WO2016126947A2 - Subcutaneous complex and single-component ecm compositing for induction of hair growth and follicular regeneration - Google Patents

Description

SUBCUTANEOUS COMPLEX AND SINGLE-COMPONENT ECM COMPOSITING FOR INDUCTION OF HAIR GROWTH AND FOLLICULAR REGENERATION

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62/111,815, filed on February 4, 2015, which is hereby incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0002] Hair loss can result from both genetic and trauma-induced causes. Current therapies for hair loss include both physical and chemical manipulations of hair follicles and associated tissues. Minoxidil is a vasodilator that has been used for the treatment of androgenic alopecia, more commonly referred to as male pattern baldness. Though only 30% effective, it remains a common treatment for hair loss in lieu of an improved treatment regimen. Transplantation is an alternative therapy, involving excision of hair follicles and surrounding tissue from a less-desired location (such as the lower occipital region) and engrafting them into the frontal hairline. Additional therapies, including subcutaneous injections, are under investigation as a replacement for chemical and surgically invasive techniques. Induction of hair growth is through activation of the hair cycle, including emergence of new hair follicles and induction of the anagen phase. Anagen is the growth phase of the hair follicle cycle and precedes an involution phase, catagen, and a quiescent phase, telogen. These phases are distinguished morphologically in histology by the cells associated with the hair shaft and it's positioning within the dermal layer and cutaneous fat. Decellularized extracellular matrix (ECM) is used as a reconstructive scaffold in both wound repair and tissue regeneration among many other applications. ECM scaffolds are prepared by treating native tissue with acids and detergents to remove cellular material leaving behind the ECM and associated macromolecules. The intricate combination of proteins creates a complex scaffold for tissue regeneration, providing both structural support and signaling from the accompanying proteins and small molecules. These scaffolds have been used in tissue reconstruction, both in volumetric tissue loss and connective tissue patching. Sheet and particulate scaffolds are used in the clinic for abdominal wall repair and dermal wound repair, respectively. SUMMARY OF THE INVENTION

[0003] In accordance with one or more embodiments, the present inventors have found that the injection of ECM from various organs and tissues into the subcutaneous space of C57BL/6 mice induced hair growth and folliculogenesis. These results show that particulate ECM compositions described herein are useful for inducing hair growth and new follicle formation.

[0004] In accordance with an embodiment, the present invention provides a method for inducing folliculogenesis in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of folliculogenic composition to the site of interest.

[0005] In accordance with an embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of folliculogenic composition to the site of interest.

[0006] In accordance with an embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of a hyaluronidase composition to the site of interest.

[0007] In accordance with an embodiment, the present invention provides a

folliculogenic composition comprising a decellularized ECM scaffold comprised of tissues derived from one or more of bone, cardiac muscle, liver, lung and/or spleen.

[0008] In accordance with a further embodiment, the present invention provides a folliculogenic composition comprising a low molecular weight hyaluronic acid and at least one additional biologically active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1C: Increased hair growth after subcutaneous ECM implantation. Decellularized extracellular matrix (ECM) was injected subcutaneously (SubQ) into C57BL/6 mice. After 1 and 3 weeks post implantation, darkened skin and hair growth was observed on and around implantation site. (A) SubQ implants in mouse after 1 week incubation. (B) Implant after partial dissection. (C) Skin under implant showing darker pigmentation than surrounding shaved skin. [0010] Figures 2A-2F: Folliculogenesis induced by 3 weeks in several complex ECM implants. (A) Cross-section of cardiac matrix implant showing a increased magnification in panel (C). (B) Bone matrix implant. (D) Spleen matrix implant. (E) Lung matrix implant from a section showing shaft of anagen hair follicles. (F) Bone matrix implant showing increased folliculogenesis focused at a point directly above the implantation and decreasing with distance away from that point.

[0011] Figures 3A-3B: Expansion of subcutaneous adipose layer. (A) PBS injected control, Hematoxylin and Eosin (H&E) and Masson's Tri chrome (MAS) of Spleenic ECM implants. Sections showing skin above and near ECM implant. (B) Quantification of subcutaneous adipose layer width. In animals with ECM implants, increased adipose width and number of follicles were observed. Error bars represent standard deviation, significance calculated by student's T-test, pO.0001.

[0012] Figure 4: Ki67 Staining of implant and PBS injected control. Scattered Ki67 positive cells are observed in PBS control. In Splenic ECM implanted mouse, immature follicles with dense Ki67 staining show active cellular growth of hair follicles. EP = epidermis. A = adipose. M = muscle.

