecia, congenital alopecia, and trichotillomania.

[0132] In any configuration of the formulations disclosed herein, the alopecia is androgenetic alopecia (AGA) or alopecia areata (AA).

[0133] Also provided is a method of treating male or female alopecia in a subject in need thereof, the method comprising topically administering to the subject a therapeutically effective amount of the formulation disclosed herein.

[0134] A method for treating or promoting hair growth on the skin or scalp of a human and / or for histomorphological remodeling of the skin, the method comprising topically administering to the subject a therapeutically effective amount of a formulation disclosed herein.

[0135] A method of treating an inflammatory, immune, and / or autoimmune skin condition in a subject, the method comprising topically administering to the subject a therapeutically effective amount of a formulation disclosed herein.

[0136] In any configuration of the formulations disclosed herein, the formulations are for use in treating inflammatory, immune, and / or autoimmune skin conditions in humans.

[0137] In any configuration of the formulations disclosed herein, the use is in the manufacture of a topical medicament for treating inflammatory, immune, and / or autoimmune skin conditions in humans.

[0138] In any configuration of the formulations disclosed herein, the use is in the manufacture of a topical medicament for the treatment or promotion of hair growth on the human skin or scalp and / or the histomorphological remodeling of the skin.

[0139] Also, nanoemulsions of CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl; and nanoemulsions of acrylic acid and C 10 ~C 30 Also provided is a formulation comprising a copolymer with an alkyl acrylate, the formulation configured for application to an area of ​​skin of a subject.

[0140] In any configuration of the formulations disclosed herein, the nanoemulsion is encapsulated in a copolymer, the CsA is at a concentration in the range of about 0.01% to about 0.5%, and the cyclic nitroxide is at a concentration in the range of about 0.1% to about 5% (wt / wt).

[0141] A formulation comprising 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl for use in a method of preventing or treating alopecia, in any configuration of the formulations disclosed herein. [Brief explanation of the drawings]

[0142] Specific embodiments will now be described, by way of non-limiting example, with reference to the following drawings:

