WO2011031977A1 - Traitements par intermittence et par administration unique, à base de lithium pour la révision des cicatrices et la cicatrisation des plaies - Google Patents

Traitements par intermittence et par administration unique, à base de lithium pour la révision des cicatrices et la cicatrisation des plaies Download PDF

Info

Publication number
WO2011031977A1
WO2011031977A1 PCT/US2010/048439 US2010048439W WO2011031977A1 WO 2011031977 A1 WO2011031977 A1 WO 2011031977A1 US 2010048439 W US2010048439 W US 2010048439W WO 2011031977 A1 WO2011031977 A1 WO 2011031977A1
Authority
WO
WIPO (PCT)
Prior art keywords
lithium
skin
treatment
wound
acid
Prior art date
Application number
PCT/US2010/048439
Other languages
English (en)
Inventor
William D. Ju
Stephen M. Prouty
Shikha P. Barman
Scott C. Kellogg
Eric Schweiger
Seth Lederman
Original Assignee
Follica, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Follica, Inc. filed Critical Follica, Inc.
Publication of WO2011031977A1 publication Critical patent/WO2011031977A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/18—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
    • A61K8/20—Halogens; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A61K8/365—Hydroxycarboxylic acids; Ketocarboxylic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/18—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves
    • A61B18/20—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser
    • A61B18/203—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by applying electromagnetic radiation, e.g. microwaves using laser applying laser energy to the outside of the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B2018/00315—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body for treatment of particular body parts
    • A61B2018/00452—Skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/81—Preparation or application process involves irradiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N5/00—Radiation therapy
    • A61N5/06—Radiation therapy using light
    • A61N5/0613—Apparatus adapted for a specific treatment
    • A61N5/0616—Skin treatment other than tanning