[0013] Figures 5A-5C: Implantation of Collagen and Low Molecular Weight Hyaluronate revealed possible role of HA in hair growth induction. (A) Subcutaneous bovine collagen injection showed minimal fat pad size increase and no robust follicle formation. Strong inflammation and encapsulation around implant suggest inflammation is not driving force in hair growth. (B) Low molecular weight hyaluronic acid (LMW-HA) injection showed full implant degradation coupled with adipose layer size increase and folliculogenesis. (C) Increased magnification of hair follicles in HA-injected animal.

[0014] Figure 6: Proposed mechanism of anagen induction after subcutaneous LMW HA injection. Tissue damage has been shown to induce hair growth and anagen transition. LMW- HA is a product of tissue damage and is recognized as a Damage- Associated Molecular Partem (DAMP) which indicates tissue damage. Through injection of LMW-HA we have activated a downstream pathway from tissue damage, which induces anagen of present hair follicles.

[0015] Figures 7A-7C: Induction of anagen-phase hair growth by low-molecular weight hyaluronic acid is dependent upon TLR2 signaling. (A) Histological evaluation of LMW-HA injections in B6.WT animals, showing induction of anagen by LMW-HA but not after immobilization with carbodiimide cross-linking (HA-NHS). (B) Anagen induction by LMW- HA is nullified in TLR2-null (Tlr2^"/_) mice. (C) Quantification of follicle counts in all 3 injections shown in figures a-b.

DETAILED DESCRIPTION OF THE INVENTION

[0016] Current treatments of alopecia include chemical and surgical intervention.

Pharmaceutical treatments often act indirectly through activating vasodilation and increasing circulation to the skin at the site of interest. This treatment is highly ineffective in most patients. Surgical treatment transplants hair follicle grafts from the patient to the site of interest, requiring a viable donor site. To further investigate the ability of ECM to induce hair growth, the inventors tested subcutaneous injections in a mouse model.

[0017] Signals for induction of anagen stage in hair follicles come from many sources, most of which are not fully understood. One mechanism is through tissue damage. This damage signals through unknown mechanisms, indirect or directly, to hair follicle cells such as keratinocytes. These signals induce the anagen phase and corresponding hair growth.

[0018] The present inventors found that subcutaneous or intradermal injection of a particulate extracellular matrix yielded a robust increase in hair growth. It is known that complex ECM scaffolds derived from whole tissues are composed of many structural and signaling molecules, such as proteoglycans, collagens and associated growth factors. It was found that injection of simple decellularized ECM scaffolds, such as hyaluronate and collagens, elucidated the participation of the different constituents of ECM scaffolds in hair growth. Induction of hair growth in LMW-HA but not collagen implants points to HA as a possible mediator of ECM scaffold-induced folliculogenesis. LMW-HA is a known damage- associated molecular partem (DAMP), which signals to resident cells that there is damage.

[0019] In accordance with an embodiment, the present invention provides a method for inducing folliculogenesis in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of folliculogenic composition to the site of interest.

[0020] In some embodiments, the site of interest is subcutaneous. In other embodiments, the site of interest is intradermal. It is understood that the term intradermal is generally used to mean injection of a composition within the epidermal and/or dermal layers of skin. It is understood that the term subcutaneous is generally used to mean injection of a composition below the layers of epidermis, including the dermis and/or adipose tissue. [0021] In some embodiments the folliculogenic composition comprises an ECM composition and/or low molecular weight hyaluronic acid. The folliculogenic composition can comprise whole tissue-derived ECM scaffolds which are decellularized and can be composed of micro and nano sized particles via cryo-milling and hydraulic sheering. The particles can range from 0.1 nm in diameter to about 500 microns in diameter.

[0022] In some embodiments, the whole tissue used to prepare the ECM scaffolds for the folliculogenic composition can be obtained from almost any tissue source in the body.

Examples of such tissues include, but are not limited to, bone, cardiac, lung, kidney, muscle, skin, and spleen tissues. Typically the tissues are prepared into small pieces, approximately 5 mm³, and exposed to a process including formic acid for sufficient time to reduce calcium levels in the tissues to near undetectable. The tissues are then treated with 3.0% peracetic acid for a sufficient number of hours, changing the solution after 2 hours of incubation, and then washed in suitable buffers until the pH was returned to 7. The tissues are then incubated in a non-ionic detergent, such as triton x-100 and 2 mM EDTA for up to 72 hours, and then washed with distilled water to remove all detergent. The tissues are then exposed to DNAse at a concentration of between 500 and 1000 U/ml and 10 mM MgCh to remove all nucleic acid material. The tissues can then washed and lyophilized and cryo-milled to micro or nano size particles. Other means of preparing particles known in the art can also be used.

