BETA3-ADRENERGIC AGONISTS FOR TREATMENT OF DISORDERS OF HAIR GROWTH

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/251 ,375, filed October 1 , 2021 , which the application is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Hair follicle stem cells are of interest for topical, as well as therapeutic, purposes. For example, androgenic alopecia is the single largest type of recognizable alopecia to affect both men and women, primarily of Caucasian origin. Androgenic alopecia or common baldness represents 99 percent of all cases of hair loss. The condition is characterized by the gradual conversion of terminal hair to short, wispy, colorless vellus hair.

[0003] In 1980, the reversal of androgenic alopecia in a male patient receiving minoxidil for hypertension was revealed and minoxidil has since been used to promote hair growth, most commonly by topical application. Minoxidil's vasodilating effect on the scalp is one of the proposed mechanisms by which minoxidil promotes hair growth. However, despite its popularity, minoxidil has not performed in a completely satisfactory fashion in promoting hair growth in all target populations. While minoxidil has been shown to stimulate some hair growth at the apex region of the scalp, hair growth at the frontal region of the scalp, for the most part, has not been shown to be improved by minoxidil treatment alone.

[0004] Each year, more than $2B is spent worldwide on surgical procedures for hair loss. The problem is not restricted to male baldness: In addition to 35M men, 20-30M women in the U.S. are also affected by hair loss. In addition, hair loss occurs as a consequence of chemotherapy, radiation, and immune suppression medications. Although hair growth usually returns when these therapies cease, some patients become permanently bald.

[0005] Current treatment options for other types of hair loss are limited; they include topical products, such as minoxidil, which retards hair shedding (effluvium) but does not stimulate new hair growth; and finasteride, which reduces testosterone conversion and also hinders effluvium. Hair transplants, where hair follicles (HF) are harvested from one part of the scalp and moved to another, are largely effective but upon harvesting of the hair follicles, a subset of them undergo apoptosis. Consequently, when those follicles are transplanted the hair falls out.

[0006] There is a continuing desire to treat hair loss. The present invention addresses this need. SUMMARY OF THE INVENTION

[0007] There exists a considerable need for alternative therapeutics that promote hair growth. The present invention addresses this need and provides additional advantages. In one aspect, the present invention provides a topical composition comprising a P3 adrenergic receptor agonist, e.g. a selective P3 adrenergic receptor agonist, in a topical formulation in an amount effective to promote hair growth. A P3 adrenergic receptor agonist of interest is ZD71 14. Another 3 adrenergic receptor agonist of interest is YM178 (mirabegron). One or a cocktail of P3 adrenergic receptor agonists can be provided in the formulation. Without limitation as to the theory, it is believed that the P3 adrenergic receptor agonist induces anagen entry and subsequent hair replacement, and stimulates progenitor populations within the hair follicle unit.

[0008] In some embodiments the topical composition further comprises one or more permeation enhancers, including without limitation DMSO. In some embodiments, the topical composition comprises from about 0.01 to about 10 mg/mL of the P3 adrenergic receptor agonist, or alternatively, from about 1 mM to about 100 mM, and may be from about 5 MM to about 50 mM, from about 10 mM to about 25 mM. In some embodiments, the dermally acceptable vehicle is selected from diluents, dispersants, and carriers or a combination thereof. In some embodiments the composition comprises nanoparticles.

[0009] In one aspect, the present invention provides a patch for application to the skin, wherein the patch comprises any topical composition disclosed herein.

[0010] In one aspect, the present invention provides a method for treating a condition of the skin, scalp or hair of a subject in need thereof, comprising applying to the skin, scalp or hair of the subject any topical composition disclosed herein. In some embodiments, the condition is alopecia, including without limitation pediatric, and adult alopecia. In some embodiments, the condition is androgenic alopecia. In some embodiments, the condition is caused by a vitamin deficiency, an iron deficiency, infection, chemotherapy, anabolic steroids, oral contraceptives or trauma.

[0011] The present invention provides topical and pharmaceutical skin and hair care compositions for enhancing the growth and appearance of mammalian hair in an individual, particularly, although not exclusively, hair of the scalp. In some embodiments of the invention the topical formulations comprise an effective dose of a P3 adrenergic receptor agonist in a formulation that provides for penetration into the scalp. The formulations of the invention may further include pharmaceutically and/or dermally acceptable vehicle(s) and/or other skin and hair conditioning agents. Additional agents to enhance skin penetration may be included in the formulation. [0012] In the methods of the invention, a topical composition comprising an effective dose of a P3 adrenergic receptor agonist is administered to the scalp, etc. of an individual for a period of time sufficient to improve the appearance of the individual’s hair. The individual may be a human suffering from androgenic alopecia. In some embodiments a topical composition comprising an effective dose of a P3 adrenergic receptor agonist is administered to the eyebrows, face, or eyelashes of an individual for a period of time sufficient to improve the appearance of the eyebrows, beard, or eyelashes.

[0013] In some embodiments, a dermally acceptable skin care composition is provided, comprising an effective dose of a topically formulated P3 adrenergic receptor agonist. The compositions of the invention can include a dermally acceptable vehicle to act as a diluent, dispersant or carrier for the active agents, so as to facilitate distribution and uptake when the composition is applied to the skin. Vehicles other than or in addition to water can include liquid or solid emollients, solvents, humectants, thickeners and powders. The dermally acceptable vehicle may range from 5% to 99.9%, preferably from 25% to 80% by weight of the composition, and can, in the absence of other topical adjuncts, form the balance of the composition. The compositions may be in the form of aqueous, aqueous/alcoholic or oily solutions; dispersions of the lotion or serum type; anhydrous or lipophilic gels; emulsions of liquid or semi-liquid consistency, which are obtained by dispersion of a fatty phase in an aqueous phase (O/W) or conversely (W/O); or suspensions or emulsions of smooth, semi-solid or solid consistency of the cream or gel type. When the compositions of the invention are formulated as an emulsion, the proportion of the fatty phase may range from 5% to 80% by weight, and preferably from 5% to 50% by weight, relative to the total weight of the composition. Oils, emulsifiers and co-emulsifiers incorporated in the composition in emulsion form are selected from among those used conventionally in the topical or dermatological field. The emulsifier and coemulsifier may be present in the composition at a proportion ranging from 0.3% to 30% by weight, and preferably from 0.5% to 20% by weight, relative to the total weight of the composition.

[0014] These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the treatment methods, and in vitro and in vivo assay methods, as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 shows the effect of [33 adrenergic receptors agonists on hair growth. Definitions

[0016] Before the present methods are described, it is to be understood that this invention is not limited to particular methods described, as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0017] It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

[0018] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges can independently be included in the smaller ranges encompassed within the invention, subject to any specifically excluded limit in the stated range. Also used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 pL” means “about 5 pL” and also “5 pL.” Generally, the term “about” includes an amount that would be expected to be within ±10% of the amount.

[0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton etal, Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994), provides one skilled in the art with a general guide to many of the terms used in the present application. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0020] P3 adrenergic receptor agonists are compounds, often small molecule, that activate P3 adrenergic receptors. In some embodiments the agonist is selective for the 3 adrenergic receptor. These receptors are generally expressed in adipose tissue and smooth muscle. A number of such agents are known and used in the art. In one embodiments, the agonist is ZD 71 14, ((S)-4-[2-[(2-hydroxy-3-phenoxypropyl)amino]ethoxy]-N-(2-met hoxyethyl) phenoxyacetamide.

[0021] Other P3 adrenergic receptor agonists of interest for use in the methods disclosed herein include, without limitation: ZD2079; YM178 (mirabegron); ICI D71 14; BRL 37344; benzyl and phenoxymethylene substituted oxadiazoles (see Biftu et al. Bioorg Med Chem Lett 2000 Jul 3;10(13):1431 -4); CL316243 and prodrugs thereof; MK-0634 and vibegron; GW427353 (solabegron) (see Hicks et aL); BMS-196085 (see Gavai et al. (2001 ) Bioorg Med Chem Lett.;1 1 (23):3041 -4; L-770,644 (see Shih et al. (1999) Bioorg Med Chem Lett.;9(9):1251 -4); and the like, which references are herein specifically incorporated by reference. A benefit of the present methods is the use of these compounds in topical formulations, which allows the use of agents that have desirable activity but undesirable side-effects when administered systemically.

[0022] Befa-adrenergic receptors are G-Protein Coupled Receptors (GPCRs), which are found on the outer cell membrane and facilitate down-stream intracellular signaling. GPCRs represent a broad group of inherently druggable receptors, owing to their cell surface presence. In the case of adrenergic receptors, the principal physiologic neurotransmitters are catecholamines, e.g., adrenaline and noradrenaline. Both alpha and befa-adrenergic receptor varieties exist, and beyond their conformational identity, possess distinct tissue expression and signaling activity (based on GPCR subtype, whether this be inhibitory, stimulatory, or associated with specific downstream pathways). Furthermore, there are three befa-adrenergic receptor subtypes, beta- 1, 2, and 3, each with distinct tissue presence and signaling activity.

[0023] Beta 1 and 2 receptors are predominantly found within cardiovascular system, whereby they function physiologically to modify cardiovascular output and vascular tone at baseline and in response to stress. As a result, these receptors have been broadly targeted for cardiovascular purposes, e.g. to lower blood pressure in patients with hypertension via antagonism, hence the colloquial term befa-blockers.

[0024] The beta3-adrenergic receptor, however, differs significantly from the beta-1 and -2 in that it is traditionally recognized to exist principally in adipose, where it serves to controls energy expenditure from this reserve. As a result, the main focus on the physiological and pharmacological implications of this receptor have related to control of metabolic effects, obesity, and diabetes. Unfortunately, no agent has successfully been translated to patients in this regard, despite promising results in animals. This has largely been attributed to species-species variability in receptor binding sites, or indeed the different metabolic capacity of adipose tissue between species.

[0025] Stem and progenitor cells possess the therapeutic potential to regenerate tissues, subject to our ability to phenotype and specify the mechanisms underpinning the functionality and fate of these populations. Herein, we identify a dermal adipose progenitor cell (APC) population and demonstrate pro-regenerative-functionality of p ₃ -adrenergic receptor (p ₃ AR) signaling in these cells via pharmacologic manipulation. We observed rapid hair regrowth following treatment in a depilated mouse model versus control, demonstrating a role for APCs and underlying mechanism behind their capacity to elicit hair regrowth. These findings possess potential to inform cell-based- or pharmacologic-approaches within regenerative medicine and serve to improve outcomes in wound healing and disorders of hair growth upon clinical translation.

[0026] "Mammalian hair," as used herein, includes hair on any part of the body of a mammal and may include facial, cranial, or body hair. Of particular interest for increased hair growth is the hair present on the human scalp. Hair of the eyelashes and eyebrows is also of interest for growth modification with the methods of the invention.

[0027] "Regulating hair growth," namely mammalian hair growth, includes reducing, modulating, inhibiting, attenuating, retarding, promoting, enhancing, and/or the diminution of hair growth, and/or reducing shaving frequency. Enhancement or promotion of hair growth is of particular interest.

[0028] “NL (Neogenic-Like) follicular structure” includes unattached primitive follicular structure, with only one of the following “small” traits: shaft, sebaceous gland, or pore. Dermal channel is absent or inconclusive. Further subcategories of NL include: NL with DP (dermal papilla)/active, NL with DP/inactive, NL without DP/active, and NL without DP/inactive.

[0029] “Nonvellus hair” includes “terminal hair.”