Definitions

  • the invention relates to intermittent lithium treatments, or a single pulse lithium treatment for scar revision and wound healing in human subjects.
  • Uses of compositions containing compounds that liberate lithium ions are described, including adjuvants and devices for administration.
  • the intermittent treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays.”
  • a dose of lithium is administered over a short period of time.
  • the lithium treatment(s) can be used in combination with other treatments for scar revision, wound healing, and hair follicle neogenesis.
  • Such combination treatments may involve mechanical or physical treatments that modulate scar revision or wound healing, or that cause integumental perturbation ⁇ e.g.
  • the combination treatment(s) may be administered concurrently with, or during the "holidays" between, cycles of intermittent lithium treatments; or concurrently with, or before and/or after the pulse lithium treatment.
  • Wound healing or wound repair, is an intricate process in which the skin (or some other organ) repairs itself after injury.
  • the epidermis (outermost layer) and dermis (inner or deeper layer) exist in a steady-state equilibrium, forming a protective barrier against the external environment. Once the protective barrier is broken, the physiologic process of wound healing is immediately set in motion.
  • the classic model of wound healing is divided into three or four sequential, yet overlapping, phases: (1) hemostasis (not considered a phase by some authors); (2) inflammation; (3) proliferation; and (4) maturation and remodeling.
  • fibrin clot When tissue is wounded, blood platelets (thrombocytes) aggregate at the injury site to form a fibrin clot. This clot acts to control active bleeding (hemostasis). Fibrin and fibronectin crosslink to form a plug that traps proteins and particles and prevent further blood loss. This fibrin-fibronectin plug, also called the extracellular matrix, is also the main structural support for the wound until collagen is deposited. Migratory cells use this plug as a matrix to crawl across, and platelets adhere to it and secrete factors. The clot is eventually lysed and replaced with granulation tissue and then later with collagen.
  • the contact of blood with collagen during wounding triggers platelets to begin secreting inflammatory factors, extracellular matrix proteins, cytokines, and growth factors.
  • the proinflammatory factors released by platelets like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine, increase cell proliferation and migration to the area, cause blood vessels to initially constrict to prevent further blood loss, and then cause blood vessels to become dilated and porous.
  • Vasoconstriction lasts five to ten minutes and is followed by vasodilation, a widening of blood vessels, which peaks at about 20 minutes post- wounding.
  • the main factor involved in vasodilation is histamine, which also causes blood vessels to become porous, allowing the tissue to become edematous. Increased porosity of blood vessels facilitates the entry of inflammatory cells into the wound site from the bloodstream.
  • PMNs polymorphonuclear neutrophils
  • helper T cells which secrete cytokines to cause more T cells to divide and to increase inflammation and enhance vasodilation and vessel permeability. T cells also increase the activity of macrophages.
  • Gamma-delta T cells are enriched in epidermis and become activated upon wounding, secreting growth factors that induce keratinocyte proliferation (Havran and Jameson, 2010).
  • Macrophages are essential to wound healing. They replace PMNs as the predominant cells in the wound by two days after injury. Attracted to the wound site by growth factors released by platelets and other cells, monocytes from the bloodstream enter the area through blood vessel walls. Numbers of monocytes in the wound peak one to one and a half days after the injury occurs. Once they are in the wound site, monocytes mature into macrophages. The main role of macrophages is to phagocytose bacteria and damaged tissue, and they also debride damaged tissue by releasing proteases.
  • Macrophages also produce factors that induce and speed angiogenesis; secrete a number of factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days, which attract cells involved in the proliferative stage of healing; and stimulate cells that re-epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix.
  • factors such as growth factors and other cytokines, especially during the third and fourth post-wounding days, which attract cells involved in the proliferative stage of healing; and stimulate cells that re-epithelialize the wound, create granulation tissue, and lay down a new extracellular matrix.
  • inflammation As inflammation dies down, fewer inflammatory factors are secreted, existing ones are broken down, and numbers of neutrophils and macrophages are reduced at the wound site. Because inflammation plays a role in fighting infection, clearing debris and inducing the proliferative phase, it is a necessary part of healing. However, inflammation can lead to tissue damage if it lasts too long. Thus the reduction of inflammation is frequently a goal in therapeutic settings. Inflammation lasts as long as there is debris in the wound. Thus the presence of dirt or other objects can extend the inflammatory phase for too long, leading to a chronic wound.
  • the proliferative phase is characterized by the overlapping steps of angiogenesis, collagen deposition, granulation tissue formation, epithelialization, and wound contraction.
  • angiogenesis neovascularization
  • new blood vessels are formed by vascular endothelial cells.
  • angiogenesis is also imperative for other stages in wound healing.
  • Endothelial growth and proliferation is stimulated by the presence of lactic acid in the wound and by hypoxia.
  • Endothelial stem cells from uninjured blood vessels attracted to the wound by fibronectin found on the fibrin scab and chemotactically by angiogenic factors released by other cells, migrate through the extracellular matrix (ECM) into the wound site to establish new blood vessels.
  • ECM extracellular matrix
  • the tissue in which angiogenesis has occurred typically looks red (is erythematous) due to the presence of capillaries.
  • Granulation tissue functions as rudimentary tissue, and begins to appear in the wound during the inflammatory phase, two to five days post wounding, and continues growing until the wound bed is covered.
  • Granulation tissue consists of new blood vessels, fibroblasts, inflammatory cells, endothelial cells, myofibroblasts, and the components of a new, provisional ECM.
  • the provisional ECM is different in composition from the ECM in normal tissue. Its components - fibronectin, hyaluronan, collagen, glycosaminoglycans, elastin, glycoproteins and proteoglycans - originate from fibroblasts. Later this provisional matrix is replaced with an ECM that more closely resembles that found in non-injured tissue.
  • Fibroblasts begin entering the wound site two to five days after wounding and their numbers peak at one to two weeks post-wounding. In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while later, they are the main cells that lay down the collagen matrix in the wound site. Fibroblasts from normal tissue migrate into the wound area from its margins. Initially fibroblasts use the fibrin scab formed in the inflammatory phase to migrate, adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed, and later collagen, which they can adhere to for migration.
  • Growth factors e.g., PDGF, TGF- ⁇
  • fibronectin encourage proliferation, migration to the wound bed, and production of ECM molecules by fibroblasts.
  • Hypoxia also contributes to fibroblast proliferation and secretion of growth factors (e.g. , growth factors that attract epithelial cells to the wound site), though too little oxygen inhibits proliferation and deposition of ECM components, which may lead to excessive, fibrotic scarring. Fibroplasia ends two to four weeks after wounding.
  • Fibroblasts begin secreting appreciable collagen by the second or third post-wounding day, and its deposition peaks at one to three weeks. Collagen deposition is important because it increases the strength of the wound; before it is laid down, the only thing holding the wound closed is the fibrin- fibronectin clot, which does not provide much resistance to traumatic injury. Also, cells involved in inflammation, angiogenesis, and connective tissue
  • Keratinocytes migrate without first proliferating. Migration of keratinocytes over the wound site is stimulated by lack of contact inhibition and by chemicals such as nitric oxide. Migration can begin as early as a few hours after wounding, although the time of onset of migration is variable. Epithelial cells require viable tissue to migrate across, so if the wound is deep it must first be filled with granulation tissue. Cells on the wound margins proliferate on the second and third day post-wounding in order to provide more cells for migration.
  • Epithelial cells climb over one another in order to migrate. This growing sheet of epithelial cells is often called the epithelial tongue. Basal and suprabasal cells become mobilized and both migrate and get “pushed” into the wound site (Usui et al, 2005). The more quickly this movement occurs, the less of a scar there will be.
  • Fibrin, collagen, and fibronectin in the ECM may further signal cells to divide and migrate.
  • migrating keratinocytes use the fibronectin cross-linked with fibrin that was deposited in inflammation as an attachment site to crawl across. As keratinocytes migrate, they move over granulation tissue but underneath the scab (if one was formed), separating it from the underlying tissue. Epithelial cells have the ability to phagocytose debris such as dead tissue and bacterial matter that would otherwise obstruct their path. Because they must dissolve any scab that forms, keratinocyte migration is best enhanced by a moist environment, since a dry one leads to formation of a bigger, tougher scab.
  • keratinocytes To make their way along the tissue, keratinocytes must dissolve the clot, debris, and parts of the ECM in order to get through. They secrete plasminogen activator, which activates plasminogen, turning it into plasmin to dissolve the scab. Cells can only migrate over living tissue, so they must secrete collagenases and proteases to dissolve damaged parts of the ECM in their way, particularly at the front of the migrating sheet. Keratinocytes also dissolve the basement membrane, using instead the new ECM laid down by fibroblasts to crawl across.
  • Keratinocytes continue migrating across the wound bed until cells from either side meet in the middle, at which point contact inhibition causes them to stop migrating. When they have finished migrating, the keratinocytes secrete proteins that form the new basement membrane and become anchored once again to the basement membrane. Basal cells begin to divide and differentiate in the same manner as they do in normal skin to re-establish the strata found in re-epithelialized skin.
  • Contraction Around a week after wounding, fibroblasts differentiate into myofibroblasts and the wound begins to contract. In full thickness wounds, contraction peaks at 5 to 15 days post wounding. Contraction can last for several weeks and continues even after the wound is completely re-epithelialized. If contraction continues for too long, it can lead to
  • Contraction reduces the size of the wound.
  • a large wound can become 40%-80% smaller after contraction.
  • Wounds can contract at a speed of up to 0.75 mm per day, depending on how loose the tissue in the wounded area is.
  • Contraction usually occurs along an "axis of contraction," which allows for greater organization and alignment of cells with collagen.
  • contraction occurs without myofibroblast involvement.
  • fibroblasts stimulated by growth factors, differentiate into myofibroblasts.
  • Myofibroblasts which are similar to smooth muscle cells, are responsible for contraction.
  • Attracted by fibronectin and growth factors they move along fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges, where they form connections to the ECM and to each other.
  • actin in the myofibroblast is linked across the cell membrane to molecules in the extracellular matrix like fibronectin and collagen.
  • Myofibroblasts have many such adhesions, which allow them to pull the ECM when they contract, reducing the wound size. In this part of contraction, closure occurs more quickly than in the first, myofibroblast-independent part. As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts lay down collagen to reinforce the wound as myofibroblasts contract. The contraction stage ends as myofibroblasts stop contracting and commit apoptosis. The breakdown of the provisional matrix leads to a decrease in hyaluronic acid and an increase in chondroitin sulfate, which gradually triggers fibroblasts to stop migrating and proliferating. These events signal the onset of the maturation stage of wound healing.
  • the maturation and remodeling phase of tissue repair is said to have begun.
  • the maturation phase can last for a year or longer, depending on the size of the wound and whether it was initially closed or left open.
  • type III collagen which is prevalent during proliferation, is gradually degraded and the stronger type I collagen is laid down in its place.
  • disorganized collagen fibers are rearranged, cross-linked, and aligned along tension lines.
  • the tensile strength of the wound increases, with the strength approaching 50% that of normal tissue by three months after injury and ultimately becoming as much as 80% as strong as normal tissue. Since metabolic activity at the wound site is reduced, the scar loses its red appearance as blood vessels that are no longer needed are removed by apoptosis.
  • wounds may heal by primary intention.
  • Such wounds may be referred to as "closed wounds.” These wounds are usually surgically closed in layers along tissue planes by a physician. In primary intention, a linear scar results at the intersection of the approximated tissues.
  • Scarring is often minimal, but can be variable depending on the size and location of the wound, the tension on tissue and other factors. Most surgical wounds are sutured closed, so they heal by primary intention. In primary intention, wound closure is usually performed with sutures, staples, or an adhesive. Other examples of wounds that heal by primary intention are well repaired lacerations, well reduced bone fractures, and wounds that heal after flap surgery (the edges of which tend to appear scarred).
  • wounds are referred to as "open wounds.”
  • wounds formed by blast injury, shrapnel e.g., from improvised explosive devices ("IEDs")
  • blunt trauma e.g., blunt trauma
  • dental wounds e.g., gingivectomy, gingivoplasty, tooth extraction sockets
  • poorly reduced fractures e.g., gingivectomy, gingivoplasty, tooth extraction sockets
  • third degree burns heal by secondary intention.
  • Healing by secondary intention follows the same basic steps as wounds that heal by primary intention, i.e., inflammation, proliferation, and remodeling, but each sequence may take much longer, especially the proliferative phase.
  • Wound care must be performed daily to encourage wound debris removal to allow for granulation tissue formation. Depending on the size and location of the wound, placement of a partial or full-thickness skin graft may be considered if no infection is present and the area is of sufficient size that healing may not be complete for at least 2 or 3 weeks. Infection and inflammation of the wound can dysregulate repair and transform the wound into a clinically non-healing wound.
  • wound healing by tertiary intention the wound is initially cleaned, debrided, and observed, and typically 4 or 5 days elapse before closure.
  • the wound is purposely left open. Examples include healing of wounds by use of tissue grafts.
  • a major component of wound healing in humans is scar formation.
  • a scar (“cicatrix”; plural, “cicatrices”) is an area of fibrous tissue that forms as part of the healing process to replace normal skin after injury.
  • a hallmark of scars is altered extracellular matrix, notably a reduction of elastin fibers (De Vries et al, 1995).
  • Scars result from damage to the dermis, and with the exception of very minor lesions, every wound results in some degree of scarring. Scars generally form in proportion to the extent of damage.
  • Scar tissue is also usually of inferior functional quality.
  • a scar is a collagen-rich, elastin-poor dermal matrix with a simple stratified epithelial covering. Deposition of such a collagen-rich matrix in the neo- dermis is prone to contracture, loss in elasticity, and reduced tensile strength. Scars in the skin are also less resistant to ultraviolet radiation. For example, scars from skin transplants are typically dysfunctional, discolored, etc. Skin flaps and grafts are common methods of achieving rapid closure of large defect wounds. Not only do these methods tend to result in scarring at the donor site, but the sites of apposition of flap or graft edges to the wound edges can also result in linear scars.
  • TGFs Transforming Growth Factors
  • RSK Ribosomal s6 kinase
  • Scarring is likely to be extensive when wounds heal by secondary intention.
  • the wound heals by granulation, wherein epithelial cells grow over the wound from all sides of the normal skin, which results in a shiny layer of epithelial cells and fibrous tissue that is rich in collagen but does not contain underlying structures ("adnexal structures," including hair follicles).
  • adnexal structures including hair follicles
  • the scar also lacks the suppleness of normal skin. This type of scar can result in contractures when it occurs over the mouth or eyes or on the skin around joints, and can be disfiguring.
  • atrophic scars In addition to scars that form by secondary intention, there are numerous other types of scars that we distinguish, including atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (which, compared with atrophic scars, also have contour abnormality, while "atrophic" scars are implied to have only thinning), ice-pick scars (a type of depressed scar), spread scars (scars that widen due to tension over a time period, and which may become somewhat atrophic in the center), fineline scars, widespread (or stretched) scars, scar contractures, and other "intermediate" types of scars that are difficult to categorize.
  • types of scars see Bayat et al, 2003, BMJ. 326:88-92, the contents of which are incorporated herein by reference in its entirety.
  • Atrophic scars are characteristic of scars that are too thin, and typically form below the plane of the skin. Atrophic scars have a pitted appearance, and are caused when underlying structures supporting the skin, such as fat or muscle, are lost. This type of scarring is commonly associated with acne, but can also be caused by chickenpox, surgery or an accident. Acne scars and striae (scars from stretched skin) are exemplary of atrophic scars. Striae are caused when the skin is stretched rapidly (for instance during pregnancy, significant weight gain, or adolescent growth spurts), or when skin is put under tension during the healing process, usually near joints. This type of scar usually improves in appearance after a few years.
  • Keloid scars are raised above the surface of the skin, but by definition they grow beyond the boundaries of the original wound. Keloids can be viewed as a tumorous (although benign) growth. Keloid scars can occur on anyone, but they are most common in dark-skinned people. Keloid scars can be caused by surgery, an accident, by acne or, sometimes, from body piercings. In some people, keloid scars form spontaneously. Although they are primarily a cosmetic problem, keloid scars can be itchy or painful in some individuals. They tend to be most common on the shoulders, chest and earlobes.
  • Human skin appendages also referred to as "adnexal” structures, include hair and hair follicles, sebaceous glands (which secrete sebum onto hair follicle to oil the hair), eccrine and apocrine sweat glands, and nails.
  • the skin of an adult human is essentially covered with hair follicles and contains approximately five million hair follicles, with approximately 100,000 - 150,000 covering the scalp. Only a minority of human hair follicles produce a hair fiber that can be appreciated visibly (a "terminal hair") and these specialized follicles are localized on specific regions of skin.
  • the portions of human skin that lack visible hair contain, for the most part, hair follicles that produce "vellus hair” while certain other hair follicles may contain or produce no hair (see Figure 1).
  • Hair follicles and particularly human hair follicles, are crypt structures comprised of distinct components, each comprised of several different specialized cells (see Figures 2 and 3).
  • the vast majority of hair follicles contain units called sebaceous glands (which produce sebum), and a minority (follicles located in specialized areas of the skin) also contain apocrine glands (which produce sweat used primarily for olfactory cues).
  • sebaceous glands which produce sebum
  • apocrine glands which produce sweat used primarily for olfactory cues.
  • the structures of the hair follicle include the follicular papilla (FP) and the germinative epithelium (GE) (together, the bulb).
  • the FP is comprised of mesenchymal cells (and connective tissue).
  • the other cells of the follicle are epithelial and include at least 8 cellular lineages including the outer root sheath (ORS), the companion layer (CL), the internal root sheath Henle's layer (He), internal root sheath Huxley's layer (Hu), the cuticle of the internal root sheath (Csth), the cuticle of the hair shaft (Csft), the cortex of the hair shaft, and the medulla of the shaft (Med).
  • ORS outer root sheath
  • CL the companion layer
  • He internal root sheath Henle's layer
  • Huxley's layer Hu
  • the cuticle of the internal root sheath Csth
  • Csft cuticle of the hair shaft
  • the mechanism for the switch from regeneration to repair with age in mammals is not fully known, but involves cytokines and an immune response that promotes rapid epidermal closure, dermal fibroplasia and increased collagen deposition.
  • hair follicles do not form, thus contributing to the lack of adnexal structures in a scar.
  • the abnormal structure of scars contributes to the their associated morbidity. Due to lack of eccrine glands, there is poor thermoregulation. Increased fibroplasia and collagen can lead to contracture and loss of mobility in affected areas of the body.
  • hair follicle keratinocytes contribute to epidermal closure, and hair follicle dermal sheath fibroblasts play central roles in dermal healing. See, Ito et al, 2005; Jahoda & Reynolds, 2001; Stenn & Paus, 2001, Physiol. Revs. 81 :449-494.
  • the only skin tissue (aside from scar tissue) that normally lacks hair follicles is the glabrous skin on palmar and plantar aspects of hands and feet, respectively, and the lips and labia.
  • human glabrous skin lacks hair follicles, it is rich in eccrine sweat glands.
  • Wound healing studies in pig have shown that sweat glands, by themselves, are capable of regenerating epidermis, which likely accounts for lack of scarring in glabrous skin wounds that spare the base of sweat glands.
  • palmar Barret at al, 2000
  • plantar Barret & Herndon, 2004 wounds can result in scarring, a sequelae related to increased depth of wound and delayed wound healing.
  • hair follicles were not understood to be capable of neogenesis and bulge cells were not proven to be a source of stem cells for hair follicle neogenesis.
  • Fathke then turned the focus of their investigation to the -catenm-independent Wnts expressed in the skin - which are not activated by lithium chloride.
  • the prolonged expression of the ⁇ -catenin independent Wnt yielded the same results - mature hair follicles were not generated in the mice and large epithelial cysts formed in the wounds.
  • Fathke interpreted their data as evidence for the restoration of tissue patterning in the adult mammalian wound epithelium - a feature not normally seen in adult cutaneous wound healing - they provided neither evidence of hair follicle neogenesis, nor the concomitant hair growth and enhanced wound healing that would be expected in connection with it.
  • mice are a poor model for human wound healing and scar formation. Mice tend to heal most wounds rapidly, with little or no scarring. In humans, however, severe wounds and burns are usually associated with cutaneous repair that results in scar tissue, no hair follicles, and the loss of regenerative capability that hair follicles may provide ⁇ see, Fathke et al, 2006, BMC Cell Biol. 7:4).
  • One reason for the difference in wound healing capability between humans and mouse may be that the biology of hair follicles in humans and mice differs in several significant respects. In the mouse, a thick fur coating is essential to healthy life (because hair plays roles in
  • Acute treatment of wounds is generally focused on hemostasis and antimicrobial considerations. The treatment depends on the type, cause, and depth of the wound as well as whether other structures beyond the skin are involved. Treatment of acute lacerations involves examination, cleaning, and closing the wound. If the laceration occurred some time ago, it may be allowed to heal by secondary intention due to the high rate of infection associated with immediate closure. Minor wounds like bruises tend to heal on their own with skin discoloration that usually disappears within 1 -2 weeks. Abrasions usually require no active treatment except keeping the area clean with soap and water, although scarring may occur. Puncture wounds may be prone to infection depending on the depth of penetration.
  • a greater amount of exudate and necrotic tissue in a wound increases likelihood of infection by serving as a medium for bacterial growth away from the host's defenses. Since bacteria thrive on dead tissue, wounds are often surgically debrided to remove the devitalized tissue. Debridement and drainage of wound fluid are an especially important part of the treatment for diabetic ulcers, which may create the need for amputation if infection gets out of control. Mechanical removal of bacteria and devitalized tissue is also the idea behind wound irrigation, which is accomplished using pulsed lavage.
  • maggot therapy the intentional introduction by a health care practitioner of live, disinfected maggots into nonhealing wounds.
  • Maggots dissolve only necrotic, infected tissue; disinfect the wound by killing bacteria; and stimulate wound healing.
  • Maggot therapy has been shown to accelerate debridement of necrotic wounds and reduce the bacterial load of the wound, leading to earlier healing, reduced wound odor and less pain. The combination and interactions of these actions make maggots an extremely potent tool in chronic wound care.
  • Negative pressure wound therapy is a treatment that improves ischemic tissues and removes wound fluid used by bacteria. This therapy, also known as vacuum- assisted closure, reduces swelling in tissues, which brings more blood and nutrients to the area, as does the negative pressure itself. The treatment also decompresses tissues and alters the shape of cells, causes them to express different mRNAs and to proliferate and produce ECM molecules.
  • Persistent chronic pain associated with non-healing wounds is caused by tissue (nociceptive) or nerve (neuropathic) damage and is influenced by dressing changes and chronic inflammation.
  • Chronic wounds take a long time to heal and patients can suffer from chronic wounds for many years.
  • Chronic wound healing may be compromised by coexisting underlying conditions, such as venous valve backflow, peripheral vascular disease, uncontrolled edema and diabetes mellitus.
  • Underlying ischemia may also be treated surgically by arterial revascularization, for example in diabetic ulcers, and patients with venous ulcers may undergo surgery to correct vein dysfunction.
  • Diabetics that are not candidates for surgery may also have their tissue oxygenation increased by Hyperbaric Oxygen Therapy, or HBOT, which can compensate for limitations of blood supply and correct hypoxia.
  • HBOT Hyperbaric Oxygen Therapy
  • higher oxygen content in tissues speeds growth factor production, fibroblast growth, and angiogenesis.
  • increased oxygen levels also means increased production of ROS.
  • Antioxidants molecules that can lose an electron to free radicals without themselves becoming radicals, can lower levels of oxidants in the body and have been used with some success in wound healing.
  • chronic wound healing may be speeded by replacing or stimulating those factors and by preventing the excessive formation of proteases like elastase that break them down.
  • VEGF vascular endothelial growth factor
  • IGF insulin-like growth factor 1-2
  • PDGF transforming growth factor- ⁇
  • EGF epidermal growth factor
  • Other treatments include implanting cultured keratinocytes into the wound to re-epithelialize it and culturing and implanting fibroblasts into wounds.
  • SLPI Secretory leukocyte protease inhibitor
  • Some patients are treated with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen to replicate skin and release growth factors.
  • skin from cadavers is grafted onto wounds, providing a cover to keep out bacteria and preventing the buildup of too much granulation tissue, which can lead to excessive scarring.
  • the allograft skin transplanted from a member of the same species
  • it encourages cellular proliferation and provides a structure for epithelial cells to crawl across.
  • allografts may not work, requiring skin grafts from elsewhere on the patient, which can cause pain and further stress on the patient's system.
  • Collagen dressings are another way to provide the matrix for cellular proliferation and migration, while also keeping the wound moist and absorbing exudate.
  • Skin grafts do not fully address the need for effective scar revision because (1) there is a limited supply of donor tissue (typically buttocks, abdomen, legs, in-front or behind the ear, etc.); (2) scarring occurs at donor sites and contractures ⁇ i.e., around the eyes and mouth); and (3) skin grafts typically (but not always) (with the possible exception of scalp) lack qualities of the donor site (porosity, vascularity, color, pigmentation, thickness, texture and overall cosmetic appearance, etc.).
  • the scar is serially excised, and a balloon is implanted at the wound site, which pushes the tissue around the scar to expand.
  • Surgical excision of hypertrophic or keloid scars is often used with other methods such as pressotherapy or silicone gel sheeting (see below). Lone excision of keloid scars shows a high recurrence rate, close to 45%.
  • Surgical excision in combination with the immunomodulator imiquimod 5% cream may also have a benefit on scar reduction.
  • Other intralesional injections can also be used.
  • collagen injections or other soft tissue fillers can be used to raise sunken scars to the level of surrounding skin. Its effects are temporary, however, and it needs to be regularly repeated. There is also a risk in some people of an allergic reaction.
  • Silicone scar treatments improve scar appearance and are often used to prevent and treat hypertrophic scarring.
  • the exact mechanism of action is unknown, though some studies suggest a manipulation of local ionic charges or a decrease in production of "proinflammatory" substances like TGF 2.
  • Dimethicone silicone gel appears to be is as effective as silicone sheeting in improving scar appearance.
  • Mustoe TA, 2008 "Evolution of silicone therapy and mechanism of action in scar management," Aesth Plast Surg 32:82-92. Polyurethane bandages are also used.
  • Pressure garments are used under supervision by a medical professional. They are most often used for burn scars that cover a large area, and is only effective on recent scars. Pressure garments are usually custom-made from elastic materials, and fit tightly around the scarring. They work best when they are worn 24 hours a day for six to twelve months. It is believed that they work by applying constant pressure to surface blood vessels and eventually causing scars to flatten and become softer.
  • Needling is an inexpensive process where the scarred area is continuously needled to promote collagen formation. Once needled the area is allowed to fully heal, and needled again if required depending on the intensity of the scar. Scarring needles and needling rollers are available for home use; however, needling should not be done on parts of the face or areas where major nerves are located without professional medical supervision. Needling at home must also be done in line with hygienic and sterilization requirements.
  • Dermabrasion involves the removal of the surface of the skin with specialist equipment and usually involves a general anesthetic. It is useful with raised scars, but is less effective when the scar is sunken below the surrounding skin.
  • Low-dose, superficial radiotherapy is used to prevent re-occurrence of severe keloid and hypertrophic scarring. It is usually effective, but only used in extreme cases due to the risk of long-term side effects.
  • Vitamin E causes contact dermatitis in up to 33% of users and in some cases it may worsen scar appearance. See, Baumann & Spencer, 1999, "The effects of topical vitamin E on the cosmetic appearance of scars," Dermatol Surg. 25:31 1-315; and Jenkins et al, 1986, “Failure of topical steroids and vitamin E to reduce postoperative scar formation following reconstructive surgery,” J Burn Care Rehabil 7: 309-312.
  • vitamin C normalizes collagen production and encourages the production of an organized, healthy collagen framework, which improves scar appearance.
  • Vitamin C and some of its esters also fade the dark pigment associated with some scars. See, Fitzpatrick & Rostan, 2002, “Double-blind, half-face study comparing topical vitamin C and vehicle for rejuvenation of photodamage,” Dermatol Surg 28:231-236; and Farris PK, 2005, “Topical vitamin C: a useful agent for treating photoaging and other dermatologic conditions,” Dermatol Surg 31 :814-818.
  • the intermittent and pulse lithium treatments can be administered in situ to acute wounds, chronic wounds, to scars, and/or surrounding skin.
  • the intermittent or pulse lithium treatments can be administered to the wound site or surrounding skin before, at the time of, and/or subsequent to, either acute wounding or, more typically, the wounding that is induced in scar revision.
  • the intermittent and pulse lithium treatments may also be administered to skin-derived cells or skin tissue ex vivo.
  • an intermittent or pulse lithium treatment may be used to enhance hair follicle neogenesis or enhance the re-association of dissociated hair follicle cells into follicles and facilitate their growth and expansion either in situ, or, alternatively, in culture for their implantation into fresh wounds and scar revisions.
  • hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors).
  • Engineered human skin, or human skin equivalents can also be used for hair follicle neogenesis and scar revision platforms.
  • Intermittent lithium treatments or a single pulse lithium treatment are used to revise scars and heal wounds in human subjects.
  • Any pharmaceutically acceptable compound that releases the lithium ion also referred to herein as lithium cation, Li+, or ionized lithium
  • lithium ion also referred to herein as lithium cation, Li+, or ionized lithium
  • compounds include, but are not limited to lithium gluconate, lithium succinate, and other organic salts/acids; and lithium chloride and other inorganic salts/acids, as described in Section 5.1, infra.
  • the intermittent lithium treatment protocol involves multiple courses of lithium treatment interrupted by lithium treatment "holidays" (periods during which no lithium treatment is administered).
  • a lithium treatment holiday is a period of time during which the patient stops the lithium treatment with the intent of resuming treatment.
  • a dose of lithium is administered over a short period of time.
  • the lithium treatment can be administered topically, transdermally, intradermally, cutaneously, subcutaneously, intramuscularly, intravenously, orally, sublingually, or can be bucchal.
  • Topical lithium treatment is a preferred embodiment because high local concentrations can be achieved while minimizing systemic exposure.
  • lithium gluconate 8% weight/weight (w/w) gel e.g., Lithioderm 8% gel
  • w/w gel commercially available in France for the treatment of seborrheic dermatitis
  • lithium is formulated into a modified release form that allows controlled release, over time, into the skin.
  • the lithium is formulated as part of a mesh scaffold that delivers lithium into the skin. More details on these and other lithium formulations and delivery methods for use in the treatment methods described herein are described in Sections 5.1-5.3 infra.
  • the intermittent and pulse lithium treatments can be administered alone to wounded skin (e.g., prior to, during, or subsequent to scar revision, or acute skin wounding, or chronic skin wounding) or in combination with other treatments to enhance wound healing or scar revision.
  • the intermittent and pulse lithium treatments can also be administered in combination with other treatments that facilitate hair follicle development and deposition into the wounded skin.
  • Embodiments of the invention include combination therapies, involving the addition of other treatment(s) concurrently with, or during the breaks between, the cycles of intermittent lithium treatments; or the addition of other treatment(s) concurrently with, or before and/or after the pulse lithium treatment.
  • Such combination therapies can include, but are not limited to, the concurrent or sequential use of other chemical agents, or mechanical or physical treatments including but not limited to, laser ⁇ e.g., Fraxel), dermatome planing, laser abrasion, electrology, intense pulsed light, or surgical treatments ⁇ e.g., skin graft or follicular unit extraction (FUE), etc.) that promote scar revision or wound healing.
  • laser e.g., Fraxel
  • dermatome planing e.g., dermatome planing
  • laser abrasion e.g., electrology, intense pulsed light
  • surgical treatments ⁇ e.g., skin graft or follicular unit extraction (FUE), etc.
  • intermittent lithium treatments or pulse lithium treatments in combination with perturbation e.g., debriding, peeling, or wounding
  • perturbation e.g., debriding, peeling, or wounding
  • methods such as laser treatment, dermabrasion, needling (using, e.g., microneedles), electromagnetic disruption, electroporation, or sonoporation; chemically ⁇ e.g., to induce inflammation); or by any other method described herein or known in the art, prior to or concurrent with administration of a lithium formulation described herein.
  • the integumental perturbation procedure can be any combination with perturbation (e.g., debriding, peeling, or wounding) of the skin and/or other tissues of the integumentary system by methods such as laser treatment, dermabrasion, needling (using, e.g., microneedles), electromagnetic disruption, electroporation, or sonoporation; chemically ⁇ e.g., to induce inflammation); or by any other method described herein or known in
  • wounding procedure used for scar revision.
  • the procedure can be controlled to limit perturbation to the epidermis, or extend deeper into the dermis and/or hypodermis.
  • the occurrence of pinpoint bleeding would indicate removal of the epidermis and portions of the upper layer of the dermis.
  • the occurrence of increased bleeding would indicate deeper penetration (and thus perturbation) into the dermis layer.
  • lasers particularly fractional lasers, and skin graft, follicular unit, and skin component transplant technologies have the capacity to induce regenerative changes in skin that mimic wounding and have applications in revision of scars.
  • laser techniques may "mimic" the plastic, embryonic-like, state of the epidermis created by other wound signals, but with laser's precision, versatility, and demonstrated efficacy in small scars.
  • intermittent lithium treatments and pulse lithium treatments administered concurrently or in sequential/alternating combination with other agents or treatments that modulate the wound healing process.
  • the intermittent and pulse lithium treatments may be administered with treatments that either promote or delay the wound healing process, such as described in Section 5.4.3 infra.
  • the intermittent lithium treatments or pulse lithium treatments described herein can be administered concurrently or alternating sequentially with one or more of the following treatments that prevent follicle senescence, for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors; stimulators of ROS breakdown, such as selenium; mTOR inhibitors, such as rapamycin; or sirtuins or activators thereof, such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
  • anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants
  • ROS reactive oxygen species
  • stimulators of ROS breakdown such as selenium
  • mTOR inhibitors such as rapamycin
  • sirtuins or activators thereof such as resveratrol
  • the intermittent lithium treatments or a pulse lithium treatment provided herein can also be administered concurrently or alternating sequentially with one or more of the following treatments that promote hair growth, in order to enhance formation of new hair follicles: minoxidil, finasteride, bimatoprost (Latisse), CaCl 2 , or adenosine, or techniques of integumental perturbation such as, e.g. , by mechanical means, chemical means,