[0143] [Figure 1] Figure 1 shows the specific morphological features of the nanoemulsions of the present invention, namely, the relatively uniform nanometer particle size and high encapsulation efficiency (EE) of CsA, as revealed by TEM and cryo-TEM images of blank nanoemulsion (A1 / A2) and 0.5% CsA nanoemulsion (B1 / B2), respectively. [Figure 2] Figure 2 shows the specific morphological features of the gel formulations of the present invention, i.e., micrometric matrix structure with improved stability and fully embedded CsA-loaded nanoemulsions, revealed by SEM images of blank gels (A1 / A2) and 0.5% CsA gels (B1 / B2) at ×10 and ×20 magnification, respectively. [Figure 3] Figure 3 shows additional physicochemical properties of the gel formulations of the present invention, namely, non-Newtonian pseudoplasticity and long-term stability, as revealed by viscosity measurements at 37°C of a blank gel (▲), a 0.1% CsA gel (□), a 0.5% Tempol gel (●), and a 0.1% CsA-0.5% Tempol gel (○). All gel formulations had relatively constant viscosities at 37°C for at least 6 weeks, with the CsA-Tempol gel having the highest viscosity (approximately 17.7 Pa·S). [Figure 4] Figure 4 shows the improved stability profile at 37°C over a period of 12 weeks (approximately 3 months), reproducing the same phenomenon of consistent stability for all gel formulations, namely, blank gel (▲), 0.1% CsA gel (■), 0.5% Tempol gel (● dashed line), and 0.1% CsA-0.5% Tempol gel (● solid line), with the highest and more favorable viscosity being for the CsA-Tempol gel. [Figure 5]Figure 5 shows the cytotoxic effect of the delivery system of the present invention as revealed by viability studies in HaCaT cells treated (24 hours) with different concentrations of blank gel (Pemulen gel), CsA gel, and free CsA and tested in the MTT assay (mean ± SD, N = 3). CsA, both in gel and free drug form, was nontoxic at concentrations below 5 μg / mL, and blank Pemulen gel was nontoxic even at higher concentrations (2 mg / mL). [Figure 6] Figures 6A-6B show the improved penetration into deeper skin layers as evidenced by comparative analysis of CsA levels in the stratum corneum (SC), epidermis, and dermis of fresh (live) and frozen human skin treated ex vivo with CsA-in-oil, CsA nanoemulsion, and gel formulations (mean ± SD, N = 9 mice × 3 donors, two-way ANOVA Dunnett's multiple comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). The behavior of the CsA gel in frozen and live tissue was similar, preferentially penetrating the epidermis and deeper layers of the dermis. [Figure 7] Figures 7A-7D demonstrate improved penetration characteristics under more controlled conditions by measuring CsA levels in the SC, epidermis, and dermis at 2, 4, 6, and 24 h time points using frozen human skin treated ex vivo with CsA-in-oil, CsA nanoemulsion, and gel formulations (mean ± SD, N = 9 mice × 3 donors, two-way ANOVA Dunnett's multiple comparisons *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Again, the CsA gel strongly and preferentially penetrated the epidermis and dermis, whereas the CsA nanoemulsion tended to remain in the SC. [Figure 8]Figures 8A-8I show the immunosuppressive effect of CsA-Tempol gel on local inflammation in vivo, as revealed by histological analysis of skin sections obtained from DNFB-induced animal models, including sham mice (uninduced) (A), mice exposed to DNFB and with induced ear inflammation (untreated) (B), mice treated with 0.1%, 0.2%, or 0.5% CsA gel (C-E), mice treated with 0.1%, 0.2%, or 0.5% CsA-5% Tempol gel (F-H), and mice treated with 5% Tempol gel alone (I). All treatment groups showed excellent recovery from local inflammation and were essentially histologically normal (see Sham 8A). [Figure 9] Figure 9 shows the immunosuppressive effects of the formulations by the profiles of three major inflammatory cytokines, TNF-α (Figure 9), IL-6 (Figure 10), and INF-γ (Figure 11), at the DNFB-induced site (mean ± SD, N = 5 mice, ANOVA Dunnett normality test, *p≦0.05, **p≦0.002, ***p<0.001). Both CsA and tempol had a potent reducing effect on cytokine levels. The 0.2% CsA-5% tempol and 0.5% CsA-5% tempol gel formulations showed significant effects on reversing inflammation, at least by the levels of TNF-α and IL-6. [Figure 10] Figure 10 shows the immunosuppressive effects of the formulations by the profiles of three major inflammatory cytokines, TNF-α (Figure 9), IL-6 (Figure 10), and INF-γ (Figure 11), at the DNFB-induced site (mean ± SD, N = 5 mice, ANOVA Dunnett normality test, *p≦0.05, **p≦0.002, ***p<0.001). Both CsA and tempol had a potent reducing effect on cytokine levels. The 0.2% CsA-5% tempol and 0.5% CsA-5% tempol gel formulations showed significant effects on reversing inflammation, at least by the levels of TNF-α and IL-6. [Figure 11]Figure 11 shows the immunosuppressive effects of the formulations by the profiles of three major inflammatory cytokines, TNF-α (Figure 9), IL-6 (Figure 10), and INF-γ (Figure 11) at the DNFB-induced site (mean ± SD, N = 5 mice, ANOVA Dunnett normality test, *p≦0.05, **p≦0.002, ***p<0.001). Both CsA and tempol had a potent reducing effect on cytokine levels. The 0.2% CsA-5% tempol and 0.5% CsA-5% tempol gel formulations showed significant effects on reversing inflammation, at least by the levels of TNF-α and IL-6. [Figure 12] Figure 12 shows the restoration effect of the formulations on hair growth in an AGA animal model, including sham, alopecia model (untreated), and groups treated with blank gel (placebo), 0.5% CsA nanoemulsion, 0.5% CsA in castor oil, 0.5% CsA gel, 5% Tempol gel, and 0.5% CsA-5% Tempol gel. Quantitative analysis of hair restoration (pixel density by area under the curve, AUC%) on day 14 (mean ± SD, N = 8 mice, ANOVA vs. Sham by Dunnett normality test; *p≦0.05, **p≦0.002, ***p<0.001) suggested superior performance of CsA-Tempol gel compared to other formulations, exceeding hair density in Sham. [Figure 13] Figures 13A-13H show the restorative effect on hair growth in histological sections (H&E stained) from the same experiment, again demonstrating the superior effect of the gel formulations CsA-Tempol (13h) and CsA gel (13c) in terms of dermal thickness and number of hair follicles, as well as overall restoration of primary skin structure, which was similar to the Sham (13a). The other treatments resulted in a thin dermis and significant loss of hair follicles, similar to the alopecia model (13b). [Figure 14]Figure 14 shows the same effect demonstrated in another experiment in an AGA animal model using only a gel formulation containing a lower concentration of active ingredient, including a sham, alopecia model (untreated), and groups treated with a 0.1% CsA gel formulation, a 0.5% Tempol gel formulation, or a 0.1% CsA-0.5% Tempol gel formulation. Figure 14 shows animals on days 2, 10, 12, and 14 after treatment. Both the sham (14a) and CsA-Tempol groups (14e) showed significant hair regrowth on day 12 and complete hair recovery on day 14, compared to the partial effect of the CsA gel (13c) and Tempol gel (13d). [Figure 15] Figure 15 shows the same effect demonstrated in another experiment in an AGA animal model using only a gel formulation containing a lower concentration of active ingredient, including: sham, alopecia model (untreated), and groups treated with 0.1% CsA gel, 0.5% Tempol gel, or 0.1% CsA-0.5% Tempol gel. Figure 15 shows quantitative analysis of hair regrowth (AUC% hair regrowth) in the same animals, again demonstrating the superior effect of 0.1% CsA-0.5% Tempol gel compared to the other gels (mean ± SD on day 14, N = 5 mice, ANOVA vs. Sham by Dunnett normality test, **p≦0.002). DETAILED DESCRIPTION OF THE INVENTION

[0144] In general, nanoemulsion-based topical drug delivery systems have attracted considerable attention due to their ability to minimize adverse effects and promote skin penetration. The inventors of this technology developed a novel composite nanoemulsion gel formulation of CsA and tempol that was shown to be highly effective for topical application to the skin. Proof of concept was demonstrated by the present example, in which CsA nanoemulsion in castor oil and tempol were incorporated into Pemulen gel. The present study included detailed characterization of this formulation and its derivatives, including physicochemical, encapsulation, recovery, and stability experiments, as well as skin penetration and clinical efficacy experiments in human keratinocytes in vitro and ex vivo in human skin samples, and in an established mouse model of androgenetic alopecia (AGA) using 5α-androstane-3β,17β-diol (DHT), where even low concentrations of CsA and tempol proved effective, the lowest concentrations reported to date for animal models of androgenetic alopecia (AGA) and AA.

[0145] More specifically, CsA nanoemulsion gels were prepared by dispersing CsA-loaded nanoemulsions in an aqueous gel (Pemulen) of a pseudoplastic cross-linked copolymer of acrylic acid and alkyl acrylate comonomers. A composite CsA-Tempol gel formulation was obtained by dissolving Tempol in the CsA nanoemulsion gel. The CsA nanoemulsions had relatively uniform particle sizes (150-200 nm, PDI < 0.20), and the particle size and zeta potential increased with increasing CsA loading ( - 25~ - 35 mV). The CsA nanoemulsion has high CsA recovery (approximately 90%) and encapsulation efficiency (EE, approximately 85%). The gel formulation exhibits increased particle size and decreased zeta potential (240-260 nm, - 45~ -The gel exhibited a voltage drop of 55 mV, indicating improved electrostatic stability and minimized droplet coalescence. Gel stability studies showed that the active ingredients, CsA and Tempol, remained stable at 37°C for at least 12 weeks (3 months), with ongoing studies testing stability for 6 months. Rheological analysis showed that the gel exhibited Newtonian pseudoplastic properties and increased viscosity (9.2-17.7 Pa·S) in the drug-loaded gel formulation.