[0030] “PEL (Pre-Existing-Like) follicular structure” includes an unattached primitive follicular structure, with one or more of the following “large” traits or two or more of the following “small” traits: shaft (large or small), sebaceous gland (large or small), or pore (large or small). Dermal channel is present. Further subcategories of PEL include: PEL with DP (dermal papilla)/active, PEL with DP/inactive, PEL without DP/active, and PEL without DP/inactive. [0031] “PELA (Pre-Existing-Like, Attached) follicular structure” includes primitive follicular structure that is attached to larger, mature, pilosebaceous unit that extends to the epidermis.

[0032] “Terminal hair” includes large, usually pigmented hairs on scalp and body. Hair shaft diameters are typically 30 pm or greater.

[0033] "Promoting hair growth" includes stimulating an increase in total hair mass and/or length. Such increase includes increased length and/or growth rate of hair shafts (i.e. follicles), increased number of hairs, and/or increased hair thickness. Some or all of the above end results can be achieved by prolonging or activating anagen, the growth phase of the hair cycle, or by shortening or delaying the catagen and telogen phases. "Promoting hair growth" may include preventing, arresting, decreasing, delaying and/or reversing hair loss.

[0034] "Anagen," as used herein, refers to the active growth phase of hair follicles. In the anagen phase, cells in the root of the hair divide rapidly, adding to the hair shaft. During this phase, the hair grows about 1 cm every 28 days. Scalp hairs stay in this active phase of growth for 2-6 years.

[0035] "Catagen," as used herein, refers to the hair growth phase that occurs at the end of the anagen phase. It signals the end of the active growth of a hair. This phase lasts for about 2-3 weeks while a club hair is formed.

[0036] "Telogen," as used herein, refers to the resting phase of the hair follicle. At any given time, 10%-15% of all hairs are in the telogen phase. This phase lasts for about 100 days for hairs on the scalp and much longer for hairs on the eyebrows, eyelashes, arms and legs. During this phase, the hair follicle is completely at rest and the club hair is completely formed. Pulling out a hair in this phase will reveal a solid, hard, dry, white material at the root. About 25-100 telogen hairs are shed normally each day.

[0037] A "growth state" of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell.

[0038] An "active agent" is an agent, drug, compound, or composition of matter or mixture thereof which provides some pharmacologic, often beneficial, effect.

[0039] "Topical application" or "topical," as used herein, means to apply or spread the compositions of the present invention onto the surface of a keratinous tissue.

[0040] "Dermatologically-acceptable," as used herein, means that the compositions or components thereof so described are suitable for use in contact with mammalian keratinous tissue without undue toxicity, incompatibility, instability, allergic response, and the like.

[0041] "Safe and effective amount" as used herein, means an amount of a compound or composition sufficient to significantly induce a positive benefit, preferably a hair growth regulating benefit, or positive hair appearance or feel benefit, including independently or in combinations the benefits disclosed herein, but low enough to avoid serious side effects, i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan.

[0042] According to the present invention "formulation" is a generic term which can encompass additives, personal care, topical, and dermopharmaceutical compositions, and additives and personal care, topical, and dermopharmaceutical compositions. Formulations can also cover additives and personal care, topical and dermopharmaceutical compositions comprising an active agent.

Detailed Description of the Embodiments

[0043] Compositions are provided for enhancing the growth of hair, where the composition comprises a P3 adrenergic receptor agonist, at a dose effective to promote mammalian hair growth, which may be formulated to enhance penetration into hair follicles.

[0044] The present invention provides topical skin and hair care compositions for enhancing the growth and appearance of mammalian hair in an individual, particularly hair of the scalp. In some embodiments of the invention the topical formulations are topical formulations. In some embodiments of the invention the topical formulations are injectable, including microinjectable. In some embodiments the topical formulations comprise a dose of a P3 adrenergic receptor agonist effective to promote anagen phase of the hair cycle, resulting in more rapid hair growth. The formulations of the invention may further include dermally acceptable vehicle(s) and/or other skin and hair conditioning agents. Additional agents to enhance skin penetration, e.g. DMSO, may be included in the formulation.

[0045] The effective dose may be at least about 0.01 [xg/dose, at least about 0.05 [xg/dose, at least about 0.1 [xg/dose, at least about 0.25 [xg/dose, at least about 0.5 [xg/dose, at least about 1 [xg/dose, at least about 2 |xg/dose, at least about 2.5 [xg/dose, at least about 5 [xg/dose, at least about 10 [xg/dose, at least about 50 [xg/dose, at least about 100 [xg/dose, at least about 250 [xg/dose, at least about 500 [xg/dose, at least about 750 [xg/dose, at least about 1 mg/dose, at least about 10 mg/dose, at least about 50 mg/dose, or at least about 100 mg/dose. In certain embodiments, the dose of P3 adrenergic receptor agonist may be administered one time per day, two times per day, three times per day, four times per day or even five times per day. In certain embodiments, the dose of P3 adrenergic receptor agonist is administered at least one time every other day, at least one time every third day, at least one time every fourth day, at least one time every fifth day, at least one time every sixth day or at least one time every seventh day. [0046] In certain embodiments, one dose of P3 adrenergic receptor agonist may be administered at two or more different locations. For example a dose of 100 pig may be administered at two or more locations on the scalp either topically or subcutaneously, such that the total dose administered at the two or more locations adds up to a total of 1 -10 mg. In certain embodiments, the P3 adrenergic receptor agonist is administered to a surface area of the scalp, such as through the use of a one or more lotions, patches or microarrays of needles in contact with said surface area. The surface area of the scalp may be a surface of from about 1 cm ² to about 100 cm ² , such as about 5 cm ² to about 50 cm ² . The patch may provide a total topical dose of P3 adrenergic receptor agonist to said surface area, wherein said dose is evenly administered or substantially evenly administered over said surface area. The microarray may provide a total dose of P3 adrenergic receptor agonist to the subcutaneous area below said surface area, wherein said dose is evenly administered or substantially evenly administered over said surface area.

[0047] The final concentration of a P3 adrenergic receptor agonist in a formulation of the disclosure may be at least about 0.1 mg/mL, at least about 0.5 mg/mL, at least about 1 mg/mL, at least about 5 mg/mL, at least about 10 mg/mL, at least about 50 mg/mL, at least about 100 mg/mL, at least about 500 mg/mL, or more.

[0048] In some embodiments, a given dosing schedule comprising one or more administrations of a P3 adrenergic receptor agonist may be repeated on a daily, weekly, biweekly, triweekly, monthly, bimonthly, annually, semi-annually, or any other period as may be determined by a medical professional. A repeated dosing schedule may be repeated for a fixed period of time determined at the start of the schedule; may be terminated, extended, or otherwise adjusted based on a measure of therapeutic effect, such as a level of reduction in the presence of detectable hair loss (e.g. a reduction of at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%); or may be terminated, extended, or otherwise adjusted for any other reason as determined by a medical professional.

[0049] A p3 adrenergic receptor agonist disclosed herein can be administered as part of a combination treatment, wherein the P3 adrenergic receptor agonist may be administered with one or more additional therapeutic agents. Such one or more additional agents can be administered simultaneously or separately with respect to the P3 adrenergic receptor agonist. Administration in combination utilizing one or more additional agents includes, for example, simultaneous administration of two agents in the same dosage form, simultaneous administration in separate dosage forms, and separate administration. For example, multiple therapeutic agents can be formulated together in the same dosage form and administered simultaneously. Alternatively multiple therapeutic agents can be simultaneously administered, wherein the agents are present in separate formulations.

[0050] In another alternative, a P3 adrenergic receptor agonist of the present invention can be administered followed by one or more additional agents, or the P3 adrenergic receptor agonist of the present invention can be administered preceded by one or more additional agents. In the separate administration protocol, a P3 adrenergic receptor agonist of the present invention and one or more additional agents may be administered a few minutes apart, or a few hours apart, or a few days apart. The term “combination treatments” also embraces the administration of the polypeptides as described herein in further combination with other biologically active compounds or ingredients and non-drug therapies.

[0051] Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the composition used for therapy, the purpose of the therapy, the target tissue being treated, and the subject being treated. Single or multiple administrations (e.g. about or more than about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or more doses) can be carried out with the dose level and pattern being selected by the treating physician.

[0052] Dosages for the P3 adrenergic receptor agonist may be determined empirically in individuals who have been given one or more administrations of a P3 adrenergic receptor agonist. Individuals may be given incremental doses of a P3 adrenergic receptor agonist and to assess efficacy of a P3 adrenergic receptor agonist, further hair loss can be monitored.

[0053] Administration of a P3 adrenergic receptor agonist according to the methods of the invention can be continuous or intermittent, depending, for example, on the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners.

[0054] In some embodiments, a P3 adrenergic receptor agonist and/or any additional therapeutic compound of the invention is administered in multiple doses. Dosing may be about once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be about once a month, once every two weeks, once a week, or once every other day.

[0055] Administration of the P3 adrenergic receptor agonist of the invention may continue as long as necessary. In some embodiments, a P3 adrenergic receptor agonist of the invention is administered for more than 1 , 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, a P3 adrenergic receptor agonist of the invention is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, a P3 adrenergic receptor agonist of the invention is administered chronically on an ongoing basis, e.g., for the treatment of chronic effects. [0056] When a combination treatment of the invention is administered as a composition that comprises one or more agents, and one agent has a shorter half-life than another agent, the unit dose forms may be adjusted accordingly.

Delivery Methods

[0057] Topical formulations comprising the P3 adrenergic receptor agonist can be administered to the region of skin comprising hair follicles, particularly skin of the scalp, but in some embodiments also comprising the skin of the eyelashes, eyebrows, beard, etc. The individual being treated may be male or female, usually a mammal, and usually a human. The individual may suffer from a hair loss condition, such as androgenic alopecia, or from alopecia resulting from various other causes. Alternatively, the individual can have normal hair growth who desires to have a promotion of hair growth or regulation of hair growth.

[0058] The compositions of the present invention can be for topical use and can be applied to the regions of skin comprising hair follicles, e.g. scalp, eyebrows, eyelashes, etc. The amounts and concentrations of the active agents in the compositions of the invention will vary depending on several different factors, including but not hereby limited to, the pH and condition of the skin; whether the skin is oily, dry, or in-between and the nature of the interaction between the various other agents to be included in the composition, but should be such to be effective while at the same time reducing the risk of untoward side effects, such as inflammation and unwanted change in the pigmentation of the hair or skin. Optimization of the concentration of the active agent(s), suitable for use with different skin types, which are used within the compositions of the invention, can be routinely determined by a skilled worker using well known methods that are commonly practiced within the art.

[0059] In general, the subject formulations may contain at least about 1 |ig/ml active agent, at least about 10 pig/ml, at least about 50 pig/ml, at least about 100 pig/ml, at least about 500 pig/ml, and not more than about 50 mg/ml, usually not more than about 10 mg/ml. In some embodiments the formulation comprises at least about 0.1 mM, at least about 0.05, at least about 1 mM, at least about 5 mM, at least about 10 mM, at least about 50 mM, and not more than about 500 mM, not more than about 250 mM active agent. The active agents of the present invention are formulated at an effective concentration within the subject formulations, meaning at a concentration that provides the intended benefit when applied.

[0060] The P3 adrenergic receptor agonist can be effective to promote anagen phase of the hair cycle, resulting in more rapid or prolonged hair growth. The P3 adrenergic receptor agonist can be used for conditions in which regulating hair growth or promoting hair growth is desired. The compositions of the invention can be used for the topical treatment of a hair loss condition, such as alopecia in a mammal. The individual being treated may be a human, or may be an animal, e.g. canine, equine, bovine, etc. suffering from hair loss. In other embodiments the individual is a laboratory animal, e.g. rabbit, mouse, rat, etc., for purposes of evaluating treatments.