  • electromagnetic means e.g. , using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCh-based, or Erbium- YAG-based, etc.
  • irradiation irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair
  • Treatments that promote hair growth, or, alternatively, treatments that prevent hair growth may also be used in combination with the intermittent lithium treatments or a pulse lithium treatment described herein in order to promote the establishment of desired hair patterning in the healed wound or revised scar, thereby improving the appearance of the treated skin.
  • treatments that regulate gender-specific specialized human hair follicles including those under the influence of sex-steroid regulation, or that regulate the differentiation of stem cells into gender-specific specialized human hair follicles, possibly resulting in follicles having features that are different from natural follicles in the target location of skin (e.g., normal sized follicles with terminal hair where previously miniaturized follicles with vellus hair were present) may be administered.
  • treatment of grafted skin with a combination of lithium and a modulator of specific hair patterning may reduce donor dominance and enhance the ability of the graft to acquire properties of the recipient site.
  • intermittent lithium treatments or a pulse lithium treatment may be used concurrently or in sequential combination with either a treatment that enhances hair growth (described above) or a cytotoxic drug, a hair growth retardant, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5-FU) (e.g., Efudex 5% cream), or other epilation or depilation methods to prevent or reduce hair growth.
  • Success of a pulse or intermittent lithium treatment described herein can be measured by one or more of the following outcomes:
  • Human subjects who are candidates for the pulse or intermittent lithium treatments described herein include any subject in need of improved wound healing, particularly wound healing without scarring, or scar revision.
  • Human subjects who are candidates for such treatments include any subject for whom improved wound healing or scar revision is desired.
  • Such human subjects include, but are not limited to, subjects with photodamaged skin, acne scars, chicken pox scars, scarring (cicatricial) alopecia, chronic non-healing wounds or scars due to, e.g., diabetes, venous or arterial disease, old age or senescence, infection, medication, chemotherapy, trauma, burns, stress, autoimmune disease, malnutrition, or endocrine dysfunction.
  • Surgical subjects who are candidates for such treatments include, but are not limited to, patients with skin graft, hair transplantation, skin cancer surgery, or Mohs surgery.
  • Subjects who are candidates for such treatments also include subjects with any other form of wounding or scarring or disease or disorder associated with wounding or scarring as discussed infra and/or known in the art.
  • the subject has a wound or scar on a cosmetically sensitive location, such as the face or neck.
  • the invention is based in part on the recognition that the timing of the administration of lithium is important for it to function as an effective modulator of wound healing (and thus, scar revision) in human subjects.
  • lithium treatment results, indirectly, in increasing Wnt signaling, but agents that increase Wnt signaling have had conflicting effects on hair follicle development and wound healing.
  • they stimulate follicle morphogenesis but also induce hair follicle tumors (Gat et al. , 1998, Cell 95: 605-614), and lead to decreased hair growth (Millar et al., 1999, Dev. Biol. 207: 133-149).
  • the invention is also based, in part, on the principle that human skin is replenished by bone-marrow derived and tissue-derived stem cells throughout life.
  • the lithium treatment(s) is used in combination with methods that mobilize tissue stem cells (e.g., using integumental perturbation) and/or methods that mobilize bone marrow-derived stem cells (e.g., growth factors such as G-CSF and/or chemical agents such as plerixafor (Mozobil®)).
  • the lithium treatments described herein are used together with methods that regulate the differentiation of these stem cells into specialized human hair follicles in order to facilitate the desired hair patterning at the acceptor site, using agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 11 -alpha-hydroxyprogesterone, ketoconazole, RU58841, dutasteride, fluridil, or QLT-7704, an antiandrogen oligonucleotide, cyoctol, topical progesterone, topical estrogen, cyproterone acetate, ru58841, combination 5 alpha reductase inhibitors, oral contraceptive pills, and others in Poulos & Mirmirani, 2005, Expert Opin.
  • agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 11 -alpha-hydroxyprogesterone, ketoconazole, RU58841, du
  • Investig. Drugs 14: 177-184 incorporated herein by reference, or any other antiestrogen, an estrogen, or estrogen-like drug (alone or in combination with agents that increase stem cell plasticity; e.g., such as valproate), etc. , known in the art.
  • Such combination treatments can further include the use of agents that modulate hair growth or that modulate wound healing.
  • Anagen Growth stage of the hair Follicle Cycle.
  • Bulb Lowermost portion of the hair follicle, containing rapidly proliferating matrix cells that produce the hair.
  • Catagen Stage of the hair cycle characterized by regression and involution of the follicle.
  • Cicatricial (scarring) Alopecia Abnormal hair loss with scarring. Caused by destruction of hair follicles and replacement with scar tissue as a result of inflammation, trauma, fibrosis, or unknown causes; examples include lichen planopilaris and discoid lupus erythematosus.
  • Exogen Phase of the hair Follicle Cycle where hair shaft is shed from the follicle.
  • Follicle cycle Hair growth in each follicle occurs in a cycle that includes the following phases: anagen (growth phase), catagen (involuting/regressing stage), telogen (the quiescent phase), exogen (shedding phase), and re-entry into anagen.
  • Integumental Pertaining to the integumentary system, which comprises the skin (epidermis, dermis, hypodermis (or subcutanea)) and all cells contained therein regardless of origin, and its appendages (including, e.g., hair and nails).
  • Kenogen Latent phase of hair cycle after hair shaft has been shed and growth is suspended in follicle.
  • Telogen effluvium Excessive shedding of hair caused by an increased proportion of follicles entering the telogen stage; common causes include drugs and fever.
  • Terminal hair Large, usually pigmented hairs on scalp and body.
  • Vellus hair Very short, nonpigmented hairs (e.g., those found diffusely over nonbeard area of face and bald scalp as a result of miniaturization of terminal hairs).
  • Figure 1 Types of human hair follicles.
  • Figure 2 Architecture of the skin.
  • Figure 3 Diagram of human hair follicle.
  • Figure 4 Cellular structure of the human hair bulb.
  • Formulation 35A' lithium chloride emulsion cream; see Table 2 is plotted over time, in hours (x-axis).
  • Figure 7 Release of Li ions through dermis (y-axis) from Formulation BX (lithium chloride gel; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • Figure 8 Release of Li ions through dermis (y-axis) from Formulation BV-001- 003A (lithium chloride hydrogel; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • FIG. 9 Release of Li ions through dermis (y-axis) from Formulation 28A (lithium chloride topical dispersion cream; see Table 2) is plotted over time, in hours (x-axis).
  • Cadaver skin was dermabraded, with a standard dermabrader to remove the stratum corneum and epidermis prior to administration of the lithium compound.
  • FIG. 12 Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg administered subcutaneously to mice dermabraded prior to dosing. "Peak samples" were taken 1 h post last dosing. [00132] Figure 13. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following DA.
  • FIG. 14 Lithium concentrations calculated in mM, in total blood (red blood cells (RBC) + plasma), as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg, administered subcutaneously to mice dermabraded prior to dosing.
  • B Skin lithium concentrations calculated in ⁇ g/kg, as a function of increasing doses in mg/kg. In the wounded groups, skin was dermabraded prior to administration of the formulation of lithium chloride dissolved in isotonic saline. Non-wounded comparisons are shown (square, diamond) with dermabrasion wounded groups (cross, triangle). * It is noted that in this experiment, dermabrasion was accomplished using a microdermabrasion device.
  • FIG. 15 Skin lithium concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg.
  • the lithium formulation was administered subcutaneously following full thickness excision (FTE) of skin. Dosing was started on the day of scab detachment (-11-14 days post-FTE). Lithium ion concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
  • FIG. 16 Plasma lithium Concentrations calculated in mM, as a function of increasing doses of a formulation of lithium chloride dissolved in isotonic saline in mg/kg.
  • the lithium formulation was administered subcutaneously following FTE. Dosing was started on the day of scab detachment ( ⁇ 11-14 days post-FTE). Lithium concentrations were measured by a validated bioanalytical ICP method (see Section 13.2 infra).
  • Figure 17 Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following FTE.
  • FIG. 1 Pharmacokinetic analysis of lithium concentrations in skin and plasma with once daily topical dosing of 8% lithium chloride or 8% lithium gluconate hydrogel ("lithium gluconate") following FTE. Lithium ion concentrations were measured by
  • ICP/MS/MS using a validated method (see Section 13.2 infra).
  • Arrows indicate peak levels of Lithium ion in skin one hour post dosing.
  • N 2 per time point— error bars denote range [00138]
  • Figure 19 Pharmacokinetic analysis of lithium concentrations in skin and plasma with twice daily topical dosing of lithium gluconate, 1%; lithium gluconate, 8%; and lithium gluconate, 16% following DA.
  • FIG. 20 Topical lithium 8% increases the proportion of mature neogenic hair follicles in healed FTE wounds, based on histologic examination.
  • A Diagrams of selected stages of hair follicle development.
  • B Percentage of stageable neogenic hair follicles at stage 5 or greater following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%. Numbers in the bars indicate the number of mice per group that were used for quantitation. Ratios above the bars indicate the number of NHF (neogenic hair follicles) > stage 5 divided by the total number of stageable NHF.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • Topical lithium 8% increases both the number and maturation of neogenic hair follicles in FTE wounds, as measured following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • Topical lithium 8% increases shaft thickness of regenerated hair follicles following DA. Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate”), 1%, 8%, or 16% or lithium chloride hydrogel (“LiCl”), 8%.
  • lithium gluconate hydrogel 1%, 8%, or 16%
  • LiCl lithium chloride hydrogel
  • FIG. 25 Topical lithium gluconate 8% results in a 16% increase thickness of regenerated hair shafts following DA.
  • LiCl lithium chloride hydrogel
  • Graph on right side shows simultaneous 90% confidence intervals, corrected for 4 comparisons by the Treatment Groups Bonferroni method.
  • FIG. 26 Healed FTE wounds treated with topical LiCl 8% have increased numbers of neogenic hair follicles, as assessed by in vivo scanning laser microscopy, imaging the wounded area approximately 60-80 ⁇ beneath the skin surface.
  • FIG. 28 Topical LiCl 8% increases the total number of neogenic hair follicles (also referred to as "HF") per FTE wound by 3-fold, based on histology of tissue sections.
  • Left graph: Median + first and third quartiles shown. Numbers above columns total number of neogenic hair follicles ("NHF”) combined from individual mice (parentheses indicate number of NHF that could not be staged).
  • NHF total number of neogenic hair follicles
  • FIG. 32 Topical LiCl 8% does not affect density of regenerated hair follicles following DA. Median + first and third quartiles shown. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.
  • FIG 35 Complexed Lithium Gluconate encapsulated within biodegradable poly (D,L-lactide-co-glycolide) ("PLG”) microspheres.
  • PLG biodegradable poly
  • FIG. 37 Synthetic Biodegradable Matrices from PLA/PLG Blends. A:
  • any compound or composition that can release a lithium ion (also referred to herein as lithium cation, Li+, or ionized lithium) is suitable for use in the compositions and methods.
  • Such compounds include but are not limited to a pharmaceutically acceptable prodrug, salt or solvate ⁇ e.g., a hydrate) of lithium (sometimes referred to herein as "lithium compounds").
  • the lithium compounds can be formulated with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof.
  • lithium- polymer complexes can be utilized to developed various sustained release lithium matrices.
  • lithium is best known as a mood stabilizing drug, primarily in the treatment of bipolar disorder, for which lithium carbonate (L1 2 CO 3 ), sold under several trade names, is the most commonly used.
  • Other commonly used lithium salts include lithium citrate (L1 3 C 6 H 5 O 7 ), lithium sulfate (L1 2 SO 4 ), lithium aspartate, and lithium orotate.
  • a lithium formulation well-suited for use in the methods disclosed herein is lithium gluconate, for example, a topical ointment of 8% lithium gluconate (LithiodermTM), is approved for the treatment of seborrheic dermatitis.
  • lithium succinate for example, an ointment comprising 8% lithium succinate, which is also used to treat seborrheic dermatitis.
  • the lithium formulation is an ointment comprising 8% lithium succinate and 0.05% zinc sulfate (marketed in the U.K. as Efalith). See, e.g., Efalith Multicenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety.
  • Examples of lithium succinate formulations and other lithium formulations for use in the intermittent lithium treatments or pulse lithium treatment described herein are also described in U.S. Patent No. 5,594,031, issued January 14, 1997, which is incorporated herein by reference in its entirety. 5.1.1 LITHIUM SALTS
  • any pharmaceutically acceptable lithium salt may be used as a source of lithium ions in the intermittent lithium treatments or a pulse lithium treatment. It will be understood by one of ordinary skill in the art that pharmaceutically acceptable lithium salts are preferred. See, e.g., Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use, Zurich, Switzerland: Wiley -VCH and VHCA; Remington 's Pharmaceutical Sciences, 1990, 18 th eds., Easton, PA: Mack Publishing;
  • the compositions used for intermittent lithium treatment or a pulse lithium treatment comprise mixtures of one or more lithium salts.
  • a mixture of a fast-dissolving lithium salt can be mixed with a slow dissolving lithium salt proportionately to achieve the release profile.
  • the lithium salts do not comprise lithium chloride.
  • the lithium salt can be the salt form of anionic amino acids or poly(amino) acids. Examples of these are glutamic acid, aspartic acid, polyglutamic acid, polyaspartic acid.
  • lithium salts of the acids set forth above, applicants do not mean only the lithium salts prepared directly from the specifically recited acids. In contrast, applicants mean to encompass the lithium salts of the acids made by any method known to one of ordinary skill in the art, including but not limited to acid-base chemistry and cation-exchange chemistry.
  • lithium salts of anionic drugs that positively affect hair growth can be administered.
  • a large anion or multianionic polymer such as polyacrylic acid can be complexed with lithium, then complexed with a cationic compound, such as finasteride, to achieve a slow release formulation of both lithium ion and finasteride.
  • a lithium complex with a polyanion can be complexed further with the amines of minoxidil, at pHs greater than 5.
  • Lithium compounds for use in the methods provided herein may contain an acidic or basic moiety, which may also be provided as a pharmaceutically acceptable salt. See, Berge et al, J. Pharm. Sci. 1977, 66: 1-19; Stahl & Wermuth, eds., 2002, Handbook of Pharmaceutical Salts, Properties, and Use Zurich, Switzerland: Wiley-VCH and VHCA.
  • the lithium salts are organic lithium salts.
  • Organic lithium salts for use in these embodiments include lithium 2,2-dichloroacetate, lithium salts of acylated amino acids ⁇ e.g., lithium -acetylcysteinate or lithium N-stearoylcysteinate), a lithium salt of poly(lactic acid), a lithium salt of a polysaccharides or derivative thereof, lithium acetylsalicylate, lithium adipate, lithium hyaluronate and derivatives thereof, lithium polyacrylate and derivatives thereof, lithium chondroitin sulfate and derivatives thereof, lithium stearate, lithium linoleate, lithium oleate, lithium taurocholate, lithium cholate, lithium glycocholate, lithium deoxycholate, lithium alginate and derivatives thereof, lithium ascorbate, lithium L-aspartate, lithium benzenesulfonate, lithium benzoate, lithium
  • the organic lithium salt for use in these embodiments is lithium (S)-2- alkylthio-2-phenylacetate or lithium (R)-2-alkylthio-2-phenylacetate ⁇ e.g., wherein the alkyl is C2-C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
  • the organic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of acetic acid, 2,2- dichloroacetic acid, acetylsalicylic acid, acylated amino acids, adipic acid, hyaluronic acid and derivatives thereof, polyacrylic acid and derivatives thereof, chondroitin sulfate and derivatives thereof, poly(lactic acid-co-glycolic acid), poly(lactic acid), poly(glycolic acid), pegylated lactic acid, stearic acid, linoleic acid, oleic acid, taurocholic acid, cholic acid, glycocholic acid, deoxycholic acid, alginic acid and derivatives thereof, anionic derivatives of polysaccharides, poly(sebacic anhydride)s and derivatives thereof, ascorbic acid, L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid
  • organic lithium salts for use in these embodiments is the lithium salt of (S)-2-alkylthio-2-phenylacetic acid or the lithium salt of (R)-2-alkylthio-2-phenylacetic acid ⁇ e.g., wherein the alkyl is C2- C22 straight chain alkyl, preferably C8-16). See, e.g., International Patent Application Publication No. WO 2009/019385, published February 12, 2009, which is incorporated herein by reference in its entirety.
  • the organic lithium salt can be modified to create sustained release lithium salts. Due to the size of the lithium ion, it is possible that the residence time of ion at the treatment site will be short. In efforts to generate sustained release lithium salts, the hydrophobicity of the salt can be enhanced and made "lipid-like," to, for example, lower the rate of ionization of the salt into lithium ions. For example, lithium chloride has a much faster rate of ionizing into lithium ions, than lithium stearate or lithium orotate.
  • the lithium salt can be that of a cholesterol derivative, or a long chain fatty acids or alcohols. Lipid complexed lithium salts of size less than 10 microns can also be effectively targeted to the hair follicles and "tethered" to the sebaceous glands, by hydrophobic-hydrophobic interactions.
  • the organic lithium salt can be in the form of complexes with anionic compounds or anionic poly(amino acids) and other polymers.
  • the complexes can be neutral, wherein all of the negative charges of the complexation agent are balanced by equimolar concentrations of Li ions.
  • the complexes can be negatively charged, with Lithium ions bound to an anionic polymer.
  • the complexes can be in the form of nano-complexes, or micro-complexes, small enough to be targeted to the hair follicles. If the complexes are targeted to the dermis, the charged nature of the complexes will "tether" the complexes to the positively charged collagen.
  • This mode of tethering holds the Li ions at the site of delivery, thereby hindering fast in-vivo clearance.
  • negatively charged polymers that can be used in this application are poly(acrylates) and its copolymers and derivatives thereof, hyaluronic acid and its derivatives, alginate and its derivatives, etc.
  • the anionic lithium complexes formed as described above can be further complexed with a cationic polymer such as chitosan, or polyethylimine form cell-permeable delivery systems.
  • the salt can be that of a fatty acid, e.g. , lithium stearate, thereby promoting absorption through skin tissues and extraction into the lipid compartments of the skin.
  • the lithium salt of sebacic acid can be administered to the skin for higher absorption and targeting into structures of the skin, such as hair follicles.
  • the lithium salts are inorganic lithium salts.
  • Inorganic lithium salts for use in these embodiments include halide salts, such as lithium bromide, lithium chloride, lithium fluoride, or lithium iodide.
  • the inorganic lithium salt is lithium fluoride.
  • the inorganic lithium salt is lithium iodide.
  • the lithium salts do not comprise lithium chloride.
  • Other inorganic lithium salts for use in these embodiments include lithium borate, lithium nitrate, lithium perchlorate, lithium phosphate, or lithium sulfate.
  • the inorganic lithium salts used for intermittent lithium treatment or a pulse lithium treatment comprise the lithium salts of boric acid, hydrobromic acid, hydrochloric acid, hydrofluoric acid, hydroiodic acid, nitric acid, perchloric acid, phosphoric acid, or sulfuric acid.
  • the lithium compounds used for intermittent lithium treatment or a pulse lithium treatment may be formulated with a pharmaceutically acceptable carrier (also referred to as a pharmaceutically acceptable excipients), i.e., a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, an encapsulating material, or a complexation agent.
  • a pharmaceutically acceptable carrier also referred to as a pharmaceutically acceptable excipients
  • each component is "pharmaceutically acceptable” in the sense of being chemically compatible with the other ingredients of a pharmaceutical formulation, and biocompatible, when in contact with the biological tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • Suitable excipients are well known to those skilled in the art, and non-limiting examples of suitable excipients are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art, including, but not limited to, the method of administration. For example, forms for topical administration such as a cream may contain excipients not suited for use in transdermal or intravenous administration. The suitability of a particular excipient depends on the specific active ingredients in the dosage form.
  • Exemplary, non-limiting, pharmaceutically acceptable carriers for use in the lithium formulations described herein are the cosmetically acceptable vehicles provided in
  • the lithium compounds suitable for use in intermittent lithium treatments or a pulse lithium treatment may be formulated to include an appropriate aqueous vehicle, including, but not limited to, water, saline, physiological saline or buffered saline (e.g., phosphate buffered saline (PBS)), sodium chloride for injection, Ringers for injection, isotonic dextrose for injection, sterile water for injection, dextrose lactated Ringers for injection, sodium bicarbonate, or albumin for injection.
  • PBS phosphate buffered saline
  • Suitable non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, lanolin oil, lanolin alcohol, linoleic acid, linolenic acid and palm seed oil.
  • Suitable water- miscible vehicles include, but are not limited to, ethanol, wool alcohol, 1,3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400), propylene glycol, glycerin, N-methyl-2-pyrrolidone ( ⁇ ), N,N-dimethylacetamide (DMA), and dimethyl sulfoxide (DMSO).
  • the water-miscible vehicle is not DMSO.
  • the lithium compounds for use in the methods disclosed herein may also be formulated with one or more of the following additional agents.
  • Suitable antimicrobial agents or preservatives include, but are not limited to, alkyl esters of p-hydroxybenzoic acid, hydantoins derivatives, propionate salts, phenols, cresols, mercurials, phenyoxyethanol, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g. , benzethonium chloride), butyl, methyl- and propyl-parabens, sorbic acid, and any of a variety of quarternary ammonium compounds.
  • Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose.
  • Suitable buffering agents include, but are not limited to, phosphate, glutamate and citrate.
  • Suitable antioxidants are those as described herein, including ascorbate, bisulfite and sodium metabisulfite.
  • Suitable local anesthetics include, but are not limited to, procaine
  • suspending and dispersing agents include but are not limited to sodium carboxymethylcelluose (CMC), hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP).
  • CMC carboxymethylcelluose
  • HPMC hydroxypropyl methylcellulose
  • PVA polyvinyl alcohol
  • PVP polyvinylpyrrolidone
  • Suitable emulsifying agents include but are not limited to, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80, and triethanolamine oleate.
  • Suitable sequestering or chelating agents include, but are not limited to, EDTA.
  • Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.
  • Suitable complexing agents include, but are not limited to, cyclodextrins, including a-cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether- ⁇ - cyclodextrin, and sulfobutylether 7-P-cyclodextrin (CAPTISOL ® , CyDex, Lenexa, KS).
  • cyclodextrins including a-cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether- ⁇ - cyclodextrin, and sulfobutylether 7-P-cyclodextrin (CAPTISOL ® , CyDex, Lenexa, KS).
  • Soothing preparations may contain sodium bicarbonate (baking soda), and coal tar based products.
  • Formulations may also optionally contain a sunscreen or other skin protectant, or a waterproofing agent.
  • a product for application to skin may additionally be formulated so that it has easy rinsing, minimal skin/eye irritation, no damage to existing skin or hair, has a thick and/or creamy feel, pleasant fragrance, low toxicity, and good biodegradability.
  • lithium gluconate - for example, 8% lithium gluconate (LithiodermTM), which is approved for the treatment of seborrheic dermatitis (see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al., 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety); or 8% lithium succinate with 0.05% zinc
  • a preparation of lithium or lithium salt comprises an anionic polymer (such as, e.g., crosslinked polyacrylic acid), which may form a gel.
  • an anionic polymer such as, e.g., crosslinked polyacrylic acid
  • a preparation provided in the examples of Sections 16-19 below may be used.
  • modified release refers to a dosage form in which the rate or place of release of the lithium or other active ingredient(s) is different from that of an immediate dosage form when administered by the same route.
  • Modified release dosage forms include, but are not limited to, delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated- and fast-, targeted-, programmed-release, and gastric retention dosage forms.
  • compositions in modified release dosage forms can be prepared using a variety of modified release devices and methods known to those skilled in the art, including, but not limited to, matrix controlled release devices, osmotic controlled release devices, multiparticulate controlled release devices, ion-exchange resins, enteric coatings, multilayered coatings, microspheres, liposomes, and combinations thereof.
  • the release rate of the active ingredient(s) can also be modified by varying the particle sizes and polymorphism of the active ingredient(s).
  • the controlled release is achieved by using an adjuvant that causes a depot effect, i.e., that causes an active agent or antigen to be released slowly, leading to prolonged exposure to a target cell or tissue (e.g., cells of the follicle, or, in the case of
  • modified release examples include those described in International Patent Application Publication No. WO 2008/1 15961, published September 25, 2008, which is incorporated herein by reference in its entirety.
  • modified release examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598, 123; 4,008,719; 5,674,533; 5,059,595;
  • the modified release dosage form can be fabricated using a matrix controlled release device known to those skilled in the art. See, Takada et al, 1999, in Encyclopedia of Controlled Drug Delivery, Mathiowitz E, ed., Vol. 2, Wiley.
  • the modified release dosage form is formulated using an erodible matrix device, which is water-swellable, erodible, or soluble polymers, including, but not limited to, synthetic polymers, and naturally occurring polymers and derivatives, such as polysaccharides and proteins.
  • Materials useful in forming an erodible matrix include, but are not limited to, chitin, chitosan, dextran, and pullulan; gum agar, gum arabic, gum karaya, locust bean gum, gum tragacanth, carrageenans, gum ghatti, guar gum, xanthan gum, and scleroglucan; starches, such as dextrin and maltodextrin; hydrophilic colloids, such as pectin; phosphatides, such as lecithin; alginates; propylene glycol alginate; gelatin; collagen;
  • cellulosics such as ethyl cellulose (EC), methylethyl cellulose (MEC), carboxymethyl cellulose (CMC), CMEC, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), cellulose acetate (CA), cellulose propionate (CP), cellulose butyrate (CB), cellulose acetate butyrate (CAB), CAP, CAT, hydroxypropyl methyl cellulose (HPMC), HPMCP, HPMCAS, hydroxypropyl methyl cellulose acetate trimellitate (HPMCAT), and ethyl hydroxyethyl cellulose (EHEC); polyvinyl pyrrolidone; polyvinyl alcohol; polyvinyl acetate; glycerol fatty acid esters; polyacrylamide; polyacrylic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT ® , Rohm America, Inc., Piscataway, NJ);
  • degradable lactic acid-glycolic acid copolymers poly-D-(-)-3-hydroxybutyric acid; and other acrylic acid derivatives, such as homopolymers and copolymers of butylmethacrylate, methyl methacrylate, ethyl methacrylate, ethylacrylate, (2-dimethylaminoethyl)methacrylate, and (trimethylaminoethyl)methacrylate chloride.
  • the compositions are formulated with a non-erodible matrix device.
  • the active ingredient(s) is dissolved or dispersed in an inert matrix and is released primarily by diffusion through the inert matrix once administered.
  • Materials suitable for use as a non-erodible matrix device include, but are not limited to, insoluble plastics, such as polyethylene, polypropylene, polyisoprene, polyisobutylene, polybutadiene,
  • the desired release kinetics can be controlled, for example, via the polymer type employed, the polymer viscosity, the particle sizes of the polymer and/or the active ingredient(s), the ratio of the active ingredient(s) versus the polymer, and other excipients or carriers in the compositions.
  • the modified release dosage forms can be prepared by methods known to those skilled in the art, including direct compression, dry or wet granulation followed by compression, and melt-granulation followed by compression.
  • the modified release dosage form can be fabricated using an osmotic controlled release device, including, but not limited to, one-chamber system, two-chamber system, asymmetric membrane technology (AMT), and extruding core system (ECS).
  • AMT asymmetric membrane technology
  • ECS extruding core system
  • such devices have at least two components: (a) a core which contains an active ingredient; and (b) a semipermeable membrane with at least one delivery port, which encapsulates the core.
  • the semipermeable membrane controls the influx of water to the core from an aqueous environment of use so as to cause drug release by extrusion through the delivery port(s).
  • the core of the osmotic device optionally includes an osmotic agent, which creates a driving force for transport of water from the environment of use into the core of the device.
  • an osmotic agent which creates a driving force for transport of water from the environment of use into the core of the device.
  • osmotic agents water- swellable hydrophilic polymers, which are also referred to as "osmopolymers" and
  • hydrogels Suitable water-swellable hydrophilic polymers as osmotic agents include, but are not limited to, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid,
  • hydrophilic vinyl and acrylic polymers polysaccharides such as calcium alginate, polyethylene oxide (PEO), polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-hydroxyethyl methacrylate), poly(acrylic) acid, poly(methacrylic) acid,
  • polyvinylpyrrolidone PVP
  • crosslinked PVP polyvinyl alcohol
  • PVA polyvinyl alcohol
  • PVA/PVP copolymers PVA/PVP copolymers with hydrophobic monomers such as methyl methacrylate and vinyl acetate, hydrophilic polyurethanes containing large PEO blocks, sodium croscarmellose, carrageenan, hydroxyethyl cellulose (HEC), hydroxypropyl cellulose (HPC), hydroxypropyl methyl cellulose (HPMC), carboxymethyl cellulose (CMC) and carboxyethyl, cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.
  • HEC hydroxyethyl cellulose
  • HPMC hydroxypropyl methyl cellulose
  • CMC carboxymethyl cellulose
  • CEC carboxyethyl
  • sodium alginate sodium carbcarbophil
  • gelatin xanthan gum
  • the other class of osmotic agents is osmogens, which are capable of imbibing water to affect an osmotic pressure gradient across the barrier of the surrounding coating.
  • Suitable osmogens include, but are not limited to, inorganic salts, such as magnesium sulfate, magnesium chloride, calcium chloride, sodium chloride, lithium chloride, potassium sulfate, potassium phosphates, sodium carbonate, sodium sulfite, lithium sulfate, potassium chloride, and sodium sulfate; sugars, such as dextrose, fructose, glucose, inositol, lactose, maltose, mannitol, raffinose, sorbitol, sucrose, trehalose, and xylitol; organic acids, such as ascorbic acid, benzoic acid, fumaric acid, citric acid, maleic acid, sebacic acid, sorbic acid, adipic acid, edetic
  • Osmotic agents of different dissolution rates can be employed to influence how rapidly the active ingredient(s) is initially delivered from the dosage form.
  • amorphous sugars such as MA OGEMTM EZ (SPI Pharma, Lewes, DE) can be used to provide faster delivery during the first couple of hours to promptly produce the desired therapeutic effect, and gradually and continually release of the remaining amount to maintain the desired level of therapeutic or prophylactic effect over an extended period of time.
  • the active ingredient(s) is released at such a rate to replace the amount of the active ingredient metabolized and excreted.
  • the core can also include a wide variety of other excipients and carriers as described herein to enhance the performance of the dosage form or to promote stability or processing.
  • Materials useful in forming the semipermeable membrane include various grades of acrylics, vinyls, ethers, polyamides, polyesters, and cellulosic derivatives that are water- permeable and water-insoluble at physiologically relevant pHs, or are susceptible to being rendered water-insoluble by chemical alteration, such as crosslinking.
  • Suitable polymers useful in forming the coating include plasticized, unplasticized, and reinforced cellulose acetate (CA), cellulose diacetate, cellulose triacetate, CA propionate, cellulose nitrate, cellulose acetate butyrate (CAB), CA ethyl carbamate, CAP, CA methyl carbamate, CA succinate, cellulose acetate trimellitate (CAT), CA dimethylaminoacetate, CA ethyl carbonate, CA chloroacetate, CA ethyl oxalate, CA methyl sulfonate, CA butyl sulfonate, CA p-toluene sulfonate, agar acetate, amylose triacetate, beta glucan acetate, beta glucan triacetate, acetaldehyde dimethyl acetate, triacetate of locust bean gum, hydroxylated ethylene-vinylacetate, EC, PEG, PPG, PEG/PPG copo
  • a semipermeable membrane can also be a hydrophobic microporous membrane, wherein the pores are substantially filled with a gas and are not wetted by the aqueous medium but are permeable to water vapor, as disclosed in U.S. Pat. No. 5,798, 1 19.
  • Such hydrophobic but water- vapor permeable membrane are typically composed of hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
  • hydrophobic polymers such as polyalkenes, polyethylene, polypropylene, polytetrafluoroethylene, polyacrylic acid derivatives, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinylidene fluoride, polyvinyl esters and ethers, natural waxes, and synthetic waxes.
  • the delivery port(s) on the semipermeable membrane can be formed post-coating by mechanical or laser drilling. Delivery port(s) can also be formed in situ by erosion of a plug of water-soluble material or by rupture of a thinner portion of the membrane over an indentation in the core. In addition, delivery ports can be formed during coating process, as in the case of asymmetric membrane coatings of the type disclosed in U.S. Pat. Nos.
  • the total amount of the active ingredient(s) released and the release rate can substantially by modulated via the thickness and porosity of the semipermeable membrane, the composition of the core, and the number, size, and position of the delivery ports.
  • An osmotic controlled-release dosage form can further comprise additional conventional excipients or carriers as described herein to promote performance or processing of the formulation.
  • the osmotic controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art. See Remington: The Science and Practice of Pharmacy, supra; Santus and Baker, J. Controlled Release 1995, 35, 1-21 ; Verma et al., Drug Development and Industrial Pharmacy 2000, 26, 695-708; and Verma et al., J. Controlled Release 2002, 79, 7-27.
  • compositions are formulated as AMT controlled- release dosage form, which comprises an asymmetric osmotic membrane that coats a core comprising the active ingredient(s) and other pharmaceutically acceptable excipients or carriers.
  • AMT controlled-release dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art, including direct compression, dry granulation, wet granulation, and a dip-coating method.
  • compositions are formulated as ESC controlled-release dosage form, which comprises an osmotic membrane that coats a core comprising the active ingredient(s), a hydroxylethyl cellulose, and other pharmaceutically acceptable excipients or carriers.
  • the lithium-containing compound can be loaded into a polymeric solution that consists of a water-soluble polymer that is a solution at room temperature (20-25°C) and below, but gels at physiological temperatures of 32-37°C.
  • the lithium-containing solution can be cooled to 2-8°C to impart a soothing effect, while being sprayed as a liquid spray on the tissue surface. Once sprayed on, the lithium- loaded solution will thicken into a gel, releasing the lithium-containing compound slowly over time.
  • the lithium-loaded solution can be injected as a liquid, to form an in situ depot within the tissue.
  • the lithium-loaded solution can be delivered as a solution, which can flow into orifices of the tissue, such as hair follicles and then, form a gel to release lithium for follicle-associated conditions, such as MPHL, folliculitis, or another condition described herein.
  • the temperature and time of gelation can be correlated to the concentration of the polymers and the length of the polymer blocks that constitute the polymers.
  • the a modified release dosage form can be fabricated as a multiparticulate controlled release device, which comprises a multiplicity of particles, granules, or pellets, ranging from about 10 ⁇ to about 3 mm, about 50 ⁇ to about 2.5 mm, or from about 100 ⁇ to about 1 mm in diameter.
  • Such multiparticulates can be made by the processes known to those skilled in the art, including microfluidization, membrane-controlled emulsification, oil-in-water, water-oil-water and oil-in oil emulsification and homogenization processes, complex coacervation, wet-and dry-granulation, extrusion/spheronization, roller-compaction, melt-congealing, and by spray-coating seed cores.
  • microfluidization membrane-controlled emulsification
  • compositions can be blended with the compositions to aid in processing and forming the multiparticulates.
  • the resulting particles can themselves constitute the multiparticulate device or can be coated by various film- forming materials, such as enteric polymers, water-swellable, and water-soluble polymers.
  • the multiparticulates can be further processed as a capsule or a tablet. 5.2.3 TARGETED DELIVERY
  • the lithium compounds for use herein may be formulated with a carrier that delivers the lithium to the site of action, for example, a follicle in a particular tissue. Such targeted delivery may be preferable in formulations for systemic administration, in order to reduce side effects associated with lithium therapy and/or ensure that the lithium reaches only follicles of particular tissues.
  • the carrier may be an aptamer targeted to a particular protein or cell type in the follicle, an antibody or antigen-binding fragment thereof, a virus, virus-like particle, virosome, liposome, micelle, microsphere, nanoparticle, or any other suitable compound.
  • compositions for use in the methods provided herein can also be formulated to be targeted to a particular tissue, follicle, or other area of the body of the subject to be treated, including liposome-, resealed erythrocyte-, and antibody-based delivery systems. Examples include, but are not limited to, those disclosed in U.S. Pat. Nos. 5,709,874; 5,759,542;