[0023] In accordance with an embodiment, the present invention provides a

folliculogenic composition comprising a decellularized ECM scaffold comprised of tissues derived from one or more of bone, cardiac muscle, liver, lung and/or spleen and other tissues.

[0024] In some embodiments, the decellularized ECM scaffold can be made from human or swine tissues.

[0025] Hyaluronan, also known as hyaluronic acid (HA) or sodium hyaluronate, is a naturally occurring linear polymer. Hyaluronan is a glycosaminoglycan (GAG) that is ubiquitously present in the extracellular matrix of all vertebrates and is also present in the capsule of some strains of Streptococci. Mammalian hyaluronan is synthesized by one of three distinct hyaluronan synthases (HAS1, 2, and 3), which produce HA polymers with different chain lengths and differ in their rates of synthesis. Whereas high molecular weight HA (> 500 kDa) is known to be anti-angiogenic, anti-inflammatory and immunosuppressive, low molecular weight HA (10 - 500 kDa) is known highly angiogenic and pro-inflammatory.

[0026] In accordance with some embodiments, the folliculogenic composition comprises low molecular weight HA (LMW-HA). For example, HA which has been degraded with hyaluronidase can produce LMW-HA. Generally, LMW-HA has a molecular weight average between about 10 kDa to about 50 kDa.

[0027] As such, in accordance with a further embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of LMW-HA to the site of interest. In some embodiments, the dosage of LWM-HA would come in a biologically compatible solution between about 1 mg/ml to about 100 mg/ml LWM-HA, preferably about 20 mg/ml, of which between 0.1 and 10 ml would be injected, preferably about 0.5 ml to about 2 ml of LMW-HA would be injected.

[0028] In accordance with a further embodiment, the present invention provides a folliculogenic composition comprising a low molecular weight hyaluronic acid and at least one additional biologically active agent.

[0029] In some embodiments LMW-HA, and/or the ECM scaffold compositions can be combined with other short chain Glycosoaminoglycans known to also activate TLR2 or TLR4, most notably chondroitin sulfate and its analogs. Further, these GAGs can be combined with short peptides of corresponding proteoglycans such as for versican, a proteoglycan molecule which increases anagen and is a chondroitin sulfate proteoglycan. Other related naturally occuring LMW which can be suitable in the inventive methods and compositions includes, for example, CS, GAG, mucopolysaccharide, collagen or

proteoglycan components, such as hyaluronic acid, heparin sulfate, glucosamines, dermatans, keratans, heparans, hyalurunan, aggrecan, and the like.

[0030] In accordance with an embodiment, the present invention provides the use of a folliculogenic composition in a subject in need thereof, comprising an effective amount of a decellularized ECM scaffold and/or low molecular weight hyaluronic acid, wherein said use comprises administering to the subject an effective amount of folliculogenic composition to the site of interest.

[0031] In accordance with another embodiment, it is contemplated that because high molecular weight HA is a component of the subcutaneous ECM in vivo, LMW-HA can be generated in situ by the administration in situ of hyaluronidase to the site of interest. There are FDA approved hyaluronidase compositions currently available, including hylenex and vitrase. Thus, in accordance with an embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of hyaluronidase to the site of interest. For example, an effective amount of hyaluronidase can be within the range of 0.1 U to 1000 U, preferably 1 U to about 300 U, and more preferably about 10 U to about 100 U.

[0032] In accordance with an embodiment, the present invention provides the use of a folliculogenic composition in a subject in need thereof, comprising an effective amount of hyaluronidase and a pharmaceutically acceptable carrier, wherein said use comprises administering to the subject an effective amount of folliculogenic composition to the site of interest.

[0033] Given that HA also modulates the immune response, HA is indicated also for conditions of immune attack against the hair follicle, such as alopecia areata. It is thought that HMW HA, such as FDA approved dermatology fillers such as Restylane, or Juvederm, can be converted also somewhat to LMW HA, either naturally or with the use of hyaluronidase.