[0061] A P3 adrenergic receptor agonist may be combined with any additional ingredient which may be active, functional, conventionally used in personal care or topical/transdermal pharmaceutical products or otherwise. A decision to include an additional ingredient and the choice of specific additional ingredients depends on the specific application and product formulation. Also, the line of demarcation between an "active" ingredient and an "inactive ingredient" is artificial and dependent on the specific application and product type. A substance that is an "active" ingredient in one application or product may be a "functional" ingredient in another, and vice versa.

[0062] In order to be effective in stimulating hair growth the composition may be formulated in such a way as to enhance the active agent’s penetration of the skin. Accordingly, the composition may be formulated in conjunction with a skin penetration enhancing agent so as to better enable the active agent to deeply penetrate the epidermis of the skin, including particularly liposomal compositions. The formulations can include other components, such as buffering agents, lipophilic agents and dermally acceptable vehicles.

[0063] The formulations may be used in the form of gels, solutions, dispersions and emulsions, or may comprise carriers such as microneedles, macrocapsules, microcapsules, nanocapsules, macrospheres, microspheres, nanospheres, liposomes, oleosomes, chylomicrons, macroparticles, microparticles, nanoparticles, macrosponges, microsponges, nanosponges, powdered organic polymers, talcs, bentonites or other inorganic carriers.

[0064] The formulations may be used in any form employed in dermopharmacy: such as lotions, sprays, gels, hair styling products, hair holding products, sunscreens, sunblocks, emulsions, dispersions, solutions, milks, suspensions, scalp treatment lotions, or sprays.

[0065] Various additional methods known in the art can be used for the delivery of P3 adrenergic receptor agonist to hair follicles, including patches, chemical enhancers, ultrasound, electroporation, cavitational ultrasound, microneedles, thermal ablation, microdermabrasion, and the like.

[0066] In some embodiments, transdermal patches store the active agent in a reservoir that is enclosed on one side with an impermeable backing and has an adhesive that contacts the skin on the other side. Some designs employ drug dissolved in a liquid or gel-based reservoir, which can simplify formulations and permit the use of liquid chemical enhancers, such as ethanol. These designs characteristically are composed of four layers: an impermeable backing membrane; a drug reservoir; a semi-permeable membrane that may serve as a rate-limiting barrier; and an adhesive layer. Other designs incorporate the drug into a solid polymer matrix, which simplifies manufacturing. Matrix systems can have three layers, by eliminating the semi-permeable membrane, or just two layers, by incorporating the drug directly into the adhesive. A variation on the transdermal patch applies a metered liquid spray, gel or other formulation to the skin that, upon evaporation or absorption, can drive drugs into the stratum corneum, which in turn serves as the drug reservoir for extended release into the viable epidermis over hours.

[0067] Transdermal delivery systems may comprise a skin permeability enhancer. Enhancement methods with patches include conventional chemical enhancers, iontophoresis and non- cavitational ultrasound. Many effective chemical enhancers disrupt the highly ordered bilayer structures of the intracellular lipids found in stratum corneum by inserting amphiphilic molecules into these bilayers to disorganize molecular packing or by extracting lipids using solvents and surfactants to create lipid packing defects of nanometer dimensions. Hundreds of different chemical enhancers have been studied, including off-the-shelf compounds and others specifically designed and synthesized for this purpose, such as Azone (1 -dodecylazacycloheptan-2-one) and SEPA (2-n-nonyl-1 ,3dioxolane).

[0068] Iontophoresis typically applies a continuous low-voltage current, providing an electrical driving force for transport across stratum corneum. Charged drugs are moved via electrophoresis, while weakly charged and uncharged compounds can be moved by electroosmotic flow of water generated by the preferential movement of mobile cations (e.g., Na ⁺ ) instead of fixed anions (e.g., keratin) in the stratum corneum. Current applications emphasize the ability of iontophoresis to provide control over drug dosing, because it scales with the amount of charge (i.e. , the product of current and time) delivered to the skin.

[0069] Ultrasound is an oscillating pressure wave at a high frequency. The dominant effect is to disrupt stratum corneum lipid structure and thereby increase permeability. The effects of non- cavitational ultrasound on skin permeability enhance transfer of small, lipophilic compounds. Ultrasound can also generate cavitation, which is the formation, oscillation and, in some cases, collapse of bubbles in an ultrasonic pressure field. Cavitation is only generated under specific conditions, e.g., low-frequency ultrasound, that differ from those of ultrasonic heating or imaging devices. The opportunity for transdermal drug delivery is that cavitation bubbles concentrate the energy of ultrasound and thereby enable targeted effects at the site of bubble activity. Cavitation preferentially occurs within the coupling medium (e.g., a hydrogel) between the ultrasound transducer and skin. [0070] Electroporation has also been shown to disrupt lipid bilayer structures in the skin. Although the electric field applied for milliseconds during electroporation provides an electrophoretic driving force, diffusion through long-lived electropores can persist for up to hours, such that transdermal transport can be increased by orders of magnitude for small model drugs, peptides, vaccines and DNA. The electric field applied during electroporation is initially concentrated in the stratum corneum.

[0071] Thermal ablation selectively heats the skin surface to generate micron-scale perforations in the stratum corneum. Transiently heating the skin’s surface to hundreds of degrees for microseconds to milliseconds localizes heat transfer to the skin surface without allowing heat to propagate to the viable tissues below. This spares these tissues from damage or pain. Mechanistically, thermal ablation may involve rapidly vaporizing water in the stratum corneum, such that the resulting volumetric expansion ablates micron-scale craters in the skin’s surface. Skin heating has been achieved using ohmic microheaters and radio-frequency ablation. The microscopic length scales of localized skin disruption caused by thermal ablation have resulted in the procedure being well tolerated.

Skin Penetration Enhancing Agent

[0072] Skin penetration agents may be included in the formulations of the invention. As used herein, a skin penetration enhancing agent is any factor that increases the penetration of the skin, preferably with minimal disruption to the acidic pH balance of the skin. Preferably, the skin penetration enhancing agent enhances the percutaneous delivery of the active agent into and through the layers of the skin, without providing substantial transdermal transmission of the active agent into the systemic circulation. The permeability enhancing agents of the invention are physio- chemically stable, do not have pharmacological effects, and have at least reduced irritancy or toxicity to the skin. When present in a composition of the invention, the amount of penetration enhancer is typically from about 1 % to about 10% by weight of the total composition weight or from about 2% to about 5% by weight. The formulation and use of skin penetration enhancers in topical formulations is set forth generally in: PERCUTANEOUS PENETRATION ENHANCERS (Eric W. Smith & Howard I. Maibach eds. 1995); Ghosh, T. K. et al. 17 PHARM. TECH. 72 (1993); Ghosh, T. K. et al. 17 PHARM. TECH. 62 (1993); and Ghosh, T. K. et al. 17 PHARM. TECH. 68 (1993), all of which are hereby incorporated herein by reference in their entirety.

[0073] Suitable skin penetration enhancing agents include those agents that are capable of reducing the resistance of the skin to the active agent and promoting the active agent partitioning from the dosage form. Penetration enhancing agents may function in a variety of ways, including via the elution of the lipid and/or lipoprotein structures of the stratum corneum, by increasing lipid fluidity (e.g., by disrupting the tightly packed lipid chains), or by engaging in various protein interactions that result in a change in protein and/or lipid configuration that creates a passage for the active agent. Suitable topical skin permeability enhancing agents can be routinely selected for a particular use by those skilled in the art, and especially with reference to one of many standard texts in the art, such as Remington's Pharmaceutical Sciences, Vol. 18, Mack Publishing Co., Easton, Pa. (1990), in particular Chapter 87, which is hereby incorporated by reference in its entirety.

[0074] Accordingly, suitable skin penetration enhancing agents include but are not hereby limited to: sulfoxides, alcohols, polyols, fatty acids, esters, amides, surface active agents (such as pluronics, sulfates, lecithin, docusate sodium, polysorbates), water, and the like. Specifically, skin penetration enhancing agents include but are not hereby limited to dimethyl sulfoxide (DMSO), N-decylmethylsulfoxide, ethanol, phenyl ethanol, propylene glycol, lauric or myristic or palmitic or steric fatty acids, lauric acid, sodium laurate, neodecanoic acid, lauryl lactate, methyl laurate, hexamethylene lauramide, leucinic acid, oleic acid, capric acid, sodium oleate, sodium caprate, dodecyl-amine, cetryl lactate, myristyl lactate, isopropyl palmitate or isopropyl myristate esters, urea and derivatives, dodecyl N,N-dimethylamino acetate, hydroxyethyl lactamide, lecithin, phyophatidylcholine, sefsol-318 (a medium chain glyceride, surfactants, including polyoxyethylene (10) lauryl ether (Brij 361 R), diethyleneglycol lauryl ether (PEG-2-L), laurocapram (Azone; 1 ,1 -dodecylazacycloheptan-2-one), acetonitrile, 1 -decanol, 2-pyrrolidone, N-methylpyrrolidone, N-ethyl-1 -pyrrolidone, 1 -methyl-2-pyrrolidone, 1 -lauryl-2-pyrrolidone, sucrose monooleate, acetone, polyethylene glycol 100-400 MW, dimethylacetamide, dimethylforamide, dimethylisosorbide, sodium bicarbonate, mentane, menthone, menthol, terpinene, D-terpinene, dipentene, N-nonalol, limonene, and various C7-16 -alkanes in amounts that are safe and effective. A vasodilator that can be used in the formulations of the present invention is niacinamide (a vitamin B ₃ compound), which aids in the penetration and uptake of active ingredients. The niacinamide may be used at a concentration of at least about 0.25% to 0.5%, more usually at least about 1%, and not more than about 5%.

Buffering Agents

[0075] The normal pH of the skin is between about 4 and about 6.5, though it varies in people of different skin types. The compositions of the invention, therefore, in certain embodiments, should be formulated in such a manner so as to reduce the effects that the actual application of the composition has on the pH barrier of the skin and/or should be formulated in a manner so as to increase the penetration of the active agent. Accordingly, in certain embodiments the typical pH ranges for the compositions of the invention include a pH of about 3 to about 8, of about 4 to about 7, and more typically about 4.5 to about 6.5 or about 5. The desired pH ranges of the compositions of the invention can be obtained in accordance with practices well known in the art, for instance, by the inclusion of various buffering agents, which should be included in an amount and concentration to optimize the flux of the active agent through the skin surface and into the dermal layer of skin, while minimizing any possibility of skin irritation due to a change in the pH of the skin.

[0076] A conventional buffering agent such as a mixture of citric acid and trisodium citrate, may be added to stabilize the desired pH. Other buffering agents include, but are not limited to, sodium phosphate, monosodium dihydrogen phosphate, and disodium monohydrogen phosphate.

[0077] Various lipophilic agents may also be included as dermal benefit agents of the present invention in amounts that are safe and effective. A lipophilic agent to be added to a composition of the invention may be, for instance, a water-insoluble (hydrophobic) organic material or mixture of materials that are miscible with a P3 adrenergic receptor agonist and are suitable for administration (e.g., topical administration) and formulated to enhance the penetration of an active agent of the invention. A lipophilic component may be in a range about 15% to about 40% by weight of the total composition weight or about 20% by weight.