[0146] Overall, incorporating CsA nanoemulsions into Pemulen's crosslinked network offered significant benefits: improved physical and chemical stability, reduced tendency of oil droplets to coalesce, and reduced risk of CsA leakage.More generally, hydrogels are the preferred type of topical formulation, accounting for over 80% of the total market.

[0147] Preliminary cytotoxicity studies in a human keratinocyte cell line demonstrated that the CsA gel formulation was safe and nontoxic at CsA concentrations below 5 μg / mL. Pemulen gel was also nontoxic at concentrations up to 2 mg / mL (relevant for topical application). Permeation studies in an ex vivo human skin model demonstrated that the gel formulation had excellent drug permeation kinetics upon topical application, with preferential distribution of the drug (CsA) to the epidermis and dermis, the target tissue where hair follicles reside. The maximum amount of CsA in the dermis (approximately 6.5 μg / g tissue) was achieved 24 hours after gel treatment, outperforming both the nanoemulsion and oil formulations.

[0148] The main problems with the topical use of nanoemulsions are: (1) they are fluid and not suitable for topical application, and (2) they tend to coalesce and increase in droplet size, thereby reducing penetration into deeper skin tissues, specifically the dermis where the drug is most needed for alopecia. The gel, with its good bioadhesive and other properties, prevented oil droplet coalescence, reduced drug leakage, and improved drug penetration into deeper layers of the skin.

[0149] Further experiments in an established animal model of inflammation using repeated topical application of DNFB to mouse ears showed that histology of inflamed ears was characterized by significant epidermal thickness and immune cell infiltration compared with sham (uninduced) controls, whereas histology of animals treated with CsA and / or Tempol gel formulations was similar to sham controls. These observations were further supported by the levels of specific cytokines, showing that treatment with CsA and Tempol gel significantly reduced levels of two important inflammatory cytokines, TNF-α and IL-6, and potentially IFN-γ. Overall, these experiments support the idea that CsA and Tempol gel formulations, alone or in combination, can act as effective immunosuppressants.

[0150] Finally, experiments in an established AGA model demonstrated that gel formulations, CsA Tempol, and the CsA-Tempol combination were more effective in inducing hair regrowth than oil control and nanoemulsion formulations. The 0.1% CsA + 0.5% Tempol combination was the most effective, demonstrating the fastest and densest hair regrowth, superior to sham treatments. These findings were further supported by histological analysis of mouse skin sections, which showed that treatments with CsA gel and CsA-Tempol gel had histological signs similar to those of sham treatments in terms of dermal thickness and hair follicle density. Furthermore, the CsA-Tempol combination gel performed better with lower concentrations of the active ingredient, 0.1% CsA and 0.5% Tempol, compared to 0.5% CsA alone, thus achieving both efficacy and safety.

[0151] Together, these findings provide proof-of-concept for the topical application of a novel gel formulation combining two known active ingredients, CsA and Tempol, for the effective treatment of alopecia. The clinical efficacy and safety of this formulation have thus far been demonstrated in an established animal model of AGA, achieving significant synergistic therapeutic effects at reduced concentrations of both active ingredients, thus reducing the likelihood of systemic toxicity or serious side effects. Importantly, the CsA and Tempol gel formulation proved effective on several levels: (1) restoring normal hair growth, (2) restoring original skin morphology, and (3) reducing local inflammation.

[0152] Regarding AA, another common clinical manifestation of alopecia, AA is an autoimmune disease characterized by non-scarring hair loss, and may involve the whole body.Hair loss is associated with the infiltration of autoreactive T cells into hair follicles, resulting in inflammation and the formation of reactive oxygen species.Based on the findings of the present invention in AGA model, it is expected that the gel formulation of CsA and Tempol is likely to be effective for AA and other rarer forms of alopecia.

[0153] Some embodiments of the present invention will now be described, by way of example, with reference to the accompanying drawings.

[0154] Example 1: Physicochemical properties of gel formulations 1.1 Materials and Methods 1.1.1 Materials CsA was from Teva (Israel); Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), polysorbate 80 (Tween® 80), sorbitan monooleate (Span® 80), Cremophor® EL, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 1-fluoro-2,4-dinitrobenzene (DNFB), and 5α-androstane-3β,17β-diol (DHT) were from Sigma-Aldrich (Israel); 3[H]-CsA was from ARC American Radiolabeled Chemicals, Inc. (USA); Ultima-Gold® liquid scintillation cocktail and Solvable® were from Perkin-Elmer (USA); macrogol 15-hydroxystearate (Solutol™ HS15) was from BASF (Germany); dimethyl sulfoxide (DMSO) was from Fluka (Switzerland); glycerol tributyrate was from Alfa Aesar (UK); castor oil was from Tamar Laboratory Supplies Ltd (Israel); anhydrous glycerol, 30% hydrogen peroxide, and all organic solvents (HPLC grade) were from JT Baker (The Netherlands); tissue culture medium was from Biological Industries Ltd (Israel); HaCaT cells (human keratinocyte cell line) were from PromoCell (Germany); Pemulen™ TR-2, a high molecular weight cross-linked copolymer of acrylic acid and a hydrophobic C10-30 alkyl acrylate comonomer, was from BF from Goodridge (USA); and Lipoid E80 from Lipoid GmbH (Ludwigshafen, Germany).