[0034] As such, in accordance with a further embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of HMW-HA and HMW-HA with hyaluronidase to the site of interest. In some embodiments, the dosage of HWM-HA would come in a biologically compatible solution between about 1 mg/ml to about 100 mg/ml HWM-HA, preferably about 20 mg/ml, of which between 0.1 and 10 ml would be injected, preferably about 0.5 ml to about 2 ml of HMW-HA would be injected. This would be combined with effective amount of hyaluronidase as described above, to create a LMW-HA composition in situ.

[0035] In accordance with some embodiments, the folliculogenic composition is administered subcutaneously to the site of interest in the subject. In an embodiment, the folliculogenic composition is administered by injection, however, other means, such as surgical implantation, for example, can also be used in the inventive methods.

[0036] It will be understood by those of ordinary skill in the art, that the folliculogenic compositions will be used to stimulate folliculogenesis and hair growth at the site of administration. The administration can be part of a larger treatment regimen, which can require repeated administration of the folliculogenic compositions are administered one or more times to the site of the subject. This can be part of a long term treatment plan, where the subject can be treated over the course of weeks or months or years.

[0037] In accordance with some embodiments, the folliculogenic composition can be combined with a pharmaceutically acceptable carrier. Preferably, the carrier is a

pharmaceutically acceptable carrier. With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use.

[0038] The choice of carrier will be determined in part by the particular inventive composition and method, as well as by the particular method used to administer the inventive composition and method. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the invention. In some embodiments, the formulations are suitable for use in cutaneous and subcutaneous administration.

[0039] Injectable formulations are in accordance with the invention. The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982), mdASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0040] The formulations and compositions of the inventive methods can be administered intradermally or subcutaneously via any known means in the art. Examples of typical means of administration include needles of various gauges and pressurized air injectors and similar means. In other embodiments, microneedle devices can be used. Conventionally, a microneedle device has been known as a device for improving transdermal absorption of drugs. A microneedle device includes a microneedle array, and microneedles provided on a base of the microneedle array are intended to pierce the stratum comeum. Further, various methods are also known in regard to a method of applying the compositions of the present invention by use of a microneedle array. Coating the surface of microneedles with compositions, forming a groove or a hollow part in microneedles through which

compositions or body components are allowed to penetrate, mixing compositions into microneedles themselves, and the like are known.

[0041] In accordance with some embodiments, the folliculogenic composition can be mixed with one or more biocompatible polymers which allow the folliculogenic composition to remain in situ for longer periods of time. [0042] "Biodegradable" is art-recognized, and includes monomers, polymers, polymer matrices, gels, compositions and formulations, such as those described herein, that are intended to degrade during use, such as in vivo. Biodegradable polymers and matrices typically differ from non-biodegradable polymers in that the former may be degraded during use. In certain embodiments, such use involves in vivo use, such as in vivo therapy, and in other certain embodiments, such use involves in vitro use. In general, degradation attributable to biodegradability involves the degradation of a biodegradable polymer into its component subunits, or digestion, e.g., by a biochemical process, of the polymer into smaller, non-polymeric subunits. In certain embodiments, two different types of biodegradation may generally be identified. For example, one type of biodegradation may involve cleavage of bonds (whether covalent or otherwise) in the polymer backbone. In such biodegradation, monomers and oligomers typically result, and even more typically, such biodegradation occurs by cleavage of a bond connecting one or more of subunits of a polymer. In contrast, another type of biodegradation may involve cleavage of a bond (whether covalent or otherwise) internal to a side chain or that connects a side chain, functional group and so on to the polymer backbone. For example, a therapeutic agent, biologically active agent, or other chemical moiety attached as a side chain to the polymer backbone may be released by biodegradation. In certain embodiments, one or the other or both general types of biodegradation may occur during use of a polymer. As used herein, the term

"biodegradation" encompasses both general types of biodegradation.

[0043] The degradation rate of a biodegradable polymer often depends in part on a variety of factors, including the chemical identity of the linkage responsible for any degradation, the molecular weight, crystallinity, biostability, and degree of cross-linking of such polymer, the physical characteristics of the implant, shape and size, and the mode and location of administration. For example, the greater the molecular weight, the higher the degree of crystallinity, and/or the greater the biostability, the biodegradation of any biodegradable polymer is usually slower. The term "biodegradable" is intended to cover materials and processes also termed "bioerodible."