[0078] Suitable lipophilic components are well known in the art and include, but are not limited to, vegetable, nut, and seed oils, such as almond oil, castor oil, coconut oil, corn oil, cotton seed oil, jojoba oil, linseed oil, grape seed oil, rape seed oil, mustard oil, olive oil, palm and palm kernel oil, peanut oil, safflower oil, sesame oil, soybean oil, sunflower-seed oil, crambe oil, wheat germ oil, and cocoa butter; animal oils and fats, such as lanolin, tallow, lard, beef fat, butterfat, mink oil, and fish oils; hydrocarbon and petroleum oils, such as petrolatum, mineral oil, and liquid paraffin. Additional lipophilic components include higher fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, 12-hydroxystearic acid, undecylenic acid, tall acid, lanolin fatty acid, isostearic acid, linoleic acid and linolenic acid.

[0079] The lipophilic component may also include a suitable stiffening agent such as isopropyl myristate, glycerol monolaurate, glycerol monooleate, glycerol monolinoleate, isopropyl isostearate, isopropyl linoleate, isopropyl myristate/fatty acid monoglyceride combination, isopropyl myristate/ethanol/L-lactic acid combination, isopropyl palmitate, methyl acetate, methyl caprate or methyl laurate. [0080] The formulation may also contain irritation-mitigating additives to minimize or eliminate the possibility of skin irritation or skin damage that may result from the penetration of the active agent or other components of the formulation. Suitable irritation-mitigating additives include, for example: a-tocopherol; monoamine oxidase blockers, particularly phenyl alcohols, such as 2- phenyl-1 -ethanol; glycerin; salicylic acids and salicylates; ascorbic acids and ascorbates; ionophores such as monensin; amphiphilic amines; ammonium chloride; N-acetylcysteine; cis- urocanic acid; capsaicin; and chloroquine. The irritant-mitigating additive, if present, may be incorporated into the formulation at a concentration effective to mitigate irritation or skin damage, typically representing not more than about 20 wt %, more typically not more than about 5 wt %, of the formulation.

Dermally Acceptable Vehicle

[0081 ] The formulation can comprise a dermally acceptable vehicle to act as a dilutant, dispersant or carrier for an active agent of the invention, so as to facilitate its distribution and uptake when the composition is applied to the skin and/or hair or scalp. Vehicles other than or in addition to water can include liquid or solid emollients, solvents, humectants, thickeners and powders.

[0082] The vehicle will usually form from about 5% to about 99.9%, preferably from about 25% to about 80% by weight of the composition, and can, in the absence of other adjuncts, form the balance of the composition.

[0083] The compositions may be in the form of aqueous, aqueous/alcoholic or oily solutions; dispersions of the lotion or serum type; anhydrous or lipophilic gels; emulsions of liquid or semiliquid consistency, which are obtained by dispersion of a fatty phase in an aqueous phase (O/W) or conversely (W/O); or suspensions or emulsions of smooth, semi-solid or solid consistency of the cream or gel type. These compositions are formulated according to the usual techniques as are well known to this art.

[0084] A topical composition of the invention will typically be formulated as a solution or gel which is prepared to be applied to the skin surface without friction, and which is typically a liquid or semiliquid preparation in which the active agent(s) of the invention are present in a lipid, alcohol or water base. Solutions are, typically, homogeneous mixtures prepared by dissolving one or more chemical substances (solute) in another liquid such that the molecules of the dissolved substance are dispersed among those of the solvent. The solution may contain other dermally acceptable chemicals to buffer, stabilize or preserve the solute. Commonly used examples of solvents used in preparing solutions are ethanol, water, propylene glycol or any other dermally acceptable vehicle, as for example, set forth below. [0085] When the compositions of the invention are formulated as an emulsion, the proportion of the fatty phase may range from about 5% to about 80% by weight, and preferably from about 5% to about 50% by weight, relative to the total weight of the composition. Oils, emulsifiers and coemulsifiers incorporated in the composition in emulsion form are selected from among those used conventionally in the dermatological field. The emulsifer and coemulsifier may be present in the composition at a proportion ranging from about 0.3% to about 30% by weight, and preferably from about 0.5% to about 20% by weight, relative to the total weight of the composition.

[0086] The compositions of the invention may also contain additives and adjuvants which are conventional in the topical, pharmaceutical or dermatological field, such as gelling agents, active agents, preservatives, antioxidants, solvents, fragrances, fillers, bactericides, odor absorbers and dyestuffs or colorants. The amounts of these various additives and adjuvants are those conventionally used in the field, and, for example, range from about 0.01% to about 10% of the total weight of the composition. Depending on their nature, these additives and adjuvants may be introduced into the fatty phase or into the aqueous phase.

[0087] Another category of functional ingredients within the topical compositions of the present invention are thickeners. A thickener will usually be present in amounts anywhere from about 0.1 to about 20% by weight, preferably from about 0.5% to about 10% by weight of the composition. Exemplary thickeners are cross-linked polyacrylate materials available under the trademark Carbopol. Gums may be employed such as xanthan, carrageenan, gelatin, karaya, pectin and locust beans gum. Under certain circumstances the thickening function may be accomplished by a material also serving as a silicone or emollient. For instance, silicone gums in excess of about 10 centistokes and esters such as glycerol stearate have dual functionality.

[0088] Powders may be incorporated into the topical composition of the invention. These powders include chalk, talc, kaolin, starch, smectite clays, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof.

[0089] Other adjunct components may also be incorporated into the topical compositions. These ingredients may include coloring agents, opacifiers and perfumes. Specifically, these ingredients may include suitable additives such as deionized water, hydrolyzed glycosaminoglycan, sodium hyaluraonate, triethanolamine, propylene glycol, methylparaben, propylparaben, acrylates, C10- C20 alkyl acrylate crosspolymers, C12-C15 alkyl benzoate, panthenol, biotin, sodium chloride, sodium phosphate and the like. Amounts of these other adjunct components may range anywhere from about 0.001 % up to about 20% by weight of the composition. [0090] The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. Many pharmaceutically acceptable carriers may be employed in the compositions of the present invention. Generally, normal saline will be employed as the pharmaceutically acceptable carrier. Other suitable carriers include, e.g., water, buffered water, 0.4% saline, 0.3% glycine, and the like, including glycoproteins for enhanced stability, such as albumin, lipoprotein, globulin, etc. These compositions may be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

Product Use, Form, and Packaging

[0091] In use, a quantity of the composition, for example from about 0.0001 ml to about 100 ml, from about 0.001 ml to about 10 ml, from about 0.01 ml to about 1 ml, typically about 0.1 ml is applied to a site of interest (i.e. , skin or hair of the eyelash, eyebrow, and/or scalp) from a suitable container or applicator and, if necessary, it is then spread over the site. The product may be specifically formulated for use as a treatment for a specific area, e.g. the eyelashes, eyebrows, the face, the hair, or the scalp.

[0092] The composition of the invention can be formulated in any form suitable for application to the site of interest). The composition can be packaged in a suitable container to suit its viscosity and intended use by the consumer. For example, a gel can be packaged in a bottle or a container fitted with a fine brush suitable controlled application to the lash line or eyebrow. The invention accordingly also provides a closed container containing a dermally acceptable composition as herein defined and may include a suitable applicator.

Therapeutic Conditions

[0093] As described herein, a dose of a P3 adrenergic receptor agonist is effective to promote anagen phase of the hair cycle, resulting in more rapid hair growth. A P3 adrenergic receptor agonist can be used for conditions in which regulating hair growth or promoting hair growth is desired. Promoting hair growth can be performed on a subject with normal hair growth or a hair loss condition. Under normal hair growth conditions on the scalp, about 88% of the hairs are in the anagen phase, about 1 % in catagen and the remainder in telogen. The compositions of the invention increase the percentage of hairs in anagen, thereby prolonging the growth phase of hair.

[0094] A hair loss condition is alopecia. “Alopecia,” as used herein is defined as loss of hair, includes, for example, alopecia areata, androgenic alopecia, etc. Hair loss is often a cause of great concern to the patient for topical and psychological reasons. Hair grows in cycles. Each cycle consists of a long growing phase (anagen), a brief transitional apoptotic phase (catagen), and a short resting phase (telogen). At the end of the resting phase, the hair falls out (exogen) and a new hair starts growing in the follicle, beginning the cycle again. Normally, about 100 scalp hairs reach the end of resting phase each day and fall out. When significantly more than 100 hairs/day go into resting phase, clinical hair loss (telogen effluvium) may occur. A disruption of the growing phase causing abnormal loss of anagen hairs is an anagen effluvium. Besides the loss of hair, the length and diameter of each hair will be reduced in the adjacent areas even though the follicles remain intact.

[0095] Conditions of hair loss include alopecia areata, traction alopecia, trichotillomania, tinea capitis (fungal infection), telogen effluvium, and androgenic alopecia ("male-pattern baldness", "female-pattern baldness"). Causes of alopecia include administration of chemotherapeutic agents and radiation, which impair or disrupt the anagen cycle. Other conditions resulting in hair loss include infection, systemic illnesses (particularly those that cause high fever, systemic lupus, endocrine disorders, and nutritional deficiencies). The compositions of the present invention find use in alleviating alopecia associated with these conditions.

[0096] Telogen effluvium is a transient, reversible, diffuse shedding of hair in which a high percentage of hair follicles enter the telogen phase prematurely as a result of physical or mental illness. Among the most important factors incriminated are childbirth, high fever, hemorrhage, sudden starvation, accidental or surgical trauma, severe emotional stress, and certain drugs.

[0097] Alopecia areata is an immunologic alopecia characterized by the abrupt onset of sharply defined areas of hair loss. In the most severe cases, the scalp will develop total hair loss (alopecia totalis) or the hair loss will involve the whole body surface (alopecia universalis). Most of the patients will run an unpredictable and relapsing course with multiple episodes of hair loss and regrowth. About 20 to 30 percent will have a single reversible episode. Regrowth of hair is common within several months, but in many instances is not complete, and relapses are common. Alopecia areata may be associated with autoimmune diseases such as vitiligo, pernicious anemia, collagen disease, and endocrinopathies.

[0098] Traumatic alopecia is induced by physical trauma, including wounds, of which the two most important groups, from the therapeutic standpoint are trichotillomania and alopecia resulting from topical procedures or improper hair care. Trichotillomania is a compulsive habit in which the individual repeatedly pulls or breaks off his or her own hair in a partially conscious state similar to thumb sucking or nail biting. Traumatic alopecia from topical procedures is done consciously in ill-advised individuals and is almost exclusively seen among females. Sometimes this type of alopecia is associated with folliculitis induced by the occlusive effect of the oily topicals used in the procedure.

[0099] Anagen effluvium is a temporary alopecia caused by the inhibition of mitosis in the hair papilla by certain cytotoxic drugs, leading to constriction of the hair shaft or to complete failure of hair formation. In particular, alopecia frequently occurs in cancer patients who are treated with chemotherapeutic drugs and/or irradiation. Such agents damage hair follicles which contain mitotically active hair-producing cells. Such damage may cause abnormally slow growth of the hair or may lead to hair loss. While various attempts have been made to protect against alopecia or abnormal rates of hair growth during such treatments, there remains a need for an agent that prevents damage to hair follicles in a safe and effective manner.

[0100] Alopecia may also result from nutritional deficiencies and metabolic defects. Caloric deprivation must be very severe to produce hair loss. Increased shedding sometimes occurs after marked weight loss for obesity. Anemia, diabetes, hyper- and hypovitaminosis, and zinc deficiency may also lead to alopecia.

[0101] In some instances, a treatment regimen described herein increases the number of hair follicles by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases the number of hair follicles by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more.