  • targeting is accomplished by the attachment of specific targeting moieties to the delivery systems containing the drug.
  • Targeting moieties can be in the form of antibodies, aptamers or small molecules that bind to specific proteins expressed in specific tissues.
  • Specific or guided targeting can "channel" the drug only to the specific tissue type, thus minimizing distribution to all tissues. This concept is especially useful if the drug causes side effects.
  • microspheres and nanospheres have been utilized, to deliver drugs into the hair follicle. Entry into the hair follicle is governed by the size of the drug-containing spheres, with microspheres of size 0.5-0.7 microns of ideal size for entry.
  • the surface of the microspheres can be functionalized with moieties that bind to specific surfaces in the follicular orifice to "retain" them at the site.
  • These moieties can be non-specific, such as hydrophobic coatings, or cationic coatings, in order to be bioadhesive to cells within the follicle.
  • the moieties can be specific and targeted to certain proteins that are expressed specifically on specific cell membranes. For example, proteins over-expressed on the follicular lymphoma cell surfaces can be targeted by delivery systems that have antibodies or aptamers designed to bind to these proteins.
  • the surface of the delivery systems can also be functionalized with cell-penetrating moieties such as cell-permeable peptides, positively charged polymers that bind to anionic cell surfaces.
  • the intermittent lithium treatments or a pulse lithium treatment described herein may be delivered locally to any part of the subject in which wound healing or scar revision is desired, including, e.g., the head (e.g., the scalp, cheek, chin, lips, ears, nose, eyelid or eyebrow), neck, abdomen, chest, breast, back, arms, armpits, stomach, genital area, buttocks, legs, hands, or feet of a subject.
  • the intermittent lithium treatment or a pulse lithium treatment is applied to wounded or scarred skin.
  • the intermittent lithium treatment or a pulse lithium treatment is applied before the skin is wounded or scarred.
  • Such local delivery of the intermittent lithium treatment or a pulse lithium treatment can be achieved by topical administration, transdermal, intradermal, subcutaneous (depot effect), or by intramuscular, intravenous and oral routes of delivery in formulations for targeting systemically delivered lithium to desired follicles. Such modes of delivery are discussed supra.
  • enhancement of wound healing or scar revision in wounded or otherwise integumentally perturbed skin is accomplished by a lithium treatment described herein in combination with a pre-designed biomaterial dressing that may serve as a substrate to encourage a step-wise attachment of keratinocytes and epithelial cells to it, such that formation of an organized extra-cellular matrix (ECM) is enhanced in order to promote wound healing.
  • ECM organized extra-cellular matrix
  • the scaffold for use in combination with lithium treatment may be comprised of a mesh of a biocompatible, bioabsorbable material that cells recognize and attach to, preferably with ease.
  • these materials can be collagen type I/III, hyaluronic acid, chitosan, alginates, or combinations and derivatives thereof or any other such material described herein or known in the art.
  • the mesh scaffold may be neutral, or charged. If the mesh is positively charged, it may permit cells (which are negatively charged) to adhere to it more effectively. If the mesh scaffold is negatively charged, it may contain signaling moieties that the cells will recognize and attach to. For example, polymers such as hyaluronic acid are present already in skin, and thus a mesh comprised of this material is thought to be compatible with cells.
  • the scaffold is pre-fabricated with a fine microstructure that is of the dimension of cells, for example, red blood cells that will initially diffuse throughout the scaffold, or epithelial cells and keratinocytes from surrounding tissue.
  • epitophelial tongue can move with greater ease and organization by crawling on the scaffold mesh.
  • the mesh scaffold has an "open-cell” structure, with the pores inter-connected, much like an open-celled foam.
  • the open, interconnecting nature of the scaffold may allow free diffusion of oxygen and cells, so that optimal organized wound healing can occur.
  • the mesh scaffold has the capacity to hydrate and remain hydrated throughout the wound healing period. This is useful because, without being bound by any theory, drying out of the wound results in a impermeable granular structure that the keratinocytes cannot "crawl upon.”
  • the mesh scaffold has moieties that act as molecular signals to the cells, for example, to aid their proliferation. These moieties include, but are not limited to, peptidoglycans and RGD integrin recognition sequences that encourage cell attachment and subsequent proliferation.
  • the mesh scaffold has incorporated within it one or more active agents, for example, a small molecule, or a nucleic acid, or a protein.
  • the additional active agent is a protein, such as noggin or WNT, or is a nucleic acid that encodes noggin or WNT.
  • a small molecule is incorporated into the scaffold, such as, e.g. , a GSK inhibitor, BMP inhibitor, or PPAR antagonist.
  • the compound incorporated in the mesh scaffold is a compound considered for use in the combination therapies described herein, for example, in Section 5.4, especially Sections 5.4.2 to 5.4.4.
  • the scaffold may incorporate superoxide dismutase, a free radical quenching molecule that functions in the reduction of inflammation.
  • compounds are included in the mesh scaffold that alter the kinetics of wound healing, for example, that slow wound healing. Such compounds are known in the art and described elsewhere herein.
  • Other compounds that may be incorporated in the mesh scaffold include growth factors that aid in cell proliferation and tissue regeneration. In some embodiments, the compounds aid in hair follicle migration or hair follicle neogenesis in the wound site.
  • the lithium compound itself is incorporated within the mesh scaffold.
  • the lithium compound is incorporated within one or more layers of a multilayered mesh scaffold.
  • the mesh scaffold contains the lithium compound in alternating layers, which may achieve a pulsatile delivery of lithium.
  • the lithium compound in incorporated in microspheres in the scaffold, enabling a controlled release of lithium from the scaffold.
  • the mesh scaffold can be fibrin gels that additionally contain lithium.
  • a fibrin network is the first scaffold that a cell encounters as it performs its role in healing wounds due to trauma or other insults to tissue.
  • the fibrin network (the "scab") assemble rapidly by a modified polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade— the result is a three-dimensional network of branching fibers, What is envisioned is a fibrin delivery matrix containing lithium, fibrinogen and thrombin, that "gels" in-situ.
  • One issue that is encountered is the ability of lithium to diffuse through the fibrin "scab” - making the drug part of the scab solves this issue.
  • the mesh scaffold is a synthetic biodegradable dressing and lithium delivery system that also acts as a "sponge" and absorbs the exudates/bloods from a wound.
  • These exudates intercalating with the synthetic scaffold contain an abundance of fibrinogen, thrombin, fibronectin, cell adhesion proteins, growth factors and hyaluronic acid, all of which create an integrated structure that is an attractive matrix for cell attachment /differentiation and delivery of lithium.
  • the release rate of lithium can be modulated by varying the composition of polymers that comprise the synthetic scaffold, or sponge.
  • a synthetic scaffold fabricated out of poly(lactide)-co-(glycolide) (PLG) and poly(lactide) (PLA) can be developed to have varied release profiles of lithium. Changing the ratio of PLA to PLG will change the release profile of the lithium from the scaffold.
  • Other polymers that can utilized to generate synthetic scaffolds are chitosan, carregenan, alginate, poly(vinyl alcohol), poly(ethylene oxide) (PEO), poly(ethylene oxide)-co-poly(propylene oxide)-co-poly(ethylene oxide) (PEO-PPO-PEO), poly(acrylates) and poly(vinyl pyrrolidone) (PVP).
  • the rate of lithium release from the formulation can be controlled, so that it takes anywhere from 2 hours to 30 days for most (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 100%) of the lithium ion to be released.
  • most of the lithium is released from the formulation within 2 hours, within 4 hours, within 8 hours, within 16 hours, within 24 hours, within 36 hours, within 48 hours, within 3 days, within 5 days, within 7 days, within 10 days, within 14 days, within 30 days, or within 2 months or more.
  • the mesh scaffold releases the aforementioned compounds in a timed release manner, acting as a controlled release formulation such as described in Section 5.2.2 above.
  • the compounds may be bound to the mesh scaffold, and are then released at a sustained release manner as a result of de-binding kinetics from the mesh.
  • the compound may be bound to a polymer, which is then incorporated to the mesh scaffold, and which may allow the compound to diffuse from the mesh at a slow rate, resulting in sustained release.
  • the mesh scaffold is extruded as a gel, with certain components of the gel precipitating out to form a mesh in situ.
  • the in situ mesh can be sprayed on the wounded or otherwise perturbed surface, such as tissue that has been extensively burned. A large area can be covered in this manner.
  • the mesh scaffold is pre-fabricated as a dressing or a wrap, to cover large areas of wounded tissue.
  • the mesh scaffold can be cut to size to fit the size of the wound to present a compatible surface for favorable movement of the epithelial tongue.
  • the scaffold is prepared by melt spinning, electrospinning, micromachining, weaving, or other methods known in the art in which open cell foams are fabricated.
  • the mesh scaffold can be fabricated by these methods, with the optional incorporation of additional compound(s) (which are optionally sterilized), then sterilized by gentle ethylene oxide sterilization.
  • the additional compounds are sterilized, and then added to the sterile mesh scaffold.
  • a combinatorial strategy that uses a biodegradable scaffold combined with administration of a lithium formulation described herein (alone or in combination with another treatment, such as described in Section 5.4, especially Sections 5.4.2 to 5.4.4) is applied, which may result in the in situ generation of embryonic stem cells or recruitment of cells required for wound healing following wounding.
  • This approach may be used together with a form of integumental perturbation described in Section 5.4.1 ⁇ e.g.
  • dermabrasion accomplished by a standard dermabrader or a laser
  • deep full-thickness excision accomplished by a bulk ablative laser
  • integumental perturbation by acute wounds, chronic wounds, or wounds generated for the purpose of scar revision.
  • integumental perturbation in combination with a scaffold that administers drug results in the in situ generation of stem cells or recruitment of other cells required for the wound healing process and may facilitate more effective wound healing with little or no scarring.
  • the scaffold is biodegradable. Placement of a 3-dimensional biodegradable scaffold in the wound assists the attachment, growth and differentiation of cells.
  • tissue repair has been by autologous cell/tissue transplantation— however, autografts are associated with donor site morbidity and limited availability.
  • An alternative is allografts, but these are susceptible to immune responses and also carry the risk of disease transfer.
  • tissue engineering has emerged as an interdisciplinary field that makes use of biomaterials, cells and factors either alone, or in combination to restore tissues. The tissue engineering strategy generally involves isolation of healthy cells from a patient, followed by their expansion in vitro.
  • a fibrin network is the natural network that forms rapidly due to a polycondensation reaction from fibrinogen, an abundant constituent of blood plasma, as soon as the protease thrombin is activated in the clotting cascade. The fibrin clot then forms a three-dimensional network for cells to attach, for re-epithelialization.
  • the biodegradability of the scaffold is modulated.
  • the biodegradability of the scaffold should be matched to the formation of the new epithelium due to wound healing.
  • One skilled in the art would know how to measure whether a synthetic matrix is biodegradable.
  • biodegradability can be measured ex vivo in implants or using rats or another animal model, by histological and HPLC analysis.
  • biodegradability by hydrolysis can be assessed.
  • the scaffold structure of choice is incubated in phosphate buffered saline, pH 7.4 and 37 °C.
  • the incubation buffer includes enzymes. The scaffolds are weighed prior to incubation.
  • the scaffolds are retrieved two-at-a-time at predetermined time points and dried in a vacuum oven.
  • the scaffolds are weighed at each time point and a plot of weight versus time is generated to develop the rate of biodegradability.
  • the biodegradability of the scaffold matrix is modulated to coincide with the healing process, and can be modulated by changing the composition of polymers utilized to fabricate the mesh. For example, a percentage of polyethylene glycol (PEG) can be included in a composition with PLG (e.g., described in the example in Section 19) to increase
  • Biodegradable synthetic matrices can be created to mimic the extra-cellular micro- environment for the enhanced cellular attachment necessary for tissue regeneration.
  • cell-recognition motifs such as RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold.
  • RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold.
  • One skilled in the art would know how to measure whether the biodegradable synthetic matrix has biomimetic properties. For example, in one embodiment, the biomimetic nature of the scaffold is judged on the basis of the content of the mesh and resultant intercalating fibrin.
  • the properties of the synthetic scaffold are dependent upon the three-dimensional geometry, matching of the modulus of the matrix with the tissue type and the porosity. It has been shown that the differentiation process can be modulated if the modulus of the tissue type is matched with the modulus of the scaffold.
  • the modulus of the scaffold is matched with the modulus of the tissue type.
  • the compressive modulus of a scaffold or hydrogel can be measured by a standard Instron instrument ⁇ e.g., using the TA Instruments DMA Q800).
  • the micro-environment created by the cells is optimally highly biocompatible to the cells present at the site, namely keratinocytes and stem cells derived from the dermal papilla.
  • this can be accomplished through the use of hydrophilic components that can absorb water.
  • hydrophobic components such as petrolatum is likely to be occlusive and prevent rapid cell proliferation.
  • the scaffold is incubated with human foreskin fibroblasts (HFF) in vitro and the scaffold is considered to be biocompatible if the cells maintain their shape and attach appropriately.
  • HFF human foreskin fibroblasts
  • the biodegradable scaffold is permeable to water, nutrients, oxygen and growth factors, enabling easy exchange of nutrients between tissues and cells (see, e.g., ASTM D39857). In some embodiments, a non-occlusive, non-permeable barrier is avoided.
  • the scaffold is used to "fill" a deep wound, as is common in a deep burn, to provide a matrix for the cells to attach, grow and differentiate - existence of the scaffold will likely minimize the scar formation normally observed in deep, large-area wounds.
  • a loose, dry, highly porous network or scaffold or mesh is placed in the bleeding site of the wound to gently absorb the blood and the cell adhesion proteins released at the site, as a result of wounding.
  • This will result in creation of a highly rich environment that consists of a combination of a 3 -dimensional scaffold combined with fibrinogen and thrombin, which will ultimately result in a highly biocompatible hydrogel suitable for cell attachment and growth.
  • inclusion of blood components and cell adhesion proteins into the network is critical for establishment of the ECM
  • extracellular matrix necessary to form continuous tissue in-growth, particularly in the case of large-area and deep wounds.
  • a dry scaffold has the added advantage of absorbing the blood at the wound site.
  • a person's own blood components can be used to create a combined synthetic/natural ECM.
  • the scaffold has an added advantage of serving as a blood absorbing gauze.
  • the scaffold has cell-recognition motifs, such as RGD peptides, to recruit cells to the site and attachment, thereof. Once attached, cells will proliferate. Without being bound by any theory, it is hypothesized that the primary attachment of cells to the scaffold is a critical step to prevent premature cell death.
  • a dry, sterile biodegradable scaffold is placed onto the freshly formed wound.
  • the properties of the scaffold will be such that it will transform into an adherent hydrogel upon water absorption.
  • Methods that may be employed to fabricate the scaffold are known in the art, and include electrospinning, micromachining, and others. Nano-fiber meshes fabricated by electrospinning, hydrogel imprint technologies have been utilized to create three-dimensional microstructures that match the supramolecular architecture of the tissue type. In situ forming scaffolds are also contemplated.
  • the active agents are administered using an active agent-containing spray-on hydrogel.
  • the active agent after placement of the biodegradable scaffold, the active agent is sprayed on the tissue.
  • the active agent (or combination of active agents, e.g., lithium and another stem cell signaling agent) may be incorporated into a spray-on hydrogel that will be sprayed on as a liquid, but which transforms into a hydrogel after it is sprayed on the tissue. This will be especially useful if the area of the wound is large and uniform coverage is needed.
  • the active agent-containing spray-on hydrogel is applied on the wound site, forming a cross-linked hydrogel that releases active agent over the time period of healing or a shorter or longer time period, as necessary.
  • the active agent will either be incorporated in microencapsulates or nano-encapsulates and suspended into the pre-hydrogel solution.
  • the active agent can also be dissolved into the pre-hydrogel solution.
  • the "pre-hydrogel” solution is defined as the solution that will be sprayed on the tissue and which also contains the active agent.
  • the active agent is contained within microspheres that can be positively charged to rapidly bind themselves to the negatively charged collagen present in the dermis. Binding the microspheres to the dermis renders the active agent-releasing moiety immobile at the site.
  • the wound may be covered with a breathable, non-occlusive spray-on hydrogel to cover the wound from infection during healing.
  • the intermittent lithium treatments or a pulse lithium treatment can be provided by administration of the lithium compound (or combination treatments, discussed in Section 5.4 infra) in forms suitable for topical ⁇ e.g., applied directly to the skin, transdermal, or intradermal), subcutaneous, intramuscular, intravenous or by other parenteral means, oral administration, sublingual administration, or bucchal administration.
  • topical ⁇ e.g. , applied directly to the skin, transdermal, or intradermal) administration is accomplished with the use of a mechanical device, such as, e.g., an iontophoretic device.
  • the lithium compounds (or combination treatment) can also be formulated as modified release dosage forms, including delayed-, extended-, prolonged-, sustained-, pulsatile-, controlled-, accelerated-, fast-, targeted-, programmed-release, and gastric retention dosage forms.
  • These dosage forms can be prepared according to conventional methods and techniques known to those skilled in the art (see, Rathbone et al, eds., 2008, Remington: The Science and Practice of Pharmacy, supra; Modified-Release Drug Delivery Technology, 2nd ed., New York, NY: Marcel Dekker, Inc.).
  • the intermittent lithium treatments or a pulse lithium treatment can be administered by a health care practitioner or by the subject. In some embodiments, the subject administers the intermittent lithium treatments or a pulse lithium treatment to him or herself.
  • topical administration is to the skin, either to the skin surface, transdermally, or intradermally. Topical administration can be with or without occlusion with a bandage or other type of dressing. In some embodiments, topical administration is to orifices or mucosa, or conjunctival, intracorneal, intraocular, ophthalmic, auricular, nasal, vaginal, urethral, respiratory, and rectal administration. The formulation used for topical administration can be designed to retain the lithium in the skin or to deliver a dose of lithium systematically. In some embodiments, topical administration of a lithium compound is combined with another treatment described herein, such as, but not limited to, a technique of integumental perturbation described in Section 5.4.1 infra.
  • Dosage forms that are suitable for topical administration for preferably local but also possible systemic effect, include emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, suspensions, tinctures, pastes, powders, crystals, foams, films, aerosols, irrigations, sprays, suppositories, sticks, bars, ointments, sutures, bandages, wound dressings, microdermabrasion or dermabrasion particles, drops, and transdermal or dermal patches.
  • the topical formulations can also comprise micro- and nano-sized capsules, liposomes, micelles, microspheres, microparticles, nanosystems, e.g., nanoparticles, nano-coacervates and mixtures thereof. See, e.g.,
  • the nano-sized delivery matrix is fabricated through a well-defined process, such as a process to produce lithium encapsulated in a polymer.
  • the lithium-releasing compound is spontaneously assembled in aqueous solutions, such as in liposomes and micelles.
  • the formulation for topical administration is a shampoo or other hair product, tanning product or sun protectant, skin lotion, or cosmetic.
  • the selected formulation will penetrate into the skin and reach the hair follicle.
  • the stratum corneum and/or epidermis have been or are removed by a method of integumental perturbation described herein (including by wounding or scar revision procedure, by laser, or by dermabrasion or microdermabrasion, which is a less vigorous form of dermabrasion), permitting application of the dosage form for topical administration directly into the exposed dermis.
  • the formulation for topical administration will be lipid-based, so that it will penetrate the stratum corneum.
  • the formulation for topical administration will contain a skin penetrant substance, such as, e.g., propylene glycol or transcutol.
  • a formulation in ointment form comprises one or more of the following ingredients: wool alcohol (acetylated lanolin alcohol), hard paraffin, white soft paraffin, liquid paraffin, and water. See, e.g., Langtry et al, supra.
  • the selected formulation is inconspicuous when applied to the skin, for example, is colorless, odorless, quickly-absorbing, etc.
  • the selected formulation is applied on the skin surface as a solution, which can crosslink into a hydrogel within a few minutes, thus creating a biocompatible dressing.
  • the hydrogel may be biodegradable.
  • the solution will absorb into the skin and crosslink into depots releasing drug.
  • the lithium ion will be used to crosslink the polymer, with release of the lithium ion controlled by the rate of degradation of the hydrogel.
  • Pharmaceutically acceptable carriers and excipients suitable for use in topical formulations include, but are not limited to, aqueous vehicles, water-miscible vehicles, nonaqueous vehicles, antimicrobial agents or preservatives against the growth of
  • microorganisms such as, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.
  • Forms for topical administration can also be in the form of ointments, creams, and gels.
  • Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including lard, benzoinated lard, olive oil, cottonseed oil, mineral oil and other oils, white petrolatum, paraffins; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water- in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, wool alcohol (acetylated lanolin alcohol), and stearic acid ⁇ see, Remington: The
  • Suitable cream base can be oil-in-water or water-in-oil.
  • Suitable cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase.
  • the oil phase is also called the "internal" phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol.
  • the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant.
  • the emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.
  • Gels are semisolid, suspension-type systems. Single-phase gels contain organic macromolecules distributed substantially uniformly throughout the liquid carrier. Suitable gelling agents include, but are not limited to, crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, and CARBOPOL ® ; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene-polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin.
  • dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.
  • lithium gluconate e.g., 8% lithium gluconate (LithiodermTM), approved for the treatment of seborrheic dermatitis ⁇ see, e.g., Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et al, 2007, Ann Dermatol Venereol 134:347-351 (abstract); and Ballanger et al, 2008, Arch Dermatol Res 300:215-223, each of which is incorporated by reference herein in its entirety); 8% lithium succinate (see, e.g., Langtry et al, 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al, 1992, Dermatology 184: 194-197, each of which is incorporated by reference herein in its entirety);
  • Each of these methods of topical administration may be used alone to administer lithium compounds or in combination with one or more other treatments as described in Section 5.4 infra.
  • topical administration is by electrical current, ultrasound, laser light, or mechanical disruption or integumental perturbation.
  • electroporation include electroporation, RF ablation, laserporation, laser ablation (fractional or non- fractional), non- ablative use of a laser, iontophoresis, phonophoresis, sonophoresis, ultrasound poration, or using a device that accomplishes skin abrasion, or microneedle or needle-free injection, such as topical spray or POWDERJECTTM (Chiron Corp., Emeryville, CA), BIOJECTTM (Bioject Medical Technologies Inc., Tualatin, OR), or JetPeelTM (from TavTech, Tel Aviv, Israel), which uses supersonically accelerated saline to remove epidermis.
  • POWDERJECTTM Chiron Corp., Emeryville, CA
  • BIOJECTTM Bioject Medical Technologies Inc., Tualatin, OR
  • JetPeelTM from TavTech, Tel Aviv, Israel
  • the device for topical administration of lithium compounds is an automatic injection device worn continuously but delivers lithium intermittently.
  • the device for topical administration of lithium compounds is an automatic injection device that is inconspicuous, for example, can be worn without undue discomfort under clothes, in the hair, under a hairpiece, etc.
  • a device for administration of the intermittent lithium treatment or a pulse lithium treatment delivers the lithium at a controlled depth in the skin so that it reaches hair follicles, but entry into the circulation is minimized.
  • the stratum corneum and epidermis is previously removed using a method of integumental perturbation (or by integumental perturbation as a result of wounding) described herein, and thus the required delivery pressures and velocities can be reduced. This reduction reduces the required complexity of the firing mechanisms.
  • a narrow firing stream is used, particularly to accomplish systemic delivery.
  • the particle injection system administers the lithium compound over a broad area of skin.
  • An exemplary particle delivery device compatible with broad-based skin delivery includes a low pressure / low velocity firing mechanism with a spray nozzle designed to deliver to a broad area.
  • a single-shot device that delivers to a 25 -cm 2 area could be fired or used multiple times on the scalp or other skin surface until the entire area is treated.
  • a dry particle spraying mechanism similar to an airbrush or miniature grit-blaster can be used to "paint" drug or drug particles onto the perturbed, wounded, or scarred area.
  • the stratum corneum and epidermis are already removed, e.g., by a method of integumental perturbation (e.g., wounding) described herein, and thus permits effective use of the mechanism using lowered pressure and velocity requirements to achieve dermal delivery.
  • the lithium compound (and/or additional drug) is present in an aqueous suspension, permitting use of standard aerosol spray can technology to deliver the lithium compound to the desired skin area.
  • dermabrasion e.g., using a mechanical device, including microdermabrasion devices that can be used to dermabrade, or alumina-, silica- or ice-based dermabrasion (as described by Webber, U.S. 6,764,493; U.S. 6,726,693; and U.S. 6,306, 119) is customized to include a drug particle delivery feature using methods readily known in the art.
  • the device fires ablation particles at the skin, it could also fire smaller drug particles that would simultaneously embed in the exposed dermis.
  • the device could switch over to firing drug particles once it is determined that adequate skin disruption has occurred. See, International Patent Application Publication No. WO 2009/061349, which is incorporated herein by reference in its entirety.
  • a standard dermabrasion device can be modified to incorporate any of the devices described above, e.g., a spraying/painting device.
  • a spray nozzle is located behind the dermabrasion wheel such that drug is sprayed into the dermis as it is exposed by the wheel.
  • the dermabrasion device via internal controls, could turn off the abrasion wheel once it is determined that adequate skin disruption has occurred, and switch on the drug spray to convert to drug painting mode.
  • a pulsed dye laser (585-595 nm) is combined with drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • a non- fractional C(3 ⁇ 4 or Erbium- YAG laser is combined with drug spraying either without or before skin disruption, in conjunction with skin disruption, or following skin disruption.
  • a fractional non-ablative laser e.g., an Erbium- YAG laser used at 1540-1550 nm
  • drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • a fractional ablative laser e.g., an Erbium-YAG laser used at 2940 nm or a CO 2 laser used at 10,600 nm
  • drug spraying either before or without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.
  • fractional ablative laser treatment of the skin e.g., an Erbium-YAG laser used at 2940 nm or a C(3 ⁇ 4 laser used at 10,600 nm
  • lithium compound delivery e.g., lithium compound delivery.
  • a fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug.
  • adequate integrated hardware and software controls are required such that the laser ablation and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug.
  • fractional ablative laser treatment of the skin e.g., an Erbium-YAG laser used at 2940 nm or a CO 2 laser used at 10,600 nm
  • lithium compound delivery e.g., lithium compound delivery.
  • a non-ablative, fractional laser could be combined with a precise delivery means such that as the laser forms a hole in the skin, the inkjet-like delivery component could fill that same hole with drug.
  • adequate integrated hardware and software controls are required such that the laser treatment and drug delivery are properly timed resulting in each newly formed hole being properly filled with drug.
  • topical administration comprises administration of lithium- containing particles.
  • the particles can be delivered to the skin in combination with any of the means above and described elsewhere infra. Additionally, the particles can be designed for intermittent or pulse delivery of lithium. In one embodiment, particles with different release properties are be delivered simultaneously to achieve pulse delivery.
  • topical administration comprises administration of a lithium-containing formulation that is delivered through channels that are created by the use of needling or micro-needle technology.
  • the formulation can be, e.g., a liquid, a gel or a dry spray.
  • topical administration may be through delivery of a lithium- containing formulation through hollow needles.
  • topical administration comprises administration of a lithium-containing formulation that is delivered into the skin by an iontophoretic patch.
  • a patch can be developed in which the lithium-containing formulation is incorporated.
  • topical administration comprises administration of a lithium-containing formulation that is incorporated into micro-needle shaped biodegradable polymers.
  • the biodegradable microneedles penetrate the targeted skin tissue, and are optionally left in place to deliver the lithium ions in a sustained fashion over time.
  • Administration can be parenterally by injection, infusion, or implantation, for local or systemic administration.
  • Parenteral administration includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, intravesical, and subcutaneous administration.
  • Compositions for parenteral administration can be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection.
  • compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.
  • aqueous vehicles water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents,
  • compositions for parenteral administration can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot.
  • the compositions are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.
  • Suitable inner matrixes include, but are not limited to, polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.
  • Suitable outer polymeric membranes include but are not limited to, polyethylene,
  • polypropylene ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
  • compositions comprising lithium compounds for oral
  • oral administration can be provided in solid, semisolid, or liquid dosage forms for oral administration.
  • oral administration also includes buccal, lingual, and sublingual administration.
  • Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewable tablets, capsules, pills, strips, troches, lozenges, pastilles, cachets, pellets, medicated chewing gum, bulk powders, effervescent or non-effervescent powders or granules, oral mists, solutions, emulsions, suspensions, wafers, sprinkles, elixirs, and syrups.
  • the pharmaceutical compositions can contain one or more pharmaceutically acceptable carriers or excipients, including, but not limited to, binders, fillers, diluents, disintegrants, wetting agents, lubricants, glidants, coloring agents, dye-migration inhibitors, sweetening agents, flavoring agents, emulsifying agents, suspending and dispersing agents, preservatives, solvents, non-aqueous liquids, organic acids, and sources of carbon dioxide.
  • Compositions for oral administration can be also provided in the forms of liposomes, micelles, microspheres, or nanosystems. Micellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
  • oral formulations approved for treating mood disorders e.g., lithium carbonate (L1 2 CO 3 ), sold under several trade names, lithium citrate (L1 3 C 6 H 5 O 7 ), lithium sulfate (Li 2 S0 4 ), lithium aspartate, or lithium orotate, may be administered in accordance with the methods described herein.
  • lithium carbonate L1 2 CO 3
  • lithium citrate L1 3 C 6 H 5 O 7
  • lithium sulfate Li 2 S0 4
  • lithium aspartate lithium orotate
  • the intermittent and pulse lithium treatments may also be administered to skin- derived cells or skin tissue ex vivo.
  • an intermittent or pulse lithium treatment may be used to enhance the re-association of dissociated hair follicle cells into follicles and their growth and expansion in culture for their implantation into fresh wounds and scar revisions.
  • hair follicles promoted by intermittent or pulse lithium treatments are added to the wound before, at the time of, and/or subsequent to, either acute wounding or, more typically, during the wounding that is induced in scar revision.
  • hair follicles can be introduced to the wound by migration or de novo hair follicle neogenesis, or by transplanting one or more of the following skin elements: full skin (xeno-; autologous human), follicular units, dissociated cells (donor dominance; recipient effects), ex vivo-expanded skin and/or follicular units, or human skin equivalents in vivo (universal donors).
  • Engineered human skin, or human skin equivalents can also be used for hair follicle neogenesis and scar revision platforms.
  • Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types (e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
  • keratinocytes hair follicle derived and non-hair follicle derived
  • dermal cells hair follicle derived and non-hair follicle derived
  • other cell types e.g. , mesenchymal stem cells
  • the lithium compound or formulation thereof can be administered topically, subcutaneously, orally, etc. Regardless of the route of administration used for lithium ion delivery, the dosing regimen should be adjusted to achieve peak concentrations of lithium in the target skin area of at least about 0.1 mM to 10 mM, and/or peak concentrations of lithium in the blood (serum or plasma samples) of at least about 1 mM (these values are sometimes referred to herein as the "target concentration").
  • ionized lithium is a monovalent cation
  • the peak concentration of lithium can be established by taking samples when peak concentrations are achieved and assaying them for lithium content using techniques well known to those skilled in the art (see, e.g., the examples of Sections 1 1 to 15 and the techniques described therein; see also Wood et al, 1986, Neuropharmacology 25: 1285-1288; and Smith, 1978, Acta Pharmacol et toxicol 43:51-54, each of which is incorporated herein by reference in its entirety).
  • samples can be taken when peak blood concentrations are typically achieved - for example, within 1 to 2 hours for standard release formulations, and 4-5 hours for sustained release formulations.
  • the peak concentration times for other formulations, including topical preparations, can be determined for the particular formulation used, and sampling can be adjusted accordingly.
  • the target concentration of lithium should be maintained in the skin and/or blood for at least 1 day; at least 2 days; at least 3 days; at least 5 days; at least 14 days; or at least 21 days; and, in certain embodiments, not more than 21 days.
  • This can be accomplished using, e.g., repeated applications of the lithium compound or a single application of a sustained release or extended release lithium formulation.
  • Either the single pulse protocol or the intermittent treatments can be used to achieve the target concentration of lithium for the shorter maintenance periods (i.e., for at least 1, 2 or 3 days). Maintenance periods longer than 3 days may require repeated application of the intermittent lithium treatments or the single pulse protocol.
  • topical administration of a lithium compound is preferred over oral or subcutaneous administration.
  • a topically administered lithium compound may achieve a higher concentration of lithium in skin than in the blood, thereby reducing the risk of toxicity associated with elevated blood levels of lithium.
  • a subcutaneously or orally administered lithium compound may be preferred in order to achieve a controlled release of lithium from the blood to the skin.
  • lithium doses should be adjusted on the basis of the blood concentration (serum or plasma) drawn (by convention) 12 or 24 hours after the last dose of the lithium compound; this trough blood concentration should be maintained below 2 mM Li+ and preferably, below about 1.5 mM Li+. In some embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • the relatively stable and characteristic pharmacokinetics of the lithium ion in individual patients makes it possible to predict dosage requirements for that individual based on the results of administration of a single test dose, followed by a skin and/or blood sample assay (plasma or serum) at the peak concentration time; followed by blood sample assays to monitor toxicity at the 12 hour or 24 hour trough concentration; and 24 hours later (when lithium is generally eliminated) which serves as the control value.
  • a skin and/or blood sample assay plasma or serum
  • blood sample assays to monitor toxicity at the 12 hour or 24 hour trough concentration; and 24 hours later (when lithium is generally eliminated) which serves as the control value.
  • a trough concentration of lithium in the skin of no less than 0.01 mM to 0.05 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of 0.05 mM to 0.1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 1 mM is preferred. In some embodiments, a trough concentration of lithium in the skin of less than 3 mM is preferred. In some embodiments, lithium concentrations at trough can be increased by twice daily dosing, or more frequent dosing. In such embodiments, topical administration of a lithium compound is preferred.