[0155] 1.1.2 Preparation and characterization of CsA nanoemulsion CsA nanoemulsions were prepared by the solvent displacement method. Briefly, cyclosporine (CsA), Tween 80, and castor oil were dissolved in acetone (9 mL). Lipoid E80 was prepared in ethanol (1 mL) and added to the acetone solution (10 mL of organic phase) and stirred at 1600 rpm for 30 minutes. The organic phase was added dropwise to DDW (20 mL) containing 0.1% w / v Solutol SH15 and stirred at 1000 rpm for 15 minutes. The acetone was evaporated using a rotary evaporator. Gel formulations were prepared based on three starting nanoemulsions with various CsA concentrations (0.05%, 0.1%, 0.2%, and 0.5% w / w) and other ingredients, as detailed in Table 1 below. TIFF2026501730000011.tif72170

[0156] 1.1.3 Preparation of CsA-Tempol gel Tempol was dissolved in CsA nanoemulsions to prepare CsA-Tempol nanoemulsions. Briefly, 20 mL of CsA nanoemulsions (containing 0.05%, 0.1%, 0.2%, and 0.5% CsA (wt / wt)) were mixed with various concentrations of Tempol (0.05%, 0.1%, 0.5%, 2%, and 5% (wt / wt)) at 1500 rpm for 3 minutes, as detailed in Table 2 below. CsA gels were prepared by dispersing the CsA nanoemulsions in Pemulen polymer. Pemulen (0.25% wt / wt) was added to the CsA nanoemulsions or CsA-Tempol nanoemulsions to prepare CsA and CsA-Tempol gel formulations, respectively. In the latter case, Pemulen (50 mg) was dispersed in CsA-Tempol nanoemulsion (20 mL) with stirring, and 1 M NaOH (18 μL) was added to produce a gel formulation. Placebo gel was prepared by dispersing Pemulen in blank nanoemulsion, and glycerol (5% w / w) was added to prevent skin drying. TIFF2026501730000012.tif51170

[0157] 1.1.4 Physicochemical characterization of the formulation The mean particle size, polydispersity index (PDI), and zeta potential of the formulations diluted in DDW (2 mL), nanoemulsions (10 μL), and gels (20 mg) were evaluated using a Zeta Sizer Nano (ZSP, 4 mW 632.8 nm "red" laser) at 25 °C in multimodal narrow modes (high resolution) with a display range of 0.6–6000 nm (average of three measurements per sample, 14 runs per measurement).

[0158] 1.1.5 Identification of CsA / Tempol Quantitative determination of CsA and tempol was performed by HPLC (Thermo Scientific, USA, equipped with an Xterra C8 column). The preparation (20 μL) was dissolved in acetonitrile (980 μL), and the mobile phase was an isocratic mixture of acetonitrile (70%) and water (30%) at a flow rate of 0.5 mL min -1 UV detection was performed at 210 nm for CsA and 234 nm for Tempol, the column temperature was maintained at 60°C, and the sample injection volume was 10 μL in partial mode.

[0159] 1.1.6 Encapsulation efficiency and recovery rate The drug recovery rate (%) was calculated as follows: Equation 1. Encapsulation efficiency (EE%) was calculated as follows: TIFF2026501730000013.tif22170

[0160] 1.1.7 Transmission Electron Microscope (TEM) The CsA and blank nanoemulsions were analyzed by TEM (JEM-1400 Plus Microscope, JEOL USA) using phosphotungstic acid staining as a contrast agent. Briefly, samples (5 μL) were placed on formvar / carbon-coated copper grids (200 mesh, EMS) and mixed with PTA (2%, 5 μL). The excess was blotted off, and the grids were air-dried.

[0161] 1.1.8 Cryo-Transmission Electron Microscopy (Cryo-TEM) Cryo-TEM allows direct imaging without the use of contrast agents. Samples were prepared by applying 2–3 μL of nanoemulsion to glow-discharge-treated TEM grids (300-mesh Cu lace substrates, Ted Pella, Ltd., Reading, USA). Excess liquid was blotted off, and the specimens were vitrified in liquid nitrogen (Vitrobot Mark IV, FEI) and examined in an FEI Tecnai 12 G2 TWIN TEM operating at 120 kV.

[0162] 1.1.9 Scanning Electron Microscope (SEM) The topography of the CsA gel and blank gel was examined by scanning electron microscopy (SEM). Briefly, the gel formulation (10 mg) was placed on a glass (1 × 1 cm 2 ) and air-dried overnight at room temperature. The gel was sputter-coated with an iridium / gold mixture before analysis. Images were taken at various low and high positions.

[0163] 1.1.10 Gel rheology experiments The rheological behavior was investigated using a rheometer (101 PP-25, Anton Paar, Austria) with a flat plate sensor. The flow curves were obtained by varying the shear rate from 0 to 1000 s in 120 s . -1 The shear stress was recorded throughout the experiment.

[0164] 1.2 Results and Discussion 1.2.1 Physicochemical characterization of CsA nanoemulsions The physicochemical properties of the nanoemulsions are summarized below in Table 3. In summary, the CsA nanoemulsions had a relatively narrow particle size distribution (PDI<0.20), a mean particle size in the range of approximately 150-190 nm, with CsA loading slightly increasing the particle size, and a CsA recovery of over 90% and an EE of 85%. TIFF2026501730000014.tif53170

[0165] TEM and cryo-TEM image analysis showed that both the blank and CsA (0.5%) nanoemulsions were highly monodisperse and spherical in shape (see Figure 1). In addition, CsA was completely dissolved in the delivery system, with no trace of CsA particles outside the spheres (no undissolved CsA).

[0166] 1.2.2 Physicochemical characterization of gel formulations SEM analysis of the blank and 0.5% CsA gel formulations showed that the nanoemulsion droplets were completely immersed in the gel matrix (Figure 2). The physicochemical properties, active ingredient loading, particle size, and zeta potential of gel formulations containing various concentrations of CsA and tempol are summarized in Table 4. The chemical stability of the two active ingredients, CsA and tempol, in selected gel formulations is shown in Table 5. TIFF2026501730000015.tif96170TIFF2026501730000016.tif131170

[0167] The results show that all gel formulations are characterized by a relatively high loading capacity of the active ingredients, a relatively uniform particle size within the range of 240-260 nm, and a zeta potential of approximately (-45) to (-55) mV. HPLC analysis of the two active ingredients in selected gel formulations showed that CsA and Tempol retained their chemical stability for at least 6 weeks. In summary, incorporating CsA nanoemulsions into the crosslinked network of Pemulen offered significant advantages: improved physical and chemical stability and a reduced risk of CsA leakage from the oil droplets during equilibration.

[0168] 1.2.3 Rheological behavior of gel formulations Rheological analysis showed that all gel formulations were non-Newtonian pseudoplastic and maintained a relatively constant viscosity at 37°C over extended periods, i.e., at least 6 weeks (Figure 3) and at least 12 weeks (Figure 4). The 0.1% CsA-0.5% Tempol gel had the highest viscosity (approximately 17.7 Pa·S) compared to the blank gel (approximately 9.2 Pa·S). Higher viscosity is advantageous for topical application.

[0169] Example 2: Safety and skin permeability of gel formulations 2.1 Materials and Methods 2.1.1 In vitro cytotoxicity assay HaCaT cells (human keratinocytes) were seeded in 96-well plates (10,000 cells / well) in DMEM supplemented with 10% FCS, streptomycin (0.1 mg / mL), and penicillin (100 units / mL) overnight at 37°C. The medium was replaced with fresh medium (100 μL) along with increasing concentrations of the test formulations. Cell viability was determined after 24 hours by MTT assay according to the manufacturer's instructions, measuring the OD at 570 nm and 690 nm.

[0170] 2.1.2 Ex vivo skin penetration analysis Human skin samples were obtained from healthy adults after elective cosmetic surgery (abdominoplasty). The skin was dissected free from the underlying fat and subcutaneous tissue, cut into small pieces (2 × 2 cm), sectioned to a thickness (0.75–1.0 mm), and stored at −80°C. For the experiments, the thawed skin samples were split into sections with a spreading area (1 cm) using receptor fluid (10% ethanol in PBS). 2 The system was maintained at 37°C, and the skin surface temperature was maintained at 32±1°C. 125 μg of CsA (non-radiolabeled) and 125 μg of [ 3 A test sample (25 μL) containing [H]-labeled CsA (1 millicurie / mL) was applied to the skin. Skin samples were removed at different time intervals (2, 4, 6, and 24 hours) and washed with 1 mL of receptor fluid. Different skin layers were obtained as follows: (1) the stratum corneum (SC) was removed using a skin sampling disk (CuDERM Corp, USA), pooled, and dissolved in DMSO; (2) the epidermal layer was scraped off with a scalpel; (3) the dermal layer was cut into 1-2 mm pieces; the separated layers were chemically dissolved with Solvable and H2O2; (4) the receptor fluid was also collected. Radioactivity was measured using a scintillation counter (Tri-CARB 2900TR, Perkin Elmer, USA).

[0171] 2.2 Results and Discussion 2.2.1 Absence of cytotoxicity The cytotoxic effects of various concentrations of CsA (2.5, 5, and 10 μg / mL) in gel form and as free drug were tested in comparison with blank Pemulen gel (2 mg / mL). Overall, CsA was found to be nontoxic at concentrations below 5 μg / mL, both in gel form and as free drug (Figure 5). Pemulen gel was also nontoxic at a concentration of 2 mg / mL, the concentration used for topical application.

[0172] 2.2.2 Skin permeability of CsA in a human ex vivo model The skin permeability of CsA was examined by topically applying CsA oil, nanoemulsion, and gel formulations to fresh (live) and frozen human skin tissues ex vivo. Preliminary experiments suggested that the CsA permeation profiles of frozen and fresh tissues were essentially similar. In both cases, the CsA gel showed preferential penetration of CsA into deeper layers of the skin tissue, i.e., the epidermis and dermis (Figures 6A-6B).

[0173] In more detailed experiments, the frozen human skin tissue described above was exposed to topical application of CsA oil, nanoemulsion, and gel formulations, and CsA penetration into the SC, epidermis, and dermis was monitored at 2, 4, 6, and 24 hours. Essentially, the results confirmed previous findings that CsA gel preferentially penetrated the epidermis and deeper skin layers of the dermis. In contrast, CsA nanoemulsion distributed primarily to the SC (Figures 7A-D).

[0174] The improved penetration of the active ingredient into deeper skin layers, characteristic of nanoemulsions and gel formulations rather than the free drug form, is further accompanied by the general benefits of gels for localized topical application. Taken together, these experiments suggest that the incorporation of CsA nanoemulsion into the Pemulen gel polymer matrix network provides a highly effective, stable, and safe CsA formulation that is particularly advantageous for topical application.

[0175] Example 3: Immunosuppressive effect of CsA-tempol in gel formulation 3.1 Materials and Methods 3.1.1 In vivo inflammation models For the inflammation experiments, we used an established local inflammation model in mice, namely, the ears of mice that developed inflammation after repeated exposure to DNFB. Briefly, 0.2% DNFB (50 μL in a 4:1 ratio of acetone and olive oil, respectively) was applied to the abdomen of shaved mice (8-week-old male C57BL / 6 mice) for three consecutive days. Subsequently, 0.2% DNFB (10 μL) was brushed onto the back of the mouse ear every other day, followed by topical application of 30 mg of Tempol gel, CsA, and CsA-Tempol gel formulations containing various concentrations of CsA. Experimental groups included sham mice (A), inflammation model mice (B) (untreated), mice treated with CsA gel (0.1%, 0.2%, and 0.5% CsA) (C)-(E), mice treated with CsA-Tempol gel (0.1%, 0.2%, and 0.5% CsA and 5% Tempol) (F)-(H), and mice treated with 5% Tempol gel (I). All mice were sacrificed on day 14, and skin tissues were analyzed by H&E staining. The experimental procedure is outlined below. TIFF2026501730000017.tif60170

[0176] H&E staining was performed using standard protocols.