[0102] In some instances, a treatment regimen described herein increases the number of activated or stimulated hair follicles (e.g., NL, PEL or PELA follicular structures) by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases the number of activated or stimulated hair follicles (e.g., NL, PEL or PELA follicular structures) by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more.

[0103] In some instances, the increase in number of hair follicles, activated or stimulated hair follicles, and/or NL, PEL, or PELA structures is observed in the treated area, for example, in an area of skin that was treated with P3 adrenergic receptor agonist. In other embodiments, the increase in number of hair follicles, activated or stimulated hair follicles, and/or NL, PEL, or PELA structures is observed adjacent to the treated area. In other embodiments, the increase in number of hair follicles, activated or stimulated hair follicles, and/or NL, PEL, or PELA structures is observed in and adjacent to the treated area.

[0104] In some instances, measurement of hair follicles in accordance with the foregoing is within 3 days, or 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 3 weeks, 4 weeks, or 1 month or longer after initiation of the treatment regimen. In one embodiment, measurement of hair follicles in accordance with the foregoing is based on a skin biopsy taken within 3 days, or 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 3 weeks, 4 weeks, or 1 month or longer after initiation of the treatment regimen. In a particular embodiment, measurement of hair follicles in accordance with the foregoing is 1 1 days, 12 days, 13 days, 14 days, or 15 days, after initiation of the treatment regimen. In a particular embodiment, measurement of hair follicles in accordance with the foregoing is based on a skin biopsy taken 1 1 days, 12 days, 13 days, 14 days, or 15 days, after initiation of the treatment regimen.

[0105] In some instances, measurement of hair follicles in accordance with the foregoing provides a means for evaluating success of a method of P3 adrenergic receptor agonist treatment (optionally in combination with other treatments). In an exemplary, non-limiting embodiment, success of a method of P3 adrenergic receptor agonist treatment is determined based on a measured increase in total hair follicles in an area of skin subjected to P3 adrenergic receptor agonist treatment, for example, compared to an area of skin that was not subjected to the P3 adrenergic receptor agonist treatment. In another embodiment, success of a method of P3 adrenergic receptor agonist treatment is determined based on a measured increase in activated or stimulated hair follicles, such as NL, PEL, or PELA follicular structures, in an area of skin subjected to P3 adrenergic receptor agonist treatment, for example, compared to an area of skin that was not subjected to the P3 adrenergic receptor agonist treatment step. In one embodiment, where a desired increase in hair follicles (or activated or stimulated hair follicles) is not observed, the treatment is discontinued. In another embodiment, where a desired increase in hair follicles (or activated or stimulated hair follicles) is not observed, P3 adrenergic receptor agonist treatment is repeated. In another embodiment, where a desired increase in hair follicles (or activated or stimulated hair follicles) is not observed, P3 adrenergic receptor agonist treatment is repeated using a different method (for example, switching from standard injections a transdermal patch or vice versa. In one embodiment, where a desired increase in hair follicles (or activated or stimulated hair follicles) is not observed, P3 adrenergic receptor agonist treatment is repeated but with a higher dose, for example, increasing the dose by a factor of 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0106] In some instances, measurement of hair follicles in accordance with the foregoing provides a means for evaluating whether a subject is a candidate for treatment, or continued treatment, with the methods described herein. In an exemplary, non-limiting embodiment, candidacy is established based on a measured increase in total hair follicles in an area of skin subjected to P3 adrenergic receptor agonist treatment, for example, compared to an area of skin that was not subjected to the P3 adrenergic receptor agonist treatment. In another embodiment, candidacy is established based on a measured increase in activated hair follicles, such as NL, PEL, or PELA follicular structures, in an area of skin subjected to P3 adrenergic receptor agonist treatment, for example, compared to an area of skin that was not subjected to the P3 adrenergic receptor agonist treatment. In one embodiment, where a desired increase in hair follicles (or activated hair follicles) is not observed, treatment of that particular subject is discontinued. In another embodiment, where a desired increase in hair follicles (or activated hair follicles) is not observed, P3 adrenergic receptor agonist treatment is repeated. In another embodiment, where a desired increase in hair follicles (or activated hair follicles) is not observed, P3 adrenergic receptor agonist treatment is repeated using a different dose. In one instance, a method of treatment is carried out over a small area of skin (e.g., 1 x1 cm, or 1.5x1.5 cm, or 2x2 cm, or 2.5x2.5 cm, or 3x3 cm or more), hair follicles are measured in accordance with these methods, and if candidacy is established, the method of treatment is carried out over a larger area of skin, such as, e.g, an entire balding area of scalp.

[0107] In some embodiments, a treatment regimen described herein increases the anagen-to- telogen ratio by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases the anagen-to-telogen ratio by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. Such an increase in the anagen-to-telogen ratio may be measured within or after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 1 1 months, or one year or longer after initiation of the treatment regimen.

[0108] In some instances, success of treatment is assessed by measuring hair count in a treated area of skin. For example, detectable hairs can be quantified by photography, e.g., by global photographic recording or phototrichographic analysis (as described in, e.g., Uno et al., 2002, Acta Venereol 82:7-12, incorporated herein by reference). Further, changes in the hair shaft thickness of photographically detectable hairs can be determined. In certain embodiments, the permanence of the hair growth is monitored over a time period of at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 4 years, or at least 5 years or more.

[0109] In some instances, a treatment regimen described herein increases hair count by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a treatment regimen described herein increases vellus hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a treatment regimen described herein increases terminal hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a treatment regimen described herein results in 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-75%, or 75-100% conversion of vellus hair to nonvellus (i.e., intermediary or terminal hair). In some embodiments, a treatment regimen described herein increases hair thickness by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a treatment regimen described herein increases hair shaft diameter by approximately 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 15, 20, 25, or 30 microns or more. In some embodiments, a treatment regimen described herein increases mean hair shaft diameter by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a treatment regimen described herein results in 1 -5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-75%, or 75-100% increase in mean hair shaft diameter. In some embodiments, a treatment regimen described herein increases the ratio of terminal to vellus hair follicles by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% a or more, by 50% or more, by 75% or more, or by 100% or more. Such an improvement in hair count, vellus hair, terminal hair, conversion of vellus hair to nonvellus (e.g., intermediate or terminal) hair, hair thickness, hair shaft diameter, or the ratio of terminal to vellus hair may be measured within or after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or one year or longer after initiation of the treatment regimen. [0110] In some instances, a P3 adrenergic receptor agonist treatment described herein increases hair count by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases vellus hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases terminal hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein results in 1 -5%, 5- 10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-75%, or 75- 100% conversion of vellus hair to nonvellus (i.e., intermediary or terminal hair). In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases hair thickness by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment herein increases hair shaft diameter by approximately 1 , 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 15, 20, 25, or 30 microns or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases hair shaft diameter by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, a P3 adrenergic receptor agonist treatment described herein increases the ratio of terminal to vellus hair follicles by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. Such an improvement in hair count, vellus hair, terminal hair, conversion of vellus hair to nonvellus (e.g., intermediate or terminal) hair, hair thickness, hair shaft diameter, or the ratio of terminal to vellus hair may be measured within or after I month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or one year or longer after initiation of the treatment regimen.

[0111] In certain of the foregoing instances, the increase in hair count, vellus hair, terminal hair, conversion of vellus hair to nonvellus (e.g., intermediate or terminal) hair, hair thickness, hair shaft diameter, and/or the ratio of terminal to vellus hair is observed in the treated area, for example, in an area of skin that was treated with P3 adrenergic receptor agonist. In other embodiments, the increase in hair count, vellus hair, terminal hair, conversion of vellus hair to nonvellus (e.g., intermediate or terminal) hair, hair thickness, hair shaft diameter, and/or the ratio of terminal to vellus hair is observed adjacent to the treated area. In other embodiments, the increase in hair count, vellus hair, terminal hair, conversion of vellus hair to nonvellus (e.g., intermediate or terminal) hair, hair thickness, hair shaft diameter, and/or the ratio of terminal to vellus hair is observed in and adjacent to the treated area.

EXAMPLES

[0112] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention. Efforts have been made to insure accuracy with respect to the numbers used e.g. amounts, temperature, concentrations, etc.) but some experimental errors and deviations should be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric. p ₃ -adrenergic receptor agonism promotes hair restoration via adipose progenitor cell KLF4 upregulation

[0113] The disclosure herein is of a beta3-adrenergic agonist (ZD71 14) that stimulates anagen/hair growth, alongside identification of the presence of this receptor/target within the hair follicle unit and a neighboring progenitor cell population in the dermis. This agent, when applied in animals which have been depilated/shaved at a specific age while their hair cycle is in the telogen/resting phase, causes rapid entry into the anagen phase, stimulating hair growth, as measured grossly and histologically, within 8 days versus the expected 2 weeks in the control group. When applied in the context of wound healing, the drug stimulates hair follicle neogenesis within the wound matrix. Mechanistically, at the level of the associated progenitor cell population, the agent increases KLF4 expression, which is notably a Yamanaka factor, a gene-transcription factor which possesses pluripotency-inducing-potential, and this helps to explain the hair follicle neogenesis phenomenon observed. Further compelling data from cluster analysis via single cell RNA sequencing includes the finding of a keratinizing/hair forming potential within the dermal progenitor cell population described, as per gene enrichment analysis. This has been further validated with cell transplant studies.

[0114] Of note, the beta-3 adrenergic receptor is widely recognized to possess physiologic implications on energy expenditure via thermogenic activation of adipose tissue. This can occur via adrenergic stimulation, for example, in times of cold exposure in order to dissipate heat from energy reserves. The association of this receptor with hair growth is not only compelling due to the above findings, but also broader implications as an extension of thermodynamic control in hair-bearing individuals.

[0115] Following cutaneous injury, collagenous scar formation is favored in lieu of regeneration of the skin and its functional constituents such as hair follicles. While scar formation may serve to readily restore the protective barrier of the skin in the subacute phase, this resolution unfortunately provides less than desirable aesthetic, functional, and biomechanical properties compared to noninjured skin. Adverse wound healing outcomes can be devastating for patients and run the spectra of under-healing, with associated chronic patent wounds, to states of excess-scarification such as hypertrophic and keloid scars. Treatment options for these maladies remain largely conservative, and while various debridement-, dressing-, mechanical- and pharmacological- treatments exist, these methods are largely ineffective in preventing or reversing adverse healing statuses. While studies focusing on fibrosis offer promise in informing potential therapeutics against evolving or existent scars, research and applications derived from stem- and progenitorpopulations stand to inform ways to enable tissue regeneration. Moreover, ways to promote regeneration versus fibrosis are even more intriguing. However, means to achieve tissue regeneration by way of functional dermal appendages have yet to be achieved clinically.

[0116] The work herein importantly describes not only a distinct progenitor population within the murine dermis, but also its capacity to produce pro-regenerative outcomes in the skin via targeting the p ₃ -adrenergic receptor (p ₃ AR) in these cells. Findings of hair follicle growth-promotion in both wounded and non-wounded states following p ₃ AR-agonist treatment addresses not only regenerative healing, but also solutions for disorders of hair growth. These results serve to inform growing evidence behind the relationship between adipose- and hair follicle- biology and homeostasis. Also relevant is the value of being able to discern and enrich specific subpopulations from the progenitor-rich, and readily available, adipose tissue, with the clinical implication of utilizing these subsets for fat grafting and regenerative medicine applications. Collectively, potential for cell-based or pharmacologic therapies exist.