  • a pulsatile effect is achieved by the multiple dosing, but the trough concentrations do not decline as much as when once daily dosing is used.
  • a trough skin concentration of lithium is maintained at 0.25 mM or higher, for example from 0.25 mM to 0.5 mM or 0.5 mM to 0.75 mM.
  • the trough concentration is maintained at approximately 0.6 mM to 1.4 mM lithium.
  • a trough skin concentration is maintained at 1 mM to 3 mM lithium. In some such
  • the trough skin concentration is maintained at less than 0.5 mM, or less than 0.75 mM, or less than 1 mM, or less than 2 mM, or less than 3 mM of lithium.
  • an effective amount of a lithium compound is administered such that the target concentration of lithium ions in plasma or serum, as measured 30 minutes to 1 hour after the lithium treatment, is 0.10-0.20 ⁇ , 0.20-0.50 ⁇ , 0.50-1.0 ⁇ , 1.0-5.0 ⁇ , 5.0-10 ⁇ , 10-20 ⁇ , 20-50 ⁇ , 50-100 ⁇ , 100-500 ⁇ , 0.1- 0.5 mM, 0.5-1.0 mM, 1.0 mM-2.0 mM, 2.0-2.5 mM, 2.5-3.0 mM, 3.0-4.0 mM, 4.0 mM-5.0 mM, 5.0-7.0 mM, or 7.0 mM or greater.
  • an effective amount of lithium is administered such that the plasma or serum lithium ion concentration measured either 8 hours, 16 hours, 1 day, 1 week, 2 weeks, or 1 month after the lithium treatment, is 0.1 to 0.5 ⁇ , 0.1 to 1.0 ⁇ , 0.5 to 1.0 ⁇ , 0.5 to 1.5 ⁇ , 1 to 10 ⁇ , 10 to 50 ⁇ , 50 to 100 ⁇ , 100 to 150 ⁇ , 150 to 200 ⁇ , 250 to 300 ⁇ , 100 to 250 ⁇ , 100 to 500 ⁇ , 200 to 400 ⁇ , 500 to 1000 ⁇ ; or 1000 to less than 1500 ⁇ .
  • the plasma or serum lithium concentration reaches at least 1 ⁇ . In one embodiment, the plasma or serum lithium concentration reaches at least 100 ⁇ .
  • the plasma or serum lithium concentration reaches at least 1 mM. In one embodiment, the plasma or serum lithium concentration does not exceed 1 mM. In another embodiments, the plasma or serum concentration of lithium does not exceed 1.5 mM. Serum lithium concentration may be measured using any technique known in the art, such as described in Sampson et ah, 1992, Trace Elements in Medicine 9:7-8.
  • an amount of a lithium compound is administered such that the target concentration of lithium in the skin is 0.01 to 0.05 ⁇ , 0.05 to 0.1 ⁇ , 0.1 to 0.5 ⁇ , 0.1 to 1 ⁇ , 0.5 to 1.0 ⁇ , 1.0 to 1.5 ⁇ , 1 to 2.5 ⁇ , 1 to 5 ⁇ , 5 to 10 ⁇ , 10 to 50 ⁇ , 50 to 100 ⁇ , 100 to 150 ⁇ , 150 to 200 ⁇ , 250 to 300 ⁇ , 100 to 250 ⁇ , 100 to 500 ⁇ , 200 to 400 ⁇ , 500 to 1000 ⁇ , 1 to 10 mM, 1 to 5 mM, 5 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM.
  • the concentration of lithium achieved in the skin is greater than 0.1 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.0 mM. In some embodiments, the concentration of lithium achieved in the skin is greater than 1.5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 1 mM to 5 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 5 mM to 10 mM. In one embodiment, the amount of lithium achieved in the skin is approximately 100 to 200 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 5 mM. In one embodiment, the amount of lithium achieved in the skin does not exceed 10 mM.
  • the amount of lithium achieved in the skin does not exceed 50 mM.
  • an amount of lithium is administered such that the concentration of lithium delivered to the stratum corneum is 0.1 to 0.5 mM, 0.5 to 1 mM, 1 to 10 mM, 10 to 100 mM, 100 to 200 mM, or 500 to 1000 mM.
  • the concentration of lithium delivered to the stratum corneum is greater than 1.5 mM.
  • the amount of lithium achieved in the stratum corneum is approximately 100 to 200 mM.
  • the amount of lithium achieved in the stratum corneum does not exceed 5 mM.
  • the amount of lithium achieved in the stratum corneum does not exceed 10 mM.
  • lithium concentrations in skin using techniques known in the art, for example, mass spectroscopy, e.g., inductively coupled plasma mass spectroscopy (ICP-MS).
  • mass spectroscopy e.g., inductively coupled plasma mass spectroscopy (ICP-MS).
  • ICP-MS inductively coupled plasma mass spectroscopy
  • concentration of lithium in skin can be measured using the method provided in the example of Section 13.2 below or equivalent methods.
  • the lithium concentration is measured in the hair shaft using techniques known in the art, e.g., Tsanaclis & Wicks, 2007, Forensic Science Intl. 176: 19-22, which is incorporated by reference herein in its entirety.
  • lithium can be applied topically, e.g., as a cream, gel, ointment, or other form for topical administration as described in Section 5.2 supra. Topical lithium may be administered to wounded or unwounded skin.
  • the lithium formulation for topical administration comprises lithium (or monovalent lithium salt) at a concentration of 50 niM, 75 niM, 100 niM, 125 niM, 150 niM, 175 niM, 200 niM, 250 niM, 300 niM, 350 niM, 400 niM, 450 niM, 500 niM, 550 niM, 600 niM, 650 niM, 700 niM, 750 niM, 800 niM, 900 niM, 1 M, 1.1 M, or 1.2 M, or more.
  • a monovalent lithium salt ⁇ e.g., lithium gluconate, lithium chloride, lithium stearate, lithium orotate, etc.
  • a divalent lithium salt ⁇ e.g., in some embodiments, lithium succinate, lithium carbonate
  • a trivalent lithium salt ⁇ e.g., in some embodiments, lithium citrate), refers to a salt form of lithium in which there are three lithium cations for each anion of the salt.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 50 mM to 200 mM is chosen for use in the embodiments described herein.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 200 mM to 400 mM is used.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 400 mM to 600 mM is used.
  • a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 600 mM to 800 mM is used.
  • concentration of lithium in a particular topical lithium formulation to deliver the intended dose of lithium will depend on the release properties of the lithium ion, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • Lithium formulations comprising the foregoing lithium (or monovalent lithium salt) concentrations may be achieved using, for example, a formulation comprising, w/w, lithium ions at a concentration of 0.10% lithium, 0.15% lithium, 0.20% lithium, 0.25% lithium, 0.30% lithium, 0.35% lithium, 0.40% lithium, 0.45% lithium, 0.50% lithium, 0.55% lithium, 0.60% lithium, 0.65% lithium, 0.70% lithium, 0.75% lithium, 0.80% lithium, 0.85% lithium, 0.90% lithium, 0.95% lithium.
  • the form of lithium for topical administration comprises, w/w, 0.1% to 0.5% lithium ions, 0.2% to 0.5% lithium ions, 0.5% to 1% lithium ions, or more.
  • the amount of a salt form of lithium to generate a topical lithium formulation with one of the aforementioned concentrations of lithium ion is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g. , the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • Lithioderm (Labcatal) comprises 8% lithium gluconate, which corresponds to 0.275% lithium ion ⁇ i.e., 274.8 mg Li+/100 g gel).
  • a formulation of topical 8% lithium gluconate, w/w contains approximately 80 mg/ml lithium gluconate, which is approximately 400 mM lithium gluconate (and, thus, 400 mM lithium ion).
  • a formulation for topical administration comprises a salt form of lithium ⁇ e.g., lithium gluconate or other form described in Section 5.1 above) at a concentration, w/w, of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 16%, 18%, 20%, or more.
  • a salt form of lithium for topical administration comprises, w/w, 1% to 2% lithium salt ⁇ e.g., lithium gluconate or other form described in Section 5.1 above), 2% to 5% lithium salt, 5% to 10% lithium salt, 10% to 15% lithium salt, 15% to 20% lithium salt, 20% to 25% lithium salt, or 25% to 50% lithium salt.
  • the form of lithium for topical administration is 1% to 20% w/w lithium salt.
  • a topical formulation of lithium comprises l%-4% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 4%-8% lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 8%-16% or more lithium gluconate (w/w). In some embodiments, a topical formulation of lithium comprises 0.2%-l%, or l%-5%, or more lithium chloride (w/w). In some embodiments, a topical formulation of lithium comprises 0.5%-2%, or 2%-4%, or 4%-8%, or 8%-16, or more lithium succinate (w/w).
  • a topical formulation of lithium comprises 0.5%-6%, 6%-12%, or 12%-25%, or more lithium stearate (w/w). In some embodiments, a topical formulation of lithium comprises l%-4%, 4%-8%, or 8%-16%, or more lithium orotate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%-0.75%, 0.75%-1.5%, or 1.5%-3%, or more lithium carbonate (w/w). In some embodiments, a topical formulation of lithium comprises 0.25%- 1.5%, 1.5%-3.0%, or 3%-6%, or more 8% lithium citrate (w/w).
  • a 50 kg patient is administered a single droplet - approximately 0.1 ml - of 8% (w/w) lithium gluconate at 3 sites, twice daily. This corresponds to approximately 8 mg lithium gluconate (0.274 mg Li+) per site, i.e., 0.16 mg/kg lithium gluconate (0.005 mg/kg Li+) per site. Over three sites twice daily, this corresponds to approximately 0.96 mg/kg lithium gluconate (0.033 mg/kg Li+) per day.
  • a patient ⁇ e.g., a 50 kg patient
  • a topical lithium formulation is administered once daily.
  • a topical lithium formulation is administered twice daily.
  • doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
  • an amount of lithium is administered such that the peak lithium concentration in skin is between 0.01 mM and 0.05 mM, 0.05 mM and 0.1 mM, 0.1 mM and 0.5 mM or between 0.5 mM and 10 mM, for example, between 0.1 and 0.5 mM, 0.5 mM and 1 mM, 1 mM and 2 mM, between 2 mM and 5 mM, 5 mM to 10 mM, or 10 mM to 50 mM.
  • the peak lithium concentration in blood may be one or more orders of magnitude lower than the peak concentration in skin (for example, 0.001 mM to 0.01 mM, 0.01 mM to 0.1 mM, or 0.1 mM to 0.5 mM, 0.5 mM to 1.0 mM, or 1.0 mM to 10 mM).
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered subcutaneous ly, to either wounded or unwounded skin.
  • the form of lithium for subcutaneous administration is administered at a dose comprising 0.001 mg lithium ion per kg of patient weight.
  • the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/kg, 6.5 mg/kg,
  • the dose does not exceed 50 mg/kg.
  • the lower ranges of dosages may be preferably used for bolus dosing.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • concentration of a salt form of lithium required to generate a subcutaneously administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • a formulation comprising lithium gluconate may be subcutaneously administered at a dosage of approximately 10 mg lithium gluconate per kg of patient weight (mg/kg), 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, or 1000 mg/kg.
  • the formulation for subcutaneous administration contains a dose of 10 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, 200 mg/kg to 400 mg/kg, 400 mg/kg to 600 mg/kg, or 100 mg/kg to 600 mg/kg of lithium gluconate.
  • the formulation for subcutaneous administration contains a dose in the range of 30 mg/kg to 150 mg/kg lithium gluconate.
  • the formulation for subcutaneous administration contains a dose in the range of about 30 mg/kg to 300 mg/kg lithium gluconate.
  • the dose for subcutaneous administration does not exceed 300 mg/kg lithium gluconate.
  • the dose for subcutaneous administration does not exceed 600 mg/kg lithium gluconate.
  • the lower ranges of dosages may be preferably used for bolus dosing.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • the lithium formulation is administered subcutaneously once daily. In some embodiments, the lithium formulation is administered subcutaneously twice daily. In some embodiments of a twice daily treatment regimen, doses are administered 6 hours apart, or 7 hours apart, or 8 hours apart, or 9 hours apart, or 10 hours apart, or 11 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.
  • an amount of lithium is administered such that the peak lithium concentration in skin is between 0.1 ⁇ and 0.2 ⁇ , 0.2 ⁇ and 0.5 ⁇ , 0.5 and 1 ⁇ , 1 ⁇ and 2 ⁇ , 2 ⁇ to 10 ⁇ , 10 ⁇ to 100 ⁇ , 100 ⁇ to 500 ⁇ , 500 ⁇ to 1000 ⁇ .
  • peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • the peak concentration in skin is 0.2 ⁇ to 1.5 ⁇ lithium.
  • the peak concentration in skin should not exceed 1 ⁇ or 1.5 ⁇ lithium. In some embodiments, the peak concentration in skin is 10 ⁇ to 100 ⁇ lithium. In some embodiments, the peak concentration in skin is 100 ⁇ to 1000 ⁇ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM.
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • a formulation of lithium described herein (by non-limiting e.g., lithium gluconate, lithium chloride, lithium succinate, lithium carbonate, lithium citrate, lithium stearate, lithium orotate, etc.) is administered orally, for example, once daily, or twice daily as determined by the medical practitioner and in accordance with Section 5.3 above.
  • an oral formulation comprising of 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or more, but preferably less than 10 mM, of lithium ions (or monovalent lithium salt) is administered.
  • an oral formulation comprising lithium ions or a monovalent lithium salt in the range of 0.1 to 0.5 mM, 0.4 to 0.6 mM, 0.5 to 1 mM, 0.6 to 1.2 mM, or 1 to 1.5 mM, is administered.
  • Administration of the foregoing amounts of lithium may be achieved by oral administration of a lithium formulation at a dosage comprising 0.001 mg lithium ion per kg of patient weight.
  • the dose is 0.001 mg/kg, 0.002 mg/kg, 0.003 mg/kg, 0.004 mg/kg, 0.005 mg/kg, 0.006 mg/kg, 0.007 mg/kg, 0.008 mg/kg, 0.009 mg/kg, 0.010 mg/ kg, 0.020 mg/kg, 0.025 mg/kg, 0.050 mg/kg, 0.075 mg/kg, 0.10 mg/kg, 0.15 mg/kg, 0.20 mg/kg, 0.25 mg/kg, 0.30 mg/kg, 0.40 mg/kg, 0.50 mg/kg, 0.75 mg/kg, 1 mg/kg, 1.5 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg, 5 mg/kg, 5.5 mg/kg, 6 mg/
  • the dose does not exceed 50 mg/kg Li+.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • concentration of a salt form of lithium required to generate an orally administered formulation that delivers lithium ions at one of the aforementioned dosages is readily deducible by one of ordinary skill in the art, and depends upon several factors including, e.g., the valency of the salt form, the stability of the salt form, the ability of the salt form to release the lithium ion, the hydrophobicity or hydrophilicity, etc.
  • a formulation comprising lithium carbonate which is a divalent lithium salt ⁇ e.g., trade names Eskalith CR, Eskalith, Lithobid
  • the oral formulation contains a dose of 2 mg/kg to 10 mg/kg, 10 mg/kg to 25 mg/kg, 25 mg/kg to 50 mg/kg, 50 mg/kg to 100 mg/kg, 100 mg/kg to 200 mg/kg, or 200 mg/kg to 500 mg/kg of lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of 5 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 5 mg/kg to 50 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose in the range of about 10 mg/kg to 100 mg/kg lithium carbonate. In one embodiment, the oral formulation contains a dose that does not exceed 300 mg/kg lithium carbonate.
  • the maximum dosage that may be administered at any one time may vary depending on the release kinetics of the lithium and the concentration of efficacy of the formulation.
  • an amount of lithium compound is administered such that the peak lithium concentration in skin is between 0.1 ⁇ and 0.2 ⁇ , 0.2 ⁇ and 0.5 ⁇ , 0.5 and 1 ⁇ , 1 ⁇ and 2 ⁇ , 2 ⁇ to 10 ⁇ , 10 ⁇ to 100 ⁇ , 100 ⁇ to 500 ⁇ , 500 ⁇ to 1000 ⁇ .
  • peak values will depend on the lithium release properties of the formulation, the hydrophobicity of the lithium salt form, the partition coefficient of the lithium salt form, etc.
  • the peak concentration in skin is 0.2 ⁇ to 1.5 ⁇ lithium.
  • the peak concentration in skin should not exceed 1 ⁇ or 1.5 ⁇ lithium. In some embodiments, the peak concentration in skin is 10 ⁇ to 100 ⁇ lithium. In some embodiments, the peak concentration in skin is 100 ⁇ to 1000 ⁇ lithium. In some such embodiments, the peak lithium concentration in blood may be several orders of magnitude higher, for example, 0.1 mM to 0.5 mM, or 0.5 mM to 1.1 mM, 1.1 to 1.5 mM, 1.5 mM to 5 mM, 5 mM to 10 mM, 10 mM to 50 mM, or 50 mM to 100 mM.
  • the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.
  • the pulse lithium treatment can be administered one time, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. For example, in the treatment of bipolar disorder, therapeutically useful amounts of lithium ( ⁇ 0.4 to 1.2 mM) are only slightly lower than toxic amounts (>1.5 mM), so the skilled practitioner knows that the blood levels of lithium must be carefully monitored during treatment to avoid toxicity.
  • a pulse lithium treatment is administered at the time of integumental perturbation. In some embodiments, a pulse lithium treatment is administered following integumental perturbation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered before scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered during scab formation. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered periscab detachment.
  • a pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered immediately after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 hour after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered up to 6 hours after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 6- 12 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 12-18 hours after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 18- 24 hours after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 1 day after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 2 days after scab detachment. In one embodiment, in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is administered 3 days after scab detachment.
  • the pulse lithium treatment in which a pulse lithium treatment is administered following an integumental perturbation that leads to formation of a scab, is administered within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the pulse lithium treatment is administered at the time of integumental perturbation and then maintained for 3 or 4 or 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, a pulse lithium treatment is administered as soon as the scab falls of and maintained for 3 or 4 or 5 days. In some embodiments, the pulse lithium treatment is administered in order to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the pulse lithium treatment is administered at the time of integumental perturbation and is maintained up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with lithium is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved.
  • a pulse treatment is combined with a form of integumental perturbation that does not lead to formation of a scab.
  • the pulse lithium treatment is administered at the time of integumental perturbation.
  • a pulse lithium treatment is administered following integumental perturbation.
  • the pulse lithium treatment is administered following an integumental perturbation that does not lead to formation of a scab
  • the pulse lithium treatment is administered within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the intermittent lithium treatment can be administered one time ⁇ e.g., using a controlled release formulation), or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations.
  • lithium can be administered daily ⁇ e.g., once, twice or three times daily) for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days.
  • Holidays can be interspersed for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 7 days; and in some embodiments not more than 14 days.
  • an intermittent lithium treatment is begun at the time of integumental perturbation. In some embodiments, an intermittent lithium treatment is begun following integumental perturbation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun before scab formation. In one embodiment, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the intermittent lithium treatment is begun during scab formation. In one embodiment, in which an intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium in the intermittent lithium treatment is periscab detachment.
  • the first administration of lithium is immediately after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is up to 6 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 6-12 hours after scab detachment.
  • the first administration of lithium is 12-18 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 18-24 hours after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 1 day after scab detachment.
  • the first administration of lithium is 2 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is 3 days after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to formation of a scab, the first administration of lithium is administered immediately after scab detachment, followed by another administration each day for several days to 1 week.
  • the pulse lithium treatment is begun within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • the intermittent lithium treatment is begun at the time of integumental perturbation and then administered daily (or twice daily) for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the intermittent lithium treatment is begun as soon as the scab falls off, and administered daily for 5 days. In some embodiments, the intermittent lithium treatment is to modulate the neoepidermis that forms underneath the scab. In some such embodiments, the intermittent lithium treatment is begun at the time of integumental perturbation and is continued with daily dosing up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with lithium is short, for example, limited to daily doses for a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of integumental perturbation and lithium administration is preferably monitored and adjusted so that optimal results are achieved.
  • an intermittent lithium treatment is combined with a form of integumental perturbation that does not lead to formation of a scab.
  • the intermittent lithium treatment is begun at the time of integumental perturbation.
  • an intermittent lithium treatment is begun following integumental perturbation.
  • the intermittent lithium treatment is begun within 15 minutes of, or 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 18 hours, 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.
  • Intermittent lithium treatment or a pulse lithium treatment in combination with other methods enhances the effectiveness of these methods.
  • the effect that each drug offers could be an additive or synergistic improvement, or a combination of two different pharmacologically defined effects, to achieve the desired end result.
  • the combined modality of treatment could involve alternating treatment of each dosage form or concurrent or simultaneous treatment. Synergism occurs when the combination has an effect that is more than would be expected from merely the additive effect of each element in the combination, for example, if branched hair follicles or more hair follicles per pore were produced by the combination and not by either alone.
  • the intermittent lithium treatments or the pulse lithium treatment described herein may be in combination with any additional treatment(s) described or incorporated by reference herein or determined to be appropriate by the medical practitioner.
  • the amount of an additional treatment(s) will depend on the desired effect and the additional compound that is selected. Dosages and regimens for administering such additional treatment(s) are the dosages and regimens commonly in use, which can be easily determined by consulting, for example, product labels or physicians' guides, such as the Physicians' Desk Reference (“PDR”) (e.g., 63rd edition, 2009, Montvale, NJ: Physicians' Desk Reference).
  • PDR Physicians' Desk Reference
  • the combination treatment comprises lithium and an additional compound(s) formulated together.
  • the lithium in such formulations may be released concurrently with or separately from the additional compound(s), or may be released and/or delivered to the tissue site with different pharmacokinetics.
  • one or more of the compounds in the formulation undergoes controlled release, whereas one or more of the other compounds does not.
  • one or more of the compounds in the formulation undergoes sustained release whereas one or more of the other compounds undergoes delayed release.
  • the combination treatment comprises lithium and an additional compound(s) formulated separately.
  • the separate formulations may be administered concurrently, sequentially, or in alternating sequence.
  • the lithium compound may be administered sequentially, or concurrently with another compound to achieve the desired effect of improved wound healing or scar revision.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more treatments selected from, e.g., cell therapy (such as a stem cell), a formulation for gene therapy (such as, e.g., a virus, virus-like particle, virosome), an antibody or antigen-binding fragment thereof, an herb, a vitamin (e.g., a form of vitamin E, a vitamin A derivative, such as, e.g., all-trans retinoic acid (ATRA), a B vitamin, such as, e.g., inositol, panthenol, or biotin, or a vitamin D3 analog), a mineral, essential oils, an antioxidant or free radical scavenger, amino acids or amino acid derivatives, a shampoo ingredient (e.g.
  • cell therapy such as a stem cell
  • a formulation for gene therapy such as, e.g., a virus, virus-like particle, virosome
  • an antibody or antigen-binding fragment thereof an
  • ammonium chloride ammonium lauryl sulfate, glycol, sodium laureth sulfate, sodium lauryl sulfate, ketoconazole, zinc pyrithione, selenium sulfide, coal tar, a salicylate derivative, dimethicone, or plant extracts or oils
  • a conditioning agent e.g., a soap product, a moisturizer, a sunscreen, a waterproofing agent, a powder, talc, or silica, an oil-control agent, alpha-hydroxy acids, beta-hydroxy acids (e.g., salicylic acid), poly-hydroxy acids, benzoyl peroxide, antiperspirant ingredients, such as astringent salts (e.g., zinc salts, such as zinc pyrithione, inorganic or organic salts of aluminum, zirconium, zinc, and mixtures thereof, aluminum chloride, aluminum
  • octachlorohydrate aluminum zirconium octachlorohydrex GLY (abbreviation for glycine), aluminum zirconium pentachlorohydrate, aluminum zirconium pentachlorohydrex GLY, aluminum zirconium tetrachlorohydrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate GLY, and aluminum zirconium trichlorohydrate GLY, potassium aluminum sulphate, (also known as alum (KAl(S0 4 ) 2 l2H20)), aluminum undecylenoyl collagen amino acid, sodium aluminum lactate+ aluminum sulphate
  • tridecyl carboxy alkylates cerulenin or a cerulenin analog, including pharmaceutically acceptable salts or solvates thereof, another fatty acid synthase inhibitor, such as triclosan or analogs thereof, a polyphenol extracted from green tea (EGCG), available from Sigma Corporation (St.
  • a massage agent e.g., an exfoliant, an anti-itch agent, a pro-inflammatory agent, an immunostimulant (e.g., interferon, cytokines, agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system, immunostimulatory nucleic acids, an adjuvant that stimulates the immune response and/or which causes a depot effect).
  • an immunostimulant e.g., interferon, cytokines, agonists or antagonists of various ligands, receptors and signal transduction molecules of the immune system, immunostimulatory nucleic acids, an adjuvant that stimulates the immune response and/or which causes a depot effect.
  • adjuvants and/or other stimulators of local cytokines are used in conjunction with the intermittent lithium treatment or pulse lithium treatment.
  • one rationale for administering adjuvants and/or other stimulators of local cytokines in conjunction with the intermittent lithium treatment or pulse lithium treatment is that the production of local cytokines may induce changes in the hair follicle cell cycle and recruit new follicle stem cells to follicles.
  • the combination ntreatment comprises lithium in ccombination with a cell cycle regulator, a hormonal agonist, a hormonal antagonist (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), an inhibitor of hormone biosynthesis and processing, a steroid (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, hydrocortisone, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), an antiandrogen (e.g., cyproterone acetate), an antiestrogen, an antihistamine (e.g., mepyramine,
  • a hormonal antagonist e.g., flutamide
  • an anti-inflammatory e.g., corticosteroids (such as, e.g., Dermatop®), NTHEs, and COX-2 inhibitors, adrenocorticoids, beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methylprednisolone, prednisolone, prednisone, hydrocortisone), an anesthetic (e.g., vocal anesthesia, lidocaine, bupivacaine, etidocaine, etc., with or without epinephrine or sodium bicarbonate) a retinoid (e.g., 13-cis-retinoic acid, adapalene, all-trans-retinoic acid, and etretinate), PMMA, Restylane, poly-L-lactic acid, collagen, hyaluronic acid, which may be present in microspheres, or other skin fillers,
  • an anti-inflammatory e.g.
  • aspirin, ibuprofen, diclofenac, and COX-2 inhibitors pain relievers, leukotreine antagonists (e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), beta2-agonists (e.g.
  • hexylresorcinol methylbenzethonium chloride, cetrimide, chlorhexidine, chlorobutanol, chlorocresol, cresol, glycerin, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium sorbate, sodium benzoate, sodium proprionate, sorbic acid, and thiomersal (thimerosal)).
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more narcotic analgesics, selected from the group of, e.g., alfentanil, benzylmorphine, codeine, codeine methyl bromide; codeine phosphate, codeine sulfate, desomorphine, dihydrocodeine,
  • one or more narcotic analgesics selected from the group of, e.g., alfentanil, benzylmorphine, codeine, codeine methyl bromide; codeine phosphate, codeine sulfate, desomorphine, dihydrocodeine,
  • hydromorphone methadone hydrochloride, morphine, morphine hydrochloride, morphine sulfate, nicomorphine, normethadone, normorphine, opium, oxycodone, oxymorphone, phenoperidine, and propiram.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more non-narcotic analgesics, selected from the group of, e.g., aceclofenac, acetaminophen, acetanilide, acetylsalicylsalicylic acid; aspirin, carbamazepine, dihydroxyaluminum acetylsalicylate, fenoprofen, fluproquazone, ibufenac, indomethacin, ketorolac, magnesium acetylsalicylate, morpholine salicylate, naproxen, phenacetin, phenyl salicylate, salacetamide, salicin, salicylamide, sodium salicylate, and tolfenamic acid.
  • Other pain treatments that may be used in combination with the lithium treatments described herein include nerve blocks or non-traditional pain medications, such as, e.g., Lyrica (pregabalin) or Neuront
  • the present invention is based, in part, on the appreciation that hair follicles play a role in wound healing.
  • Inducing the formation of new hair follicles in wounds, or enhancing the entry of hair follicles into wounds (for example, by transplanting hair follicles into wounds) harnesses their regenerative capacity and provides a transformational approach to scar revision and the management of wounds.
  • the approaches described herein permit scar revision under sterile and controlled conditions that recreates and harnesses the fetal skin's plastic and regenerative capacity.
  • Fractional laser treatment of scarred tissue creates areas of small micro-injuries with intact epidermis in- between, and the integumental perturbation of the laser activates hair follicle deposition into the injury sites, either by migration from the intact epidermis or by inducing hair follicle neogenesis in the wound.
  • laser- induced wounding of columns (the non- ablative coagulum is a preferred embodiment) triggers the regenerative capacity of the intervening normal skin stem cells.
  • This technique may have utility in, for example, revising small scars (to improve texture, pigmentation and other features).
  • one advantage of using combinations comprising integumental perturbation is that the perturbation provides a signal for hair follicle deposition and/or deposition of other adnexal structures into the wound site, e.g., by their migration and/or by generation of new hair follicles (hair follicle neogenesis) or adnexal structures.
  • hair follicle neogenesis new hair follicles
  • a wound heals by scarring may depend on the efficiency of hair follicle or other adnexal structure deposition into the wound. If these structures, e.g., hair follicles, are not timely deposited into the healing wound, the process will result in a scar.
  • wound healing without scarring may be effected by improving the efficiency of adnexal structure ⁇ e.g., hair follicles) deposition into the wound or by slowing wound healing in order to allow sufficient time for deposition of these structures into the wound site.
  • adnexal structure e.g., hair follicles
  • enhancement of wound healing or scar revision is accomplished by lithium treatment alone, for example, in acutely wounded skin or skin affected by a chronic non-healing wound, i.e., skin already subjected to integumental perturbation.
  • the lithium treatment is administered to skin that has been damaged and which no longer contains follicles.
  • the lithium treatment may restore follicle production in that area of skin.
  • an area of skin containing a wound that has not healed correctly, such as a scar ⁇ e.g., a keloid scar) is administered a lithium treatment in order to restore hair follicles and/or hair growth to that area of skin.
  • enhancement of wound healing or scar revision is accomplished by a combination of integumental perturbation and a pulse or intermittent lithium treatment.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with integumental perturbation or, optionally, also comprises another treatment known in the art or described herein.
  • Combinations comprising integumental perturbation are preferred for skin that is not already acutely wounded, since wounding itself is a form of integumental perturbation.
  • Integumental perturbation can be achieved by any means known in the art or described herein, such as, for example, using chemical or mechanical means.
  • integumental perturbation comprises disrupting the skin of the subject (for example, resulting in the induction of re-epithelialization of the skin of the subject).
  • a certain area of the epithelium is partially or wholly disrupted.
  • a certain area of both the epithelium and stratum corneum are partially or wholly disrupted.
  • Integumental perturbation can be used to induce, for example, a burn, excision, dermabrasion, full-thickness excision, or other form of abrasion or wound.
  • the combination of integumental perturbation and lithium treatment is administered to skin that has been damaged and which no longer contains hair follicles.
  • the combination of integumental perturbation and lithium treatment restores follicle production in that area of skin.
  • an area of skin containing a wound that has not healed correctly such as a scar ⁇ e.g. , a keloid scar
  • a combination treatment of integumental perturbation and lithium is administered in order to restore hair follicles and/or growth of hair to that area of skin.
  • Mechanical means of integumental perturbation include, for example, use of sandpaper, a felt wheel, ultrasound, supersonically accelerated mixture of saline and oxygen, tape-stripping, spiky patch, or peels.
  • Chemical means of integumental perturbation can be achieved, for example, using phenol, trichloroacetic acid, or ascorbic acid.
  • Electromagnetic means of integumental perturbation include, for example, use of a laser (e.g., using lasers, such as those that deliver ablative, non-ablative, fractional, non-fractional, superficial or deep treatment, and/or are CC based, or Erbium-YAG-based, etc.).
  • Integumental perturbation can also be achieved through, for example, the use of visible, infrared, ultraviolet, radio, or X-ray irradiation.
  • integumental perturbation is by light energy, such as described in Leavitt et al, 2009, Clin. Drug. Invest. 29:283-292.
  • Electrical or magnetic means of disruption of the epidermis can be achieved, for example, through the application of an electrical current, or through electroporation or RF ablation.
  • Electric or magnetic means can also include the induction of an electric or a magnetic field, or an electromagnetic field. For example, an electrical current can be induced in the skin by application of an alternating magnetic field.
  • a radiofrequency power source can be coupled to a conducting element, and the currents that are induced will heat the skin, resulting in an alteration or disruption of the skin. Integumental perturbation can also be achieved through surgery, for example, a biopsy, a skin transplant, skin graft, follicular unit extraction, hair transplant, cosmetic surgery, open- heart surgery, etc.
  • integumental perturbation is by laser treatment, as discussed below.
  • Exemplary laser treatments for integumental perturbation include Fraxel, laser abrasion, Erbium- YAG laser, Ultrapulse CO 2 fractional laser, Ultrapulse CO 2 ablative laser, Smooth Peel Full-ablation Erbium laser (Candela), as described, for example, in the examples of Section 8 below.
  • a laser treatment is chosen in which the integumental perturbation achieved most resembles that achieved by dermabrasion (for example, a dermabrasion method described herein).
  • integumental perturbation by laser treatment is by a fractional laser.
  • a fractional laser treatment is treatment with an Erbium- YAG laser at around 1540 nm or around 1550 nm (for example, using a Fraxel® laser (Solta Medical)). Treatment with an Erbium-YAG laser at 1540 or 1550 nm is typically non-ablative, and pinpoint bleeding typical of laser treatment is not observed since the stratum corneum is left in tact.
  • integumental perturbation by laser treatment is by a fractional laser, using, e.g. , a CO 2 laser at 10,600 nm. Treatment with a CO 2 laser at 10,600 nm is typically ablative, and typically leads to the appearance of pinpoint bleeding.
  • a standard CO 2 or Erbium- YAG laser can be used to create superficial and, optionally, broad based, integumental perturbation similar to dermabrasion (discussed below). Although the pinpoint bleeding clinical endpoint may not be achieved due to the coagulation properties of (particularly non-ablative) lasers, use of a laser has an advantage making it possible to select the specific depth of skin disruption to effectively remove the stratum corneum and epidermis, or portions thereof.
  • the laser treatment is ablative.
  • full ablation of tissue is generated by the targeting of tissue water at wavelengths of 10,600 nm by a CO 2 laser or 2940 nm by an Erbium- YAG laser.
  • the epidermis is removed entirely and the dermis receives thermal tissue damage.
  • the depth of tissue ablation may be a full ablation of the epidermis, or a partial ablation of the epidermis, with both modes causing adequate wounding to the skin to induce the inflammatory cascade requisite for regeneration.
  • the depth of ablation may extend partially into the dermis, to generate a deep wound.
  • a lithium composition described herein is delivered by a sustained release depot that is comprised of biocompatible, bioabsorbable polymers that are compatible to tissue.
  • the standard full thickness excision model is created using scissors or with a scalpel in animal models (see, also, the examples of Section 16 infra). This procedure, while contemplated for use herein, carries with it the risk of scarring.
  • various fractional laser modalities could be used to achieve a similarly deep disruption on a grid pattern.
  • a fractional laser can be use to "drill," for example, 1-mm diameter holes with a 1-mm hole spacing (the fractional laser can make holes of smaller dimensions). Although the skin is completely removed within the 1-mm hole, the surrounding intact skin prevents scarring and therefore the full thickness excision model is invoked within each hole.
  • the laser treatment is ablative and fractional.
  • fractional tissue ablation can be achieved using a CO2 laser at 10,600 nm or an Erbium- YAG laser at 2940 nm ⁇ e.g., the Lux 2940 laser, Pixel laser, or Pro fractional laser).
  • the lasing beam creates micro-columns of thermal injury into the skin, at depths up to 4 mm and vaporizes the tissue in the process.
  • Ablative treatment with a fractional laser leads to ablation of a fraction of the skin leaving intervening regions of normal skin intact to rapidly repopulate the epidermis. Approximately 15%— 25% of the skin is treated per session.
  • micro thermal zones can be varied to create a dense "grid" of injury columns surrounded by intact skin and viable cells.
  • the density of the grid on the treatment area plays an important role. The denser the grid, the more the thermal injury and the type of injury begins to approximate full ablation. Therefore, it is appreciated that there may be an "optimum" MTZ density that is appropriate for use in the methods disclosed herein.
  • a lithium composition described herein is delivered into the dermis immediately after wounding, or after initial re-epithelialization has occurred.
  • the mode of laser treatment is non-ablative, wherein the stratum corneum and the epidermis are intact after treatment, with the dermis selected for the deep thermal treatment required for the requisite injury to tissue.
  • This can be accomplished by cooling the epidermis during the laser treatment.
  • the depth of treatment may be 1 mm to 3 mm into the skin.
  • contact cooling such as a copper or sapphire tip.
  • Lasers that are non- ablative have emission wavelengths between 1000-1600 nm, with energy fluences that will cause thermal injury, but do not vaporize the tissue.