[0044] In certain embodiments, polymeric formulations of the present invention biodegrade within a period that is acceptable in the desired application. In certain embodiments, such as in vivo therapy, such degradation occurs in a period usually less than about five years, one year, six months, three months, one month, fifteen days, five days, three days, or even one day on exposure to a physiological solution with a pH between 6 and 8 having a temperature of between about 25 and 37° C. In other embodiments, the polymer degrades in a period of between about one hour and several weeks, depending on the desired application. In some embodiments, the polymer or polymer matrix may include a detectable agent that is released on degradation.

[0045] Biocompatible polymer, biocompatible cross-linked polymer matrix and biocompatibility are art-recognized. For example, biocompatible polymers include polymers that are neither themselves toxic to the host (e.g., and animal or human), nor degrade (if the polymer degrades) at a rate that produces monomeric or oligomeric subunits or other byproducts at toxic concentrations in the host. In certain embodiments of the present invention, biodegradation generally involves degradation of the polymer in an organism, e.g., into its monomeric subunits, which may be known to be effectively non-toxic. Intermediate oligomeric products resulting from such degradation may have different toxicological properties, however, or biodegradation may involve oxidation or other biochemical reactions that generate molecules other than monomeric subunits of the polymer. Consequently, in certain embodiments, toxicology of a biodegradable polymer intended for in vivo use, such as implantation or injection into a patient, may be determined after one or more toxicity analyses. It is not necessary that any subject composition have a purity of 100% to be deemed biocompatible; indeed, it is only necessary that the subject compositions be biocompatible as set forth above. Hence, a subject composition may comprise polymers comprising 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75% or even less of biocompatible polymers, e.g., including polymers and other materials and excipients described herein, and still be biocompatible.

[0046] "Gel" refers to a state of matter between liquid and solid, and is generally defined as a cross-linked polymer network swollen in a liquid medium. Typically, a gel is a two- phase colloidal dispersion containing both solid and liquid, wherein the amount of solid is greater than that in the two-phase colloidal dispersion referred to as a "sol." As such, a "gel" has some of the properties of a liquid (i.e., the shape is resilient and deformable) and some of the properties of a solid (i.e., the shape is discrete enough to maintain three dimensions on a two-dimensional surface).

[0047] Hydrogels consist of hydrophilic polymers cross-linked to from a water-swollen, insoluble polymer network. Cross-linking can be initiated by many physical or chemical mechanisms. Photopolymerization is a method of covalently crosslink polymer chains, whereby a photoinitiator and polymer solution (termed "pre-gel" solution) is exposed to a light source specific to the photoinitiator. On activation, the photoinitiator reacts with specific functional groups in the polymer chains, crosslinking them to form the hydrogel. The reaction is rapid (3-5 minutes) and proceeds at room and body temperature.

Photoinduced gelation enables spatial and temporal control of scaffold formation, permitting shape manipulation after injection and during gelation in vivo. Cells and bioactive factors can be easily incorporated into the hydrogel scaffold by simply mixing with the polymer solution prior to photogelation.

[0048] Alternatively, the reactants can contain complementary reactive groups, as an imide and an amide, that yield cross-linking without the need of an external initiator.

[0049] Hydrogels of interest can be semi-interpenetrating networks that promote cell, tissue and organ repair while discouraging scar formation. The hydrogels of interest also are configured to have a viscosity that will enable the gelled hydrogel to remain affixed on or in the cell, tissue or organ, or surface. Viscosity can be controlled by the monomers and polymers used, by the level of water trapped in the hydrogel, and by incorporated thickeners, such as biopolymers, such as proteins, lipids, saccharides and the like. An example of such a thickener is hyaluronic acid or collagen.

[0050] "Incorporated," "encapsulated," and "entrapped" are art-recognized when used in reference to a therapeutic agent, dye, or other material and a polymeric composition, such as a composition of the present invention. In certain embodiments, these terms include incorporating, formulating or otherwise including such agent into a composition that allows for sustained release of such agent in the desired application. The terms may contemplate any manner by which a therapeutic agent or other material is incorporated into a polymer matrix, including, for example, attached to a monomer of such polymer (by covalent or other binding interaction) and having such monomer be part of the polymerization to give a polymeric formulation, distributed throughout the polymeric matrix, appended to the surface of the polymeric matrix (by covalent or other binding interactions), encapsulated inside the polymeric matrix, etc. The term "co-incorporation" or "co-encapsulation" refers to the incorporation of a therapeutic agent or other material and at least one other therapeutic agent or other material in a subject composition.