[0117] Through flow assisted cell sorting and immunofluorescent histology, we sought to address the existence of a hypothesized mesenchymal-like subset of fibroblasts within the skin as defined by CD marker expression, and likewise validate the presence of beta-adrenergic receptors in these cells. After defining this cutaneous population by its ability to form adipose cells in cell culture, and validating the presence of the p ₃ AR as a highly expressed druggable target in these cells, we sought to probe the role of this population and associated receptor in the skin. Agonism of the p ₃ AR was performed through topical treatment in non-wounded and wounded skin following depilation. Using an established murine wound healing model with serial monitoring and harvest of specimens across multiple timepoints, we evaluated the gross, histological, and transcriptional output at single cell resolution, between treated- and control-wounds. Following revelation of significant upregulation of the transcription factor KLF4 within treatment-group wound-derived cells via RNA sequencing, we further validated the relationship of p ₃ AR signaling to KLF4 levels in this population via selective local knockout using kenpaullone, a small molecule inhibitor of KLF4, with and without concomitant p ₃ AR-agonist for rescue, followed by FACS analysis- and immunofluorescent histological- validation. Additionally, transplant studies utilizing sorted green fluorescent protein (GFP)-positive cells were performed via direct injection as well as collagen gel matrix delivery, with monitoring the anatomic fate of these cells at baseline-skin and in the context of wound healing. Furthermore, through use of a non-wounded model of depilation during the extended second telogen phase, we evaluated the effects of treatment on hair growth at baseline, and determined the ability of p ₃ AR-agonist to induce anagen entry and subsequent pelage replacement. Ancillary studies employing p ₃ AR-antagonism and evaluation for opposing effects were used to discern the p ₃ AR-specific nature of findings.

[0118] As shown in Figure 1 , localized treatment with a beta3-adrenergic agonist, restricted to wells formed by fastening silicon rings to the dermis, demonstrated localized hair growth confined to the treatment area, with more extensive hair growth exhibited upon higher and repeated dosing. Monitoring of hair cycle stage through serial harvest with histological assessment of hair follicle morphology demonstrated rapid entry into the anagen phase in response to treatment with an exemplary beta3-adrenergic agonist, ZD71 14. Broader application of topical ZD7114 treatment to the dorsum of mice depilated in the extended second telogen phase resulted in rapid anagen (as noticeable by initial pigmentation of the skin) and rapid full repair of the pelage compared to control. Immunofluorescent histology reveals that the beta3-, rather than 1 - or 2- adrenergic receptor is highly localized to the hair follicle unit. Furthermore, a direct indicator of beta3 adrenergic receptor activity, Uncoupling protein-1 (UCP-1 ) is also highly expressed in the same distribution in anagen hair follicles as per immunofluorescent histology. Likewise, Tyrosine Hydroxylase (TH), which is the rate limiting step in the production of catecholamines (the neurotransmitter for adrenergic receptors), is present in anagen hair follicles which exhibit rapid cell division as per the marker ki67. Collectively, these results demonstrate that topical treatment with a beta3-adrenergic receptor agonist stimulates hair growth following topical application, and this receptor and its key signaling molecules and downstream effects are localized constituents of the hair follicle. [0119] A distinct /3-adrenergic receptor-rich Adipose Progenitor Cell population exists within the dermis. Fibroblasts are commonly characterized as a population of cells from the dermis which are negative for lineage markers (CD45, Teri 19, CD324(e-Cadherin), Tie2, epCAM, and CD31 ), and while some markers may be used to positively-identify fibroblasts, to date no one marker can be declared as entirely fibroblast-specific. Following lineage-negative gating, we isolated a subset of fibroblasts through positive gating for PDGFRa, Seal , and CD34, as we rationalized these markers to be a combination of broad fibroblast- yet also mesenchymal stromal cell-like indicators, and thus may represent a pro-regenerative fibroblast cohort, per se. Following sorting, these incidences represented around 0.5-1.5% of all lineage negative cells. Sorted cells, when plated and cultured in standard media, gave rise to adipose following confluence, as confirmed by LipidTox and oil-red-o staining. As such, the population described are denoted as adipose progenitor cells (APCs). Immunofluorescent (IF) histology of skin specimens demonstrated areas of co-localizing immunoreactivity between PDGFRa, Seal , and CD34 cells, alongside DAPI counterstaining. Distinct anatomic localization of these occurrences was noted within the perifollicular region of the dermis.

[0120] Following the validation of this population’s existence via skin harvest and FACS plus cell culture expansion of adipose forming cells, and IF histology placing these cells within the dermis, we sought to identify a cell surface marker that could then be targeted to influence the activity of these cells. We postulated that these APCs possessed adrenergic receptors, especially given the known role of p ₃ adrenergic signaling in influencing adipose phenotype and metabolic activity, particularly at the level of progenitor cells. Indeed, this was found to be the case, with both FACS- and immunofluorescent histology co-staining verifying the presence of p-adrenergic receptors in APCs. The percentage of p 1 , 2, and 3 subsets were also quantified by FACS and histology, alongside the finding of the P3AR, but not pi or 2, around the epithelial lining inclusive of the hair follicle.

[0121] The cutaneous-derived Lin-, Sca1 +, PDGFRa+, CD34+ population demonstrates adipogenic potential and perifolicular localization within the dermis, alongside exhibition of beta- 1 , -2 and -3 adrenergic receptors: The Lin-; Seal , PDGFRa, CD34+ cell population as sorted by FACS demonstrates in vivo adipogenic potential in cell culture with standard media one week post plating, as verified by subsequent bright field (BF) and LipidTox 594 staining imaging. Histologically, the markers for this population co-localize in the dermis at the perifolicular region as per immunofluorescent staining, beta-adrenergic receptors are highly expressed within this population as per FACS and IF histology. [0122] We further hypothesized that the p ₃ AR held importance in this population over the p- 1 and 2 subtypes which were also expressed, particularly from a wound healing perspective. Various investigations have demonstrated the detrimental effects of increased catecholamines (the stress-hormone and principal neurotransmitter in adrenergic stimulation) by way of delaying wound healing. To this end, in vitro studies of wound healing-relevant cells such as keratinocytes and fibroblasts have implicated p- 1 and 2 adrenergic receptors in delayed wound healing, as blockade of these receptors has proven beneficial in promoting keratinocyte migration and fibroblast proliferation, which are otherwise hindered by agonism. However, the p ₃ AR has been largely under-elucidated in this domain, despite evidence of its beneficial effects in glucose, triglycerides, and weight-status in mouse models of diabetes and obesity, factors which have inherent clinical connotations with adverse wound healing outcomes. As pharmacological properties of the p ₃ AR facilitate a higher threshold to desensitization than the p- 1 and 2 ARs, and many functional effects of the p ₃ AR are contrary to that of its counterparts, it is possible that the p ₃ AR may be implicit in negative feed-back regulation in states of overstimulation by catecholamines.

[0123] Wounds treated with /3 ₃ AR-agonist exhibit increased rates of epithelialization and hair follicle neogenesis. Given the preponderance of the p ₃ AR within APCs and its recognized role as the principle adrenergic receptor in adipose tissue, we specifically targeted this receptor pharmacologically. The effects of ZD71 14, a potent and selective p ₃ AR agonist, were evaluated on APCs in vivo using a murine model of wound healing.

[0124] Gross analysis of wound healing phenotype revealed rapid epithelialization and reduced time to wound closure in treated wounds. To determine the specific implications of treatment at the level of APCs, we sorted these cells from wound tissue harvested at serial timepoints using FACS. APCs were upregulated in response to wound healing around day 7, congruent with the patterns of response to wound healing exhibited by fibroblasts or stem cells. Furthermore, this phenomenon was augmented in treatment wounds as per FACS analysis and histological IR.

[0125] Upon histological evaluation, the interesting finding of hair follicle formation was noted within the matrix of ZD7114-treated wounds. These instances featured a high degree of local APC marker expression histologically and exhibited positive staining for the cytokeratin CK5/14 and cadherin p-Cad.

[0126] Wounds treated with ZD7114 exhibit increased re-epithelialization and wound matrix-folliculogenesis: Using a wound healing model, the response of wound tissue to treatment was monitored grossly and histologically, with notable rapid re-epithelialization and reduced time to wound closure in the treatment group. Treated wounds featured hair follicle formation within the wound matrix. Immunoreactivity for the p ₃ AR, tyrosine hydroxylase (TH), Uncoupling Protein-1 (UCP1 ), cytokeratin5 and 14 (CK5/14) and P-cadherin (p-cad) were also demonstrated within the wound matrix as evidence of this target and mechanistic response elements following treatment.

[0127] RNA sequencing of wound derived-APCs reveals KLF4 upregulation upon /3 ₃ AR-agonist- treatment. Sorted APCs from non-wounded, day 7, and day 14 time points across treatment and control groups were subjected to single cell RNA-sequencing. Results revealed distinct transcriptional clusters within APCs as denoted by gene enrichment profiling, encompassing an immune, angiogenic, and interestingly, an epithelializing cohort. Furthermore, treatment-specific incidences of gene-upregulation were identified.

[0128] Single-cell RNA sequencing of APCs reveals distinct clusters and gene enrichment profile inclusive of an epithelializing subset: Single cell RNA-sequencing reveals distinct clusters upon UMAP presentation, with use of a heatmap to signal the top 10 genes expressed within each cluster, as defined by enrichment analysis with EnrichR. Immune, fibroblast-like, angiogenic and keratinizing subsets were present within APCs, and shared-signaling pathways arising from single cell RNAseq analysis of both APCs and fibroblasts from wounds were identified via cell chat.

[0129] Intriguingly, we found that the transcription- and Yamanaka-factor- Kruppel-like Factor 4 (KLF4) was upregulated in APCs upon p ₃ AR-treatment, a finding which was further confirmed by co-localization with APC markers upon IF histology. KLF4 was expressed characteristically along the epidermis yet was enhanced within the wound matrix of ZD71 14-treated wounds.

[0130] KLF4 is upregulated in APCs following ZD7114 treatment: Violin plots demonstrated a relative increase in expression within APCs from treated- versus control-wounds. Immunofluorescent histology revealed a high degree of colocalization between KLF4 and APC markers. Additionally, local KLF4 inhibition via kenpaullone was found to impair wound healing and hair follicle maturation. Topical application of kenpaullone to non-wounded and wounded skin impaired wound healing rate and hair growth both grossly and histologically, as well as limited KLF4 positivity within tissue derived APCs as per FACS versus control. Maturation of the hair follicle was impaired within the kenpaullone group, which featured minute telogen-phase follicles versus advanced control- and treatment-group counterparts. Co-administration of P3AR- treatment attenuated these effects grossly and histologically, and restored KLF4 positivity.

[0131] Kenpaullone-mediated inhibition of KLF-4 impairs wound healing and hair follicle maturation and can be rescued by concomitant AR-treatment. Further validation of the role of KLF4 was achieved via application of kenpaullone, a selective KLF4-inhibitor, which acted to reduce KLF4-positivity amongst APCs as per FACS analysis, as well as decrease healing rate in vivo, whilst impairing hair follicle maturation at both baseline and in the context of wound healing. These findings were effectively reversed grossly and histologically upon concomitant treatment with ZD7114.

[0132] GFP+ transplanted APCs assume a perifollicular residence in the dermis and enter the wound matrix upon healing, plus contribute to folliculoneogenesis. To further confirm the relationship of APCs and hair follicle neogenesis, we conducted GFP-positive-APC transplant studies via both direct injection and collagen gel scaffold-delivery. Here, we found GFP+ cells assumed a similar perifollicular anatomic destiny within the dermis of unwounded- and wounded- APC-injected-skin, and furthermore resulted in de novo GFP positivity within hair follicles themselves.