  • the non-ablative lasers can be bulk, wherein a single spot beam can be used to treat a homogenous section of tissue. In some embodiments, multiple treatments are required to achieve the desired effect.
  • a lithium composition described herein is delivered deep into the dermis in polymeric micro-depots and released in a sustained fashion.
  • Lasers that are non-ablative include the pulsed dye laser (vascular)(at, e.g., 585-595 nm), the 1064 Nd:YAG laser, or the Erbium- YAG laser at 1540 nm or 1550 nm ⁇ e.g., the Fraxel® laser).
  • the mode of laser treatment is fractional and non-ablative. Treatment with a fractional, non-ablative laser leads to perturbation of a fraction of the skin, leaving intervening regions of normal skin intact to rapidly repopulate the epidermis.
  • Approximately 15%— 25% of the skin is treated per session.
  • the skin barrier function is maintained, while deep thermal heating of dermis can occur.
  • zones of dermis and epidermis are coagulated and the stratum corneum is left essentially intact.
  • This process has been coined "fractional photothermolysis" and can be accomplished, e.g., using the Erbium-YAG laser with an emission at or around 1540 nm or 1550 nm.
  • a lithium composition described herein is delivered immediately after the tissue injury, deep into the dermis.
  • a combination of bulk and fractional ablation modes of tissue injury are used.
  • a combination treatment comprising use of a laser includes administration to the skin of a compound absorbing light at wavelengths between 1000-1600 nm for the purpose of efficient channeling of light to heat energy.
  • This method of channeling energy may cause micro-zones of thermal injury within the skin.
  • the compound may be delivered to the skin homogenously in the treatment zone, then subsequently irradiated with a non-ablative laser to efficiently capture the vibrational energy of the infrared beam. This method may result in evenly distributed and deep thermal injury, without causing tissue vaporization.
  • a combination treatment comprising use of a laser includes administration of a lithium compound formulation that is encapsulated in matrices that are highly hydrophilic and charged, for example, linked to the dermis by covalent or ionic bonding to prevent the matrices from being cleared by phagocytosis, as part of the wound healing process.
  • a combination treatment comprising use of a laser includes the step of placing a biocompatible, synthetic skin substitute on the newly created wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function.
  • the biocompatible synthetic skin substitute is be impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic.
  • other molecules are also co-eluted at the site through the skin substitute, such as, e.g.
  • the skin substitute in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
  • a fractional like hole pattern (similar to that achieved with a fractional laser or full thickness excision) is achieved with using an array of punch biopsy needles.
  • 1 -mm punch biopsies can be arranged with 1 -mm hole spacing.
  • the cored skin samples can be removed and, thus, an effect approximating the full thickness excision model is invoked within each hole.
  • microneedles e.g., 19 or 21 gauge needles
  • micro-coring needles could be used.
  • integumental perturbation is by dermabrasion (also referred to herein as "DA"), a well-established dermatological procedure that has been used for decades as a skin resurfacing technique (Grimes, 2005, Microdermabrasion. Dermatol Surg 31 : 1351-1354). While the popularity of mechanical DA has decreased in recent years with the advent of laser-based procedures, DA is still used for removing facial scars resulting from acne and other trauma. Small, portable mechanical dermabrasion equipment uses interchangeable diamond firaises operated at different rotation speeds to remove the epidermis and dermis to differing skin depths levels.
  • Dermabrasion may be carried out using any technique known in the art or as described herein, e.g., in the examples of Sections 9, 10 and 16 infra.
  • dermabrasion may be carried out using standard DA with aluminum oxide crystals using the Aseptico Econo-Dermabrader, Advance Microderm DX system, or M2-T system; standard DA with Bell Hand Engine with diamond fraize; etc.
  • DA is carried out using an abrasive wheel.
  • DA with an abrasive wheel is used in order to achieve pinpoint bleeding.
  • DA may be carried out using an abrasive wheel to achieve larger globules of bleeding and frayed collagen.
  • non-powered devices such as abrasive cloths can also be used to achieve the DA, with the optional achievement of the same endpoint(s).
  • DA is accomplished using a device typically used for microdermabrasion.
  • a microdermabrasion device is used to remove a greater depth and/or area of skin than is typical for microdermabrasion (also referred to herein as "MDA").
  • MDA microdermabrasion
  • the microdermabrasion device is used under sterile conditions.
  • DA is achieved by using a device typically used for microdermabrasion to the point where treatment is stopped upon the observation of pinpoint bleeding, which signals the removal of the stratum corneum and epidermis into the papillary dermis.
  • DA is achieved by using a device for
  • this extended use is reduced by using a microdermabrasion device with increased output pressure and increased abrasion particle size, which may accelerate the skin removal process.
  • DA is accomplished by removal of surface skin by particle bombardment (also referred to herein as "particle mediated dermabrasion" (“PMDA”)), for example, with alumina-, ice- or silica-based particles.
  • particle bombardment also referred to herein as "particle mediated dermabrasion” (“PMDA)
  • PMDA particle mediated dermabrasion
  • micron- sized particles are propelled toward the surface of the skin via short strokes of a handpiece, such as a particle gun, as known in the art.
  • the velocity of particles is controlled through positive or negative pressure.
  • the depth of skin removed by particle bombardment DA ⁇ e.g., PMDA) is a function of the volume of particles impacting the skin, the suction or positive pressure, the speed of movement of the handpiece, and the number of passes per area of the skin.
  • integumental perturbation by one or more of the aforementioned methods achieves removal of part or all of the epidermis. In some embodiments, integumental perturbation removes the entire epidermis. In some
  • integumental perturbation disrupts the papillary dermis.
  • integumental perturbation removes the papillary dermis.
  • integumental perturbation removes the reticular dermis.
  • the depth of integumental perturbation depends on the thickness of the skin at a particular treatment area. For example, the skin of the eyelid is significantly thinner than that of the scalp.
  • the occurrence of pinpoint bleeding indicates that the epidermis and portions of the dermis have been removed. Deeper penetration can results in much more bleeding, and the perturbation can go as deeps as the hypodermis.
  • integumental perturbation by one or more of the aforementioned methods is to a skin depth of 60 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 60-100 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 100 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 150 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 100-500 ⁇ . In some embodiments, integumental perturbation is to a skin depth of less than 500 ⁇ . In some embodiments, integumental perturbation is to a skin depth of 500- 1000 ⁇ .
  • integumental perturbation is to a skin depth of 1 mm or more. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 3 mm. In some embodiments, integumental perturbation is to a skin depth of 1 mm to 5 mm.
  • Integumental perturbation such as occurs during wounding, produces in the affected skin tissue an increase in the number of hair follicle stem cells and in the plasticity of hair follicle cells.
  • the pulse or intermittent lithium treatments cause formation of new hair follicles or enhanced branching, division, or differentiation of existing hair follicles or hair follicle progenitors. Accordingly, and without being bound by any theory for how the invention works, integumental perturbation (or wounding) in combination with a pulse or intermittent lithium treatment provides an environment for the formation of a large number of follicles to enhance wound healing and, preferably, wound healing with reduced scarring.
  • integumental perturbation techniques described herein for example, laser techniques or surgical dissection of follicles, etc., lead to hair follicle transection in vivo. It is thought that at least 40% of follicles, after bisection, form two follicles (which would result if all the "bottoms” from the -30% of failed "tops” successfully yielded a hair fiber and in addition, if all the "tops” from the -30% of failed bottom bisected follicles successfully yielded a hair fiber.) Therefore, the percentage of bisected follicles that produce two new follicles is in the range of -40-70% (with the maximum -70% being the result of all bisections producing two new follicles and no bisections resulting in either a top or a bottom (but not both) producing a follicle). It is expected that this efficiency will be increased when pulsatile lithium is applied because it induces differentiation.
  • FSCs originate from one or more of the following: (i) existing follicles ("follicle derived follicle stem cells” or “FDFSC”) (see, e.g., Toscani et al, 2009, Dermatol Surg.
  • FDFSC follicle derived follicle stem cells
  • TDFSC tissue derived follicle stem cells
  • BMDFSC bone marrow derived follicle stem cells
  • FSCs generate new hair follicles that preserve the type of hair follicle that is typical for each location of skin or scalp.
  • FSCs from the coronal scalp of a male with MPHL typically generate atrophic follicles with vellus or club hairs.
  • FSCs from the occipital scalp of the same male typically generate follicles with terminal hair that are not subject to involution in response to DHT
  • FSCs responsible for follicle formation may be reprogrammed.
  • FSCs in the process of asymmetric division and subsequent differentiation are susceptible to signals (such as estrogen or testosterone) that alter the determinism of their differentiation program.
  • a pulse or intermittent lithium treatment in combination with integumental perturbation provides a window during which a third treatment that alters the follicle development program may be administered in order to significantly change the number and quality of follicles in a particular area of skin.
  • any treatment that enhances hair growth or, alternatively, that prevents hair growth or removes excessive hair, that is known in the art or yet to be developed is contemplated for use in such combination treatments.
  • treatments that promote hair growth include minoxidil, finasteride, bimatoprost (Latisse), CaCl 2 , or adenosine, or techniques of integumental perturbation such as by mechanical means, chemical means, electromagnetic means (e.g., using a laser such as one that delivers ablative, non-ablative, non-fractional, superficial, or deep treatment, and/or are CCVbased, or Erbium- YAG-based, etc.), irradiation, radio frequency (RF) ablation, or surgical procedures (e.g., hair transplantation, strip harvesting, follicular unit extraction (FUE), scalp reduction, etc.).
  • RF radio frequency
  • treatments that remove unwanted hair or prevent hair growth include, e.g., cytotoxic drugs, hair growth retardants, such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • cytotoxic drugs such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • hair growth retardants such as eflornithine HC1 (Vaniqa), 5-fluorouracil (5- FU) (e.g., Efudex 5% cream), or other epilation or depilation methods.
  • a third treatment may comprise treatment with an estrogen or testosterone modulator, such as those described in Poulos & Mirmirani, 2005, Expert Opin. Investig. Drugs 14: 177
  • the third treatment is administered simultaneously with integumental perturbation. In some embodiments, the third treatment is administered after integumental perturbation. In some embodiments, the third treatment is administered 1 day, 2 days, 3 days, 5 days, 7 days, 10 days, or 2 weeks after integumental perturbation. In one embodiment, the third treatment is administered at the time of integumental perturbation and then daily for 5 days thereafter (in some embodiments, a scab forms during this time). In some embodiments, the third treatment is administered daily for 5 days beginning as soon as the scab falls off. In some embodiments, the third treatment is administered in order to modulate the neoepidermis that forms underneath the scab.
  • the third treatment is administered at the time of integumental perturbation and up to some time after scab falls off, for example, between 5 - 14 days following integumental perturbation.
  • the course of treatment with the third treatment is short, for example, limited to a few days just following scab detachment, or even continued only for as long as the scab is still attached.
  • the timing of the integumental perturbation, lithium administration, and the third treatment is preferably monitored and adjusted so that optimal results are achieved.
  • This invention is based, in part, on the discovery that there is a correlation between the extent of wound contraction and the deposition of adnexal structures, such as new hair follicles, in wounded areas.
  • adnexal structures such as new hair follicles
  • intermittent and pulse lithium treatments may promote wound healing and scar revision by, at least in part, promoting the entry of hair follicles into the wound as it heals. This may occur by inducing the generation of new hair follicles and/or promoting migration of hair follicles into the wound site.
  • the intermittent and pulse lithium treatments described herein may (i) promote hair follicle neogenesis ⁇ e.g. , de novo formation of hair follicles from tissue or bone-marrow derived stem cells or disintegration of preexisting follicles into cells that mix together and reform the hair follicle); and/or (ii) promote branching ⁇ e.g., with the assistance of stem cells from dissociated hair follicles) and division of existing hair follicles.
  • hair follicle neogenesis e.g. , de novo formation of hair follicles from tissue or bone-marrow derived stem cells or disintegration of preexisting follicles into cells that mix together and reform the hair follicle
  • branching e.g., with the assistance of stem cells from dissociated hair follicles
  • a superficial wound is healed by the assistance of hair follicles remaining in the wound.
  • the hair follicles disintegrate and are reorganized and reformed by the presence of stem cells that enter the wound.
  • hair follicle neogenesis promotes wound healing.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that increase the number of hair follicles or that counteract hair follicle cell senescence (also referred to herein as "anti-senescence agents"), for example, anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants); inhibitors of reactive oxygen species (ROS) generation, such as superoxide dismutase inhibitors;
  • agents that increase the number of hair follicles or that counteract hair follicle cell senescence also referred to herein as "anti-senescence agents”
  • anti-oxidants such as glutathione, ascorbic acid, tocopherol, uric acid, or polyphenol antioxidants
  • ROS reactive oxygen species
  • ROS breakdown such as selenium
  • mTOR inhibitors such as rapamycin
  • sirtuins or activators thereof such as resveratrol, or other SIRT1, SIRT3 activators, or nicotinamide inhibitors.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents that induce an immune response or cause inflammation, such as, e.g., tetanus toxoid, topical non-specific irritants (anthralin), or sensitizers (squaric acid dibutyl ester [SADBE] and diphenyl cyclopropenone [DPCP]). While not intending to be bound by any theory, it is thought that by contacting these agents to the skin, lymphocytes and hair follicle stem cells may be recruited to skin.
  • agents that induce an immune response or cause inflammation such as, e.g., tetanus toxoid, topical non-specific irritants (anthralin), or sensitizers (squaric acid dibutyl ester [SADBE] and diphenyl cyclopropenone [DPCP]). While not intending to be bound by any theory, it is thought that by contacting these agents to the skin, lymphocytes
  • the combination treatment comprises a pulse or intermittent lithium treatment together with a cytokine thought to regulate the activity of Dermal Papillae, which is believed to be the target of androgen regulation of hair growth.
  • Interleukin-1 alpha decreases responses to androgen in cultured dermal papilla cells (Boivin et al, 2006, Exp Dermatol. 15:784-793).
  • TGF- ⁇ may mediate androgen-induced hair growth suppression, since in culture, human dermal papilla cells (DPCs) from androgenetic alopecia (AGA) subjects that transiently expressing androgen receptor were co-cultured with keratinocytes (KCs), and secreted TGF- ⁇ that inhibited KC growth (Inui et al., 2003, J Investig Dermatol Symp Proc. 8:69-71). Thus, a TGF-B1 inhibitor may be used in a combination treatment.
  • DPCs human dermal papilla cells
  • AGA androgenetic alopecia
  • KCs keratinocytes
  • TGF-B1 inhibitor may be used in a combination treatment.
  • Melatonin is a protein hormone secreted by the pineal gland that modulates hair growth, pigmentation and/or molting in many species. Human scalp hair follicles in anagen are important sites of extra-pineal melatonin synthesis. Melatonin may also regulate hair Follicle Cycle control, since it inhibits estrogen receptor-alpha expression (Fischer et al, 2008, Pineal Res. 44: 1-15). Melatonin and the other treatments described herein can be administered, for example, during the lithium treatment "holidays.” Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a chemical or mechanical (such as those discussed infra) treatment that induces an inflammatory process in the skin. While not intending to be bound by any theory, inducing inflammation in the site where hair growth is desired helps to recruit stem cells to the tissues that drive the formation of new follicles.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with an antiapoptotic compound.
  • the antiapoptotic compound is not a Wnt or a Wnt agonist.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more of stem cell therapy, hair cloning or hair plugs, follicular unit extraction, hair or skin transplantation, massage, a skin graft, or any surgical procedure aimed at skin or hair restoration.
  • the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with use of a laser device or other mode of accomplishing "photo-biostimulation" of the hair follicles.
  • a laser device or other mode of accomplishing "photo-biostimulation" of the hair follicles.
  • the Hairmax Lasercomb or the Leimo laser are non-limiting examples of devices that can be used in combination with the methods described herein.
  • intermittent lithium treatment or a pulse lithium treatment alone or in combination with other treatments described herein, synchronizes hair follicle cells in the cell cycle.
  • lithium is administered to arrest hair follicle cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their re-entry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle stem cells in G2/M phase, which synchronizes them; then the lithium treatment is removed; and then their re-entry in to the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles and an increased number of follicles).
  • the lithium treatment arrests hair follicle stem cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their reentry into the cell cycle and mitotic division is stimulated with other drugs (which leads to anagen follicles, and an increased number of follicles).
  • intermittent lithium treatment or a pulse lithium treatment alone or in combination with the aforementioned combination treatments, synchronizes hair follicle cells in the Follicle Cycle.
  • the treatment regimen induces follicles to enter anagen.
  • the treatment regimen prevents follicles from entering catagen.
  • the treatment regimen induces follicles in telogen to enter exogen, or induces follicles in exogen to enter anagen.
  • a combination treatment comprises a pulse or intermittent lithium treatment in combination with another treatment that modulates wound healing, including any treatment described herein or known in the art to modulate wound healing.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that enhances one or more of the steps of wound healing discussed in Section 2.1.1 above, including any treatment described herein or known in the art to enhance wound healing.
  • enhancement of a step of wound healing or enhancement of wound healing is meant the hastening of healing, improvement of healing, or reduction of scarring, etc.
  • the pulse or intermittent lithium treatment is administered in combination with a wound dressing or skin replacement, such as, for example, gauze, calcium-alginates, impregnated gauzes, films, foams, hydrogels, hydrocolloids, adsorptive powders and pastes, silicone, mechanical vacuum, dermal matrix replacements, dermal living replacements, or skin living replacements, a collagen dressing, cadaveric skin, or other matrix useful to promote healing of the wound such as described herein or known in the art. See, e.g., Table 10.3 in Lorenz & Longaker, which is incorporated by reference herein in its entirety.
  • the pulse or intermittent lithium treatment is administered in combination with a pain reliever, antibiotic and antibacterial use or other anti-infectives (such as, e.g., tea tree oil), debridement, drainage of wound fluid, mechanical removal of bacteria, removal of devitalized tissue (such as, e.g., by surgery or maggot therapy), irrigation (e.g., by pulsed lavage), vacuum-assisted closure (otherwise referred to as negative pressure wound therapy), warming, oxygenation (e.g., using hyperbaric oxygen therapy), antioxidant therapy, revascularization therapy, moist wound healing, removing mechanical stress, use of elastase inhibitors, or adding cells or other materials to secrete or enhance levels of healing factors.
  • a pain reliever such as, e.g., tea tree oil
  • debridement such as, e.g., tea tree oil
  • debridement such as, e.g., tea tree oil
  • drainage of wound fluid such as, e.g., by surgery or maggot therapy
  • the pulse or intermittent lithium treatment is administered in combination with the upregulation of endogenous growth factors or exogenous application of growth factors, which may accelerate normal healing and improve healing efficacy.
  • growth factors include, but are not limited to, vascular endothelial growth factor (VEGF), insulin-like growth factor 1-2 (IGF), PDGF, transforming growth factor- ⁇ (TGF- ⁇ ), epidermal growth factor (EGF), EGF -receptor, members of the FGF family, and others described herein and listed in, e.g., Table 10.2 in Lorenz & Longaker, which is incorporated by reference herein in its entirety.
  • Such growth factors can be applied exogenously or may be applied by spreading onto the wound a gel of the patient's own platelets, implanting cultured keratinocytes into the wound, or treating the wound with artificial skin substitutes that have fibroblasts and keratinocytes in a matrix of collagen.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that reduces the time it takes for a wound to heal or that reduces the extent of the wound.
  • treatments include, for example, periodic rotation of the patient or wounded tissue or use of an air mattress, use of a lower pressure cast or relieving excessive suture tension, cleansing of the wound, debridement of tissue, particularly necrotic tissue, improvement of circulation and oxygen delivery to the tissue by, e.g., hyperbaric oxygen therapy or other oxygen administration, whirlpool therapy, ultrasound therapy, electrical stimulation, magnetic therapy have been utilized to aid the body in healing wounds coverage of wound with vascularized tissue, revascularization of the wounded tissue, treatment of circulatory obstruction or other treatment that improves circulation, treatment of ischemia, edema, or hypoxia, or improvement of the hematocrit (e.g., to at least 15%).
  • Treatment of tissue necrosis treatments or prevention of infection (e.g., with antibiotics such as povidone-iodine, chlorhexidine gluconate, hexachlorophene, or silver sulfadiazine and others described herein (particularly for burn wound care), irrigation (e.g., with saline), and/or debridement), improvement of nutrition ⁇ e.g., increasing intake of vitamins, e.g., vitamin A, C, Bl, B2, B5, or B6, or trace metals, such as, e.g., zinc and copper, amino acids such as arginine, glutamine, or Bromelain, Curcumin, etc.), herbal supplements (e.g., Aloe Vera, Centella), diabetes treatment (for example, to improve vascular conditions, or by administering glucose), skin graft, treatment with hormones (such as estrogen) or treatment with growth factors (e.g., epigallocate, hematoma, hematoma, hematoma, hematoma
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that slows the natural adult wound healing process.
  • such combination treatments are used in the presence of a sterile wound dressing that obviates the need to heal the wound quickly (for example, in natural wound healing, the wound heals quickly in order to avoid infection).
  • the pulse or intermittent lithium treatment is administered in combination with a treatment that causes the postnatal wound healing process to resemble the fetal wound healing process. In some embodiments, this is accomplished by placing the wounded skin into a womb-like environment, for example, using a dressing and/or heat.
  • the pulse or intermittent lithium treatment is administered in combination with an agent that reduces or inhibits the inflammatory phase of wound healing, using, e.g. , an anti-inflammatory agent such as a NSAID or a topical glucocorticoids, an anti- androgen, or an antagonist of TNFa, TGF , NFkB, IL-1, IL-6, IL-8, IL-10, IL-18, or an antagonist of one or more other proinflammatory cytokines.
  • the pulse or intermittent lithium treatment is administered in combination with an agent that slows the wound healing process by extending the inflammatory phase, e.g., an androgen (see, e.g., Gilliver et al, 2007, Clin.
  • the treatment is administered in combination with an agent that suppresses the proliferative phase of wound healing, or the maturation and remodeling phase of wound healing.
  • the treatment is administered in combination with an agent that slows or interferes with fibrin deposition, clotting caused by fibrin, or fibrin-induced immunity.
  • the treatment is administered in combination with a treatment that inhibits the activity of fibrinogen.
  • the treatment is administered in combination with an agent that decreases the activity of myofibroblasts.
  • the treatment is administered in combination with a treatment that reduces collagen synthesis, deposition, or accumulation, for example, collagenases.
  • the treatment is administered in combination with a treatment that maintains the wound in an open state for a longer than normal period of time.
  • a treatment is administered in combination with rapamycin or corticosteroids.
  • a biocompatible, synthetic skin substitute is placed on the wound, especially if the wound is deep, covers large area, and is bulk ablated. This process can help minimize or prevent the rapid wound contraction that occurs after loss of a large area of tissue, frequently culminating in scar tissue formation and loss of skin function.
  • the biocompatible synthetic skin substitute is impregnated with depots of a slow releasing lithium formulation described herein. This method of treatment may enable treating a large area in one session at the treatment clinic.
  • other molecules are also co-eluted at the site through the skin substitute, such as, e.g., anesthetics and antibiotics, to prevent further pain and minimization of infection, or any other compound described herein.
  • the skin substitute in the presence or absence of a lithium compound and/or other compounds described herein, may also be pre-cooled and applied to the wound to provide a feeling of comfort to the patient. This mode of lithium or other compound application may prevent the lithium or other compound from being cleared away from the wound site as the wound heals.
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that improves wound healing, in order to reduce the appearance or extent of scarring.
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that improves the appearance and/or function of scarred skin, including any such treatment described herein or known in the art.
  • a pulse or intermittent lithium treatment is administered in combination with scar revision, such as by skin graft, serial expansion of surrounding skin, or laser treatment as described in Section 5.4 above.
  • a pulse or intermittent lithium treatment is administered in combination with re-excision with subsequent healing by primary intention, treatment with steroids (e.g., corticosteroid injection), silicone scar treatments (e.g.
  • porcine fillers or other cosmetic fillers e.g., inserted under atrophic scars
  • ribosomal 6 kinase (RSK) antagonists e.g., ribosomal 6 kinase (RSK) antagonists
  • RSK ribosomal 6 kinase
  • antagonists of pro-inflammatory cytokines such as TGF 2 or TNF
  • osteopontin antagonists the use of pressure garments, needling, dermabrasion, collagen injections, low-dose radiotherapy, or vitamins (e.g., vitamin E or vitamin C or its esters).
  • a pulse or intermittent lithium treatment is administered in combination with a treatment that reduces surgical scarring, e.g. , by placement of elective incisions parallel to the natural lines of skin tension (Langer's lines) or by applying sutures in a "zigzag" pattern.
  • the pulse or intermittent lithium treatment is administered in combination with a treatment of wounds that minimizes scarring, by, for example, administering physical therapy to a subject (e.g., range-of-motion exercises), reducing infection, reducing separation of wound edges, minimizing collagen synthesis, deposition, or accumulation or otherwise causing the process of healing by secondary intention to better resemble healing by primary intention.
  • intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for enhancing scar revision or wound healing improves the effectiveness of these methods, making the treatment more effective, efficient, cost-effective, pain-free, and/or user friendly. For example, fewer treatments may be required.
  • one of the previously described wound healing or scar revision treatments on its own is not cosmetically satisfactory, does not adequately restore function of the skin, or the benefits are too short-lived.
  • the intermittent lithium treatment or a pulse lithium treatment can be administered prior to, concurrently with, or subsequent to the administration of a second (or third, or more) treatment.
  • the intermittent lithium treatment or a pulse lithium treatment is administered to a subject at reasonably the same time as the other treatment.
  • This method provides that the two administrations are performed within a time frame of less than one minute to about five minutes, or up to about sixty minutes from each other, for example, at the same doctor's visit.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered at exactly the same time.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a sequence and within a time interval such that the intermittent lithium treatment or a pulse lithium treatment and the other treatment can act together to provide an increased benefit than if they were administered alone.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered sufficiently close in time so as to provide the desired outcome.
  • Each can be administered simultaneously or separately, in any appropriate form and by any suitable route.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered by different routes of
  • each is administered by the same route of administration.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment can be administered at the same or different sites of the subject's body.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment may or may not be administered in admixture or at the same site of administration by the same route of administration.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered less than 1 hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 1 1 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
  • the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one month apart, 1 to 2 months apart, 2 to 3 months apart, 3 to 4 months apart, 6 months apart, or one year or more apart.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered in a time frame where both are still active. One skilled in the art would be able to determine such a time frame by determining the half life of each administered component.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered within the same patient visit. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered prior to the administration of the other treatment. In an alternate embodiment, the intermittent lithium treatment or a pulse lithium treatment is administered subsequent to the administration of the other treatment.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are cyclically administered to a subject.
  • Cycling treatment involves the administration of the intermittent lithium treatment or a pulse lithium treatment for a period of time, followed by the administration of the other treatment for a period of time and repeating this sequential administration.
  • the first treatment may be with the intermittent lithium treatment or a pulse lithium treatment or with the other treatment, depending on the subject's prior treatment history and the intended outcome.
  • cycling treatment can also reduce the development of resistance to one or more of the treatments, avoid or reduce the side effects of one of the treatments, and/or improve the efficacy of the treatment.
  • alternating administration of the intermittent lithium treatment or a pulse lithium treatment may be followed by the administration of another treatment (or vice versa) 1 year later, 6 months later, 3 months later, 1 month later, 3 weeks later, 2 weeks later, 1 week later, 4 to 6 days later, 2 to 4 days later, or 1 to 2 days later, wherein such a cycle may be repeated as many times as desired.
  • the intermittent lithium treatment or a pulse lithium treatment and the other treatment are alternately administered in a cycle of 3 weeks or less, once every two weeks, once every 10 days or once every week.
  • Such time frames can be extended or reduced depending on whether a controlled release formulation of either the lithium compound or the other treatment formulation is used, and/or depending on the progress of the treatment course.
  • an area of skin that was pre-treated with lithium is used as a source for transplanted follicles.
  • treatment with lithium at the wounds(s) from which transplanted tissue was obtained and/or the site of implantation is initiated for one week, and then discontinued and optionally followed by another treatment.
  • a candidate subject for intermittent lithium treatment i.e., alternating lithium treatment with "vacation/holiday” periods
  • a pulse lithium treatment for promoting hair growth is any subject at risk for, has, or has had a wound or scar.
  • the subject may be any subject, preferably a human subject, including male, female, intermediate/ambiguous ⁇ e.g., XO), and transsexual subjects.
  • a human subject including male, female, intermediate/ambiguous ⁇ e.g., XO), and transsexual subjects.
  • the subject is a Caucasian subject. In certain embodiments, the subject is an African subject or an African-American subject. In certain embodiments, the subject is a human adolescent. In certain embodiments, the subject is undergoing puberty. In certain embodiments, the subject is a young adult. In certain embodiment, the subject is a middle- aged adult. In certain embodiments, the subject is a premenopausal adult. In certain embodiments, the subject is undergoing menopause. In certain embodiments, the subject is postmenopausal. In certain embodiments, the subject is elderly.
  • the subject is a human of 1 year old or less, 2 years old or less, 2 years old, 5 years old, 5 to 10 years old, 10 to 15 years old, e.g., 12 years old, 15 to 20 years old, 20 to 25 years old, 25 to 30 years old, 30 years old or older, 30 to 35 years old, 35 years old or older, 35 to 40 years old, 40 years old or older, 40 to 45 years old, 45 to 50 years old, 50 years old or older, 50 to 55 years old, 55 to 60 years old, 60 years old or older, 60 to 65 years old, e.g., 65 years old, 65 to 70 years old, 70 to 75 years old, 75 to 80 years old, 80 to 85 years old, 85 to 90 years old, 90 to 95 years old or 95 years old or older.
  • the subject is a male 20 to 50 years old. In some embodiments, the subject is a male or female 12 to 40 years old. In some embodiments, the subject is not a female subject. In some embodiments, the subject is not pregnant or expecting to become pregnant. In some embodiments, the subject is not a pregnant female in the first trimester of pregnancy. In some embodiments, the subject is not breastfeeding.
  • the intermittent lithium treatment or a pulse lithium treatment is delivered to an area in which enhanced wound healing or scar revision is desired, for example, the scalp, face (e.g., the eyebrow, eyelashes, upper lip, lower lip, chin, cheeks, beard area, or mustache area) or neck, or another part of the body, such as, e.g., the chest, breasts, sternum, abdomen, arms, armpits (site of axillary hair), legs, hands, feet, or genitals.
  • a wounded or scarred part of the skin is treated.
  • the wounded or scarred part of the skin is a flexion surface or involves the extremities, breasts, sternum, face, or neck.
  • Wounds treatable by the methods described herein include, but are not limited to, any form of wound known in the art or to be discovered.
  • wounds treatable by the methods described herein include acute wounds (surgical and non-surgical), chronic or non-healing wounds, pressure sores (also referred to as decubitus ulcers or bed sores), pressure necrosis, lower extremity ulcers, radiation injury (such as, e.g., caused by radiation overdose), an erythema, skin abrasion, or a non-healing wound caused by wounding (e.g., a surgical incision) of irradiated skin.
  • the methods described herein are used to enhance healing of wounds caused by blisters, cutaneous trauma, and surgery, such as described in Mulvaney & Harrington, 1994, Chapter 7, "Cutaneous trauma and its treatment," in Textbook of Military Medicine: Military Dermatology, Office of the Surgeon General, Department of the Army, Virtual Naval Hospital Project, which is incorporated by reference herein in its entirety.
  • the methods described herein are used to enhance (e.g., hasten, improve, minimize scarring, etc.) healing of wounds by primary intention.
  • the methods described herein are used to enhance healing of wounds by secondary intention.
  • the methods described herein are used to enhance healing of wounds by tertiary intention.
  • the wound to be treated by the methods described herein has wound dehiscence, which is the premature "bursting" open of a wound along surgical suture.
  • the patient is at risk for wound dehiscence, based on one or more of the following risk factors: age, diabetes, obesity, poor knotting or grabbing of stitches, and trauma to the wound after surgery, or inadequate ability to form scars.
  • the methods described herein are used to treat a radiation scar, acne scar, curettage scar, spread scar, split-thickness scar, flap necrosis, scarring following infection, leg ulcer, burn scar, sternotomy scar, or as treatment to minimize scarring following curettage, following surgical excision, following follicular unit transplantation, or following Cesarean section, as exemplified in the examples of Section 7.
  • the methods described herein are used to enhance healing of transplanted skin at recipient sites ⁇ e.g., skin grafts or hair transplantation, such as long- term frontal hair scalp or eyebrow plugs), so that, for example, the skin blends in with the skin at the recipient site with regard to thickness, pigmentation, hair patterning, etc.
  • a scar that results from skin grafting where the graft edges join the host skin, common in battlefield wounds is treated by the methods described herein. In general any "flap" surgery or "free flap” graft will result in these scars.
  • the methods described herein are used to enhance healing of a split thickness skin graft.
  • the split-thickness donor skin tissue for grafting of wound sites is taken from the scalp, as described in Weyandt, et al, 2009, Dermatol. Surg. 35: 1873- 1879, which is incorporated herein by reference in its entirety.
  • lithium treatment may benefit this process by facilitating the "recipient dominance" phase (that temporally follows "donor dominance”). It is postulated that pulse or intermittent lithium treatment can make skin grafts (even pinch grafts) take on attributes of the recipient site by stimulating "local" tissue stem cells to form site-appropriate follicles.
  • Such an intervention can help not only autologous grafts, but also allogeneic grafts, fetal cell grafts (like placenta stem cell “bandaids”), and also stem cell grafts ⁇ ex vivo expanded
  • Scars treatable by the methods described herein include, but are not limited to, any form of scar known in the art or to be discovered.