[0051] More specifically, the physical form in which any therapeutic agent or other material is encapsulated in polymers may vary with the particular embodiment. For example, a therapeutic agent or other material may be first encapsulated in a microsphere and then combined with the polymer in such a way that at least a portion of the microsphere structure is maintained. Alternatively, a therapeutic agent or other material may be sufficiently immiscible in the polymer of the invention that it is dispersed as small droplets, rather than being dissolved in the polymer. Any form of encapsulation or incorporation is contemplated by the present invention, in so much as the sustained release of any encapsulated therapeutic agent or other material determines whether the form of encapsulation is sufficiently acceptable for any particular use.

[0052] Suitable hydrophilic polymers to serve as the polymers combined with the folliculogenic compounds include synthetic polymers such as poly(ethylene glycol), poly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol),

poly(vinylpyrrolidone), poly(ethyloxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamers and meroxapols), poloxamines, carboxymethyl cellulose, and hydroxyalkylated celluloses such as hydroxy ethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as polypeptides, polysaccharides or carbohydrates such as Ficoll™, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin, carboxy methyl starch, or copolymers or blends thereof. As used herein, "celluloses" includes cellulose and derivatives of the types described above; "dextran" includes dextran and similar derivatives thereof.

[0053] In accordance with an embodiment, the folliculogenic composition is administered in combination with one or more additional biologically active agents. The terms "active agent" and a "biologically active agent" are used interchangeably herein to refer to a chemical or biological compound that induces a desired pharmacological and/or

physiological effect, wherein the effect may be prophylactic or therapeutic. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of those active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the terms "active agent,"

"pharmacologically active agent" and "drug" are used, then, it is to be understood that the invention includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs etc. The active agent can be a biological entity, such as a virus or cell, whether naturally occurring or manipulated, such as transformed.

[0054] Further examples of biologically active agents include, without limitation, enzymes, receptor antagonists or agonists, hormones, growth factors, autogenous bone marrow, antibiotics, antimicrobial agents, and antibodies. The term "biologically active agent" is also intended to encompass various cell types and genes that can be incorporated into the compositions of the invention.

[0055] In accordance with another embodiment, the present invention provides a method for inducing hair growth in a cutaneous site of interest in a subject comprising administering to the subject an effective amount of folliculogenic composition to the site of interest. In some embodiments, the folliculogenic composition comprises an ECM composition and/or low molecular weight hyaluronic acid. In other embodiments, the folliculogenic composition is administered subcutaneously to the site of interest in the subject. In addition, the folliculogenic composition can be administered in combination with one or more additional biologically active agents.

[0056] In some embodiments, the additional active agent can include Toll-Like Receptor (TLR) agonists. Examples of TLR agonists include imiquimod. Currently, two TLR agonists are FDA approved for use in cancer patients in addition to imiquimod, the TLR4 agonist monophosphoryl lipid A (MPL), and the TLR2/4 agonist bacillus Calmette-Guerin (BCG). Also of interest is the TLR7/8 agonist, resiquimod, which is an imidazoquinoline like imiquimod, all of which can be used in the inventive methods.

[0057] In accordance with the inventive methods for inducing hair growth, the folliculogenic composition can be administered one or more times, as part of a treatment regimen, as needed to continue growth of hair at the site of interest.

[0058] It will be understood by those of ordinary skill in the art, that the inventive compositions and methods described herein can be useful for the treatment of alopecia or hair loss from a number of different causes. Examples of alopecia which may be treated with the inventive compositions and methods described herein, include, but are not limited to, alopecia areata, alopecia totalis, alopecia universalis, lipedematous alopecia, androgenetic alopecia, chemotherapeutic agents, ectodermal dysplasia, radiation, endocrine disorders, acne keloidalis nuchae, central centrifugal cicatricial alopecia, cellulitis of the scalp, burns, skin cancer, sarcoidoisis, and trauma.

[0059] As used herein, the terms "treating" or "treatment" are art-recognized terms which includes curing as well as ameliorating at least one symptom of any condition or disease. Treating includes reducing the likelihood of a disease, disorder or condition from occurring in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, e.g., causing any level of regression of the disease; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder or condition, even if the underlying pathophysiology is not affected or other symptoms remain at the same level.

[0060] The subject referred to in the inventive methods is a mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. It is preferred that the mammals are from the order Carnivora, including Felines (cats) and Canines (dogs). It is more preferred that the mammals are from the order

Artiodactyla, including Bovine (cows) and Swine (pigs) or of the order Perssodactyla, including Equine (horses). It is most preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0061] The kits disclosed herein will include a container means for the folliculogenic composition of interest. The kit may include a delivery device. The kit optionally will include a container means for a second folliculogenic composition of interest, and/or one or more additional active agents. Instructions for their use can be included.

[0062] Uses for such kits include, for example, therapeutic applications. The invention provides kits for use in treating a disease or condition. For example, the kit may comprise an LMW-HA, and ECM matrix, a syringe and instructions for administration.

EXAMPLES

[0063] ECM Scaffold Preparation.

[0064] Native tissue (bone, cardiac muscle, liver, lung and spleen) was knife-milled to form small 5 mm³ pieces then washed thoroughly with distilled water to remove blood. Bone was pre-treated by incubation in 10% Formic acid (Sigma) for 1 week, changing daily until the solubilized calcium levels were undetectable by a colorimetric calcium test. Tissues were then treated with 3.0% peracetic acid for 4 hours, changing the solution after 2 hours of incubation. The pH was returned to 7 by successive washes with IX PBS. Samples were then incubated in 1% Triton XI 00 + 2 mM EDTA for 3 days. Thorough washing with distilled water removed all residual Triton-X100 (Sigma). The last step included 600 U/ml DNase + 10 mM MgCh to remove residual nucleic acids. Final wash with distilled water preceded lyophilization and cryo-milling (SPEX SamplePrep Freezer Mill) to form micron-scale particles. Bovine collagen (Sigma) was milled in the same manner as the tissue-derived ECM to form a fine powder. Sodium Hyaluronate (13 kD, LifeCore Biomedical) was used without additional processing.

[0065] Subcutaneous scaffold injection.

[0066] Female 6 week old C57BL/6 mice were shaved using an electric razor and injected with 0.2 cc of a PBS-ECM suspension containing 200 mg dry-weight ECM.

Collagen was prepared in a similar manner to the complex ECM implants. Sodium hyaluronate was hydrated at 300 mg per 0.1 cc implant. The implants were allowed to incubate for 1 and 3 weeks prior to dissection. PBS controls were injected in the same manner with 0.2 cc 1XPBS. Protocols for all in vivo experiments were approved in compliance with the NIH Guide for Care and Use of Laboratory Animals.

[0067] Histology.

[0068] Implants were fixed in 10% Formalin (Sigma) overnight prior to ethanol dehydration and mounting in paraffin. 5 μιτι sections were stained directly with Hematoxylin and Eosin or Masson's Tri chrome. Antigen retrieval for Ki67 (Abeam) immunostaining was performed in a sodium citrate antigen retrieval buffer for 30 minutes in a vegetable steamer. Samples were blocked in 1% BSA for 1 hour at room temperature, stained for Ki67 at 1 : 100 dilution in PBS + 1% BSA overnight at 4 °C in a humidity chamber. Primary antibody was washed off and staining was visualized using SuperPicTure HRP-DAB Chromogen Kit (Invitrogen).

EXAMPLE 1

[0069] Injection of complex extracellular matrix (ECM) scaffolds results in hair growth in C57BL/6 mice.

[0070] C57BL/6 mice received 0.2 cc extracellular matrix (ECM) implants via subcutaneous injection. After 1 week implant incubation, animals were sacrificed and their implants and surrounding tissue were removed for analysis. Prior to dissection, darkened skin and hair growth were observed above and around ECM implant (Figure 1). Bone, cardiac, lung, and spleen matrices induced hair growth at 3 weeks post injection. This effect was least apparent in the lung matrix, but still visible. After 1 week implants displayed localized hair growth surrounding the injection site, whereas after 3 weeks, the hair growth in several implants had spread outside of the surrounding area.

EXAMPLE 2

[0071] Histological analysis shows increased folliculogenesis and fat pad width in animal injected with complex ECM scaffolds.

[0072] Various ECM-derived scaffolds, including Bone (Figure 2B), Cardiac (Figure 2A, C), Lung (Figure 2D), and Spleen (Figure 2E) matricies increased hair follicle formation. The skin above several implants, such as bone matrix, contained anagen follicles and tapered off to a more telogen-dominant population further from the implant (Figure 2F). Folliculogenesis was not induced in animals injected with liver derived ECM. Mice that displayed hair growth phenotype also displayed a large increase in white adipose (Figure 3B). This was

accompanied by an increase in follicles, which were present in both the dermal connective tissue and adipose pad (Figure 3 A). In saline-injected controls this white adipose pad was four times smaller, and follicles were only present in dermal connective tissue. This increase in adipogenesis has been associated with induction of anagen in hair follicles.

EXAMPLE 3

[0073] Ki67 immunostaining reveals proliferating follicles in white adipose pad.

[0074] Ki67 immunostaining of ECM- and saline-injected animals confirmed the presence of active cellular proliferation (Figure 4). Saline control showed scattered Ki67+ cells in existing follicles in the dermal connective tissue. After ECM injection, there was an induction of folliculogenesis, which is also marked by large clusters of Ki67+ cells in the white adipose pad. This staining, along with morphology of follicles and surrounding tissue, suggests an induction of anagen through subcutaneous ECM injection.

EXAMPLE 4

[0075] Injection of Hyaluronic Acid but not Collagen yields folliculogenesis.

[0076] To test the role of differing ECM molecules in hair growth, we injected single molecule ECM implants, including bovine Collagen (Figure 5A) and low molecular weight hyaluronate (LMW-HA) (Figures 5B, 5C). LMW-HA implant fully absorbed 7 days post injection, whereas a thick capsule formed around the Collagen implant. LMW-HA induced folliculogenesis, whereas Collagen promoted an inflammatory response without resulting hair growth. This suggests that inflammation due to ECM injection is not the sole contributor to hair growth. Torsional strain on skin caused by implantation did not induce the hair growth seen in complex ECM implants, as the collagen implant remained without degradation and no hair growth was seen whereas the degraded HA implant caused growth. This also confirms that residual cellular materials did not cause hair growth observed in complex ECM implants.

[0077] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0078] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0079] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

Claims:

1. Use of a folliculogenic composition in a subject in need thereof, comprising an effective amount of a decellularized ECM scaffold and/or low molecular weight hyaluronic acid, wherein said use comprises administering to the subject an effective amount of folliculogenic composition to the site of interest.

2. The use of claim 1, wherein the folliculogenic composition comprises a decellularized ECM scaffold and low molecular weight hyaluronic acid.

3. The use of claim 1, wherein the folliculogenic composition is administered subcutaneously to the site of interest in the subject.

4. The use of claim 1, wherein the folliculogenic composition is administered intradermally to the site of interest in the subject.

5. The use of either of claims 3 or 4, wherein the folliculogenic composition is administered one or more times.

6. The use of either claims 1 or 2, wherein the folliculogenic composition is administered in combination with one or more biocompatible hydrophilic polymers.

7. The use of either claims 1 or 2, wherein the folliculogenic composition is administered in combination with one or more additional biologically active agents.

8. The use of either claims 1 or 2, wherein the folliculogenic composition is administered using a needle or microneedle.

9. The use of claim 1 , wherein the ECM composition is a decellularized ECM composition from at least one tissue and/or organ from a swine or human source.

10. The use of claim 1 , wherein the ECM composition is in a particulate form with diameters from 0.1 nm to 500 μηι.

1 1. The use of either claims 1 or 2, wherein the folliculogenic composition induces hair growth in a cutaneous site of interest in the subject.

12. Use of a folliculogenic composition in a subject in need thereof, comprising an effective amount of hyaluronidase and a pharmaceutically acceptable carrier, wherein said use comprises administering to the subject an effective amount of folliculogenic composition to the site of interest.

13. The use of claim 12, wherein the folliculogenic composition further comprises an effective amount of high molecular weight hyaluronic acid and a biologically compatible carrier.

14. The use of either of claim 12 or 13, wherein the folliculogenic composition is administered subcutaneously to the site of interest in the subj ect.

15. The use of either of claim 12 or 13, wherein the effective amount of hyaluronidase administered is between 0.1 U to 1000 U, preferably 1 U to about 300 U, and more preferably about 10 U to about 100 U.

16. The use of either of claim 12 or 13, wherein the effective amount of high molecular weight hyaluronic acid administered is between 1 mg/ml to about 100 mg/ml HWM-HA, preferably about 20 mg/ml.

17. The use of either of claim 12 or 13, wherein the folliculogenic composition is administered one or more times.

18. The use of either of claim 12 or 13, wherein the folliculogenic composition is administered in combination with one or more biocompatible hydrophilic polymers.

19. The use of either of claim 12 or 13, wherein the folliculogenic composition is administered in combination with one or more additional biologically active agents.

20. The use of either of claim 12 or 13, wherein the folliculogenic composition is administered using a needle or microneedle.

21. The use of either claims 12 or 13, wherein the folliculogenic composition induces hair growth in a cutaneous site of interest in the subject.