[0133] GFP+ transplanted APCs provide further evidence of this population ’s role in adipose-hair follicle maturation: Collagen gels were used as conduits for APC transplant from UBC-GFP mice into CD1 -nude mice recipients, with evidence of improved wound healing in the cell-treated group and tracking of GFP positivity in the treatment group in a hair follicle distribution as per UV- dermatoscopy and histological evaluation for GFP. Further direct injection of APCs from UBC- GFP mice also supported the aforementioned findings histologically, with enhanced GFP signal with the wound matrix following wounding after transplant as well as localization of these cells around the hair follicle and within nascent hair shafts.

[0134] /3 ₃ AR-treatment induces anagen and restoration of pelage in a depilation-hair regrowth model. We reinforce the existence of a relationship between the p ₃ AR and hair growth through ancillary studies in unwounded mice, whereby skin was depilated in age-matched mice during the extended second telogen phase, and hair regrowth monitored grossly at regular intervals, alongside serial histological hair cycle-staging, and compared between treated and control groups. Demonstrably rapid hair growth was observed in the p ₃ AR agonist treatment group alongside rapid entry into anagen following treatment. Furthermore, hair growth was notably delayed upon treatment with p ₃ AR antagonist, suggesting this phenomenon is p ₃ AR-activity- dependant.

[0135] ZD7114 stimulates hair cycle progression: Using a mouse model to evaluate hair regrowth, C57BI/6 mice were depilated in the extended second telogen phase (7 weeks old) and subjected to ZD71 14 treatment versus control, with gross and histologic evidence demonstrating rapid anagen entry and restoration of the entire pelage in the treatment group. (See figure 1 and further description.) [0136] These results provide evidence that a relationship between adipose and hair follicle cycling exists, which supports the role of adrenergic receptors in stimulating progenitor populations within the hair follicle unit as a mechanism underlying this homeostatic relationship. These finding provide a basis for the use of |33-adrenergic agonists to elicit hair regrowth.

[0137] While attempts to achieve regenerative outcomes in wound healing have yet to be realized clinically, stem cell therapies are poised as promising candidates for this. However, clinical translation in this regard has been stifled by inherent teratogenicity of multipotent-cell-treatment options, alongside the considerable practical concerns of identifying, sourcing, harvesting, expanding, and transplanting alternative more-specific progenitor populations for use as treatments, not to mention associated ethical and regulatory hurdles in their path to clinical translation. Controlling the activity and fate of native stem-cell populations as opposed to relying on exogenous sources may obviate many of the above challenges. Thus, identification of progenitor populations and the means to pharmacologically target and manipulate these in vivo are prudent in regenerative medicine.

[0138] In attempts to inform novel therapeutic options to improve adverse wound healing outcomes, fundamental studies have aimed to leverage a better understanding of both fibrosis and stem cell biology. Investigations into the role of fibroblast heterogeneity and functionality during wound healing have sought to reveal mechanisms underlying the process of fibrosis. Although this may effectively inform potential therapeutic means to manipulate evolving or established scars, alternative lines of investigation are required to elucidate the function of stem and progenitor populations and associated queues which enable tissue regeneration versus scarring during healing. Recently there has been a focus on exploring the possibility of transdifferentiation between progenitors and fibroblasts, which would otherwise be considered disparate entities. This work is intriguing as it indicates existence of a potential axis of proregeneration vs. pro-fibrosis and revelation of associated mechanisms may prove useful to effectively promote one fate over the other. As such, a better collective understanding of both fibroblasts and progenitor populations and their relationships are likely required to both abrogate scarring while promoting the holy-grail of tissue regeneration. As the progenitor population of interest in this study was a subset of lineage negative cells from the dermis, it is effectively a subset of fibroblasts, as per the commonly employed means of lineage negative gating to identify fibroblasts.

[0139] Hence an axis can be studied assuming progenitor vs fibroblast fate can be manipulated, and that |33AR is implicit in dictating this, showing that agonism stimulated hair follicle neogenesis. Materials and Methods

[0140] Experimental Design: This study was designed to evaluate wound tissue grossly, histologically and at a cellular- and gene-level using a commonly employed murine wound healing model to generate and evaluate wound progression through harvest of wound tissue at multiple timepoints throughout wound closure. Considerations to the individual components of this study design are contained below.

[0141] Mice. C57BI/6 mice (Jackson Labs, ME, USA), aged 6-8 weeks were utilized for wound healing studies. Equal distribution of male and female mice was employed in accordance with NIH Policy on Sex as a Biological Variable in Research. C57BL/6-Tg(UBC-GFP)30Scha/J Ubiquitous GFP (Jackson Labs, ME, USA) mice were utilized for fluorescent cell harvest and transplant studies. Equal distribution of male and female mice was employed in accordance with NIH Policy on Sex as a Biological Variable in Research. Homozygous CDI -Foxnf™ immunodeficient nude mice (Charles River Laboratories, Wilmington, MA) were utilized as recipients for cell transfer studies. Equal distribution of male and female mice was employed in accordance with NIH Policy on Sex as a Biological Variable in Research.

[0142] Wound Healing Model. The dorsum of C57BI/6 mice were prepared for wounding by depilation under anesthesia. This was carried out mechanically using hair clippers (Wahl, Sterling, IL, USA) followed by 60 seconds of chemical depilation creme (Nair, Church & Dwight, Ewing, NJ, USA). After allowing overnight recovery following depilation, mice were prepped with alternating betadine and alcohol swab cleansing of the skin under anesthesia and subjected to bilateral, full thickness, dorsal cutaneous wounds, 8mm in diameter. Silicon rings were then fastened into place surrounding the wound using superglue and 8 x 5-0 nylon sutures in simple interrupted fashion for each ring. This aspect of the wound healing model ensures wounds remain stented open, preventing premature contracture of the mouse panniculus carnosus skin layer, while allowing for wound closure to occur at day 14, akin to the rate of cutaneous wound healing observed in humans. Wounds were dressed using tegaderm (3M, St Paul, MN, USA) dressings wrapped circumferentially around the torso of the mouse. Wounds were monitored regularly alongside dressing changes every 48 hours, with local topical pharmacological agent applied to treatment group mice upon these time points. Mice were sacrificed at various timepoints throughout wound healing immediately prior to harvesting wound tissue for subsequent analysis.

[0143] Anesthesia. Isoflurane (VetOne Las vegas, NV, USA) was vaporized at 2%W/V and administered via 100% oxygen at 2L/min. Anesthetic induction was carried out within a chamber, with maintenance throughout surgical procedures achieved via continuous flow through a nose cone (30ml syringe fastened to rubber anesthetic vaporizer outlet tube). [0144] Analgesia. Buprenorphine 0.1 mg/kg was administered intraperitoneally prior to surgery. Animals were monitored for any signs of pain or distress in the post operative period and issued further analgesia as necessary.

[0145] Adrenergic Pharmacologic Agents. B3AR antagonist: SR5230A (Tocris Bioscience, Bristol, UK), a selective beta3-AR antagonist, soluble in diH20 or DMSO was prepared in a 10 mM stock concentration and aliquoted by volumes for 10 doses at 5mg/kg per dose, i.e. 34.5 pl per dose.

[0146] B3AR agonist: ZD71 14 (Tocris Bioscience, Bristol, UK), a potent and selective beta3-AR agonist soluble in DMSO was prepared in a 10 mM stock concentration and aliquoted by volumes for 20 doses at 5 mg/kg per dose, i.e. 27.5 pl per dose.

[0147] Euthanasia. Mice were euthanized via CO ₂ gas chamber poisoning, in which CO ₂ was titrated up after mice were enclosed in the chamber. The mice spent at least 8 minutes in the chamber and after this time secondary confirmation of death was performed via cervical dislocation.

[0148] Wound Tissue Harvest for histology. Harvested wound tissue was immediately placed in 10% neutral buffered formalin for fixation in preparation for further tissue processing.

[0149] Wound Tissue Harvest for FACS. Harvested wound tissue was immediately placed onto petri dishes on ice and minced for subsequent tissue digest.

[0150] Tissue Processing and Embedding for paraffin-based histology. Following fixation in formalin for 14-24hours, tissue was washed in PBS x 3 times prior to serial dehydration. Tissue was dehydrated in ascending EtOH concentrations, 30% x 40 mins, 50% x 40 mins, 70% x 60 mins, 95% x 60 mins, and 100% for 60 mins. Tissue was then prepped in a solution of 100% EtOH and 100% Xylene in a 50:50 mix for 20 mins, followed by 100% xylene for 20 mins, followed by transfer to an oven at 58°C into a solution of 100% xylene and 100% paraffin in a 50:50 mix for 30 mins, followed by 3 washes in paraffin for 30 mins each. Tissue specimens were then orientated and embedded in paraffin blocks utilizing a Leica HistoCore Arcadia H embedding machine (Leica, Wetzlar, Germany). Embedded blocks were placed on a Leica HistoCore Arcadia C cool plate (Leica, Wetzlar, Germany) overnight to harden.

[0151 ] Tissue Processing and Embedding for cryosection histology. Following fixation in formalin for 14-24hours, tissue was washed in PBS x 3 times prior to transfer into a solution of 70% sucrose in diH20. After one week homogenizing in sucrose at 4 °C, tissue was flash frozen in OCT (TissueTek). This was carried out via orienting tissue specimens in plastic blocks which were filled with OCT and partially submerged in a container of dry ice and 95% EtOH. After freezing, blocks were stored at -40 °C overnight prior to cryo-sectioning. [0152] Paraffin-based Histology Sectioning. Paraffin embedded tissue samples were sectioned at 8um, unless otherwise specified, using a Leica RM2255 sectioning device (Leica, Wetzlar, Germany). Sections were immediately transferred to water bath at 40 °C prior to transfer onto Fisherbrand Superfrost Plus Microscope Slides (Thermofisher, Waltham, MA) and placed on a hotplate at 37 °C for 12hours to fix and dry.

[0153] Cryo/OCT-based Histology Sectioning. OCT -embedded tissue samples were sectioned at 8um, unless otherwise specified, using a ThermoFisher CryoStar NX70 Cyro-sectioning device (Thermofisher, Waltham, MA). Sections were immediately transferred onto Fisherbrand Superfrost Plus Microscope Slides (Thermofisher, Waltham, MA) and placed into slide boxes on a tray of dry ice plus stored at -20 °C prior to staining.

[0154] Haematoxylin & Eosin Staining. Select slides were placed in slide racks for transfer across serial buckets throughout the deparaffinization, rehydration, and staining process. Deparaffinization and rehydration was carried out via serial steps of: Xylene x 20 mins, 100% EtOH x 20 mins, 95% EtOH x 10 mins, 70% EtOH x 10 mins, 50% EtOH x10 mins, 30% EtOH x 10 mins, and running tap water for 10 mins. Slides were then placed in haematoxylin for 4 mins, washed in running tap water for 10 mins, dipped twice in 1% ammonia and placed in running tap water for 5 minutes, 95% EtOH for 5 mins, dipped 10 times in Eosin followed by placement in running tap water until eosin stopped leaking (~7 mins), 95% EtOH x 5 mins, 100% EtOH x 10 mins, and 8 dips in xylene before mounting with permount (Fisher Scientific) and a Richard-Allan Scientific Coverglass (Thermofisher, Waltham, MA).

[0155] Trichrome Staining. Select slides were placed in slide racks for transfer across serial buckets throughout the deparaffinization, rehydration, and staining process. Deparaffinization and rehydration was carried out via serial steps of: Xylene x 20 mins, 100% EtOH x 20 mins, 95% EtOH x 10 mins, 70% EtOH x 10 mins, 50% EtOH x10 mins, 30% EtOH x 10 mins, and running tap water for 10 mins. Following this the slides were placed in Bouins solution for 1 hour followed by a 5 minute rinse in running tap water for 5 mins, then Iron Haematoxylin for 5 mins, another wash in running tap water for 5 mins, Acid Fuschin for 4 mins, running tap water for 5 mins, Phosphotungistic acid for 45 mins, Aniline blue for 2 mins, running tap water for 5 mins, 1 % Acetic Acid for 5 mins, 15 dips in tap water, 15 dips in 95% EtOH, 15 dips in 100% EtOH, 8 dips in Xylene, and mounting with permount (Fisher Scientific) and a Richard-Allan Scientific Coverglass (Thermofisher, Waltham, MA).

[0156] Immunofluorescent Staining for Paraffin-based slides. Select slides were positioned in a slide rack and placed face up in an oven at 50°C for 1 hour prior to transfer to xylene for 20 mins followed by serial rehydration in descending alcohol concentrations until reaching diH2O for at least 5 mins. Slides were then covered in wash buffer (0.5% tween in PBS) for 10 mins, and incubated for 5-10 mins at 37°C with 1 :1 Trypsin Concentrate:Trypsin Buffer for antigen retrieval. Slides were then washed with PBS x3, followed by washing with 0.025% Triton X in PBS x 2 (the latter serves to dissolve none- specific fluorescein isothiocyanate). Slides were then blocked with 1 x Powerblock (diluted in PBS) for 1 hour. Upon removal of powerblock, specimens were isolated on the slide with a PAP pen-drawn border. Antibody mastermix was created for dilution of antibodies, and consisted of 0.1x Powerblock and 0.1x PBS Triton in a 1 :1 ratio. For primary antibody mix, antibodies were pipetted into the antibody mastermix at a 1 :100 concentration, or at the manufacturer’s recommendation. The overall volume of antibody mix needed was anticipated with the estimation that 100-150ul would be needed per slide, dependent on the number of specimens on said slide. For antibody incubation, slides were placed face up in a humidified chamber (i.e. damp paper towel lined slide box) for 1 hour at room temp. Primary antibody was then removed, and slides washed twice with 0.1x Tween to remove unbound antibodies, followed by a rinse with PBS. Secondary antibody, at a 1 :200 concentration in antibody mastermix (or at the concentration recommended by the manufacturer), was performed for 1 hour. Slides were then washed twice with 0.1x Tween followed by a PBS rinse. Slides were mounted with fluoromount with DAPI (Thermofisher) and a coverslip, fastened into place on either side with clear nail polish. Slides were stored at 4 °C prior to imaging.

[0157] Immunofluorescent Staining for OCT-based slides. Slides were washed with 0.1 x Tween x 2, followed by a PBS wash. Slides were then blocked with 1 x Powerblock (diluted in PBS) for 1 hour. Upon removal of powerblock, specimens were isolated on the slide with a PAP pen-drawn border. Antibody mastermix was created for dilution of antibodies, and consisted of 0.1 x Powerblock and 0.1x PBS Triton in a 1 :1 ratio. For primary antibody mix, antibodies were pipetted into the antibody mastermix at a 1 :100 concentration, or at the manufacturers recommendation. The overall volume of antibody mix needed was anticipated with the estimation that 100-150ul would be needed per slide, dependent on the number of specimens on said slide. For antibody incubation, slides were placed face up in a humidified chamber (i.e. damp paper towel lined slide box) for 1 hour at room temp. Primary antibody was then removed, and slides washed twice with 0.1 x Tween to remove unbound antibodies, followed by a rinse with PBS. Secondary antibody, at a 1 :200 concentration in antibody mastermix (or at the concentration recommended by the manufacturer), was performed for 1 hour. Slides were then washed twice with 0.1 x Tween followed by a PBS rinse. Slides were mounted with fluoromount with DAPI (Thermofisher) and a coverslip, fastened into place on either side with clear nail polish. Slides were stored at 4°C prior to imaging. [0158] Imaging. Microscopy imaging was performed with a Leica DMI4000B microscope with Leica DFC7000T camera attachment, and Leica CTR4000 light box for brightfield microscopy or lumencor sola light engine for laser-fluorescence microscopy. Where appropriate, for fluorescent microscopy of thicker specimens, higher resolution and higher magnification, a Leica SP8 confocal microscope was utilized (Leica, Wetzlar, Germany).

[0159] Tissue Preparation for FACS. Tissue was both mechanically and chemically digested. Initially, harvested tissue was minced using surgical scissors on a petri dish placed on ice. This tissue was then transferred to a conical tube with 1 :1 collagenase II and IV (Thermofisher), sealed with parafilm (Pechiney Co.) and placed on a shaker at 150rpm at 37°C for 90 minutes. Resultant digested tissue homogenate was filtered through 10Oum filters into 50ml conical tubes, with FACS buffer added to deactivate the collagenase and help rinse any remaining cells through the filter. The resultant cells in suspension were then centrifuged for 5 mins at 1500 RPM to produce cell pellets. Following this, the liquid portion was discarded and cells resuspended in FACS buffer and transferred to FACS Eppendorf tubes plus spun down again as a wash prior to removal of supernatant and resuspension in primary antibody at 1 :100 ratio. This was left to incubate on a mobile plate for 30mins. Following this, the cells were spun down, supernatant discarded and stained in secondary antibody at 1 :200 for 20mins plus filtered into FACS Falcon tubes prior to FACS analysis.

[0160] Flow Assisted Cell Sorting. A BD FACS Aria II Flow cytometer Machine (BD Biosciences) was used alongside BD FACS DIVA software. Sample tubes were loaded and gated by respective fluorescent markers and were analyzed for events, with incidences/cell populations of interest sorted into tubes for further analysis. FlowJo Software (FlowJo LLC) was used to analyze flow cytometry output.

[0161] Cell Culture. Cells of interest (as sorted by FACS) were plated onto 48 well plates for cell culture and maintained with standard culture media (from a stock consisting of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS), and anti- biotic/anti-mycotic (anti-anti) in a 40:5:5 ratio). Cells were monitored and media was extracted and replaced every 48 hours. Alternatively, extracted media was sent for cytokine analysis. Upon reaching confluence, cells were fixed in PFA for future adipogenic or immunofluorescent staining, or in the event of validating purity of the population, were harvested for staining and repeat FACS analysis.

[0162] RNA sequencing. Single cell RNA-sequencing was carried out as per the methods of Leavitt et aL: “Prrxl Fibroblasts Represent a Pro-fibrotic Lineage in the Mouse Ventral Dermis” on samples hashtagged by treatment group and harvest timepoint, with separate tubes for fibroblasts and APCs. In brief, the 10x Genomics Chromium Instrument was used, with barcoding performed via the 10x Chromium Single Cell platform, library preparation according to this manufacturers protocol, and sequencing of cDNA libraries carried out on a HiSeq 4000 Illumina platform. Cell ranger’s mkfastq (10x Genomics) was used to convert base calls to reads, which were then aligned against the mouse genome and evaluated for quality as delineated by apoptotic cell and background RNA thresholding. Data was normalized, hashtags were oligo demultiplexed, and cell subpopulations were identified following uniform manifold approximation projection (UMAP) analysis, with annotations ascribed using SingleR. Enrichment analysis was performed using EnrichR.

[0163] Statistical Analysis. Statistical analyses was carried out using GraphPad Prism sand Microsoft excel.

[0164] LipidTox. LipidTOX™ Deep Red (594) staining was carried out as per the manufacturer’s instructions. Following fixation of cells with 4% formaldehyde in buffer, this solution was removed and fixed cells rinsed with PBS buffer before staining with 1 :200 LipidTOX™ neutral lipid stain in buffer with incubation for 30minutes prior to image acquisition.

[0165] Pharmacological Vehicles. Dimethyl sulfoxide (DMSO) was the vehicle for ZD7114, as this agent is miscible in DMSO, and makes it suitable for storage as well as application. Of note, DMSO aids dermal penetrance thus providing benefit in delivery. DMSO was filtered through a vacuum conical tube filter under cell culture hood to ensure sterility prior to use for drug reconstitution.

[0166] SR59230A was miscible in diH20 obtained from a UV sterilized and filtered miliQ (sigma aidrich) device with an additional pass of filtration through a vacuum conical tube filter under a cell culture hood to ensure sterility.

[0167] Antibodies (Primary, Secondary and Conjugated, for Immunofluorescent Histology and FACS). Primary

Mouse Anti-PDGF Receptor alpha/PDGFR-a Antibody (C-9) (SantaCruz Biochem, Santa Cruz, CA, USA)

Rat Anti-Sca1 / Ly6A/E antibody [E13 161 -7] (abeam, Cambridge, MA, USA)

Goat (OCT/frozen) CD34 Antibody in IHC (F) (Thermofisher, Waltham, MA) Mouse Anti-UCP1 Antibody (A-6) (SantaCruz Biochem, Santa Cruz, CA, USA) Rabbit polyclonal anti mouse IgG antibody to UCP-1 (abeam, Cambridge, MA, USA) (Goat) Anti-beta 1 Adrenergic Receptor antibody (abeam, Cambridge, MA, USA) (Mouse) P2-AR Antibody (E-3) (SantaCruz Biochem, Santa Cruz, CA, USA) (Rabbit) Anti-beta 3 Adrenergic Receptor antibody (abeam, Cambridge, MA, USA) Rabbit polyclonal anti mouse IgG antibody to UCP-1 (abeam, Cambridge, MA, USA) Mouse Ki67 Antibody (Ki-67) (SantaCruz Biochem, Santa Cruz, CA, USA)

Collagen I Monoclonal Antibody (5D8-G9) 100ug mouse (Thermofisher, Waltham, MA) Anti-Tyrosine Hydroxylase antibody (abeam, Cambridge, MA, USA)

Anti-Neuropeptide Y antibody (abeam, Cambridge, MA, USA)

Secondary

Goat anti-Chicken IgY (H+L) Cross-Adsorbed Secondary Antibody, DyLight 755 (Thermofisher, Waltham, MA)

Donkey anti-Rat IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (Thermofisher, Waltham, MA)

Conjugated

CD140a (PDGFRA) Monoclonal Antibody (APA5), PE (eBioscience, San Diego, CA, USA) Brilliant Violet 605™ anti-mouse Ly-6A/E (Sca-1 ) Antibody (108133) (BioLegend, San Diego, CA, USA)

CD34 Antibody (ICO1 15) PerCP-Cy5.5 (SantaCruz Biochem, Santa Cruz, CA, USA)

CD45 Monoclonal Antibody (30-F11 ), eFluor 450, (eBioscience, San Diego, CA, USA)

TER-119 Monoclonal Antibody (TER-119), eFluor 450, (eBioscience, San Diego, CA, USA)

CD324 (E-Cadherin) Monoclonal Antibody (DECMA-1 ), Biotin (eBioscience, San Diego, CA, USA)

CD202b (TIE2) Monoclonal Antibody (TEK4), Biotin (eBioscience, San Diego, CA, USA) CD326 (EpCAM) Monoclonal Antibody (G8.8), eFluor 450 (eBioscience, San Diego, CA, USA) Pacific Blue CD31 Antibody (Thermofisher, Waltham, MA)