  • Non-limiting examples of scars that can be revised or otherwise treated by the methods described herein include scars that form by secondary intention, atrophic scars, hypertrophic scars, keloid scars, hypopigmented scars, hyperpigmented scars, depressed scars (including ice-pick scars), and spread scars.
  • Scars form following a variety of causes including, e.g., cosmetic procedures and skin transplants are not really clinical categories of scars.
  • scars caused by a disease or disorder such as scarring (cicatricial) alopecia, scars caused by excessive wound healing, scars caused by joint contracture, or scars caused by burns or wounds.
  • the methods described herein may also be used to treat wounded skin, or skin that may become wounded, in order to prevent, minimize, or reduce scar formation.
  • the scar is caused by surgery, such as a open heart surgery, joint surgery, face lift, skin graft, or hair transplant, etc.
  • the subject for whom pulse or intermittent lithium treatment is intended is a patient who has scarring (cicatricial) alopecia, a condition of permanent hair loss in which the hair follicle is destroyed by inflammation and replaced with scar tissue.
  • scarring alopecia is moderate to severe.
  • the subject has wounding or scarring caused by, exacerbated by, or associated with medication, such as corticosteroid use, chemotherapy ⁇ e.g., anti-cancer therapy or cytotoxic drugs or other antiproliferative agents), thallium compounds, vitamins ⁇ e.g., vitamin A), retinoids, anti-viral therapy, or psychological therapy.
  • medication such as corticosteroid use, chemotherapy ⁇ e.g., anti-cancer therapy or cytotoxic drugs or other antiproliferative agents), thallium compounds, vitamins ⁇ e.g., vitamin A), retinoids, anti-viral therapy, or psychological therapy.
  • the subject has wounding or scarring caused by, exacerbated by, or associated with radiation (including therapeutic radiation treatment or radiation overdose), trauma (chronic or acute, mild or severe), physical trauma, endocrine dysfunction, surgery (including, for example, face lift, hair transplant, cosmetic surgery, and surgery of flexion surfaces, the extremities, breasts, sternum, and neck), sutures, x-ray atrophy, burning or other wound or injury, stress, aging, an inflammatory disease or condition (acute or chronic), an autoimmune disease or disorder, malnutrition (including, e.g., vitamin or trace metal deficiency, scurvy), anemia, diabetes, obesity, a circulatory disorder, such as, e.g., arterial or venous insufficiency, occlusive vascular disease, microvascular occlusive disease, vasoconstriction, hypovolemia, venous valvular disease, impaired oxygen delivery or tissue perfusion, caused by, e.g., ischemia, hypoxia, stroke,
  • radiation including
  • a human skin xenograft (without skin appendages) can be considered as similar to a scar, and can be wounded and then treated pharmacologically to induce hair follicles and/or monitor revision of the scar.
  • Xenografts can also be combined with inducible genetically modified cells to activate pathways know to form hair follicles.
  • the safety and efficacy of a pulse or intermittent lithium treatment is tested in a full thickness or a split thickness human skin xenograft ⁇ e.g., obtained surgically from scar revisions; from foreskin; or cadaveric), or may be tested in a three-dimensional organotypic human skin culture on SCID mice.
  • Success of a pulse or intermittent lithium treatment can be measured by:
  • any method known in the art may be used to evaluate the safety and efficacy of an intermittent lithium protocol or pulse lithium protocol, or of the combination treatments described in Section 5.4.
  • a human skin xenograft model is used.
  • an intermittent lithium treatment or pulse lithium treatment may be administered with a full thickness excision, laser, inflammatory stimulus, or dermabrasion procedure for integumental perturbation.
  • a synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for enhancing wound healing or scar revision may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone).
  • Another animal model for use in evaluating treatment that may more closely mimic the biology of human skin and hair is a guinea pig model (see, Stenn & Paus, 2001, Physiol. Revs. 81 : 449-494).
  • the methods for evaluating treatment in animals described elsewhere in this section and in the example in Section 16 below may be applied to guinea pigs according to methods known in the art. See also, e.g., Kramer et al, 1990, Dermatol Monatsschr. 176:417-20; and Simon et al, 1987, Ann Plast Surg 19:519-23.
  • Other animal models that may be of use in evaluating the treatments described herein include pig, cat, or stumptailed macaque models.
  • success of a pulse or intermittent lithium treatment can be measured by:
  • hair follicle neogenesis or regeneration • increased proportion of hair follicles in anagen or decreased proportion of follicles in telogen
  • VSS Vancouver Scar Scale
  • the intermittent lithium treatment or pulse lithium treatment improves one of the foregoing measures 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 may be measured after 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or one year or longer after initiation of the intermittent lithium treatment or pulse lithium treatment.
  • a synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment described herein may be measured as an improvement over a control subject receiving only one of the two treatments (i.e., the intermittent lithium treatment or pulse lithium treatment alone or the second treatment alone).
  • Human skin equivalents can be grown and assembled in vitro, with the advantage that they can be grown to theoretically to any size/shape; can be comprised of different types of cells, including keratinocytes (hair follicle derived and non-hair follicle derived), dermal cells (hair follicle derived and non-hair follicle derived), other cell types ⁇ e.g. , mesenchymal stem cells); can contain cells that are genetically modified to include, e.g., markers or "inducible" signaling molecules; provide an unlimited and uniform source of human cells; from normal skin based on histology and marker studies; are generally devoid of skin appendages; and can be wounded and show similar wound healing events as in vivo.
  • keratinocytes hair follicle derived and non-hair follicle derived
  • dermal cells hair follicle derived and non-hair follicle derived
  • other cell types ⁇ e.g. , mesenchymal stem cells
  • the intermittent lithium treatments or pulse lithium treatments may facilitate wound healing and scar revision by:
  • epithelial stem cells located in the epidermis and in the hair follicle, giving rise to keratinocytes that re-epithelialize the wound (see, e.g., Lau et al, 2009, Exp. Dermatol. 18:921-933).
  • the invention is based, in part, on the principle that the lithium ion (Li+) is an inhibitor of the polyphosphoinositide cycle that can reversibly arrest cells in cell cycle.
  • the lithium ion has been shown to cause metaphase arrest that can be reversed by the addition of CaC12 or myo-inositol. (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293).
  • the lithium ion has also been shown to arrest cancer cell lines at certain stages of the cell cycle (see, e.g., Wang JS, 2008, World J. Gastroenterol. 14:3982-3989).
  • the invention is based in part on the inventors' recognition that the lithium ion can be used in a pulse or intermittent treatment regimen to synchronize groups of hair follicle cells or hair follicle stem cells that are in various stages of cell cycle (cycling asynchronously).
  • the lithium ion may cause hair follicle stem cells to stop dividing and more readily differentiate into hair follicles and thereby improve wound healing. Restarting the cell cycle at the termination of a pulse lithium treatment, or during the "holidays" between intermittent lithium treatments should restart cell cycle synchronously.
  • the synchronization phenomenon can be described by analogy to traffic lights: periodically arresting the motion of individual cars generates synchronization because cars pile up behind stop lights. Similarly, by introducing a signal that periodically arrests cell division, synchronization is generated because when the "stop" signal is removed, cells initiate division at the same time.
  • Such synchronization of cell cycles in the hair follicle cells results in relative synchronization of hair follicle cycle stage in groups of follicles that otherwise have a stochastic distribution of stages of follicle cycle (asynchronous follicle cycle).
  • the Follicle Stem Cells that are thought to be involved can be derived from (1) other Follicle Stem Cells (e.g., from the bulge or crypt), (2) from other tissue stem cells, termed "pre-Follicle Stem Cells" (from the interfollicular skin), (3) from bone marrow- derived stem cells ("BMST", such as hematopoietic stem cells), (4) uncommitted epithelial progenitor cells; and/or (5) from mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells.
  • BMST bone marrow- derived stem cells
  • mesenchymal stem cells such as hair follicle dermal sheath cells and adipocyte stem cells.
  • BMST bone marrow derived stem cells
  • their differentiation into Follicle Stem Cells requires intact follicles, whose cells can play the role of "nurse cells” and provide appropriate signals to guide the differentiation of bone marrow derived stem cells into Follicle Stem Cells.
  • Integumental perturbation (by wounding, e.g., during scar revision, or by the induction of inflammation) (1) provides signals for Follicle Stem Cells to divide symmetrically to begin the process of forming new follicles; (2) mobilizes tissue stem cells ("pre-Follicle Stem Cells”) from interfollicular skin to
  • intermittent or pulse lithium treatment organizes the normally asynchronous state of human hair follicle cells in Cell Cycle and human hair follicles in Follicle Cycle into relatively more
  • This protocol is adapted from the IACUC VA protocol. Specifically, 4 week old male SCID mice are obtained from Charles River and allowed to acclimate for at least 1 week. In preparation for surgery, mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia. Full thickness adult human skin (measuring approximately 1.5 cm x 2 cm; removed during surgical procedures from the CHTN, NDRI or cadaver scalp skin from ABS) is sutured into a full thickness skin excision site on the dorsal surface of the mouse. The grafts are bandaged and allowed to heal for at least 5 weeks.
  • mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia.
  • the epidermis of the human skin is removed using a microdermabrasion device to dermabrade as described above.
  • mice may be required to confirm and/or optimize these settings for human scalp xenografts. Additionally, some mice may be required to test the differences between full thickness and split thickness human scalp xenografts. Furthermore, reducing the overall thickness of the human skin may improve the "take" rate of the grafts, which is
  • mice receive vehicle alone or the lithium composition, delivered systemically or topically, or neither vehicle nor lithium composition, for 5 consecutive days, (the lithium composition chosen is the one determined to be most efficacious in the
  • C57BL/6J model with efficacy determined to be increased number and/or size of neogenic hair follicles.
  • One dose of the lithium composition is delivered, using the most efficacious dose as described above, systemically and, in a separate experiment, a dose is delivered topically. Additionally, histology and/or photography is performed daily (until the end of the experiment) following scab detachment in order to monitor hair follicle neogenesis.
  • An additional set of mice are treated with the lithium composition or vehicle or neither, with the exception that the xenografted mice are not wounded, in order to assess the effect of the lithium composition in the absence of wounding.
  • mice At approximately 2 weeks post-scab detachment, all mice are anesthetized with ketamine (80 mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of ⁇ 100 ⁇ , and monitored by toe pinch to determine the surgical plane of anesthesia. Subsequently, they have a terminal blood draw (to detect drug in the plasma), and are euthanized. The wound is then removed, which is trisected with one-third taken for biochemistry, one third for determination of lithium levels in the skin using mass spectrometry, and one third for histology/immunohistochemistry.
  • mice are needed for the optimization of microdermabrasion settings and split thickness versus full thickness xenografts. Considering that the "take" rate of human skin xenografts is approximately 50%, the total number of mice to optimally receive human skin grafts is approximately 500.
  • a female human subject 75 years old, underwent treatment of a basal cell carcinoma of the nose five years prior to presentation.
  • the resulting scar is atrophic, hypo- pigmented and lacking normal pore pattern.
  • the scar is mechanically disrupted by excision, dermatome planing, dermabrasion, laser abrasion, or Fraxel, and treatment with topical lithium gluconate 8% is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 28 years old, presents with extensive, broad, shallow, acne scars.
  • the scars are atrophic, hypo-pigmented and lack normal pore pattern.
  • the scars are dermabraded using a moderate grit diamond fraise and treatment with 8% topical lithium is initiated.
  • Response to treatment is determined by measuring skin thickness of the scar, return of pigmentation and re-establishment of adnexal structures.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 75 years old, underwent excision of a large malignant melanoma of the forehead with subsequent reconstruction using a split-thickness skin graft. After one year, the resulting graft demonstrates depression, skin atrophy, hyper-pigmentation and loss of normal pore pattern.
  • the graft is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, establishment of normal pigmentation and re-establishment of adnexal structures.
  • a female human subject undergoes excision of a large congenital nevus of the cheek. Due to the tension of closure, the incision line spreads and widens over time resulting in a scar that is atrophic, hypo-pigmented and lacking normal pore pattern. The scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a female human subject 65 years old, underwent a face-lift procedure.
  • a portion of the cheek flap subsequently became necrotic and healed by secondary intent.
  • the resulting scar was atrophic, hypo-pigmented and lacked normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with topical 8% lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a female human subject 37 years old, underwent a phenol chemical peel of her lips to decrease rhytides.
  • the patient developed a staphylococcal (herpetic) infection of the treated area.
  • the infected area healed with an atrophic, hypo- pigmented scar that lacked the normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 70 years old, developed non-healing leg ulcers involving the tibial aspects of both legs.
  • the affected area was pre-treated with 8% topical lithium carbonate and subsequently grafted using small pinch grafts harvested from the thighs.
  • Response to treatment is determined by measuring skin thickness and re- establishment of adnexal structures over the grafted area following graft take.
  • a female human subject 40 years old, underwent split thickness grafting to reconstruct a facial defect following excision of a squamous cell carcinoma.
  • the graft donor site healed with a hypo-pigmented scar that lacked the normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a male human subject 30 years old, suffered a burn of the left cheek that healed with a contracted, hypo-pigmented scar that lacked normal pore pattern.
  • the scar is mechanically disrupted by excision, dermabrasion, laser abrasion, or Fraxel, and treatment with 8% topical lithium gluconate is initiated.
  • Response to treatment is determined by measuring skin thickness, return of pigmentation and re-establishment of adnexal structures.
  • a 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 new pigmented nodular pearly lesions of 1 cm on his left (A) and right (B) scapula. Shave biopsies reveal both lesions to be nodular BCCs.
  • Lesion (A) is treated with aggressive 3 pass curettage then application of aluminum chloride.
  • Lesion (B) is treated with aggressive 3 pass curettage then application of aluminum chloride.
  • Lesion B is post-treated with topical 8% lithium gluconate daily for 5 days post-procedure (in addition to routine wound care).
  • the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 76 year old fair skin male with a history of multiple basal cell carcinomas presents with 2 biopsy proven BCC lesions of 1 cm on his left scapula (A) and 1 cm on his right scapula(B). Conservative surgical excision is performed on both lesions .
  • Lesion (A) is treated with then treated with routine wound care.
  • Lesion (B) is treated with pre-treated with 5 days topical 8% lithium gluconate daily prior to surgery followed by application for 5 days post-procedure (in addition to routine wound care).
  • the lesions are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 74 year old black male with history CAD undergoes CABG surgery requiring median sternotomy. Following the procedure the scar is found to measure 14 cm in diameter. Starting Day 1 thru 7 he applies topical 8% lithium gluconate to the 7 cm superior portion of the scar (in addition to routine wound care).
  • the superior and inferior aspects of the sternotomy scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • a 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Strip Harvesting Method" with donor area located at the posterior scalp along the occipital protuberance.
  • Surgery requires a long scar on the posterior scalp measuring 28 cm.
  • Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the 14 cm “left” portion of the scar (in addition to routine wound care).
  • the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
  • a 51 year old white male with history Androgenic Alopecia undergoes Hair Transplant with the "Follicular Unit Extraction" with donor area located at the posterior scalp above and below the occipital protuberance.
  • punch graft were taken from the posterior scalp and left to heal with secondary intention.
  • Starting Day 1 thru 7 topical 8% lithium gluconate is applied only to the "left" portion of the donor area (in addition to routine wound care).
  • the right and left aspects of the scar are compared with respect to skin thickness, return of pigmentation, re-establishment of adnexal structures and global assessment the physician and patient's satisfaction regarding the cosmetic result of the 2 distinct lesions using a Visual Analogue Scale (VAS).
  • VAS Visual Analogue Scale
  • hair that is regenerated in the treated donor area may be used as a source of future, repeated hair transplants in accordance with the foregoing method.
  • VAS Visual Analogue Scale
  • a patient has a 5 cm scar resulting from surgery. Two to three months after surgery, the scar is treated with fractional laser. Half the scar is treated with lithium and the whole scar is treated with laser. In another variation, a patient has two surgical scars, one of which is treated with the lithium and laser combination and the other of which is treated with laser alone.
  • the subject is administered a fractional and non-ablative laser therapy using an Erbium- YAG laser with an emission at 1540-1550 nm (set to 50-70 J/cm 2 , treatment level of 8-10 (density of the "dots"), and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to apply the Lithium gluconate 8% gel to the treated area of the ear for one week. After one week, treatment with lithium gluconate is discontinued and he is evaluated after three weeks.
  • Lithium gluconate 8% gel Lithium gluconate 8% gel
  • the treatments may alternatively be accomplished by applying an ablative laser treatment in place of the non-ablative laser treatment.
  • the application of Lithium gluconate is sterile and, optionally, the treatment area is covered by a bandage.
  • ablative laser treatment may accomplished using an Erbium- YAG laser at 2940 nm or a CO 2 laser at 10,600 nm.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • An Ultrapulse (fractional mode) CO 2 laser is used to disrupt the epidermis and dermis to approximately 100 to 500 ⁇ in depth.
  • the Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring fractional ablation.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • the Ultrapulse is set to deliver up to 350 mJ, up to 52.5 Watts, using pattern size #8, density #4, and fill the square treatment site with up to 5 passes.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • An Ultrapulse CO 2 laser (ablative mode) is used to disrupt the epidermis and dermis to approximately 100 to 500 ⁇ in depth.
  • the Ultrapulse laser produces an effect that is similar to that of dermabrasion yet the disruption produced delivers a greater amount of energy deeper into the skin in a non-scaring ablation that resembles the dermabrasion.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • Ultrapulse is set to deliver up to 500 mJ in 1 msec, 1 Watts, using a spot size of 3 mm at 2 Hz to fill the square treatment site, which may require up to 15 passes.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 are injected to anesthetize the surface of the area to be treated.
  • the ablative erbium laser is set to deliver up to 5 Joules 240 msec in of energy at level 3 so that in up to 15 passes it will produce a disruption up to 500 ⁇ deep.
  • the treated area is a 1.5 cm x 1.5 cm square.
  • the dermabrasion treatments provided in the examples of Sections 7.1-1.7, 7.9, and 7.10 may alternatively be accomplished by applying one of the following dermabrasion treatments.
  • Lidocaine HCL 2% with Epinephrine 1 : 100,000 is injected to anesthetize the surface of the area to be treated.
  • Standard dermabrasion using the Aseptico Econo-Dermabrader from Tiemann and Company, is performed to a depth of approximately 150 ⁇ , that includes removal the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pools of blood in the treated area.
  • Each dermabraded area is a 1.5 cm x 1.5 cm square.
  • a Bell Hand dermabrasion device may be used.
  • Section 11-16 which present mouse studies using dermabrasion, and the protocols for use in humans in the examples of Sections 7, 9 and 10.
  • dermabrasion was carried out using a microdermabrasion device. While dermabrasion in humans may also be carried out using a microdermabrasion device, where sterile conditions are preferential, a dermabrasion device is preferably used.
  • the following example provides a protocol for demonstrating the importance of timing of lithium gluconate treatment for the optimization of follicular neogenesis and wound healing/scar revision after integumental perturbation.
  • patients are treated with a pulse lithium of 8% lithium gluconate (topical gel) in combination with dermabrasion.
  • patients may be treated with the intermittent lithium treatment or a pulse lithium treatment alone (as described infra), dermabrasion alone (or with a vehicle, e.g., petrolatum), or may not receive any treatment.
  • any patient population may be treated, preferred patients are Caucasian males 20-50 years of age. Patients for whom treatment may be contraindicated (particularly at the clinical trial stage) are those who are currently participating in or have participated in any clinical study with an investigational drug within the thirty (30) days immediately preceding treatment, with current or recent use ( ⁇ 1 y) of isotretinoin (Accutane), currently taking hormone therapy, or steroids or other immunomodulators or have taken these medications within the past thirty (30) days (inhaled steroids are acceptable), currently using Rogaine or Propecia or used them in the past forty- five (45) days, immune compromised or undergoing therapy to treat an immune disorder, have a clinically significant medical condition that may interfere with the protocol described herein, have other active skin diseases (such as actinic keratosis or psoriasis) or skin infections (bacterial, fungal or viral, esp.
  • other active skin diseases such as actinic keratosis or psoriasis
  • skin infections bacterial, fungal or viral
  • HSV infection in the area to be treated, have a history of keloids or hypertrophic scarring, hypersensitivity to lidocaine, poor wound healing, diabetes, or coagulopathy, undergoing current drug or alcohol abuse, psychiatric dysfunction, or other factors that would limit compliance, have sunburned skin, or who are currently taking anti-platelet agents other than aspirin.
  • Dermabrasion using alumina particles is performed on Day 0. Dermabrasion is performed to a depth of approximately 100 ⁇ , which includes removal of the entire epidermis and disruption of the papillary dermis (detectable by a shiny, whitish appearance) inducing the formation of small pinpoints of blood in the treated area. Dermabrasion is performed in two sites of the skin. The area is then allowed to heal without manipulation. A 4 mm punch biopsy is performed on days 1 1 and 14, and the presence of new hair follicles is examined in these subjects based on histological assessment. A third biopsy is optionally performed on Day 14 on an untreated area 1 cm away from the treated area to serve as histologic control.
  • the protocol may be amended in accordance with the findings. For example, if dermabrasion causes presence of neogenic hairs in a 4 mm punch biopsy in, for example, at least three of the first 15 patients, then additional patients will be treated.
  • the procedure begins with shaving/clipping of the existing hair in the area to be treated followed by a thorough cleaning with antiseptic cleansing agent.
  • Numbing agents such as lidocaine HCL 2% and Epinephrine 1 : 100,000, are injected to anesthetize the surface to be treated.
  • Standard dermabrasion is performed to a depth of approximately 100 ⁇ , which includes removal the entire epidermis and disruption of the papillary dermis
  • Each dermabraded area is approximately a 1.5 cm x 1.5 cm square.
  • Suitable dermabrasion devices are the ASEPTICO ECONO-DERMABRADER from
  • sterilized sandpaper may be used for dermabrasion.
  • Adhesive ocular shields are worn by the patient during the procedure to avoid complications due to aluminum crystals entering the eye (chemosis, photofobia, punctuate keratitis) and the doctor should wear safety goggles.
  • the dermabrasion tool is carefully maneuvered over the area to carefully remove layers of skin until the desired level is reached. The procedure usually takes only a few minutes.
  • Pre-dermabrasion patients should be asked to: not wear contact lenses during the procedure, discontinue use of over the counter exfoliation products such as Retinol, Glycolic or other hydroxy acids, Salicylic acid, Beta hydroxyl acids 3 days prior to treatment, discontinue use of retinoids 30 days prior to treatment, not receive Botox or collagen injections for 2 weeks prior to treatment.
  • over the counter exfoliation products such as Retinol, Glycolic or other hydroxy acids, Salicylic acid, Beta hydroxyl acids 3 days prior to treatment, discontinue use of retinoids 30 days prior to treatment, not receive Botox or collagen injections for 2 weeks prior to treatment.
  • the treated skin will be red, swollen and tender, and the wound should be cared for as follows until new skin starts to grow; this usually takes 7-10 days: 1) Keep the area clean and dry for today.
  • the area should either be covered with Vaseline and bandaged after or covered with duoderm or a similar covering. Alternatively, it may be preferable to not cover, bandage, or otherwise manipulate the treated area; 2) Avoid touching the area when washing hair; 3) Pat the area dry. Do not cover, bandage, or otherwise manipulate the treated area.
  • the treated are may itch as the new skin grows and may be slightly swollen, sensitive, and bright pink for several weeks after dermabrasion.
  • the procedure begins with thoroughly cleaning the area to be biopsied with antiseptic cleansing agent.
  • Lidocaine HCL 2% and Epinephrine 1 : 100,000 (approximately 0.5 cc to each site) are injected to anesthetize the site that will be biopsied.
  • 4 mm punch biopsy is performed. Biopsied site is closed with 2 4.0 Ethilon sutures. Vaseline and band-aid are applied. Tissue samples are stored in formalin for histological analysis.
  • This example provides a protocol for characterizing and comparing the percutaneous absorption pharmacokinetics of four formulations containing a lithium salt, in human cadaver skin, using the in vitro skin finite dose model.
  • This model is a well- established tool for the study of percutaneous absorption and the determination of the pharmacokinetics of topically applied drugs.
  • the model uses human cadaver skin mounted in specially designed diffusion chambers allowing the skin to be maintained at a temperature and humidity that match typical in vivo conditions.
  • a dose (e.g., 0.1 gram) of formulation is applied to the top of the partial thickness skin or dermis and drug absorption is measured by monitoring its rate of appearance in the reservoir solution bathing the other surface of the skin.
  • compositions of the formulations are provided in Table 2 below.
  • the formulations were tested initially for stability in solution at 4 °C, 25 °C and 40 °C. All formulations were stable solutions or emulsions at the temperatures tested.
  • the excipients selected for the formulations were based on levels approved for topical drug formulations and each excipient was selected for its viscosity-enhancing properties or its ability to enhance permeation through tissues. Methylparaben was added to the formulations for its preservative activity.
  • hydrogel Carbopol 980 1.5%
  • Dermal tissue was prepared by heating the full thickness skin at 40 °C for 20 minutes in de- ionized water and removing the epidermis using sterile forceps. All cells were mounted in a diffusion apparatus in which the dermal bathing solution was stirred magnetically at approximately 600 RPM and its skin surface temperature maintained at 32.0° ⁇ 1.0 °C.
  • PBS Human Receptor Solution
  • the formulation BX is a neutral hydrogel, with its gel-like consistency produced by the presence of high molecular weight hydroxyethyl cellulose (HEC).
  • HEC high molecular weight hydroxyethyl cellulose
  • the diffusion of lithium through the hydrogel and through the dermis is slower than 35A', with 80% released in approximately 8 hours.
  • Use of an anionic hydrogel (formulation BV-001-003A) slowed down release even further, with 80% released in 12 hours. It is possible that complexation of lithium ions with the anionic polymer Carbopol 980 slows down the release of lithium ions from the hydrogel.
  • This example provides an assessment of the rate of permeation and residence time of lithium ions provided in various formulations in an in vivo mouse model developed for follicle neogenesis. Based on the data, appropriate formulations are selected for an in vivo mouse experiment to assess neogenesis. Formulations that have an adequate rate of permeation through the dermis and longest residence time are selected as formulations to enroll in an in vivo model for neogenesis. It is postulated that lithium ions can induce differentiation of stem cells into neogenic hair follicles.
  • Formulations selected in this experiment were : 35A', 35BX and BV-001-003A with their respective compositions as shown in Table 2 supra.
  • mice 24 C57/BL 6 mice were enrolled in each group. There were 6 groups in total, with 3 groups enrolled for dermabrasion (DA) and 3 for FTE treated skin. A different formulation was enrolled in each of the three groups for DA and FTE.
  • DA dermabrasion
  • FTE FTE
  • Dosing for the DA groups was started at day 0, immediately after debriding the mouse skin with dermabrasion, and continued to day 5. Scab formation on the wound occurs approximately at day 1 and thus the formulations are delivered on top of scabbed wounds.
  • Dosing for the FTE groups was started at approximately day 7, or when the scab detached from the wound. The formulations were delivered to the re-epithelialized skin for five days.
  • Each wound was dosed with a formulation volume of 0.1 ml, or 0.1 g since the density of each formulation was determined to be approximately 1 g/ml. Dosing was accomplished with a 100 microliter Wiretrol device. Post-dosing, the wound was covered with a non-stick Tegaderm bandage.
  • Blood levels were an order of magnitude lower than in skin, possibly because the formulation used, in which the Li ion is complexed with CarboPol 980 to form a polymer, enhances its residence in the skin, in contrast to Li ion in, for example, saline, which is expected to be highly water soluble.
  • Dermabrasion by any other means such as full-thickness or partial-thickness excision, micro-needle roller perturbation, laser fractional, non-fractional or ablative, are alternate means of integumental perturbation, prior to administration of lithium.
  • the skin and the corresponding plasma concentrations of Li ions were determined following subcutaneous administration of lithium chloride at increasing dose concentrations.
  • This protocol can also be adapted to determine follicular neogenesis as a function of increasing dose concentrations of lithium.
  • mice were treated with either DA or FTE or unwounded (see Table 3 below), and dosed subcutaneously with 0.1 ml of a formulation containing increasing concentrations of lithium chloride in isotonic saline.
  • DA mice received 42 mg/kg, 127 mg/kg, or 381 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5 th day.
  • Lithium treatment of FTE mice started the day of scab detachment (at day 10-11). FTE mice received 64 mg/kg, 150 mg/kg, or 240 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5 th day.
  • mice were sacrificed, and the entire wound area of skin was analyzed for Li concentration and blood was drawn and centrifuged into red blood cells (RBC) and plasma. Then, at the 21 st day, a section of skin was biopsied and analyzed for Li concentration, along with the corresponding plasma and RBC concentrations.
  • RBC red blood cells
  • mice that received FTE treatment as a mode of wounding were dosed on the day of scab detachment (e.g., on day 10-15 post-wounding) with a single dose of lithium chloride for 5 days.
  • the mice were sacrificed and the wound was biopsied and analyzed for Li concentration.
  • blood was drawn and centrifuged into RBC and plasma and assayed for lithium levels.
  • Lithium concentrations were measured by the validated bioanalytical ICP method provided below.
  • LOQ for Li in the assay 50 mM.
  • This method was developed to quantify lithium in murine skin, plasma and pellet of red blood cells (RBC).
  • RBC red blood cells
  • Known quantities of lithium were added to matrices collected from control mice that were not exposed to any lithium as part of preclinical testing. Processing of samples is described below and involved digestion with hot nitric acid to reduce interference by organic matter and to convert particulate-associated metals to a form that could be measured by ICP/MS. Acid digests were cooled and then filtered prior to injection into the ICP /MS.
  • the calibration process involved using lithium standards that were dissolved in purified water.
  • the primary lithium standard was diluted to make a set of lithium working standards.
  • One set of these working standards was used to generate the calibration curve.
  • a separate set was used to prepare the QC samples described in the section entitled "Accuracy.”
  • the method was validated for all three murine matrices.
  • the final calibration curve covered the concentration range from 0.05-50 ⁇ g/L.
  • Igepal-70 is a non-ionic detergent and was added to dissociate erythrocyte membranes.
  • the solution was triturated with a pipettor and then transferred to a microwave vessel. The mass of transferred solution was measured before adding known amounts of lithium working standard and 1.0 mL concentrated nitric acid. Mixture was heated in a microwave for approximately fifteen minutes. After digestion was complete, the digest was cooled and purified water was added to achieve a total volume of 25 mL. Once the solution was mixed and passed through a 0.45- ⁇ nylon filter, it was ready for injection into the ICP/MS.
  • LLOQ Precision at Lower Limit of Quantitation
  • Precision was measured as inter-injection variability, by analyzing six separate injections of the same QC sample, removed from the same vial. Analyte concentration for this test was near the mid-point of the calibration range. Results are presented in Table 8. In terms of %CV, precision was less than 1% for all matrices.
  • Precision was based on six replicate injections of the QC standard, the concentration of which corresponds to the mid-point of the calibration range.
  • the %CV for plasma was 0.642 for skin 0.631 and for RBC 0.540. The results are presented in Table 8.
  • Li concentrations in skin and blood increased in a dose-related fashion.
  • the concentrations of Li in RBC were negligible (data not shown).
  • Li concentrations in skin at trough were 0.0001-0.0009 mM Li.
  • Li concentrations in blood at peak (1 hr post dosing on day 5) were between 0.695 mM - 1.059 mM Li (see Figures 16 and 17), and trough concentrations in blood were 0.02-0.09 mM Li.
  • the data show that when lithium chloride is delivered subcutaneously, lithium ions extract to the skin and the plasma in a linear dose-related fashion, although plasma concentrations were many-fold higher than in skin.
  • the lithium concentration in skin at 5 days correlates in a linear dose-related fashion, with no difference observed between wounded skin and non-wounded skin (see Figure 14B).
  • the skin samples were obtained 1 hour post-dosing, in other words at "peak" skin concentrations.
  • the data demonstrate that lithium does distribute to the skin, where it may play a role in stem cell modulation toward differentiation into de novo hair follicles.
  • the purpose of the experiment in this example was to evaluate the absorption of Li ions into the skin and blood compartments in an in vivo mouse model developed for follicle neogenesis, with once/day topical administration of two lithium formulations, a Lithium Gluconate Hydrogel and a Lithium Chloride Hydrogel (see Table 10 below).
  • This example provides a protocol for and assessment of the rate of permeation and residence time of lithium ions provided in the formulations, which can be adapted for an in vivo experiment to assess HF neogenesis.
  • Hydrogel "HC730”; also referred to Hydrogel, "BV-001-003A”; also
  • lithium gluconate referred to herein as “lithium chloride”
  • CarboPol 980 CarboPol 980
  • mice Twenty-four (24) C57/BL 6 mice were enrolled in each group. There were 4 groups in total, with 2 groups enrolled for DA and 2 for FTE treatment. Dosing for the DA groups was started at day 1 , immediately after debriding the mouse skin with dermabrasion, and continued once daily to day 4 (i.e., single dose administered at Oh, 24 h, 48 h, 72 h). A thick scab forms on the wound after day 1 , and thus the formulations are delivered prior to scabbing of the wounds.
  • Dosing for the FTE groups was started at approximately day 10-15 post-FTE wound, when the scab detached from the wound (this is referred to herein as "Day 1"), and continued to day 4 post-scab detachment (i.e., single dose administered at Oh, 24 h, 48 h, 72 h post scab detachment).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Medicinal Chemistry (AREA)
  • Inorganic Chemistry (AREA)
  • Dermatology (AREA)
  • Birds (AREA)
  • Engineering & Computer Science (AREA)
  • Emergency Medicine (AREA)
  • Materials Engineering (AREA)
  • Hematology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne les traitements par intermittence à base de lithium, ou un traitement par administration unique à base de lithium pour la révision des cicatrices et la cicatrisation des plaies chez l'homme. L'invention concerne des compositions contenant des composés qui libèrent des ions lithium, y compris des adjuvants et des dispositifs prévus pour cette administration. Le protocole de traitement par intermittence implique plusieurs cures de traitement à base de lithium interrompues par des « pauses » thérapeutiques. Le protocole par administration unique consiste à administrer une dose de lithium sur une courte durée. Le ou les traitements à base de lithium peuvent être utilisés en association avec d'autres traitements pour la révision des cicatrices, la cicatrisation des plaies et la néoformation des follicules pileux. De telles polythérapies peuvent impliquer des traitements mécaniques ou physiques qui modulent la révision des cicatrices ou la cicatrisation des plaies, ou qui entraînent la perturbation des téguments, et/ou des traitements chimiques qui modulent la cicatrisation des plaies, la révision des cicatrices ou la néoformation des follicules pileux ou qui entraînent la perturbation des téguments ou la stimulation immunitaire pour le traitement des cicatrices ou la révision des cicatrices.
PCT/US2010/048439 2009-09-11 2010-09-10 Traitements par intermittence et par administration unique, à base de lithium pour la révision des cicatrices et la cicatrisation des plaies WO2011031977A1 (fr)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US24185709P 2009-09-11 2009-09-11
US61/241,857 2009-09-11
US33025010P 2010-04-30 2010-04-30
US61/330,250 2010-04-30
US35653410P 2010-06-18 2010-06-18
US35653110P 2010-06-18 2010-06-18
US61/356,531 2010-06-18
US61/356,534 2010-06-18