[0177] 3.1.3 Biochemical analysis The levels of inflammatory cytokines (TNF-α, IL-6, and IFN-γ) in ear tissue were assessed using ELISA. Briefly, 20–30 mg samples were homogenized (Bertin Technologies, USA) in PBS (1 mL) containing protease inhibitors (EZBlock, USA) at 4500 rpm for eight cycles of 30 seconds with a 40-second break between cycles. The levels of TNF-α, IFN-γ, and IL-6 in the lysates were determined according to the manufacturer's instructions (DuoSet ELISA, USA).

[0178] 3.2 Results and Discussion 3.2.1 Histological analysis of local inflammation in vivo Histological results showed that all treatment groups, including CsA, CsA-Tempol gel with various CsA concentrations, and Tempol gel, prevented or significantly attenuated signs of inflammation, namely, epidermal thickness and immune cell infiltration into the skin tissue (Figures 8A-8I). The histological manifestations of the treatment groups were essentially similar to those of the sham group and significantly different from those of the inflammatory model (untreated group), which showed a marked increase in epidermal thickness and massive infiltration of immune cells. These results suggest that both CsA and Tempol in the gel formulation are effective inhibitors of local inflammation.

[0179] 3.2.2 Analysis of major inflammatory cytokines Subsequent analysis of key inflammatory cytokines in ear tissue further supported the histological observations (Figures 9-11). Treatment with both CsA and Tempol gel formulations successfully restored TNF-α, IL-6, and, to a lesser extent, INF-γ to normal or near-normal levels at the DNFB-induced site, up to levels comparable to those in sham mice.

[0180] Example 4: Enhanced effect of Tempol-CsA gel on hair growth restoration 4.1 Materials and Methods 4.1.1 In vivo alopecia model A well-established animal model of androgenetic alopecia (AGA) was used in hair restoration experiments. Briefly, mice (8-week-old female C57BL / 6 mice, telogen effluvium) were subjected to AGA induction by intraperitoneal injection of 5α-androstane-3β,17β-diol (DHT, 20 mg / kg). In this case, 10 mg of 5α-androstane-3α,17β-diol was dispersed in an equal volume of ethanol, 1,2-propanediol, and saline solution (1 mL). The injection consisted of 60 μL of DHT dispersion and 140 μL of 2-hydroxypropyl-β-cyclodextrin solution. Prior to the experiment, the coal hair on the back was removed, and all mice had telogen hair (no dark skin) before treatment. Each group was housed separately and provided with pelleted food and water ad libitum.

[0181] In the preliminary experiment, sham mice (a), AGA model (untreated) (b), and treatment groups (N = 8 per group) with 0.5% CsA gel (c), blank gel (d), 0.5% CsA nanoemulsion (e), 0.5% CsA in castor oil (f), 5% Tempol gel (g), or 5% CsA-5% Tempol gel (h) were included. Treatments were performed on the shaved back (4 cm 2 ) was applied daily (50 mg of each formulation), and DHT was injected once weekly. Mice were photographed every other day, and hair growth was quantified using Fiji ImageJ software. After the treatment period (2 weeks), the mice were sacrificed, and only mice that developed AGA (80%) were included in further experiments (N=8).

[0182] Subsequent experiments tested treatment with selected gel formulations containing clinically relevant concentrations of 0.1% CsA and 0.5% Tempol, and experimental groups included sham (a), AGA model (untreated) (b), and groups treated with 0.1% CsA gel (c), 0.5% Tempol gel (d), or 0.1% CsA-0.5% Tempol gel (e) (N=5 per group).

[0183] 4.1.2 Histological analysis Dorsal skin and ear samples were fixed overnight in 4% formaldehyde (pH 7.4) and stored in 70% ethanol for 1 week, then embedded in paraffin, sectioned, and stained (H&E, hematoxylin and eosin staining).

[0184] 4.2 Results and Discussion 4.2.1 In vivo observation of AGA model In preliminary experiments, formulations containing a higher concentration of CsA active ingredient (0.5% CsA) were tested (CsA nanoemulsion, CsA in castor oil, CsA gel, Tempol (B gel), and CsA-Tempol gel). Hair growth was assessed by visual evaluation on days 8, 10, 12, and 14 (not shown). Results indicated that only the sham and CsA-Tempol groups showed significant hair regrowth on day 12 and complete recovery on day 14, whereas treatment with a single active ingredient (CsA or Tempol) showed only partial hair recovery. The AGA model (untreated) and blank gel treatment did not show any hair growth recovery even on day 14.

[0185] Quantitative analysis of hair regrowth (pixel density by AUC%) confirmed the superior performance of CsA-Tempol gel compared to Tempol gel, CsA gel, and other treatments (Figure 12). When statistical analysis was performed in comparison to the AGA model, the effect of 0.5% CsA-5% Tempol gel on hair growth recovery was essentially similar to Sham (no statistically significant difference). Further statistical analysis comparing the effect of each formulation with CsA gel suggested that the recovery effect of the CsA / Tempol gel combination may be superior to Sham and other Tempol and CsA gels.

[0186] 4.2.2 Histological analysis of in vivo AGA models Further histological analysis of skin tissue obtained in preliminary experiments confirmed the strong immunosuppressive effect of the gel formulation (Figure 13). In contrast to the AGA model, which has a thin dermis and lacks hair follicles, the sham model exhibited a thick dermis and numerous hair follicles, and the effect of the 0.5% CsA-5% Tempol gel was closest to the sham model in terms of restoration of primary skin morphology. The 0.5% CsA gel also demonstrated a relatively high degree of skin preservation. Other treatments, including 0.5% CsA nanoemulsion, 0.5% CsA in castor oil, and blank gel, were essentially ineffective.

[0187] In another experiment, formulations containing clinically relevant concentrations of 0.1% CsA and 0.5% Tempol (CsA, Tempol, and CsA-Tempol Gel) were tested in an AGA model. Animals were subjected to visual evaluation (FIG. 14) and quantitative analysis of hair regrowth (FIG. 15) on days 2, 10, 12, and 14. The results of this experiment replicated the superior efficacy of the CsA-Tempol gel formulation even at lower concentrations of the active ingredient, suggesting the surprisingly improved properties of the CsA-Tempol combination in a gel formulation, as opposed to gel formulations containing a single active ingredient.

[0188] Overall, the in vivo animal model observations suggest that the CsA-Tempol combination in a gel formulation is surprisingly effective and safe for the topical treatment of alopecia and for promoting hair growth in general, as well as for reducing local skin inflammation and reconstituting primary skin tissue morphology. Furthermore, the effects of the CsA-Tempol gel formulation were superior to, and potentially synergistic with, gel formulations containing single active ingredients, and even at relatively low concentrations of 0.1% CsA and 0.5% Tempol, the effects were sufficiently significant in terms of hair regrowth. This last point is particularly important in light of the known adverse reactions associated with topical and systemic use of CsA.

Claims

1. A topical formulation comprising a matrix polymeric emulsifier composed of a crosslinked copolymer of acrylic acid and acrylate, and a nanoemulsion comprising an immunosuppressant and a cyclic nitroxide.

2. 10. The formulation of claim 1, which is a gel formulation selected from an aqueous gel formulation, an emulsion gel formulation, and a non-aqueous gel formulation.

3. 10. The formulation of claim 1, wherein the matrix polymer emulsifier is a high molecular weight copolymer of acrylic acid and an acrylate.

4. The acrylate is C １０ ~C ３０ 4. The formulation of claim 3, which is an alkyl acrylate.

5. 5. A formulation according to any one of claims 1 to 4, wherein the matrix polymer emulsifier is selected from high molecular weight copolymers of acrylic acid and acrylates, the acrylic acid and the acrylates being crosslinked with allylpentaerythritol.

6. A formulation according to any one of claims 1 to 5, comprising at least one thickening agent.

7. 7. The formulation of claim 6, wherein the thickening agent is selected from polyacrylates and their derivatives, carbopol, polycarbophil, poloxamer, polypropylene glycol, waxes, polysaccharides, cellulose, hyaluronic acid, chitosan and its derivatives, natural polyproteins, gelatin, collagen, polyamino acids, mineral clays, magnesium aluminum silicate, and polymeric silicon.

8. The formulation of any one of claims 1 to 7, wherein the nanoemulsion and the matrix polymer emulsifier are provided as a stable gel.

9. 9. The formulation of any one of claims 1 to 8, wherein the immunosuppressant is selected from agents that reduce or suppress the activity of the immune system in vivo, and optionally is selected from steroids, cell growth inhibitors, antibodies, immunophilins, mycophenolates, and tumor necrosis factor (TNF-α) inhibitors.

10. 10. The formulation of claim 9, wherein the immunosuppressant is selected from azathioprine (Imuran), cyclosporine A (CsA), mercaptopurine (Purinethol, 6-MP), rapamycin, fujimycin, and methotrexate.

11. 11. The formulation of claim 10, wherein the immunosuppressant is CsA.

12. The formulation - Acrylic acid and C １０ ~C ３０ a matrix of a crosslinked copolymer with an alkyl acrylate; a nanoemulsion comprising a lipophilic immunosuppressant and a cyclic nitroxide, said cyclic nitroxide being a compound of formula I: During the ceremony, A represents a carbon atom or a carbon chain containing up to three carbon atoms, one of which is substituted with an oxygen atom or an oxygen-containing group, or one or more of which is substituted with one or two bromine atoms; R １ , R ２ , R ３ , and R ４ each independently represents H and C １ ~C ５ alkyl; or R １ and R ２ together with the carbon atoms to which they are attached form a 3- to 7-membered ring, and / or R ３ and R ４ together with the carbon atoms to which they are attached form a 3- to 7-membered ring; R ５ represents aldehydes, ketones, carboxylic acids, carbonyl groups, -O-, -S-, -OH, -SH, -COOH, and -COONH. ２ , —CN, and a group selected from a primary amine, a secondary amine, a tertiary amine, or a quaternary amine; and O represents an oxygen radical; Nanoemulsion and The formulation according to any one of claims 1 to 11, comprising:

13. The formulation of claim 12, wherein the cyclic nitroxide compound is a five-membered heterocyclic structure.

14. The formulation of claim 12, wherein the cyclic nitroxide compound is a six-membered heterocyclic structure.

15. The formulation of claim 12, wherein the cyclic nitroxide compound is a five-membered heterocyclic structure containing an endocyclic double bond.

16. In the cyclic nitroxide compound of formula (I), R １ , R ２ , R ３ , and R ４ Each of the groups independently represents C １ ~C ５ 13. The formulation of claim 12, wherein the alkyl group is alkyl.

17. In the cyclic nitroxide compound of formula (I), R １ , R ２ , R ３ , and R ４ 13. The formulation of claim 12, wherein each of is independently selected from methyl, ethyl, propyl, isopropyl, butyl, and pentyl.

18. In the cyclic nitroxide compound of formula (I), R １ , R ２ , R ３ , and R ４ 13. The formulation of claim 12, wherein each of is a methyl group.

19. The cyclic nitroxide compound of formula (I) is a compound of formula (II): In the formula, A and R ５ 13. The formulation of claim 12, wherein each of is as defined in claim 12.

20. 20. The formulation of claim 12 or 19, wherein in the cyclic nitroxide compound of formula (I) or formula (II), A is a carbon group containing 1, 2, or 3 carbon atoms, and at least one of the carbon atoms is bonded to an oxygen-containing group.

21. The oxygen-containing group is a hydroxyl group or an ether group, or the oxygen-containing group is R ５ 20. The formulation of claim 19, wherein the oxygen-containing group is selected from:

22. The A-R ５ The groups are A=O, A-OH, A-COOH, and A-COONH ２ 20. The formulation of claim 19, wherein the group is selected from:

23. A-R ５ But -CHR ５ --, --CHR ５ -CH ２ --, --CHR ５ -CH ２ -CH ２ - or -CH ２ -CHR ５ -CH ２ The formulation of claim 19, wherein

24. The A-R ５ The group is —CHR ５ --, --CHR ５ -CH ２ -CH ２ - or -CH ２ -CHR ５ -CH ２ - and R ５ 20. The formulation of claim 19, wherein is an oxygen-containing group.

25. The A-R ５ The group is —CH(OH)—, —CH(OH)—CH ２ -CH ２ - or -CH ２ -CH(OH)-CH ２ The formulation of claim 24, wherein

26. The A-R ５ The group is —CH ２ -CH(OH)-CH ２ The formulation of claim 24, wherein

27. The cyclic nitroxide compound has a five-membered heterocyclic structure, and the A-R ５ The group is —CH ２ 20. The formulation of claim 19, wherein the compound is -CH(OH)-.

28. The cyclic nitroxide compound has a 6-membered heterocyclic structure, and the A-R ５ The group is —CH ２ -CH(OH)-CH ２ The formulation of claim 19, wherein

29. The formulation of claim 1 , wherein the cyclic nitroxide is tempol.

30. 2. The formulation of claim 1, wherein the cyclic nitroxide is a compound of the following formula:

31. 2. The formulation of claim 1, wherein the cyclic nitroxide is a compound of the following formula:

32. 2. The formulation of claim 1, wherein the cyclic nitroxide is selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

33. 33. The formulation of any one of claims 1 to 32, wherein the nanoemulsion comprises CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl.

34. At least one of the following: a matrix of a crosslinked copolymer of acrylic acid and a C10 to C30 alkyl acrylate, and - a nanoemulsion comprising CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl, The formulation according to any one of claims 1 to 33, comprising at least one of:

35. 35. The formulation of any one of claims 1 to 34, wherein the nanoemulsion has a particle size in the range of about 200 nm to about 300 nm.

36. The nanoemulsion comprises acrylic acid and C １０ ~C ３０ A formulation according to any one of claims 1 to 35, which is encapsulated in a matrix of a cross-linked copolymer with an alkyl acrylate to an extent of 60% to 100%.

37. 37. The formulation of any one of claims 1 to 36, which is stable at 37°C for a period of at least 3 months.

38. A formulation according to any one of claims 1 to 37, which distributes preferentially into deeper skin layers.

39. 39. The formulation of any one of claims 1 to 38, which has increased penetration into the epidermal and dermal layers of the skin compared to penetration of the formulation into the stratum corneum.

40. 10. The formulation of claim 1, comprising CsA and tempol.

41. 41. The formulation of claim 40, wherein the CsA is at a concentration in the range of about 0.01% to about 0.5% and the Tempol is at a concentration in the range of about 0.1% to about 5% (w / w).

42. 42. The formulation of claim 41, wherein the concentration of CsA is about 0.1% and the concentration of Tempol is about 0.5% (wt / wt).

43. A formulation according to any one of claims 1 to 42 for use in treating or promoting hair growth on the human skin or scalp and / or for the histomorphological reconstruction of the skin.

44. 43. A formulation as claimed in claim 41 or 42, for use in treating or promoting hair growth on the skin or scalp of a human and / or for the histomorphological remodelling of the skin, wherein the effects of CsA and Tempol on the treatment or promotion of hair growth on the skin or scalp of the human and / or for the histomorphological remodelling of the skin are synergistic.

45. Use of a formulation according to any one of claims 1 to 44 in the treatment of alopecia.

46. 46. ​​The use according to claim 45, wherein the alopecia is selected from androgenic alopecia, androgenetic alopecia (AGA), toxic alopecia, alopecia areata (AA), telogen effluvium, alopecia caused by endocrine disorders, metabolic disorders, and nutritional disorders, drug-induced alopecia, mechanical alopecia, alopecia caused by skin diseases, cicatricial alopecia, congenital alopecia, and trichotillomania.

47. 47. The use according to claim 46, wherein the alopecia is androgenetic alopecia (AGA) or alopecia areata (AA).

48. 43. A method of treating male or female alopecia in a subject in need thereof, comprising topically administering to said subject a therapeutically effective amount of the formulation of any one of claims 1 to 42.

49. 43. A method for treating or promoting hair growth on the skin or scalp of a human and / or for histomorphological remodeling of the skin, comprising topically administering to a subject a therapeutically effective amount of a formulation according to any one of claims 1 to 42.

50. 43. A method of treating an inflammatory, immune, and / or autoimmune skin condition in a subject, comprising topically administering to said subject a therapeutically effective amount of the formulation of any one of claims 1 to 42.

51. A formulation according to any one of claims 1 to 42 for use in the treatment of inflammatory, immune and / or autoimmune skin conditions in humans.

52. 43. Use of a formulation according to any one of claims 1 to 42 in the manufacture of a topical medicament for the treatment of inflammatory, immune and / or autoimmune skin conditions in humans.

53. Use of a formulation according to any one of claims 1 to 42 in the manufacture of a topical medicament for the treatment or promotion of hair growth on the human skin or scalp and / or the histomorphological remodelling of the skin.

54. a nanoemulsion of CsA and a cyclic nitroxide selected from 3-carbamoyl-proxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrroline-1-oxyl; and a nanoemulsion of acrylic acid and C １０ ~C ３０ a copolymer with an alkyl acrylate, and configured for application to an area of ​​skin of a subject.

55. 55. The formulation of claim 54, wherein the nanoemulsion is encapsulated in the copolymer, the CsA is at a concentration in the range of about 0.01% to about 0.5%, and the cyclic nitroxide is at a concentration in the range of about 0.1% to about 5% (wt / wt).

56. 56. A formulation according to claim 54 or 55, comprising 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl for use in a method for preventing or treating alopecia.

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