Publications (1)

Publication Number Publication Date
WO2011031977A1 true WO2011031977A1 (fr) 2011-03-17

Family

ID=43732814

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/048439 WO2011031977A1 (fr) 2009-09-11 2010-09-10 Traitements par intermittence et par administration unique, à base de lithium pour la révision des cicatrices et la cicatrisation des plaies

Country Status (1)

Country Link
WO (1) WO2011031977A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3551111A4 (fr) * 2016-12-07 2020-06-24 Sciton, Inc. Traitement laser de plaies
US20210322785A1 (en) * 2018-12-10 2021-10-21 C.P. Medical Corporation Compositions and methods for treating wounds
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020013298A1 (en) * 1996-12-02 2002-01-31 William L. Hunter Compositions and methods for treating or preventing inflammatory diseases
US20070002497A1 (en) * 2001-10-30 2007-01-04 Toshihiko Shimizu Carriage arm assembly for locating magnetic head, and magnetic disk apparatus using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020013298A1 (en) * 1996-12-02 2002-01-31 William L. Hunter Compositions and methods for treating or preventing inflammatory diseases
US20070002497A1 (en) * 2001-10-30 2007-01-04 Toshihiko Shimizu Carriage arm assembly for locating magnetic head, and magnetic disk apparatus using the same

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11207511B2 (en) 2010-12-06 2021-12-28 Follica, Inc. Methods for treating baldness and promoting hair growth
EP3551111A4 (fr) * 2016-12-07 2020-06-24 Sciton, Inc. Traitement laser de plaies
US20210322785A1 (en) * 2018-12-10 2021-10-21 C.P. Medical Corporation Compositions and methods for treating wounds

Similar Documents

Publication Publication Date Title
US11207511B2 (en) Methods for treating baldness and promoting hair growth
WO2012065065A1 (fr) Procédés et compositions pour moduler la pousse de cheveux, la cicatrisation de plaie et la révision de cicatrice
JP7150120B2 (ja) 侵襲的且つ非侵襲的な処置上のスキンケアのための組成物及び方法
Fabbrocini et al. Acne scars: pathogenesis, classification and treatment
KR20120113211A (ko) 모발 성장을 조절하기 위한 간헐 리튬 치료 및 펄스 리튬 치료
Lee et al. Erbium–yttrium–aluminum–garnet laser irradiation ameliorates skin permeation and follicular delivery of antialopecia drugs
WO2016154017A1 (fr) Efficacité améliorée et administration topique ciblée de médicaments sur la peau avec des nanoparticules
Camacho Medium‐depth and deep chemical peels
WO2012125941A1 (fr) Traitement au lithium pour infections microbiennes
WO2004100923A1 (fr) Composition a usage externe administree par voie percutanee
JP2004059482A (ja) 組織再生促進剤
WO2011031977A1 (fr) Traitements par intermittence et par administration unique, à base de lithium pour la révision des cicatrices et la cicatrisation des plaies
Kalil et al. Treatment of acne scars using the microneedling and drug delivery technique
US20200289441A1 (en) Methods for promoting hair growth using valproic acid
Simao et al. Combining superficial chemical peels
RU2702347C2 (ru) Лечение или профилактика себорейного кератоза с применением артемизинина и его производных
Tenenbaum et al. Cosmetic Dermatology
Bajaj The Seboblastic Propagation: Sebaceous Hyperplasia
Skroza et al. Mechanic Resurfacing, Needling, Dermoabrasion and Microdermoabrasion
Roenigk et al. Benign Soft Tissue Tumors/Richard M. Rubenstein and Kevin Spohr
Evans et al. Lasers, intense pulsed light and photodynamic therapy
MX2007006059A (es) Composicion de crema desmanchadora de piel.
Johnson et al. Facial Resurfacing: Dermabrasion, Chemical Peel, and Laser Ablation
Ghersetich et al. Pyruvic acid

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10816160

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 10816160

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE