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Title:
INTERMITTENT AND PULSE LITHIUM TREATMENTS FOR MODULATING HAIR GROWTH
Kind Code:
A1
Abstract:
The invention relates to intermittent lithium treatments, or a single pulse lithium treatment for modulating hair growth 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." For the single pulse protocol, 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 the enhancement or inhibition of hair growth. Such combination treatments may involve mechanical or physical treatments that cause integumental perturbation (e.g. such as electrology (electrolysis), laser, intense pulsed light, dermabrasion, etc.); immune stimulation (e.g., such as adjuvants, antigens, cytokines, growth factors, etc. ); and/or chemical treatments (e.g., that cause integumental perturbation or that enhance or inhibit hair growth); and/or surgical treatments (e.g., hair transplant) for the enhancement or inhibition of hair growth. 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.


Inventors:
JU, William, D. (435 Bernardsville Road, Mendham, NJ, 07945, US)
PROUTY, Stephen, M. (236 Windsor Way, Doylestown, PA, 18901, US)
BARMAN, Shikha, P. (2 Bonnievale Ave, Bedford, MA, 01730, US)
KELLOGG, Scott, C. (PO Box 1773, Mattapoisett, MA, 02739, US)
SCHWEIGER, Eric (166 East 34th Street, #10BNew York, NY, 10016, US)
LEDERMAN, Seth (200 E. 72nd Street - 35K, New York, NY, 10021, US)
Application Number:
US2010/048457
Publication Date:
03/17/2011
Filing Date:
09/10/2010
Assignee:
FOLLICA, INC. (222 Berkeley Street, Suite 1040 Rm. 302, Boston MA, 02116, US)
JU, William, D. (435 Bernardsville Road, Mendham, NJ, 07945, US)
PROUTY, Stephen, M. (236 Windsor Way, Doylestown, PA, 18901, US)
BARMAN, Shikha, P. (2 Bonnievale Ave, Bedford, MA, 01730, US)
KELLOGG, Scott, C. (PO Box 1773, Mattapoisett, MA, 02739, US)
SCHWEIGER, Eric (166 East 34th Street, #10BNew York, NY, 10016, US)
LEDERMAN, Seth (200 E. 72nd Street - 35K, New York, NY, 10021, US)
International Classes:
A61K8/65
View Patent Images:
Attorney, Agent or Firm:
CORUZZI, Laura, A. et al. (Jones Day, 222 East 41st StreetNew York, NY, 10017-6702, US)
Claims:
WHAT IS CLAIMED IS:

1. A method for stimulating hair growth in a human subject, comprising administering to a human subject in need thereof a treatment of (i) integumental perturbation by fractional laser: (ii) followed by a pulse of a lithium composition that delivers an effective amount of lithium ions; (iii) followed by minoxidil and finasteride, wherein the treatment stimulates hair growth.

Description:
INTERMITTENT AND PULSE LITHIUM TREATMENTS

FOR MODULATING HAIR GROWTH

[0001] This application claims priority to U.S. provisional application Serial No.

61/241.857, filed September 1 1 , 2009, U.S. provisional application Serial No. 61/330,250. filed April 30, 2010, and U.S. provisional application Serial No. 61/356,534, filed June 18, 2010, the entire contents of each of which is incorporated herein by reference in its entirety.

1. INTRODUCTION

[0002] The invention relates to intermittent lithium treatments, or a single pulse lithium treatment for modulating hair growth 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." For the single pulse protocol, 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 the enhancement or inhibition of hair growth. Such combination treatments may involve mechanical or physical treatments that cause integumental perturbation (e.g. such as electrology (electrolysis), laser, intense pulsed light, dermabrasion, etc.); immune stimulation (e.g., such as adjuvants, antigens, cytokines, growth factors, etc. ); and/or chemical treatments (e.g., that cause integumental perturbation or that enhance or inhibit hair growth); and/or surgical treatments (e.g., hair transplant) for the enhancement or inhibition of hair growth. 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.

2. BACKGROUND

[0003] 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. 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). Essentially, only the glaborous skin on palmar and plantar aspects of hands and feet, respectively, and the lips and labia lack hair follicles. 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. Accordingly, both the presence and absence of visible hair on human non-glaborous skin is mediated by regulation of activity of specialized follicles.

[0004| 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). In addition to the cells and structures associated with making and anchoring the hair shaft, the vast majority of hair follicles contain units called sebaceous glands (which produce sebum). Some hair follicles have apocrine glands attached to them, and are located in the axilla and other specific areas of the body.

[0005] In addition to the hair shaft, 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). (Stenn & Paus, 2001 , Physiol. Revs. 81 : 449-494.) (See also Figures 2-4.)

[0006J Scalp and certain other hair in humans tend to grow in follicular units. A follicular unit of scalp hair is typically composed of one to four terminal hair follicles; one to two vellus hair follicles; their associated sebaceous glands, neurovascular plexus, an erector pilorum muscle and a circumferential band of adventitial collagen, termed the

"perifolliculum" (Headington JT, 1 84, Arch. Dermatol. 120:449-456; Bernstein RM. 2005, "Follicular Unit Hair Transplantation," Ch. 34 in Surgery of the Skin, Robinson et ai, eds., St. Louis: Mosby, pp. 549-574).

[0007] Hair follicles are believed to produce approximately 20 individual hair shafts over the life of the follicle as the follicle progresses through cycles of hair production, shedding (ejection), involution and new growth. The regulation of hair growth and follicle

regeneration have been investigated in murine systems. However, the biology of hair follicles in the mouse is different from those of the human in several important aspects. In the mouse, a thick fur coating is essential to healthy life (because hair plays roles in thermoregulation and other functions.) Mouse skin is covered with hair follicles that produce terminal hair (fur), whereas significant regions of human skin are covered with hair follicles that produce vellus hair, which is invisible. Mouse and other non-primate mammals have synchronous Follicle Cycles, whereas human follicles progress through the Follicle Cycle in an asynchronous fashion. While the mouse has certain specialized follicles (e.g., whiskers, guard, awl, auchene, and zigzag hair), mouse follicles are generally not subject to developmental and gender-specific hair patterning. In contrast, a significant number of human follicles are individual participants in choreographed hair patterning that affects the type, length and color of shaft produced at different times in development and aging and in a gender specific manner.

2.1 HAIR FOLLICLE MORPHOGENESIS AND REGENERATION

[0008] It is believed that follicle formation occurs but once in a lifetime (in utero), so that a mammal, and particularly a human, is born with a fixed number of follicles, which does not normally increase thereafter. It has been proposed, however, that follicle neogenesis can be associated with wound healing in animals (e.g., rabbits, mice). See, Stenn & Paus, 2001 , Physiol. Revs. 81 :449-494. Nevertheless, 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).

[0009] In a mouse study, Cotsarelis showed that physically disrupting the skin and existing follicles, in a defined fashion, can lead to follicle neogenesis (Ito et ai, 2007, Nature 447:316-321). Despite earlier suggestions of the regenerative capacity of the adult mammalian skin to recreate the embryonic follicle, follicle neogenesis was never proven because of an incomplete understanding of the fundamental biology of the follicle and the lack of tools needed to demonstrate the occurrence or hair follicle neogenesis (see, Argyris et al . 1959, Dev. Biol. 1 : 269-80; Miller, 1973, J. Invest. Dermatol. 58: 1-9; and ligman, 1959, Ann NY Acad Sci 83: 507-51 1 ). More recently, a series of murine experiments definitively showed that hair follicle-derived epithelial stem cell progenitors migrate out of the follicle and contribute to the re-epithelialization of injured skin (see, Morris et al, 2004, Nature Biotechnology 22:41 1 -417; Ito et ai, 2004, Differentiation 72:548-57: and Ito et ai , 2005, Nature Medicine 1 1 : 1351-1354.)

[0010] Some of the molecular signals involved in the regulation of follicle regeneration and hair growth have been investigated in murine systems. Based on experiments performed on murine cells in culture, Morgan implicated a role for Wnt signaling to the dermal papillae (DP; which also may be referred to as "follicular papillae") (e.g., resulting from β-catenin accumulation in the epidermis) in the coordination of hair follicle development (Kishimoto et al. , 2000, Genes & Dev. 14: 1 181 - 1 185; see also U.S. Patent Application Publication No. US 2006/0134074, which postulates a role for Wnt produced in three-dimensional tissue cultures in vitro in promoting hair growth). Previous reports were conflicting. One group, consistent with Morgan's theory, showed that expression of stabilized β-catenin in the epidermis of transgenic mice resulted in hair follicle morphogenesis, but unfortunately led to the development of hair follicle tumors (Gat et al. , 1998, Cell 95:605-614). Another group found that continuous activation of a hyperstabilized and highly amplified (12-21 copies) form of β- catenin in mice also resulted in hair follicle tumors, but that transient activation of this hyperstabilized and highly amplified form of β-catenin led to normal hair follicle patterning, although no hair growth was observed in mice harboring a single copy of the hyperstabilized form of β-catenin (Lo Cclso et al. , 2004, Development 131 : 1787- 1799). These results are in contrast to the results of another study, in which a reduction of β-catenin signaling was found to cause hair follicles to develop into cysts in postnatal mice (Niemann et al., 2002,

Development. 129:95-109). Moreover, there are examples in which increased β-catenin was found to decrease hair growth and hair follicle formation. For example, one group showed that forced expression of β-catenin dependent Wnt 3a in murine skin decreased hair growth (Millar et al, 1999, Dev. Biol. 207: 133-149). In another study, continuous expression of β- catenin during embryogenesis was found to induce placodes, but they became aborted and did not produce a hair shaft (Narhi et al.,. 2008, Development. 135: 1019-1028). In yet another study, increased β-catenin expression during embryogenesis was found to alter cell fate toward formation of hair follicles, but continued presence of β-catenin prevented further development of the hair follicles (Zhang et al , 2008, Development. 135 :2161-2172). Finally, overexpression of Lef-1 (a transcription factor in Wnt pathway) was found to lead to abnormal hair follicle growth, as reported in Zhou et al , 1995, Genes Dev. 9:700-713.

[0011] Despite this confusion about a role of β-catenin in hair growth, Morgan speculated that exogenous Wnt could extend the hair cycle and promote hair growth, and further, that "lithium chloride or similar small ions" that inhibit the activ ity of glycogen synthase kinase - 3β (ΰ8Κ-3β) may induce Wnt signal transduction (through the accumulation of β-catenin), and thus, may be used to promote hair growth (US Patent 6,924, 141 ; US Patent 7, 175,842; and US Patent Application Publication 2008/0286261 ).

[0012] Fathke's experiments tested this theory. In animal studies designed to explore the role of Wnt, Fathke showed that prolonged activation of β-catenin dependent Wnt signaling (i.e.. using lithium chloride as suggested by Morgan) during wound healing in mice resulted in generation of rudiments of hair follicles but did not result in the formation of hair follicles or growth of more hair. (Fathke et al, 2006, BMC Cell Biol. 7:4). In this regard, it is of interest to note that lithium treatment in human subjects is associated with pathological hair loss (for a review, see Mercke et al, 2000, Ann. Clin. Psych. 12:35-42). Fathke turned the focus of his investigation, instead, to the -catenin-independent Wnts expressed in the skin - which are not activated by lithium chloride. Unfortunately, the prolonged expression of the β-catcnin independent Wnt yielded the same results - hair follicles were not generated in the mice.

[0013) As noted by Fathke, cutaneous repair in adult mammals following full thickness wound is understood to result in scar tissue and the loss of the regenerative capability of the hair follicle. However, Cotsarelis showed, in mice, that following wound closure of large healed wounds created by full thickness excision (FTE) ( I cm2 square wounds) new hairs are formed at the center of the wound (Ito et al. , 2007, Nature 447:316-321 ). Here again the role of Wnt was examined. In these experiments, inhibition of Wnt signaling at the time of wounding decreased the number of new hairs formed in the healed wound. While increasing Wnt expression increased the number of new hairs formed in the healed wound, Cotsarelis noted that "the hair follicle dermal papilla of these mice contain -25-38% more cells compared with normal. It is possible that the larger number of dermal papilla cells contributes to the greater number of hair follicles that form after wounding" (Ito et al. 2007, Nature 447:316-321 , full Methods, available at vvww.nature.com/nature). This suggests that these Wnt-overexpressing mice were already predisposed to growing more hair. Thus, Cotsarelis' recognition that "... to date there has been no evidence that extracellular Wnt ligands can promote actual hair follicle neogenesis in adult skin" (Ito et al. 2007, Nature 447:316-321 , at p. 319) remains correct.

[0014] A recent study reported that androgen stimulated DP cells from males with male pattern hair loss (MPHL) (but not DP control cells) inhibit Wnt3a-induced keratinocyte proliferation. Androgens also inhibited Wnt signaling in MPHL DP cells, but not control DP cells. These findings suggest that Wnt agonists would not be able to reverse androgen induced hair loss, since MPHL alopecia DP cells inhibit potent Wnt agonists (Kitagawa et al, 2009, J. Clin. Endocrinol. Metab. 94: 1288-1294).

[0015] Thus, while Wnt signaling has generated interest, its role in hair follicle stem cell and follicle development and hair growth is, at best, unclear.

2.2 THE HAIR FOLLICLE CYCLE

[0016] Hair growth in each follicle occurs in a cycle that includes the following principal phases: anagen (growth phase), catagen (involuting/regressing stage), telogen (the quiescent phase), exogen (shedding phase), and re-entry into anagen (sometimes referred to herein as the "Follicle Cycle"). In addition there is a latent phase, termed kenogen, which is a persistent suspension of growth after a hair follicle has been shed. The Follicle Cycle has been relied on to explain many phenomena associated with hair growth and hair loss.

However, it is an incomplete explanation for the regulation of human hair growth.

2-2.1 ASYNCHRONOUS CYCLING IS UNIQUE TO HUMANS

[0017] In fur-covered terrestrial, non-primate mammals the hair cycle is typically synchronized across large areas of the skin, sometimes in "waves" across the skin and sometimes the entire fur coat sheds in response to environmental or other triggers. See, e.g., Stenn & Paus, 2001. Physiol Rev 81 :449-494.

[0018] Human hair follicles are relatively unique among mammals (and particularly non- primates) since hair follicles in a region of skin are not synchronized. On an adult human scalp, at any particular time approximately 90% are in anagen; 10-14% in telogen and 1 -2% in catagen.

2.2.2 SPECIALIZED FOLLICLES IN HUMANS

[0019] While certain other mammals do have specialized hair follicles (e.g., whiskers and eyelashes on canines), humans are unique in the number and variety of their specialized hair follicles. Hair patterning in humans is mediated by distinct specialized hair follicles that play out their predetermined programs over the life of the human.

[0020] It is believed that humans are born with their complement of hair follicles and that each hair follicle belongs to a class of follicles that accounts for the distinctive length, temporal appearance, regulation by sex hormones, etc., of the hair shaft it produces. The duration of the Follicle Cycle length is believed to be a characteristic of each distinct specialized human hair follicle that accounts for the length of hair produced and other aspects of the follicle function. For example, the anagen stage for eyebrow hair follicles is approximately 4 months and for scalp hair follicles is approximately 3-4 years. It has been proposed that eyebrow hairs have a shorter length compared to scalp hairs because the former have a shorter anagen phase.

[0021 J It also has been proposed that in humans, sex hormones regulate the length of anagen in the Follicle Cycle of certain specialized hair follicles that participate in hair patterning: decreasing anagen in programmed hair loss and increasing anagen in programmed hair growth. 10022] However, while current descriptions of the hair Follicle Cycle arc powerful for explaining features of fur covered terrestrial mammals, certain features of human hair follicle biology remain unexplained.

2.2.3 FOLLICLE CYCLE AND PATHOLOGICAL HAIR LOSS

[0023] The Follicle Cycle has also been used to distinguish two different types of pathologic hair loss (effluvium): Anagen effluvium and Telogen effluvium.

[0024] Anagen is associated with intense mitotic activity; therefore, follicles in anagen arc sensitive to cancer chemotherapeutic agents. Anagen effluvium is believed to be the process in which certain hair follicles undergo hair loss and involution during chemotherapy because these agents typically target cells with high metabolic or mitotic activity (e.g. , 5- fluorouracil, methotrexate, cyclophosphamide, vincristine), in addition to chemotherapy drugs, Anagen effluvium can be caused by other toxins, radiation exposure (e.g. , radiation overdose), endocrine diseases, trauma, pressure, and certain diseases like alopecia areata (an autoimmune disease that attacks anagen follicles.)

[0025] Telogen effluvium is a premature interruption of anagen and early entry of anagen follicles into the Telogen (or resting) phase. The proportion of telogen hairs on the scalp increases to 25-50% and telogen follicles remain in telogen for more than the usual 3-6 months. Telogen effluvium is caused frequently by drugs like lithium and other drugs like valproic acid and carbamazepine and numerous other drugs including the commonly used beta blockers and oral contraceptives. In addition to psychiatric drugs, telogen effluvium can be induced by childbirth, traction, febrile illnesses, surgery, stress, or poor nutrition (see Mercke et al, 2000, Ann. Clin. Psych. 12:35-42).

2.3 HUMAN HAIR PATTERNING IS MEDIATED BY DISTINCT

HAIR FOLLICLE TYPES WITH SPECIFIC FEATURES

[0026] At a microscopic level, human skin is essentially covered with hair follicles. The portions of human skin that lack visible hair contain, for the most part, hair follicles that produce "vellus hair" which is thin and short (i.e., less than 2 cm in length) and often colorless. Certain other hair follicles may contain or produce no hair. 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. Another follicle type is the "sebaceous" follicle, which has a large sebaceous gland and a vellus-like hair shaft localized in the acne-prone areas. Accordingly, both the presence and absence of visible hair on human skin is mediated by regulation of the activity of specialized hair follicles.

[0027| The spatial and temporal aspects of human hair patterning are believed to depend on the localization of specialized hair follicles with unique features during embryogenesis. It is further believed that this complement of hair follicles is maintained throughout life without renewal or replacement. Human fetus follicles may produce lanugo hair during gestation, which is intermediate between vellus and terminal hair in thickness and length and is typically shed by the time of normal birth. By the time of birth, distinct specialized follicle types are positioned in specific areas of the skin where they will each play a programmed role in hair patterning over the life of the human individual, producing various hair types (lanugo, vellus or terminal hair) either constitutively or depending on certain signals, such as sex hormones or other factors (e.g., lanugo hair can reappear in starvation or in eating disorders such as anorexia nervosa and bulimia).

[0028] Gender is associated with specific patterning of human hair. The growth and loss of visible hair in specific areas of the skin, in stereotypical gender dimorphic patterns, are regarded as "Secondary Sexual Characteristics/' This terminology relates "secondary" features such as hair patterning to the genitals and reproductive organs, which are termed "Primary Sexual Characteristics." The distinctive genitals and reproductive organs of males and females acquired during embryonic development undergo further changes in puberty and menopause/andropause. In addition to hair growth and loss, breasts in females are also considered Secondary Sexual Characteristics.

10029] Certain human hair follicles are targeted to specific skin areas and develop specialized characteristics during embryogenesis under the influence of sex hormones such as testosterone and dihydrotestosterone ("androgens") and/or estrogens. Further, certain human hair follicles are driven to change activity by sex hormones during puberty and in

menopause/ andropause .

[0030] The appearance and intensity of secondary sex characteristics can be described as being regulated by ratios of androgens and estrogens, since to a certain extent either of these groups of hormones (androgens and estrogens) can act to induce certain activities or to inhibit the effect of the other group (i.e.. androgens inhibit estrogen effects and estrogens inhibit androgen effects). For example, androgens induce male characteristics and suppress female characteristics while estrogens induce female characteristics and suppress male

characteristics. Male and female, as used herein, refer to the extremes of genetic gender dimorphism and include by reference the various conditions and states that represent a spectrum of male and female features (such as XO syndromes or conditions that result from exogenous sex steroid administration).

[0031 J Specialized human hair follicles have quantitative variation in activity as well as qualitative variation. For example, sex steroids have qualitative effects on hair patterning either in embryogenesis or in adult life or both (e.g., males have beard hair follicles that produce terminal hair after puberty whereas females do not). Males and females also vary in the amount of gender-specific hair patterning (e.g., a higher density of leg hair follicles produce terminal hair on male rather than female legs). Also, individuals of the same gender exhibit quantitative variation. For example, male chest and back hair presents in different individuals as a spectrum from almost hairless to dense hair and from small regions of follicles producing terminal hair to large regions.

[0032] Gender specific human hair patterning highlights the distinct biological programming of specific hair follicles. Distinct hair follicles in relative proximity on the male scalp and face respond to high androgen/estrogen ratios in diametrically opposite ways: high androgen/estrogen ratios induce vellus to terminal hair transformation in male moustache/beard skin (particularly during puberty), but induce terminal to vellus follicle transformation change in male frontal/temporal scalp (progressively post puberty) in MPHL.

[0033] The effects of androgen/estrogen levels on other regions evidences further variations in the biological programming of specific hair follicles. Hair follicles on the occipital scalp are relatively insensitive to high androgen/estrogen ratios (but later, after more prolonged androgen exposure, undergo age-related thinning). Hair follicles in the axillary and pubic regions (anogenital region) appear to be more sensitive to androgen than moustache/beard follicles; since terminal hair in axillac/pubis grows: (a) in females with relatively low levels of androgen; (b) early in male puberty before beard/moustache; and (c) in patients with genetic 5-alpha-Reductase Type II deficiency.

2.3.1 MALE PATTERN HAIR LOSS:

[0034] Male pattern hair loss (MPHL) is a type of "androgenetic alopecia."

Androgenetic alopecia is a genetically-mediated disorder that occurs in approximately 50% of men by the age of 50 years (see review, Stough ct al, 2005). in women, the histological features of the condition are the same as in men, but susceptibility, age at onset, rate of progression and pattern of hair loss differ between genders (Dinh and Sinclair, 2007).

J0035] After puberty, males begin to lose the scalp hair over the vertex, crown and frontal/parietal areas in a relatively characteristic pattern that is a continuum (described by the Hamilton Norwood scale; see Figure 5). The process of hair loss occurs at the level of the hair follicles by "'miniaturization" through which the hair follicle becomes progressively smaller both in depth and circumference, and the hair shaft produced becomes shorter and thinner. The ratio of terminal-to-vellus-like hairs may be reduced from approximately 7: 1 to less than 2: 1 . Miniaturization results in club hair shafts or vcllus hair shafts. The loss of scalp hair in men is known to be a process driven by the androgen, DHT, which can be inhibited and to some extent reversed by finasteride, which inhibits 5-alpha-reductase II (which converts testosterone to DHT). In advanced stages of MPHL, the affected hair follicles on the bald vertex or temples are considered to be atrophied, or perhaps involuted irreparably ("senescent"). The process by which this occurs is not completely understood. One theory holds that androgens change the length of anagen and telogen phases, so that a normal ratio of anagen to telogen ratio of approximately 12: 1 becomes approximately 5: 1 in MPHL. Telogen hairs are more loosely anchored and prone to shedding or being pulled out (for example, by combing or brushing hair). At the end of telogen, a club hair is produced that is a fully keratinized hair. The hair follicles on MPHL affected areas also undergo follicular miniaturization in which a growing proportion of terminal follicles become vcllus follicles. Additionally, androgenetic alopecia is thought to involve the progressive conversion of hair follicle units with 3 or more terminal hairs to follicular units having fewer terminal hairs (e.g., units with 2 terminal hairs progress to units with 1 terminal hair).

[0036| MPHL is associated with specific polymorphisms of the androgen receptor, the EDA2R gene. Men who are genetically deficient in Type II 5-alpha-reductase do not experience MPHL (see Jenkins et al, 1992, J Clin Invest 89:293-300).

[0037] Several lines of investigation have elucidated mechanistic aspects of the sensitivity to androgen of male frontal parietal and coronal hair follicles. Androgen activity may be mediated by a co-factor to the androgen receptor Hic-5/ARA55 (Inui, 2007, J Invest Dermatol 127:2302-2306). Hic-5/ARA55 mRNA expression was high in dermal papilla cells from the beard and bald frontal scalp but low in cells from the occipital scalp. Another androgen receptor coactivator ARA70/ELE 1 had decreased expression of a splice variant form ( ARA70beta/ELE 1 beta) in the dermal papilla of balding recipient areas than non- balding areas (Lee et al, 2005, J Cutan Pathol 32:567-571 ).

2.3.2 FEMALE PATTERN HAIR LOSS

{0038] In addition to the progression of MPHL, both males and females develop diffuse hair loss in the frontal/parietal scalp called "thinning," which begins between 12 and 40 years of age. Collectively, MPHL and diffuse thinning in males and females is termed

"'androgenetic alopecia." Perhaps more than males, females notice (and complain ol) diffuse hair thinning progressively in middle age more than males, perhaps because diffuse alopecia is more noticeable and problematic for females because they do not suffer from MPHL and retain the frontal hairline. In females, thinning is known as "'Female Pattern Hair Loss (FPHL)" and may be caused or exacerbated by androgens (Price, 2003, J. Investig. Dermatol. Symp. Proc. 8:24-27).

[0039] Mechanistically, FPHL is thought to share some features with MPHL in terms of progressive reduction in the duration of anagen and progressive follicular miniaturization, although recent studies have found a prolongation of kenogen.

2.3.3 DONOR DOMINANCE

|0040J The unique features of specialized human hair follicles continue to show the characteristics of the donor site when skin, hair follicles, or hair follicle units are

transplanted, which has been refen-ed to as "donor dominance" (Orentreich N, 1959, Ann NY Acad Sci. 83:463-479). This principle is evidenced by the results of the commonly performed procedure of transplanting scalp hair (skin, follicles or follicle units) in males from areas that are not subject to androgen-triggered, MPHL (e.g. occipital scalp) to areas in which specialized follicles have begun producing vellus hair or have stopped producing hair under the influence of androgens (e.g. frontal/temporal; crown or vertex scalp. The transplanted follicles retain the programmed terminal hair producing features from their original location. However, more recent studies suggest that the recipient site may affect some characteristics of transplanted hairs. See Hwang et al , 2002, Dermatol. Surg. 28:795-799.

2.4 CURRENT TREATMENTS FOR HAIR LOSS IN HUMAN

SUBJECTS

[00411 Human hair loss can be categorized as ( 1 ) gender specific hair patterning, (2) pathological hair loss, or (3) hair loss after wounding, all which can be associated with effects on self-esteem and self-image, and many individuals explore whether their hair loss process can be treated. Current treatments offered involve a limited selection of agents and regimens, such as chemical and surgical approaches that either stimulate or transplant pre-existing hair - none are associated with true follicular neogenesis.

[0042] Chemical treatments involve the use of drugs for the treatment of certain MPHL. These include, for example, minoxidil (an antihypertensive drug that opens the + channel); and antiandrogens such as finasteride, dutasteride or ketoconazole. While these types of treatments are reasonably effective in preventing or delaying MPHL, they are less effective in stimulating the growth of significant terminal hair in scalp of MPHL after baldness has been present for 6 months or more. Moreover, minoxidil and finasteride require continuous treatment for lasting effects. Consequently, patients with advanced MPHL may express dissatisfaction with even statistically significant, but cosmetically insignificant increase in hair counts and such frustration may contribute to poor compliance and further unsatisfactory outcomes.

[0043] Recently, bimatoprost (a prostaglandin analog used to control the progression of glaucoma in the management of ocular hypertension) has been FDA approved to lengthen eyelashes and is marketed under the name Latisse®, with the claim of growing eyelashes, making them longer, thicker and darker.

[0044] A device that uses low level light energy directly on the scalp (the HairMax Lasercomb) has received FDA clearance as a 51 OK device. Although the device is advertised as a "Laser," it operates by applying low level monochromatic light energy directly to the scalp, which is thought to stimulate hair growth through "photo-biostimulation" of hair follicles. Various types of devices operating on similar principles were referenced as the predicate for HairMax (see, Lolis et ai, 2006, J. Cosmetic Dermatol. 5:274-276).

[0045] Finally, more drastic measures for treating hair loss involve hair transplantation— in which scalp strips, hair follicles or follicular units from the occipital scalp (which are resistant to the effects of androgens in inducing MPHL type alopecia) are excised and transplanted to a person's balding or thinning areas. Another surgical method that has been used is scalp reduction; in this procedure, the skin in the balding area of the scalp is surgically excised and the surrounding skin (with hair) is pulled together and sutured. Surgical methods are best for focal hair loss, and are less effective for diffuse hair loss, are less effective for women and younger patients are not ideal candidates because the pattern and extent of hair loss is variable, and may be inconvenient because of the expense of the surgery, duration of time to show a cosmetic effect, creation of scarring. Despite surgical advances in hair transplantation, cosmetic coverage is constrained by the area of and the number of hairs in a patient's donor sites.

[0046] Because of limited effective treatment options, there is substantial interest among individuals for novel, safe and effective treatments for hair loss, including those that lead to true follicular neogenesis. 2.5 CURRENT TREATMENTS FOR REMOVAL OF UNWANTED HAIR IN HUMAN SUBJECTS

[0047] Current methods for removing unwanted hair involve depilation and epilation with or without the use of hair growth retardants.

[0048] Depilation affects the part of the hair above the surface of the skin. The most common form of depilation is shaving. Another popular option is the use of chemical depilatories (e.g., Nair®), which work by breaking the disulfide bonds that link the protein chains that give hair its strength, making the hair disintegrate.

[0049] Epilation is removal of the entire hair, including the part below the skin, and is believed to be longer-lasting. Methods include plucking with tweezers, waxing, sugaring, epilation devices, threading, home pulsed light, and can include the use of hair growth retardants (e.g., Vaniqa® (eflornithine)).

[0050] Electrology (electrolysis), laser and intense pulsed light are used for permanent hair removal. However, permanent hair removal is an imperfect process. For example, laser hair removal does not work well on light hair and/or on dark skin. Moreover, multiple sessions with trained medical personnel are required.

3. SUMMARY OF THE INVENTION

[0051] Intermittent lithium treatments or a single pulse lithium treatment are used to modulate the growth of hair in human subjects. Any pharmaceutically acceptable compound that releases the lithium ion (also referred to herein as lithium cation, Li+, ionized lithium) can be used for the lithium treatment; such 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.

[0052] 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. For the single pulse protocol, a dose of lithium is administered over a short period of time.

[0053] The lithium treatment can be administered topically, transdermally, intradermal ly, cutaneously, subcutaneously, intramuscularly, intravenously, orally, sublingually, or can be bucchal. Adjuvants that target the lithium to the desired hair follicles may be included in the formulations used. Topical lithium treatment is a preferred embodiment because high local concentrations can be achieved while minimizing systemic exposure. In one such embodiment, lithium gluconate 8% weight/weight (w/w) gel (e.g. , Lithioderm 8% gel) commercially available in France for the treatment of seborrheic dermatitis (Dreno B, 2007, Ann Dermatol Venereol. 134:347-351 , incorporated herein by reference) can be used in the treatment methods described herein. Other lithium formulations, combinations, and delivery methods for use in the treatment methods described herein are described in Sections 5.1 -5.3 infra. For example, the lithium formulations, including various modified release forms, may be delivered topically as additives to shampoos and other hair products, as a lotion, cream, or ointment, may be delivered using devices such as iontophoresis, micro-needle injection arrays, or auto-injector devices.

[0054] 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 treatments ) 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 electrology, laser, intense pulsed light, dermabrasion, or surgical treatments (e.g., hair transplant, strip harvesting, follicular unit extraction (FUE), scalp reduction, etc. ) that either promote or inhibit the growth of hair.

[0055] In one embodiment, intermittent lithium treatment or a pulse lithium treatment can be in combination with perturbation (e.g. , debriding, peeling, or wounding) of the skin and/or other tissues of the integumentary system by methods such as dermabrasion, microneedles, laser treatment, 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 the lithium formulation. 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.

[0056] In one embodiment, intermittent lithium treatment or a pulse lithium treatment can be used concurrently or in sequential/alternating combination with other agents or treatments that stimulate hair growth to increase overall hair density in a human subject. For example, intermittent lithium treatments or a pulse lithium treatment administered concurrently or alternating sequentially with one or more of the following agents can be used to promote hair growth: minoxidil, finasteride, bimatoprost (Latisse), CaCl2, or adenosine. Moreover, the lithium treatment, with or without an additional agent for promoting hair growth, can be used in combination with integumental perturbation to promote hair growth, such as by, e.g. , 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. ). For example, a low-level laser therapy treatment (e.g. , HairMax) can be applied concurrently with the intermittent lithium treatment or pulsed lithium treatment to stimulate hair growth. In another embodiment, intermittent lithium treatments or a pulse lithium treatment administered concurrently or alternating sequentially with one or more agents that prevent hair follicle cells from sencscing can be used to promote hair growth, 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.

[0057] Success of a pulse or intermittent lithium treatment can be measured by:

• increased terminal hair formation

• increased hair density

• hair cuttings

• increased hair count

• increased hair weight

• increased hair shaft thickness (diameter)

• increased hair length

• follicle synchronization so that the overall hair density appears to be greater compared to previous asynchronous hair growth

• increased proportion of follicles in anagen or decreased proportion of follicles in telogen

• follicle regeneration

• increased numbers of follicular units with 3 or more hair follicles

• subjective patient measures of increased hair.

[0058] Human subjects who are candidates for such treatments include any subject for whom increased hair growth is desired including, but not limited to, subjects with

nonscarring (noncicatricial) alopecia, such as androgenetic alopecia (AGA), including MPHL or FPHL, or any other form of hair loss caused by androgens, toxic alopecia, alopecia areata ( including alopecia universalis), scarring (cicatricial) alopecia, pathologic alopecia (caused by, e.g. , medication, chemotherapy, trauma, wounds, burns, stress, autoimmune diseases), senescence (age-related hair loss), malnutrition, or endocnne dysfunction), or hypotrichosis, or any other disease, disorder, or form of hair loss as discussed infra and/or known in the art.

[0059] In an alternative embodiment, intermittent lithium treatments or a pulse lithium treatment is used concurrently or in sequential combination with a cytotoxic drug, a hair growth retardant, epilation or depilation methods to reduce unwanted hair growth. For example, intermittent lithium treatments or a pulse lithium treatment alternating sequentially with one or more of the following agents or treatments can be used to inhibit unwanted hair growth: etlornithine HC1 (Vaniqa), 5-fluorouracil (5-FU) (e.g., Efudex 5% cream), and/or epilation. Success of treatment can be measured by:

• decreased terminal hair formation

• follicle synchronization so that synergies are achieved when the hair growth retardant is sequentially applied

• decreased proportion of follicles in anagen or increased proportion of follicles in telogen

• inhibition of follicle regeneration

• decreased hair length

• decreased hair thickness

• decreased pigmentation, which will make thinner hairs less visible

• decreased hair weight

• subjective patient measures of decreased hair.

[0060] Human subjects who are candidates for such treatment include any subject for whom elimination of unwanted visible hair is desired including, but not limited to, those afflicted with hypertrichosis, excess hair in androgen-dependent areas of the skin, idiopathic hirsutism, female post-menopausal facial hair, axillary hair, leg hair, back hair, ear hair, nares or nose hair, or any other disease, disorder, or form of unwanted hair or excessive hair as discussed infra and/or known in the art.

[0061] The invention is based in part on the realization that human hair follicles and follicular units are relatively unique (among other mammals, particularly non-primates) in that they enter and progress through different stages of the hair Follicle Cycle relatively independently of each other, even independently of neighboring follicles or follicular units. Consequently, the normal biology of human hair patterning is based on a probability distribution of the hair cycle stage that follicles will be in. generated by a stochastic (random) process by which follicles cycle independently.

[0062] The object of the invention is to synchronize hair follicle growth in the treated area to more effectively promote the growth of terminal hair (in preference to vellus hair); and/or promote the branching of pre-existing hair follicles (seen as an increased number of hair shafts per pore); and/or increase the width of hair follicles (thereby promoting growth of an increased shaft width); and /or promote regeneration of hair follicles or generation of new hair follicles ("follicle neogenesis"); and/or delay or prevent follicle senescence. In the alternative, the object is to inhibit the growth of unwanted hair (as measured by, e.g., decreased terminal hair formation or inhibition of follicle regeneration or generation of new follicles). Without being bound to any theory, the intermittent lithium treatments or pulse lithium treatments may achieve these results by:

• regulating the unique human processes that regulate visible hair growth.

• regulating the activity of specialized human hair follicles.

• regulating specific activities of specialized human hair follicles.

• regulating gender-specific specialized human hair follicles, including those under the influence of sex-steroid regulation.

• altering the activity of specialized human hair follicles, sometimes in conjunction with transplantation.

• regulating the differentiation of stem cells into gender-specific specialized human hair follicles, that may result 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).

• altering, delaying or preventing programmed senescence of hair follicles.

[0063] A number of molecular targets have been proposed for lithium action, including but not limited to inositol monophosphatase, a family of structurally related

phosphomonoesterases, and the protein kinase glycogen synthase kinase-3 (GSK-3) (for a review see Phiel & Klein, 2001 , Annu. Rev. Pharmacol. Toxicol. 41 :789-813). While not bound by any theory of how the invention works, 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. In plant cells which demonstrate remarkable precision in the timing of mitotic events, the lithium ion has been shown to cause metaphase arrest that can be reversed by the addition of CaCl2 or myo-inositol. (Wolniak, 1 87, 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 that are in various stages of cell cycle (cycling asynchronously). More particularly, a pulse lithium treatment or intermittent lithium treatments can be used to reversibly arrest mitosis in hair follicle cells. 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), as is the case with the human scalp.

[0064] Thus, 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 hair growth in human subjects. For example, lithium treatment results, indirectly, in increasing Wnt signaling, but agents that increase Wnt signaling have had conflicting effects on follicle development. When continuously present, they stimulate follicle morphogenesis but also induce hair follicle tumors (Gat et ai, 1998, Cell 95: 605-614), leading to decreased hair growth (Millar et ai , 1999, Dev. Biol. 207: 133-149). In the case of lithium, it has been shown to arrest mitosis (Wolniak, 1987, Eur. J. Cell Biol. 44: 286-293; and Wang, 2008, World J. Gastroenterol. 14:3982-3989), cause pathological hair loss when systematically administered (see, e.g. , Mercke et al , 2000, Ann. Clin. Psych. 12:35-42), or, at best, stimulate the generation of only rudiments of hair follicles (Fathke et ai , 2006, BMC Cell Biol. 7:4). These apparently discrepant roles of lithium as a stimulator of Wnt signaling and a negative regulator of the cell cycle are resolved in the present invention. By using lithium in formulations for intermittent or pulse treatments described herein, for example, before, concurrently with, or after treatment with another hair growth modulator, it functions as an effective modulator of hair growth in humans. It is thus also possible that the timing of exposure to other compounds with Wnt agonist activity may be important for modulating hair growth.

[0065] 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. In some embodiments, 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®)); and/or methods that regulate the differentiation of these stem cells into gender-specific specialized human hair follicles (e.g., using agents such as finasteride, fluconazole, spironolactone, flutamide, diazoxide, 1 l -alpha-hydroxyprogesterone, ketoconazole, U58841 , dutasteride, fluridil, or QLT-7704, an antiandrogen oligonucleotide, cyoctol, topical progesterone, topical estrogen, cyproterone acetate, ru 8841 , combination 5 alpha reductase inhibitors, oral contraceptive pills, and others in Poulos & Mirmirani, 2005, Expert Opin. 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), that can result in the appearance of specialized follicles having features that arc different from natural follicles in the target location of skin. Such combination treatments can further include the use of agents that enhance the growth of hair (e.g., minoxidil, finasteride, bimatoprost (Latisse), CaCl2, adenosine, and others described herein) or aid in the removal of hair (e.g. , 5- fluorouracil, eflornithine (Vaniqa), or others described herein). The Follicle Stem Cells involved can be derived from (1 ) other Follicle Stem Cells, (2) from other tissue stem cells, termed "pre-Follicle Stem Cells" (from the interfollicular skin), (3) from bone marrow- derived stem cells ("BMST"). and/or (4) from mesenchymal stem cells such as adipocyte stem cells. In the case of bone marrow derived stem cells (BMST), 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 (for example, by the induction of inflammation, wounding, or laser treatment) (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 differentiate into stem cells and (3) increases the trafficking of bone marrow derived stem cells to affected areas of skin and promotes their differentiation into Follicle Stem Cells by nurse cells in existing follicles. When used in combination with such procedures, 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 synchronous states of human hair follicle cells in Cell Cycle and human hair follicles in Follicle Cycle.

[0066] The methods of the invention are illustrated by the examples described in Sections 6 to 37.

3.1 GLOSSARY OF TERMS FOR HAIR

AND DISORDERS OF HAIR GROWTH

[0067] The following terms are used herein consistently with their art-accepted meanings summarized below.

[0068] Alopecia: Abnormal hair loss:

[0069] Alopecia areata: Hair loss in patches, thought to be caused by an autoimmune response to hair follicles in the anagen stage; extensive forms of the disorder are called alopecia areata totalis (hair loss over the entire scalp) and alopecia areata universalis (hair loss over the entire body).

[0070] Anagen: Growth stage of the hair-Follicle Cycle.

[0071] Anagen effluvium: Abrupt shedding of hair caused by interruption of active hair- follicle growth (e.g., in patients undergoing chemotherapy).

[0072] Androgenetic alopecia (AGA): Baldness caused by miniaturization of genetically predisposed follicles in the MPHL pattern (frontal recession and thinning at the vertex) or the FPHL pattern (loss of hair primarily over the crown, with sparing of frontal hair).

10073] Bulb: Lowermost portion of the hair follicle, containing rapidly proliferating matrix cells that produce the hair.

[0074] Bulge: Portion of the outer-root sheath of the hair follicle, located at the region of the insertion of the arrector pili muscle; thought to contain epithelial stem cells responsible for regenerating follicles in the anagen stage.

[0075] Catagen: Stage of the hair cycle characterized by regression and involution of the follicle.

[0076] Club hair: Fully keratinized, dead hair— the final product of a follicle in the telogen stage; 50 to 150 club hairs are shed daily from a normal scalp.

[0077] Female Pattern Hair Loss (FPHL): form of gender specific hair patterning in females (also sometimes referred to as female pattern alopecia). [0078| 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.

[0079] Kenogen: Latent phase of hair cycle after hair shaft has been shed and growth is suspended in follicle.

10080] Hirsutism: Excessive hair growth in androgen-dependent areas in women.

[0081) Hypertrichosis: Excessive hair growth (usually diffuse) beyond that considered normal according to age, race, sex, and skin region.

[0082] 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).

[0083] Lanugo hair: Fine hair on the body of the fetus, usually shed in utero or within weeks after birth.

[0084] Male Pattern Hair Loss (MPHL): form of gender specific hair patterning in men (also sometimes referred to as male pattern alopecia).

[0085] With regard to the concentrations of lithium (including its concentration in formulations, in tissue, in serum, etc. , and as a salt form, as ionized lithium in solution, etc.) described herein, since ionized lithium is a monovalent cation, the concentration of lithium expressed in millimolar units (niM) is equal to its concentration expressed in

millicquivalents (mEq) (i.e. , to avoid any doubt, 1 mM Li+ = 1 mEq Li+), as is sometimes used in the art.

[0086] Miniaturization: Primary pathological process in androgenetic alopecia, resulting in conversion of large (terminal) hairs into small (vellus) hairs.

[0087] Permanent alopecia: Caused by destruction of hair follicles as a result of inflammation, trauma, fibrosis, or unknown causes; examples include lichen planopilaris and discoid lupus erythematosus. Include diseases referred to as scarring alopecia.

[0088] Telogen: Resting stage of the hair cycle; club hair is the final product and is eventually shed.

[0089] Telogen effluvium: Excessive shedding of hair caused by an increased proportion of follicles entering the telogen stage; common causes include drugs and fever.

[0090] Terminal hair: Large, usually pigmented hairs on scalp and body.

[0091] 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 ). 4. DESCRIPTION OF THE FIGURES

[0092| Figure 1. Types of human hair follicles.

[0093] Figure 2. Architecture of the skin.

[0094J Figure 3. Diagram of human hair follicle.

[0095J Figure 4. Cellular structure of the human hair bulb.

[0096] Figure 5. Hamilton Norwood classification of male pattern hair loss (MPHL).

[0097] Figure 6. Permeation of lithium ions (also referred to as "Li ions") through the dermis (y-axis) from Formulation 35A' (lithium chloride emulsion 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.

[0098] Figure 7. Permeation of Li ions through intact cadaver skin (y-axis) from

Formulation 35 A' (lithium chloride emulsion cream; see Table 2) is plotted over time, in hours (x-axis).

[0099] Figure 8. 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.

[00100] Figure 9. Release of Li ions through dermis (y-axis) from Formulation BV-001 - 003A (lithium chloride hydrogei; 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.

[00101] Figure 10. 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.

[00102] Figure 11. Pharmacokinetic analysis of lithium concentrations in skin and plasma with once daily topical dosing with lithium gluconate hydrogei ("lithium gluconate") 8% and lithium chloride hydrogei ("lithium chloride") 8% following dermabrasion (DA). Lithium ion concentrations were measured by ICP/MS/MS, using a validated method (see Section 29 infra). Shown are lithium concentrations in skin (top) and blood (bottom), with once daily topical dosing with 80 mg/ml lithium on dermabraded mouse skin. Doses were given at Time (T)=0, T=24h, T=48h, and T=72 h. Skin and Blood samples were taken at T=0, 4-6h, 24h, 25h, 28h, 48h. 49h, 52h, 72h, 73h, 76h, and 96h. Arrows indicate peak levels of Lithium ion in skin one hour post dosing. N=2 per time point— error bars denote range. To obtain peak levels, mice were sacrificed and skin and blood obtained - 1 hour after dosing on the 5th day. To obtain trough levels, mice were sacrificed and skin and blood obtained - 1 hour without dosing, on the 5th day.

[00103] Figure 12. Linearity of calibration curves, validation of bioanalytical ICP method described in Section 29 infra.

[00104] Figure 13. 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.

[00105] Figure 14. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following DA.

[00106] Figure 15. A: 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.

[00107] Figure 16. 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 (-1 1 -14 days post-FTE). Lithium ion concentrations were measured by a validated bioanalytical ICP method (see Section 29 infra),

[00108] Figure 17. 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 (-1 1- 14 days post-FTE). Lithium concentrations were measured by a validated bioanalytical ICP method (see Section 29 infra). [00109] Figure 18. Comparison of Peak lithium concentrations in plasma and skin upon subcutaneous administration of a formulation of lithium chloride dissolved in isotonic saline following FTE.

[00110) Figure 19. 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 20 infra). Single dose administered at Oh, 24 h, 48 h, 72 h. Tissue samples (skin and blood taken at T=0, 4-6h, 24h, 25h, 28h, 48h, 49h, 52h, 72h, 73 h, 76h, 96h). Arrows indicate peak levels of Lithium ion in skin one hour post dosing. N=2 per time point— error bars denote range

[00111] Figure 20. 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.

[00112] Figure 21. 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 ("LiCr"), 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.

[00113] Figure 22. Topical lithium 8% increases maturation of neogenic hair follicles as shown by histology ( 13x = mouse ID; l Ox magnification). Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCl"), 8%.

[00114] Figure 23. 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%.

[00115] Figure 24. No adverse systemic effects of topical lithium as indicated by equal weight gain. Weight gain profile of mice administered topical lithium gluconate hydrogel ("lithium gluconate"), 1 %, 8%, or 16%, or lithium chloride hydrogel ("LiCl"), 8%, following FTE. [00116] Figure 25. 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%.

[00117] Figure 26. Topical lithium gluconate 8% results in a 16% increase thickness of regenerated hair shafts following DA. Tissues analyzed following administration of topical lithium gluconate hydrogel ("lithium gluconate"), 1%, 8%, or 16% or lithium chloride hydrogel ("LiCF*), 8%. Median + first and third quartile shown. P-values for comparisons of lithium treatments to placebo are for 1-sided tests for superiority, and should < 0.0125 for statistical significance with a family-wise error rate of alpha = 5% adjusted by the Bonferroni method for 4 comparisons to placebo. Graph on right side shows simultaneous 90% confidence intervals, corrected for 4 comparisons by the Treatment Groups Bonferroni method.

[00118] Figure 27. Healed FTE wounds treated with topical LiCT 8% have increased numbers of neogenic hair follicles, as assessed by in vivo scanning laser microscopy, imaging the wounded area approximately 60-80 μιη beneath the skin surface.

[00119] Figure 28. Topical LiCl 8% results in a 1.82 fold increase in number of neogenic hair follicles per FTE wound.

[00120] Figure 29. 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 C'NHF") combined from individual mice (parentheses indicate number of NIIF that could not be staged). Numbers below columns =total number of slides analyzed where one slide =one mouse (parentheses indicate slides not analyzed due to technical issues), p-value is for one-sided Wilcoxon test for superiority to placebo, p < 0.0125 for statistical significance with a family-wise error rate of a =5% corrected by the Bonferroni method for 4 comparisons to placebo. Right graph: Hodges-Lehman estimate of median difference. Simultaneous 90% confidence intervals, corrected for 4 comparisons by the Bonferroni method.

[00121J Figure 30. Topical LiCl 8% increase the percentage of FTE wound covered with neogenic hair follicles, based on in vivo scanning confocal microscopy.

[00122] Figure 31. Topical LiCl 8% results in a 1.57 fold increase over placebo in coverage of the FTE wound with neogenic hair follicles. [00123] Figure 32. Following FTE, topical LiCl 8%, as compared to placebo, does not affect the density of neogenic hair follicles in the region where follicles are forming.

[00124] Figure 33. Topical LiCl 8% does not affect density of regenerated hair follicles following DA.

[00125] Figure 34 Complexed Lithium Gluconate encapsulated within biodegradable poly (D,L-lactide-co-glycolide) ("PLG") microspheres.

[00126] Figure 35. Non-Complexed Lithium Gluconate encapsulated within

biodegradable PLG microspheres.

[00127] Figure 36. Synthetic Biodegradable Matrices from PLA PLG Blends. A:

Scanning electron micrograph (SEM) of a 100% PLA matrix. B: SEM of a 100% PLG matrix. C: In vitro profiles of lithium ion release from biodegradable matrices comprised of different ratios of PLA and PLG.

5. DESCRIPTION OF THE INVENTION

5.1 LITHIUM COMPOSITIONS

[00128] 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"). Optionally, the lithium compounds can be formulated with a pharmaceutically acceptable vehicle, carrier, diluent, or excipient, or a mixture thereof. Additionally, lithium- polymer complexes can be utilized to developed various sustained release lithium matrices.

[00129] Any form of lithium approved for pharmacological use may be used in the intermittent lithium treatments or a pulse lithium treatment. For example, lithium is best known as a mood stabilizing drug, primarily in the treatment of bipolar disorder, for which lithium carbonate (L12CO3), sold under several trade names, is the most commonly used. Other commonly used lithium salts include lithium citrate (L13C6H5O7), lithium sulfate (L12SO4), 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 (Lithioderm™), is approved for the treatment of seborrheic dermatitis. See, e.g. , Dreno and Moyse, 2002, Eur J Dermatol 12:549-552; Dreno et ai , 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. Another lithium formulation for use in the methods disclosed herein is lithium succinate, for example, an ointment comprising 8% lithium succinate, which is also used to treat seborrheic dermatitis. See, e.g., Langtry et al.. 1996, Clinical and Experimental Dermatology 22:216-219; and Cuelenaere et al.. 1992, Dermatology 1 84: 1 4-1 7, each of which is incorporated by reference herein in its entirety. In one embodiment, 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 Mullicenter 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. 5,594,03 1 , issued January 14, 1997. which is incorporated herein by reference in its entirety.

5.1.1 LITHIUM SALTS

[00130] 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, 18th eds., Easton, PA: Mack Publishing;

Remington: The Science and Practice of Pharmacy, 1995, 19th eds., Easton, PA: Mack Publishing.

[00131] In some embodiments, the compositions used for intermittent lithium treatment or a pulse lithium treatment comprise mixtures of one or more lithium salts. For example, a mixture of a fast-dissolving lithium salt can be mixed with a slow dissolving lithium salt proportionately to achieve the release profile. In certain embodiments, the lithium salts do not comprise lithium chloride.

[00132] In some embodiments, 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.

[00133] By reciting 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. [00134J In another embodiment, lithium salts of anionic drugs that positively affect hair growth, such as prostaglandins can be administered. In another embodiment, 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. Similarly, a lithium complex with a polyanion can be complexed further with the amines of minoxidil, at pHs greater than 5.

[00135] 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.

5.1.2 ORGANIC LITHIUM SALTS

[00136] In some embodiments, 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 N-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 4- acetamidobenzoate, lithium (+)-camphoratc, lithium camphorsulfonate, lithium (+)-( lS camphor-10-sulfonate, lithium caprate, lithium caproate, lithium caprylate, lithium cinnamate, lithium citrate, lithium cyclamate, lithium cvclohexanesulfamate, lithium dodecyl sulfate, lithium ethane- 1 ,2-disulfonate, lithium ethanesulfonate, lithium 2-hydroxy- ethanesulfonate, lithium formate, lithium fumarate, lithium galactarate, lithium gentisate, lithium glucoheptonate, lithium D-gluconate, lithium D-glucuronate, lithium L-glutamate, lithium a-oxoglutarate, lithium glycolate, lithium hippurate, lithium (+)-L-lactate, lithium (±)-DL-lactate, lithium lactobionate. lithium laurate, lithium (-)-L-malate, lithium maleate, lithium malonate, lithium (±)-DL-mandelate, lithium methanesulfonate, lithium naphthalene- 2-sulfonate, lithium naphthalene- 1 ,5-disulfonate, lithium 1 -hydroxy-2-naphthoate, lithium nicotinate, lithium oleate, lithium orotate, lithium oxalate, lithium palmitate, lithium pamoate, lithium L-pyroglutamate, lithium saccharate, lithium salicylate, lithium 4-amino-salicylate, sebacic acid, lithium stearate, lithium succinate, lithium tannate, lithium (+)-L-tartarate, lithium thiocvanate, lithium p-toluenesulfonate, lithium undecylenate, or lithium valerate. In some embodiments, 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.

[00137] In some embodiments, 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, (+)-camphoric acid, camphorsulfonic acid, (+)-( 15)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, cyclohexanesulfamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy-ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, D- glucuronic acid, L-glutamic acid, a-oxoglutaric acid, glycolic acid, hippuric acid, (+)-L-lactic acid, (±)-DL-lactic acid, lactobionic acid, lauric acid, maleic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene- 1 ,5-disulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, L-pyroglutamic acid, saccharic acid, salicylic acid, 4-amino- salicylic acid, sebacic acid, stearic acid, succinic acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, or valeric acid. Other 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.

SUSTAINED RELEASE ORGANIC LITHIUM SALTS [00138| In some embodiments, 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. In that regard, 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.

[00139] In some embodiments, 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. Examples of 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. In one variation, 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.

[00140] In some embodiments, particularly for administration of the lithium formulation to the skin, 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. In another example, 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.

5.1.3 INORGANIC LITHIUM SALTS

[00141] In some embodiments, 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. In one embodiment, the inorganic lithium salt is lithium fluoride. In another embodiment, the inorganic lithium salt is lithium iodide. In certain embodiments, 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.

[00142] In some embodiments, 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.

5.2 LITHIUM FORMULATIONS AND MODES OF DELIVERY

5.2.1 LITHIUM FORMULATIONS

[0 143J 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. In one embodiment, 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. See. Remington: The Science and Practice of Pharmacy, 2005, 21st ed., Philadelphia, PA: Lippincott Williams & Wilkins; Rowe et ai , eds., 2005, Handbook of Pharmaceutical Excipients, 5th ed., The Pharmaceutical Press and the American

Pharmaceutical Association; Ash & Ash eds., 2007, Handbook of Pharmaceutical Additives, 3rd ed., Gower Publishing Company; Gibson ed., 2009, Pharmaceutical Preformidation and Formulation, 2nd ed., Boca Raton, FL: CRC Press LLC, each of which is incorporated herein by reference.

[00144] 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 transdennal 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

International Patent Application Publication No. WO 2005/120451 , which is incorporated herein by reference in its entirety.

[00145] 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. 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 (ΝΜΡ), NN-dimefhylacetamide (DMA), and dimethyl sulfoxide (DMSO). In one embodiment, the water-miscible vehicle is not DMSO.

[001461 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 metabi sulfite. Suitable local anesthetics include, but are not limited to, procaine

hydrochloride, lidocaine and salts thereof, benzocaine and salts thereof and novacaine and salts thereof. Suitable suspending and dispersing agents include but are not limited to sodium carboxymethylcelluose (CMC), hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP). 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, β-cyclodextrin, hydroxypropyl-P-cyclodextrin, sulfobutylether-p-cyclodextrin, and sulfobutylether 7-β- cyclodextrin (CAPTISOL®, CyDex, Lenexa, KS).

[00147] Soothing preparations, e.g., for topical administration, 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.

[00148] A product for application to the scalp or face may additionally be formulated so that it has easy rinsing, minimal skin/eye irritation, no damage to existing hair, has a thick and/or creamy feel, pleasant fragrance, low toxicity, good biodegradability, and a slightly acidic pi I (pi I less than 7), since a basic environment weakens the hair by breaking the disulfide bonds in hair keratin.

[00149] In particular embodiments, commercially available preparations of lithium can be used, such as, e.g., lithium gluconate - for example, 8% lithium gluconate (Lithioderm™), 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 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). In some embodiments, a preparation of lithium or lithium salt comprises an anionic polymer (such as, e.g., crosslinked polyacrylic acid), which may form a gel. For example, a preparation provided in the Examples of Sections 27, 30, 33, 34, or 37 below may be used.

5.2.2 MODES OF ADMINISTRATION

[00150] The intermittent lithium treatments or a pulse lithium treatment can be provided by administration of the lithium compound (or combination treatments, discussed in Section 5.3 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. In some embodiments, the 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.

5.2.2.1 TOPICAL ADMINISTRATION

[00151] In a preferred embodiment, 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 5.3.3 infra.

[00152] 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, 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., International Patent Application Publication Nos. WO 2005/107710, published November 17, 2005, and WO 2005/020940, published March 10, 2005, each of which is incorporated herein by reference in its entirety. In one embodiment, the nano-sized delivery matrix is fabricated through a well- defined process, such as a process to produce lithium encapsulated in a polymer. In another embodiment, the lithium-releasing compound is spontaneously assembled in aqueous solutions, such as in liposomes and micelles. In some embodiments, the formulation for topical administration is a shampoo product, hair conditioner, hair dye, hair styling product, or skin lotion or cosmetic.

[00153] The selected formulation will penetrate into the skin and reach the hair follicle. Thus, in some embodiments, the stratum corneum and/or epidermis are removed by a method of integumental perturbation described herein (or by microdcrmabrasion, a less vigorous form of dermabrasion), permitting application of the dosage form for topical administration directly into the exposed dermis. In some embodiments, the formulation for topical administration will be lipid-based, so that it will penetrate the stratum corneum. In some embodiments, the formulation for topical administration will contain a skin penetrant substance, such as, e.g., propylene glycol or transcutol. See, e.g., International Patent Application Publication No. WO 2004/103353, published December 2, 2004, which is incorporated herein by reference in its entirety. The ability to penetrate into the skin can be tested using any method known in the art, such as, e.g. , the method described in International Patent Application Publication No. WO 2005/107710, which is incorporated herein by reference in its entirety. In one embodiment, 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 ai, supra. In some embodiments, the selected formulation is inconspicuous when applied to the skin, for example, is colorless, odorless, quickly-absorbing, etc. In some embodiments, 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. In one application, the hydrogel may be biodegradable. In another embodiment, the solution will absorb into the skin and crosslink into depots releasing drug. In another embodiment, 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.

[00154] Pharmaceutically acceptable earners 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, 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.

[00155] 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, hydroxysteann 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 Science and Practice of Pharmacy, supra). These vehicles are emollient but generally require addition of antioxidants and preservatives.

[00156] 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.

[00157] 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. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and/or stirring.

[00158] In particular embodiments, commercially available preparations of lithium can be used for topical administration in the methods described herein. These include, e.g., lithium gluconate, e.g., 8% lithium gluconate (Lithioderm™), 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 sulfate (marketed in the U.K. as Efalith; see, e.g. , Efalith ulticenter Trial Group, 1992, J Am Acad Dermatol 26:452-457, which is incorporated by reference herein in its entirety).

Other means of topical administration, including mechanical means

[00159] Other means of topical administration of lithium compounds are also

contemplated. 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 5.3.3 infra.

[00160] In some embodiments, topical administration is by electrical current, ultrasound, laser light, or mechanical disruption or integumental perturbation. These 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 POWDERJECT™ (Chiron Corp., Emeryville, CA), BIOJECT™ (Bioject Medical Technologies Inc., Tualatin, OR), or JetPeel™ (from TavTech, Tel Aviv, Israel), which uses supersonically accelerated saline to remove epidermis. Means of topical administration that can be used in accordance with the methods described herein are known in the art and are described in, e.g., U.S. Patent Nos. 5.957,895, 5,250.023, 6,306, 1 19, 6,726,693, and 6,764.493, and International Patent Application Publication Nos. WO

2009/061349, WO 1999/003521, WO 1996/017648, and WO 1998/01 1937. each of which is incorporated herein by reference in its entirety.

[00161] In some embodiments, the device for topical administration of lithium compounds is an automatic injection device worn continuously but delivers lithium intermittently. In some embodiments, 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. In some embodiments, 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. [00162] Other methods for administration of the lithium compounds described herein, used alone or in combination with other treatments described in Section 5.3.3 (e.g.. in combination with integumental perturbation methods such as dermabrasion, laser treatment, or partial thickness or full thickness excision) include use of a transdermal particle injection system, such as, e.g., a "gene gun.'' Such systems typically accelerate drug or drug particles to supersonic velocities and "shoot" a narrow stream of drug through the stratum corneum. In some embodiments, the stratum corneum and epidermis is previously removed using a method of integumental perturbation described herein, and thus the required delivery pressures and velocities can be reduced. This reduction reduces the required complexity of the firing mechanisms. In some embodiments, a narrow firing stream is used, particularly to accomplish systemic delivery. In other embodiments, 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 (in some embodiments, for use in conjunction with integumental perturbation, wherein the surface of skin to which drug is administered corresponds to the perturbed area) includes a low pressure / low velocity firing mechanism with a spray nozzle designed to deliver to a broad area. For example, a single- shot device that delivers to a 25-cm2 area could be fired or used multiple times on the scalp or other skin surface until the entire area is treated.

[00163] In another embodiment, 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 area. In some embodiments, the stratum corneum and epidermis are already removed, e.g., by a method of integumental perturbation described herein, and thus permits effective use of the mechanism using lowered pressure and velocity requirements to achieve dermal delivery.

[00164] In another embodiment, 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.

[00165] Specific embodiments of modes of administration using a device that combines integumental perturbation and lithium compound delivery follow. An advantage of using such a device is that it offers a convenient one step process for administration of the lithium compound.

[00166] In one embodiment, 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, 1 19) is customized to include a drug particle delivery feature using methods readily known in the art. As the device tires ablation particles at the skin, it could also fire smaller drug particles that would simultaneously embed in the exposed dermis. Alternatively, via an internal valve control, ihe device could switch over to firing dmg 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.

[00167] In another embodiment, a standard dermabrasion device can be modified to incorporate any of the devices described above, e.g., a spraying painting device. In one embodiment, a spray nozzle is located behind the dermabrasion wheel such that drug is sprayed into the dermis as it is exposed by the wheel. Alternatively, 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.

[00168] In another embodiment, a non-fractional C02 or Erbium- YAG laser is combined with drug spraying either without skin disruption, in conjunction with skin disruption, or following skin disruption.

[00169] In another embodiment, a fractional non-ablative laser (e.g. , an Erbium- YAG laser used at 1540-1550 nm) is combined with drug spraying either without skin perturbation, in conjunction with skin perturbation, or following skin perturbation. In another embodiment, a fractional ablative laser (e.g. , an Erbium- YAG laser used at 2940 nm or a C02 laser used at 10,600 nm) is combined with drug spraying either without skin perturbation, in conjunction with skin perturbation, or following skin perturbation.

[00170) In another embodiment, fractional ablative laser treatment of the skin (e.g., an Erbium- YAG laser used at 2940 nm or a C02 laser used at 10.600 nm) is combined with lithium compound delivery. For example, by invoking inkjet technology, 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. One of skill in the art would appreciate that 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. In another embodiment, fractional ablative laser treatment of the skin (e.g., an Erbium- YAG laser used at 2940 nm or a CO2 laser used at 10,600 nm) is combined with lithium compound delivery. For example, by invoking inkjet technology, use of 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. One of skill in the art would appreciate that adequate integrated hardware and software controls are required such that the laser treatment and drug delivery arc properly timed resulting in each newly formed hole being properly filled with drug.

[00171] m some embodiments, 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.

[00172] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is delivered through channels that are created by the use of micro-needle technology. The formulation can be, e.g. , a liquid, a gel or a dry spray. In another variation, topical administration may be through delivery of a lithium-containing formulation through hollow needles.

[00173] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is delivered into the skin by an iontophoretic patch. In one example of this embodiment, a patch can be developed in which the lithium-containing formulation is incorporated.

[00174] In another embodiment, topical administration comprises administration of a lithium-containing formulation that is incorporated into micro-needle shaped biodegradable polymers. In one such embodiment, 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.

5.2.2.2 PARENTERAL ADMINISTRATION

[00175] Administration can be parenterally by injection, infusion, or implantation, for local or systemic administration. Parenteral administration, as used herein, 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. Such dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra). Compositions intended for parenteral administration can include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, vater-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. All such compositions must be sterile, as known in the art. The compositions for parenteral administration can be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot. In one embodiment, 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, polydimethyl siloxanes, 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 ethylcne/vinyloxyethanol copolymer.

5.2.2.3 ORAL ADMINISTRATION

[00176J Pharmaceutical compositions comprising lithium compounds for oral

administration can be provided in solid, semisolid, or liquid dosage forms for oral administration. As used herein, oral administration also includes buccal, lingual, and sublingual administration. Suitable oral dosage forms include, but are not limited to, tablets, fastmelts, chewablc 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. In addition to the active ingredient(s), 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.

[00177] In particular embodiments, oral formulations approved for treating mood disorders, e.g., lithium carbonate (L12CO3), sold under several trade names, lithium citrate L13C H5O7), lithium sulfate (L12SO4), lithium aspartate, or lithium orotate, may be administered in accordance with the methods described herein.

5.2.3 MODIFIED RELEASE FORMS

[00178] The lithium compounds for use in intermittent lithium treatments or a pulse lithium treatment can be formulated as a modified release dosage form. As used herein, the term "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. The 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). In some embodiments, 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

immunostimulatory adjuvants, prolonged exposure to the immune system).

[00179] Examples of formulations for modified release to skin or hair 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. Other examples of modified release 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;

5,591.767; 5, 120.548; 5,073,543; 5,639,476; 5.354,556; 5,639.480; 5,733,566; 5,739, 108: 5,891 ,474; 5.922,356; 5,958,458; 5,972,891 ; 5,980,945; 5,993,855; 6,045,830; 6,087.324; 6, 1 13,943; 6, 1 7,350; 6,248,363; 6,264,970; 6,267,981; 6,270,798; 6,375,987; 6,376,461 ; 6,419,961 ; 6,589,548; 6,613,358; 6,623,756; 6,699,500; 6,793.936; 6.827,947; 6,902.742; 6,958, 161 ; 7.255,876; 7.416,738: 7,427,414; 7,485.322; Bussemer et al, rit. Rev. Ther. Drug Carrier Syst. 2001 , 18, 433-458; Modi ied-Release Drug Delivery Technology, 2nd ed.; Rathbone et al., Eds.; Marcel Dekker AG: 2005; Maroni et al., Expert. Opin. Drug Deliv. 2005, 2, 855-871 ; Shi et al , Expert Opin. Drug Deliv. 2005, 2, 1039-1058; Polymers in Drug Delivery; Ijeoma et al., Eds.; CRC Press LLC: Boca Raton, FL, 2006; Badawy et al. , J.

Pharm. Sci. 2007, 9, 948-959; Modified-Release Drug Delivery Technology, supra; Conway, Recent Pat. Drug Deliv. Formul. 2008, 2, 1-8; Gazzaniga et al., Eur. J. Pharm. Biopharm. 2008, 68, 1 1- 18; Nagarwal et al, Curr. Drug Deliv. 2008, 5, 282-289; Gallardo et al, Pharm. Dev. Techno!. 2008, 13, 413-423; Chrzanowski. AAPS PharmSciTech. 2008, 9, 635-638; Chrzanowski, AAPS PharmSciTech. 2008, 9, 639-645; alantzi et al, Recent Pat. Drug Deliv. Formul 2009, 3, 49-63; Saigal et al, Recent Pat. Drug Deliv. Formul. 2009, 3, 64-70; and Roy et al., J. Control Release 2009. 134, 74-80, each of which is incorporated by reference herein in its entirety.

5.2.3.1 MATRIX CONTROLLED RELEASE DEVICES

[00180] 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.

[00181| In certain embodiments, 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; polyacryiic acid; copolymers of ethacrylic acid or methacrylic acid (EUDRAGIT®, Rohm America, Inc., Piscataway, NJ); poly(2-hydroxyethyl- methacrylate); polylactides; copolymers of L-glutamic acid and ethyl-L-glutamate;

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-dimethylaminoethyi)methacrylate, and (trimethylaminoethyl)methacrylate chloride.

[00182] In certain embodiments, 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,

polymethylmethacrylate, polybutylmethacrylate, chlorinated polyethylene, polyvinylchloride, methyl acrylate-methyl methacrylate copolymers, ethylene-vinyl acetate copolymers, ethylene/propylene copolymers, ethyl ene/ethyl aery late copolymers, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubbers, epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, ethylene/vinyloxyethanol copolymer, polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, silicone rubbers, polydimethylsiloxanes, and silicone carbonate copolymers; hydrophilic polymers, such as ethyl cellulose, cellulose acetate, crospovidone, and cross- linked partially hydrolyzcd polyvinyl acetate; and fatty compounds, such as carnauba wax, microcrystalline wax, and triglycerides.

[00183] In a matrix controlled release system, 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.

[00184] 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. 5.2.3.2 OSMOTIC CONTROLLED RELEASE DEVICES

[00185] 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). In general, 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).

[00186] In addition to the active ingredient(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. One class of osmotic agents is 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,

polyvinylpyrrolidone (PVP), crosslinked PVP, polyvinyl alcohol (PVA), 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 carboxvethyi, cellulose (CEC), sodium alginate, polycarbophil, gelatin, xanthan gum, and sodium starch glycolate.

[00187] 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 acid, glutamic acid, p-toluenesulfonic acid, succinic acid, and tartaric acid; urea; and mixtures thereof. [00188J Osmotic agents of different dissolution rates can be employed to influence how rapidly the active ingredient(s) is initially delivered from the dosage form. For example, amorphous sugars, such as MANNOGEM 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. In this case, the active ingredients ) is released at such a rate to replace the amount of the active ingredient metabolized and excreted.

[00189] 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.

[00190] 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. Examples of 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 copolymers, PVP, HEC, HPC, CMC, CMEC, HPMC, HPMCP, HPMCAS, HPMCAT, poly(acrylic) acids and esters and poly- (methacrylic) acids and esters and copolymers thereof, starch, dextran, dextrin, chitosan, collagen, gelatin, polyalkenes, polyethers, polysulfones, polyethersulfones, polystyrenes, polyvinyl halides, polyvinyl esters and ethers, natural waxes, and synthetic waxes.

[00191 J 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 arc permeable to water vapor, as disclosed in U.S. Pat. No. 5,798,1 1 . 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.

[00192] The deliver 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.

5,612,059 and 5,698,220.

[00193] 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.

[00194] 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 αί , Drug Development and Industrial Pharmacy 2000, 26, 695-708; and Verma et al. , ,1. Controlled Release 2002, 79, 7-27.

[00195] In certain embodiments, the 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. See, U.S. Patent No. 5,612,059 and International Publication No. WO 2002/17918. The 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. In certain embodiments, the 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.

5 2.3.3 N SITU GELLING DRUG DELIVERY SYSTEMS

[00196] In one embodiment, 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. In one application 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. Examples of these thermo-gelling polymers are polyiisopropyl acrylamidc), poIy(EO)x-(PO)y-(EO)x and poly(PO)x-(EO)y-(PO)x, wherein EO=ethylene oxide and PO=propylene oxide. Other examples include, but are not limited to, PLA-PEO-PLA polymers, wherein PLA=polylactic acid, PEO=polyethylene oxide, poly(sebacic anhydride)- poly(ethylene oxide)-poly(sebacic anhydride) and poly(stearate)-poly(ethylene oxide)- poly(stearate). In a variation of the idea, the lithium-loaded solution can be injected as a liquid, to form an in-situ depot within the tissue. In another variation of the concept, 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.

5.2.3.4 MULTIPARTICULATE CONTROLLED RELEASE DEVICES

[00197] 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 μηι to about 3 mm, about 50 μπι to about 2.5 mm, or from about 100 μηι 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. See, for example, Ghebre-Sellassie, ed., 1994, Multiparticulate Oral Drug Delivery, Marcel Dekke; and Ghebre-Sellassie ed., 1989, Pharmaceutical Pelletization Technology, Marcel Dekker.

[00198] Other excipients or carriers as described herein 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.

TARGETED DELIVERY [00199] 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.

[00200] 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;

5,840,674; 5,900,252; 5,972,366; 5,985,307; 6,004,534; 6,039,975; 6,048,736; 6,060,082; 6,071,495; 6, 120,751 ; 6,131,570; 6,139,865; 6,253,872; 6,271 ,359; 6,274,552; 6,316,652; and 7,169,410.

[00201] In some embodiments, 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. For hair follicle drug delivery, 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. However, out-flux of sebaceous fluid from the hair follicle can result in a short residence time of the delivery systems in the follicle. To minimize this, 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. 5.2.3.6 LOCA DELIVERY

[00202] Common side effects of systemic lithium treatment include muscle tremors, twitching, ataxia, and hypothyroidism. Long term use has been linked to

hyperparathyroidism, hypercalcemia (bone loss), hypertension, kidney damage, nephrogenic diabetes insipidus (polyuria and polydipsia), seizures, and weight gain. There also appears to be an increased risk of Ebstein (cardiac) Anomaly in infants born to women taking lithium during the first trimester of pregnancy. In order to circumvent these side effects, the dosage of systemically administered lithium is tightly controlled. The intermittent lithium treatments or a pulse lithium treatment described herein may have a decreased risk of such side effects because of the intermittent or temporary nature of the treatment. Another way in which such side effects may be circumvented is to deliver the intermittent lithium treatment or a pulse lithium treatment locally to the site where hair growth modulation is desired.

[00203] The intermittent lithium treatments or a pulse lithium treatment described herein may be delivered locally to any part of the subject in which modulation of hair growth is desired, including, e.g., the head (e.g. , the scalp, cheek, chin, upper lip, lower lip, ears, nose, eyelashes, or eyebrow), neck, abdomen, chest, breast, e.g., the nipples, back, arms, armpits (axillary hair), stomach, genital area, buttocks, legs, hands, or feet of a subject. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is applied to wounded or scarred skin.

[00204] 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.

5.2.4 DELIVERY VIA SCAFFOLDS

[00205] In some embodiments, a lithium treatment described herein is delivered 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. Delivery of lithium in combination with, or via, such a "scaffold" may be of particular benefit when the lithium is administered in connection with integumental perturbation (see, e.g. , Section 5.3.4 infra), including but not limited to techniques that involve skin excision (such as full thickness excision), surgical techniques for hair transplantation, or other techniques in which the skin is wounded as described herein.

[00206] 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. For example, 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.

[00207] In some embodiments, 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. In some embodiments, the mesh scaffold has an "open-cell" structure, with the pores interconnected, much like an open-celled foam. The open, interconnecting nature of the scaffold may allow free diffusion of oxygen and cells. In some embodiments, the mesh scaffold has the capacity to hydrate and remain hydrated throughout a wound healing period. In some embodiments, 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.

[00208] In some embodiments, the mesh scaffold has incorporated within it one or more additional active agents, for example, a small molecule, or a nucleic acid, or a protein, such as described in Section 5.3.4 infra. In some embodiments, the additional active agent is a protein, such as noggin or WNT, or is a nucleic acid that encodes noggin or NT. In some embodiments, a small molecule is incorporated into the scaffold, such as, e.g., a GS inhibitor. BMP inhibitor, or PPAR antagonist. In some embodiments, the compound incorporated in the mesh scaffold is a compound considered for use in the combination therapies described herein, for example, in Section 5.3.4. In some embodiments, the compounds aid in hair follicle migration or hair follicle neogenesis in the wound site. In some embodiments, the scaffold may incorporate superoxide dismutase, a free radical quenching molecule that functions in the reduction of inflammation. (00209J In some embodiments, the lithium compound itself is incorporated within the mesh scaffold. In some embodiments, the lithium compound is incorporated within one or more layers of a multilayered mesh scaffold. For example, in one embodiment the mesh scaffold contains the lithium compound in alternating layers, which may achieve a pulsatile delivery of lithium. In some embodiments, the lithium compound in incorporated in microspheres in the scaffold, enabling a controlled release of lithium from the scaffold.

[00210] In another embodiment, the mesh scaffold can be fibrin gels that additionally contain lithium. For example, the fibrin delivery matrix may contain lithium, fibrinogen and thrombin, that '"gels" in situ. One issue that may be encountered when lithium is administered to integumental perturbed skin is the ability of lithium to diffuse through the fibrin "scab" - making the drug part of the scab may help solve this issue.

[00211] In another embodiment, the mesh scaffold is a synthetic biodegradable dressing and lithium delivery system that also acts as a "sponge" and absorbs the exudates/bloods from integumental perturbed skin. These exudates intercalating with the synthetic scaffold may contain an abundance of fibrinogen, thrombin, fibronectin, cell adhesion proteins, growth factors and hyaluronic acid, all of which create an integrated staicture that is an attractive matrix for cell attachment /differentiation and delivery of lithium.

[00212] The release rate of lithium can be modulated by varying the composition of polymers that comprise the synthetic scaffold, or sponge. For example, 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(ethylcne oxide) (PEO), poIy(ethylene oxide)-co-poly( propylene oxide)-co-poly(ethylene oxide) (PEO-PPO-PEO), poly(acrylates) and polyvinyl pyrrolidone) (PVP). By varying the composition of polymers, the rate of lithium release from the formulation (e.g., scaffold or sponge) 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. In some embodiments, 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. For a specific example of such formulations that may be used in accordance with such embodiments, see the example of Section 37 infra. [00213] In some embodiments, the mesh scaffold releases the aforementioned compounds in a timed release manner, acting as a controlled release formulation such as described in Section 5.2.3 above. For example, 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. In some embodiments, 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.

[00214) In some embodiments, the mesh scaffold is extruded as a gel, with certain components of the gel precipitating out to form a mesh in situ. Alternatively, in some embodiments, the in situ mesh can be sprayed on the integumentally perturbed surface. A large area can be covered in this manner.

[00215] In some embodiments, the mesh scaffold is pre- abricated as a dressing or a wrap, to cover large areas of skin. In some such embodiments, the mesh scaffold can be cut to size to fit the size of the area of the integumental perturbed skin.

[00216] In some embodiments, the scaffold is prepared by melt spinning, electrospinning, micromachining, weaving, or other methods known in the art in which open cell foams are fabricated. Using starting materials that are United States Pharmacopeia (USP)-approved, 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. In some embodiments, the additional compounds are sterilized, and then added to the sterile mesh scaffold.

[00217] In a particular embodiment, 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 5.3.4), is applied. This approach may be used together with a form of integumental perturbation described in Section 5.3.4 (e.g. , dermabrasion accomplished by a standard dermabrader or a laser, or full-thickness excision accomplished by a bulk ablative laser) or integumental perturbation by wounding.

5.2.4.1 B QPEQKAPARlfl PROPERTIES Q¥ THE

[00218] In one embodiment, the scaffold is biodegradable. Placement of a 3-dimensional biodegradable scaffold on the integumentally perturbed site assists the attachment, growth and differentiation of cells. Historically, 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. Thus, 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. These expanded cells are then seeded onto three-dimensional biodegradable frameworks that provide structural support for the cells and allow cellular infiltration, attachment, proliferation and growth ultimately- leading to new tissue.

[00219] In some embodiments, the biodegradability of the scaffold is modulated. Ideally, the biodegradability of the scaffold should be matched to the formation of the new epithelium as the integumentally perturbed skin heals. One skilled in the art would know how to measure whether a synthetic matrix is biodegradable. For example, biodegradability can be measured ex vivo in implants or using rats or another animal model, by histological and HPLC analysis. In one embodiment, biodegradability by hydrolysis can be assessed. In such an embodiment, the scaffold structure of choice is incubated in phosphate buffered saline, pH 7.4 and 37 °C. For degradation by enzymolysis, 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. In one embodiment, 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 37) to increase biodegradation (for example, see ASTM El 279 - 89, 2008, Standard Test Method for Biodegradation By a Shake-Flask Die-Away Method).

5.2.4.2 BIOMIMETIC PROPERTIES OF THE SCAFFOLD

[00220] Biodegradable synthetic matrices can be created to mimic the extra-cellular micro- environment for the enhanced cellular attachment necessary for tissue regeneration. In some embodiments, cell-recognition motifs such as RGD peptides may be incorporated to encourage cells to attach themselves to the scaffold.

[00221] One skilled in the art would know how to measure whether the biodegradable synthetic matrix has biomimetic properties. For example, in one embodiment, the biomirnetic nature of the scaffold is judged on the basis of the content of the mesh and resultant intercalating fibrin.

5.2.4.3 PHYSICAL PROPERTIES OF THE SCAFFOLD

[00222J 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.

[00223] One skilled in the art would know how to measure whether the biodegradable synthetic matrix has optimal physical properties. For example, in one embodiment, the modulus of the scaffold is matched with the modulus of the tissue ty pe. In general, the compressive modulus of a scaffold or hydrogel can be measured by a standard Instron instrument (e.g. , using the TA Instruments DMA Q800).

5.2.4.4 INCOMPATIBILITY OF THE SCAFFOLD

[00224] Further, 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. In one embodiment, this can be accomplished through the use of hydrophilic components that can absorb water. Use of hydrophobic components such as petrolatum is likely to be occlusive and prevent rapid cell proliferation.

[00225] One skilled in the art would know how to measure whether the biodegradable synthetic matrix is biocompatible. For example, in one embodiment, 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. See, e.g., the following reference for studies on the biocompatibility of materials: Altankov et al , 1996, Journal of Biomedical Materials Research Part A; 30:385-391, which is incorporated by reference herein in its entirety.

5.2.4.5 OXYGEN PERMEABILITY OF THE SCAFFOLD

[00226] In some embodiments, 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. 5.2.4.6 COMBINED BIOLOGICAL/SYNTHETIC MESH

[00227] In another embodiment, a loose, dry, highly porous network or scaffold or mesh is placed in a bleeding site of integumentally perturbed skin to gently absorb the blood and the cell adhesion proteins released at the site. 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. In some embodiments, 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.

[00228] A dry scaffold has the added advantage of absorbing the blood at the perturbed site. Thus, a person's own blood components can be used to create a combined

synthetic/natural ECM. In practical terms, the scaffold has an added advantage of serving as a blood absorbing gauze.

[00229J In another embodiment, 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.

[00230) In one embodiment, a dry, sterile biodegradable scaffold is placed onto the freshly integumentally perturbed (e.g. , wounded) skin. The properties of the scaffold will be such that it will transform into an adherent hydrogel upon water absorption.

5.2.4.7 FABRICATING AND APPLYING THE SCAFFOLD

[00231] 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 and 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.

[00232] In some embodiments, the active agents (e.g., lithium alone or in a combination described herein) are administered using an active agent-containing spray-on hydrogel. In one such embodiment, 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 integumentally perturbed site is large and uniform coverage is needed.

[00233] In some embodiments, the active agent-containing spray-on hydrogel is applied on the integumentally perturbed 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. Depending upon the release characteristics that are required, the active agent will either be incorporated in micro-encapsulates 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.

[00234] In some embodiments, 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.

[00235] In a variation of the foregoing embodiments, the integumentally perturbed site may be covered with a breathable, non-occlusive spray-on hydrogel to cover the site during healing.

5.3 LITHIUM TREATMENT REGIMENS

[00236] In the embodiments described herein, 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"). It is noted that, with regard to the concentrations of lithium (including its concentration in formulations, in tissue, in serum, etc. , and as a salt form, as an ionized atom in solution, etc. ) described herein, since ionized lithium is a monovalent cation, the concentration of lithium expressed in millimolar units (mM) is equal to its concentration expressed in milliequivalents (mEq), as is sometimes used in the art (i.e., 1 mM Li+ = 1 mEq Li+). 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 27-31 and the techniques cited therein; see also Wood et ai, 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). For example, when using oral formulations, 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.

[00237] In some embodiments, 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. In some embodiments, it is preferable to allow the concentration of lithium to decline between dosages, in order to achieve a pulsatile effect.

[00238] In some embodiments, topical administration of a lithium compound is preferred over oral or subcutaneous administration. Depending on the formulation used, 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. Conversely, and depending on the formulation used, 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.

[00239] Regardless of the route of administration, care should be taken to avoid toxicity. In this regard, 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. Once the dose is established for a patient, routine monitoring for toxicity is recommended. For a rev iew of the pharmacokinetics and monitoring of lithium concentrations, see Amdisen, 1980, Ther. Drug. Monit. 2:73-83;

Goodman & Gilman, 1980, "The Pharmacological Basis of Therapeutics" at pp. 430-434; Grandjean & Aubry, 2009, CNS Drugs 23:331-349; and the APA Practice Guideline for the Treatment of Patients with Bipolar Disorder, Second Edition, 2002, each of which is incorporated by reference herein in its entirety.

[00240J In some embodiments, 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. In this regard, 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. In some embodiments, 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. In some embodiments, the trough concentration is maintained at approximately 0.6 mM to 1.4 mM lithium. In some such embodiments, a trough skin concentration is maintained at 1 mM to 3 mM lithium. In some such

embodiments, 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.

[00241) In specific embodiments, 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 μΜ, 0.20-0.50 μΜ, 0.50- 1.0 μΜ, 1 .0-5.0 μΜ, 5.0-10 μΜ, 10-20 μΜ, 20-50 μΜ, 50- 100 μΜ, 100-500 μΜ, 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. In some embodiments, 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 μΜ, 0.1 to 1.0 μΜ, 0.5 to 1 .0 μΜ, 0.5 to 1.5 μΜ, 1 to 10 μΜ, 10 to 50 μΜ, 50 to 100 μΜ, 100 to 150 μΜ, 150 to 200 μΜ, 250 to 300 μΜ, 100 to 250 μΜ, 100 to 500 μΜ, 200 to 400 μΜ, 500 to 1000 μΜ; or 1000 to less than 1500 μΜ. In one embodiment, the plasma or serum lithium concentration reaches at least 1 μΜ. In one embodiment, the plasma or serum lithium concentration reaches at least 100 μΜ. In one embodiment, 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 al, 1992, Trace Elements in Medicine 9:7-8.

[00242] In specific embodiments, an amount of a lithium compound is administered such that the target concentration of lithium in the skin is 0.01 to 0.05 μΜ, 0.05 to 0.1 μΜ, 0.1 to 0.5 μΜ, 0.1 to 1 μΜ. 0.5 to 1.0 μΜ, 1.0 to 1.5 μΜ, 1 to 2.5 μΜ, 1 to 5 μΜ, 5 to 10 μΜ, 10 to 50 μΜ, 50 to 100 μΜ, 100 to 150 μΜ, 150 to 200 μΜ, 250 to 300 μΜ, 100 to 250 μΜ, 100 to 500 μΜ, 200 to 400 μΜ, 500 to 1000 μΜ, 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. In some embodiments, 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. In one embodiment, the amount of lithium achieved in the skin does not exceed 50 mM. In some embodiments, an amount of lithium is administered such that the concentration of lithium delivered to the stratum comeum 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. In some embodiments, the concentration of lithium delivered to the stratum corneum is greater than 1.5 mM. In one embodiment, the amount of lithium achieved in the stratum corneum is approximately 100 to 200 mM. In one embodiment, the amount of lithium achieved in the stratum corneum does not exceed 5 mM. In one embodiment, the amount of lithium achieved in the stratum corneum does not exceed 10 mM. One of skill in the art would be able to measure lithium concentrations in skin using techniques known in the art, for example, mass spectroscopy, e.g. , inductively coupled plasma mass spectroscopy (ICP-MS). For example, the concentration of lithium in skin can be measured using the method provided in the example of Section 29.2 below or equivalent methods. [00243] In other embodiments, 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.

[00244] Specific, non-limiting, formulations of lithium for topical, subcutaneous, and oral administration are provided in Sections 5.3.1.1-5.3.1.3 below.

5.3.1.1 TOPICAL FORMS FOR ADMINISTRATION

[00245] In the embodiments described in the subsections that follow, lithium can be applied topically, e.g. , as a cream, gel, ointment, or other form for topical administration as described in Section 5.2.2.1 and 5.3 supra. Topical lithium may be administered to wounded or unwounded skin.

[00246] In some embodiments, the lithium formulation for topical administration (e.g. , gel, cream, ointment, salve, etc.) comprises lithium (or monovalent lithium salt) at a concentration of 50 mM, 75 mM, 100 raM, 125 mM, 150 mM, 175 mM, 200 mM, 250 mM, 300 niM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 900 mM, 1 M, 1.1 M. or 1.2 M, or more. As used herein, a monovalent lithium salt (e.g. , lithium gluconate, lithium chloride, lithium stearate, lithium orotate, etc.) refers to a salt form of lithium in which there is one lithium cation for each anion of the salt. A divalent lithium salt (e.g. , in some embodiments, lithium succinate, lithium carbonate) refers to a salt form of lithium in which there are two lithium cations for each anion of the salt. 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. In some embodiments, 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. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 200 mM to 400 mM is used. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 400 mM to 600 mM is used. In some embodiments, a lithium formulation comprising lithium (or monovalent lithium salt) at a concentration in the range of 600 mM to 800 mM is used. The 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.

[00247] 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. In some embodiments, 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.

[00248] 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. For example, Lithioderm (Labcatal) comprises 8% lithium gluconate, which corresponds to 0.275% lithium ion (i.e. , 274.8 mg Li+/100 g gel). It is noted that a formulation of topical 8% lithium gluconate, w/w, contains approximately 80 mg ml lithium gluconate, which is approximately 400 raM lithium gluconate (and, thus, 400 mM lithium ion). Thus, in some exemplary embodiments, 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. In some embodiments, 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. In one embodiment, the form of lithium for topical administration is 1 % to 20% w/w lithium salt.

[00249J In some embodiments, 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%- 1%, 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). In some embodiments, 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).

[00250] In an exemplary embodiment, 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. Thus, in some embodiments, a patient (e.g., a 50 kg patient) is administered about 30-50 mg, about 50-75 mg, or about 75-100 mg topical lithium gluconate/day, which is equivalent to about 1 -1.7 mg, 1.7-2.2 mg, or 2.2-3.5 mg, respectively, Li+/day. In some embodiments, a topical lithium formulation is administered once daily. In some embodiments, a topical lithium formulation is administered 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 1 1 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.

[00251] In some embodiments when lithium is administered topically, 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. In some such embodiments, 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). In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.

5.3.1.2 sypcUTATW S FQR fl OR AP fWSTRATIW

[00252] In some embodiments, 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 subcutaneously, to either wounded or unwounded skin.

[00253] In some embodiments, the form of lithium for subcutaneous administration is administered at a dose comprising 0.001 mg lithium ion per kg of patient weight. In some embodiments, 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, 7 mg/kg, 7.5 mg/kg, 8 mg/kg, 8.5 mg/kg, 9 mg/kg. 9.5 mg kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg kg, or 50 mg/kg or more of lithium ions. In some embodiments, the dose does not exceed 50 mg/kg. The lower ranges of dosages may be preferably used for bolus dosing. For a controlled release (e.g., a delayed release or a sustained release) dosage form, 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.

[00254J The 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. For example, to achieve an equivalent dosage of lithium ions, 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. In some embodiments, 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. In one embodiment, the formulation for subcutaneous administration contains a dose in the range of 30 mg/kg to 150 mg/kg lithium gluconate. In one embodiment, the formulation for subcutaneous administration contains a dose in the range of about 30 mg/kg to 300 mg/kg lithium gluconate. In one embodiment, the dose for subcutaneous administration does not exceed 300 mg/kg lithium gluconate. In another embodiment, 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. For a controlled release {e.g., a delayed release or a sustained release) dosage form, 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. [00255] In some embodiments, 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 1 1 hours apart, or 12 hours apart. In a particular embodiment, the doses are administered 7 to 8 hours apart.

[00256] In some embodiments when lithium is administered subcutaneously (for example, once daily, although smaller doses may be administered more than once daily), an amount of lithium is administered such that the peak lithium concentration in skin is between 0.1 μΜ and 0.2 μΜ, 0.2 μΜ and 0.5 μΜ, 0.5 and 1 μΜ, 1 μΜ and 2 μΜ, 2 μΜ to 10 μΜ, 10 μΜ to 100 μΜ, 100 μΜ to 500 μΜ, 500 μΜ to 1000 μΜ. These 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. In some embodiments, the peak

concentration in skin is 0.2 μΜ to 1.5 μΜ lithium. In some embodiments, the peak concentration in skin should not exceed 1 μΜ or 1.5 μΜ lithium. In some embodiments, the peak concentration in skin is 10 μΜ to 100 μΜ lithium. In some embodiments, the peak concentration in skin is 100 μΜ to 1000 μΜ 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. These 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. In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.

5.3.1.3 ORAL FORMS FOR ADMINISTRATION

[00257] In some embodiments, 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 5.3.1 above.

[00258] In some embodiments, 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. In some embodiments, 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.

[00259] 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. In some embodiments, 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, 7 mg/kg, 7.5 mg kg, 8 mg/kg, 8.5 mg kg, 9 mg/kg, 9.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, or 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, or 50 mg/kg or more of lithium ions. In some embodiments, the dose does not exceed 50 mg/kg Li+. For a controlled release (e.g. , a delayed release or a sustained release) dosage form, 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.

[00260] The 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. For example, to achieve an equivalent dosage of Li+, a formulation comprising lithium carbonate, which is a divalent lithium salt (e.g. , trade names Eskalith CR, Eskalith, Lithobid), may be orally administered at a dosage of approximately 2 mg lithium carbonate per kg of patient weight (mg/kg), 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 75 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, or 250 mg/kg or more is administered. In some embodiments, 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. For a controlled release (e.g., a delayed release or a sustained release) dosage form, 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.

[00261 | In some embodiments when the lithium formulation is for oral administration (for example, for once daily administration, although smaller doses may be administered more than once daily), an amount of lithium compound is administered such that the peak lithium concentration in skin is between 0.1 μΜ and 0.2 μ , 0.2 μΜ and 0.5 μΜ, 0.5 and l μΜ, 1 μΜ and 2 μΜ, 2 μΜ to 10 μΜ, 10 μΜ to 100 μΜ, 100 μΜ to 500 μΜ, 500 μΜ to 1000 μΜ. These 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. In some embodiments, the peak concentration in skin is 0.2 μ to 1.5 μΜ lithium. In some embodiments, the peak concentration in skin should not exceed 1 μΜ or 1 .5 μΜ lithium. In some embodiments, the peak concentration in skin is 10 μΜ to 100 μΜ lithium. In some embodiments, the peak concentration in skin is 100 μΜ to 1000 μΜ 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. These 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. In some such embodiments, the steady state blood concentration of lithium should not exceed a maximum of 1.5 mM to 2 mM.

5.3.2 PULSE TREATMENT

[00262] 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 v ary 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. [00263] In some embodiments, 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. Γη 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. 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 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. 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 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. 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 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. In some embodiments, 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 within 3 days. 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.

[00264] In one embodiment, 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. In some embodiments, 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.

[00265] In some embodiments, a pulse treatment is combined with a form of integumental perturbation that does not lead to formation of a scab. In one such embodiment, the pulse lithium treatment is administered at the time of integumental perturbation. In some embodiments, a pulse lithium treatment is administered following integumental perturbation. In some embodiments, in which a 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.

[00266] The Examples in Sections 6 to 37 provide exemplary protocols for carrying out the aforementioned embodiments, although the invention is not meant to be so limited.

5.3.3 INTERMITTENT TREATMENTS

[00267] 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.

[00268J In one embodiment, 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.

[00269] In some embodiments, 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 detacliment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to fonnation of a scab, 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. 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 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 1 8-24 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 I day after scab detachment. In one embodiment, in which the intermittent lithium treatment is administered following an integumental perturbation that leads to fonnation of a scab, 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. In some embodiments, in which an intermittent lithium treatment is begun following an integumental perturbation that leads to formation of a scab, the pulse lithium treatment is begun within 3 days, 5 days, 7 days, 10 days, 2 weeks, or 3 weeks after integumental perturbation.

[00270] In one embodiment, 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. In some embodiments, 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.

[00271] In some embodiments, an intermittent lithium treatment is combined with a form of integumental perturbation that does not lead to formation of a scab. In one such embodiment, the intermittent lithium treatment is begun at the time of integumental perturbation. In some embodiments, an intermittent lithium treatment is begun following integumental perturbation. In some embodiments, in which an intermittent lithium treatment is begun following an integumental perturbation that does not lead to formation of a scab, 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.

[00272] The Examples in Sections 6 to 37 provide exemplary protocols for carrying out the aforementioned embodiments, although the invention is not meant to be so limited. 5.3.4 COMBINATION TREATMENTS

[00273] intermittent lithium treatment or a pulse lithium treatment in combination with conventional methods for enhancing hair growth or removal of unwanted hair 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. Most drugs for hair loss aim to retain the existing hair follicles in their active cycling states, or to rejuvenate telogen hair follicles to actively cycling anagen states. However, a dosage form that encourages the growth of "new" hair follicles, combined with one that retains the hair follicles in their actively cycling states offer significant value to the individual who is balding. The combined modality of treatment could involve alternating treatment of each dosage form or concurrent or simultaneous treatment.

[00274] While not intending to be bound by any theory, a reason for this enhancing effect, or synergism, is that intermittent lithium treatment or a pulse lithium treatment synchronizes the hair/Follicle Cycle. Since all the follicles are at the same stage in their growth cycle, a treatment intended to enhance hair growth or remove hair will be more effective, efficient, cost-effective, and user friendly. For example, fewer treatments may be required.

[00275] 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).

[00276] In one embodiment, 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. For example, in some embodiments, one or more of the compounds in the formulation undergoes controlled release, whereas one or more of the other compounds does not. For example, one or more of the compounds in the formulation undergoes sustained release whereas one or more of the other compounds undergoes delayed release. [00277] In another embodiment, the combination treatment comprises lithium and an additional compound(s) formulated separately. The separate formulations may be administered concurrently, sequentially, or in alternating sequence. For example, the lithium compound may be administered sequentially, or concurrently with another compound such as finasteride or minoxidil, to achieve the desired effect of hair retention and growth.

[00278] In some embodiments, 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, vims-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. , 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, 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

chlorohydrate, aluminum chlorohydrex, aluminum chlorohydrex PEG, aluminum

chlorohydrex PG, aluminum dichlorohydrate, aluminum dichlorohydrex PEG. aluminum dichlorohydrex PG, aluminum sesquichlorohydrate, aluminum sesquichlorohydrex PEG, aluminum sesquichlorohydrex PG, aluminum sulfate, aluminum zirconium

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 ( A1(S04)212H20)), aluminum undecylenoyl collagen amino acid, sodium aluminum Iactate+ aluminum sulphate

(Na2HAl(OOCCHOHCH3)2-(OH)6) + A12(S04)3), sodium aluminum chlorohydroxylactate, aluminum bromohydrate (Al2Br(OH)5nH20), aluminum chloride (A1C1 6H20), complexes of zinc salt and of sodium salt, complexes of lanthanum and cerium, and the aluminum salt of lipoamino acids (R— CO— NH— CHR'— CO— OA1— (OH)2 with R = C6-n and R'=amino acid), retinoids (e.g., retinoic acid, retinol, retinal, or retinyl esters), sunscreens (e.g., derivatives of para-aminobenzoic acid (PABA), cinnamate and salicylate, avobenzophenone (Parsol 1789®). octyl methoxycinnamate (Parsol™ MCX) and 2-hydroxy-4-methoxy benzophenone (also known as oxybenzone and available as Benzophenone™ , and preservatives), an anti-age cream, a sebum production inhibitor and/or pore size reducing agent (e.g., carboxyalkylates of branched alcohols and/or alkoxylates thereof, e.g., 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. Louis, Missouri), or -methylene-y-butyrolactone), a massage agent, an exfoliant, an anti-itch agent, a pro-inflammatory agent, an immunostimulant (e.g., 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), a cell cycle regulator, a hormonal agonist, 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, mineralocorticoids, estrogen, testosterone, progestins), antigestagens (e.g., mifepristone, onapristone), an antiandrogen (e.g., cyproterone acetate), an antiestrogen, an antihistamine (e.g., niepyramine, diphenhydramine, and antazoline), an anti-inflammatory (e.g., corticosteroids, NTHEs, and COX-2 inhibitors), a retinoid (e.g., 13-cis-retinoic acid, adapalene, all-trans-retinoic acid, and etretinate), an immunosuppressant (e.g. , cyclosporine, tacrolimus, rapamycin, everolimus, and

pimecrolimus), an antibiotic, an anti-cancer agent (such as, e.g., fluorouracil (5-FU or f5U) or other pyrimidine analogs, methotrexate, cyclophosphamide, vincristine), a mood stabilizer (e.g., valproic acid or carbamazepine) an antimetabolite, an anti-viral agent, and an antimicrobial (e.g., benzyl benzoate, benzalkonium chloride, benzoic acid, benzyl alcohol, butylparaben, ethylparaben, methylparaben, propylparaben, camphorated metacresol, camphorated phenol, 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)). [00279| In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with integumental perturbation and, optionally, also comprises another treatment known in the art or described herein.

Integumental perturbation can be achieved by any means known in the art or described herein, such as, for example, using chemical or mechanical means. In one embodiment, integumental perturbation comprises disrupting the skin of the subject (for example, resulting in the induction of re-epithelialization of the skin of the subject ). In some embodiments, a certain area of the epithelium is partially or wholly disrupted. In some embodiments, a certain area of both the epithelium and stratum corneum are partially or wholly disrupted. For a discussion of skin disruption and re-epithelialization, including methods for disrupting skin and inducing and detecting re-epithelialization, see PCT Publication Nos. WO

2008/042216 and WO 2006/105109, each of which is incorporated herein by reference. Integumental perturbation can be used to induce, for example, a burn, excision,

dermabrasion, full-thickness excision, or other form of abrasion or wound.

[00280] 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 C02-based, or Erbium- Y AG-based, etc.). Integumental perturbation can also be achieved through, for example, the use of visible, infrared, ultraviolet, radio, or X-ray irradiation. In one embodiment, 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, hair transplant, cosmetic surgery, etc.

[00281] In some embodiments, integumental perturbation is by laser treatment.

Exemplary laser treatments for integumental perturbation include Erbium- YAG laser, Ultrapulse C02 fractional laser. Ultrapulse C02 ablative laser, Smooth Peel Full-ablation Erbium laser (Candela), as described, for example, in the examples of Sections 7, 9, 1 1 , 13, 15, 17, 1 , 21. 23, and 24, below. In one embodiment, a laser treatment is chosen in which the integumental perturbation achieved most resembles that achieved by dermabrasion (for example, a dermabrasion method described herein). In a preferred embodiment, integumental perturbation by laser treatment is by a fractional laser. See, e.g., the laser treatments described in U.S. Provisional Application Nos. 61/262,820, 61/262,840, 61/262,831 , each of which is incorporated herein by reference in its entirety. One example of 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. The column of dead (epidermal and/or dermal) cells in the path of the laser treatment is termed a "coagulum." In another embodiment, integumental perturbation by laser treatment is by a fractional laser, using, e.g., a CO2 laser at 10,600 nm. Treatment with a C02 laser at 10,600 nm is typically ablative, and typically leads to the appearance of pinpoint bleeding.

[00282] A standard C02 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.

[00283] In one embodiment, the laser treatment is ablative. For example, full ablation of tissue is generated by the targeting of tissue water at wavelengths of 10,600 nm by a C02 laser or 2940 nm by an Erbium-YAG laser. In this mode of laser treatment, 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. In another variation, the depth of ablation may extend partially into the dermis, to generate a deep wound. The denuded skin surface is then treated with a lithium composition described herein; alternatively, the lithium composition can be delivered into the skin after the initial re-epithclialization has occurred already, to prevent clearance and extrusion of the lithium-containing depots from the tissue site by the biological debris- clearance process. In one embodiment, a lithium composition described herein is delivered by a sustained release depot that is comprised of biocompatible, bioabsorbable polymers that are compatible to tissue.

[00284J The standard full thickness excision model is created using scissors or with a scalpel in animal models (see, also, the examples of Section 28-30, and 32 infra). This procedure, while contemplated for use herein (see, e.g., the example of Section 36 infra), carries with it the risk of scarring. However, 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.

[00285] In some embodiments, the laser treatment is ablative and fractional. For example, fractional tissue ablation can be achieved using a C02 laser at 10,600 nm or an Erbium- YAG laser at 2940 nm (e.g., the Lux 2940 laser, Pixel laser, or Profractional laser). In some such embodiments, 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. The density of micro thermal zones (MTZ) 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. In one embodiment, a lithium composition described herein is delivered into the dermis immediately after wounding, or after initial re-epithelialization has occurred.

[00286] In another embodiment, 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. For example, one could use the timed cooling of the epidermis with a cryogen spray while the laser delivers deep thermal damage to the dermis. In this application, the depth of treatment may be 1 mm to 3 mm into the skin. One could also use 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. In one embodiment, 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), the 1064 Nd:YAG laser, or the Erbium- YAG laser at 1540 nm or 1550 nm (e.g., the Fraxel® laser). Use of an Erbium- YAG laser at around 1540 run or around 1550 nm, as opposed to its use at 2940 nm, "'coagulates" zones of dermis and epidermis (forming a "coagulum") and leaves the stratum corneum essentially intact.

[00287] In another embodiment, 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. As in any non-ablative process, the skin barrier function is maintained, while deep thermal heating of dermis can occur. Thus, 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. In one embodiment, a lithium composition described herein is delivered immediately after the tissue injury, deep into the dermis. In another embodiment, a combination of bulk and fractional ablation modes of tissue injury are used.

[00288] In a specific embodiment, the mode of laser treatment for, e.g., a Caucasian male 30-50 years old, is fractional and non-ablative using an Erbium- YAG laser at 1550 nm, with the following settings: 50-70 J/cm2, treatment level 8-10 (density of the "dots"), with 8 passes. In this regard, the laser device can be equipped with a touch pad screen that offers the operator a menu of options for setting the parameters for operating the laser to promote hair growth. For example, the device can be programmed to offer the operator selections for hair growth vs. removal, choice of skin color, hair follicle density, power settings, etc.

[00289] In another embodiment, 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. [00290] In another embodiment, 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.

[00291 ] In another embodiment, 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. In one embodiment, 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 bald area on the scalp in one session at the treatment clinic. In some embodiments, 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.

[00292] In some embodiments, 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. For example, 1 -mm punch biopsies can be arranged with 1-mm hole spacing. When inserted into the scalp or other area of skin to be treated, the cored skin samples can be removed and, thus, an effect approximating the full thickness excision model is invoked within each hole. Similarly, and for smaller holes, microneedles (e.g., 19 or 21 gauge needles) and/or micro-coring needles could be used.

[00293] In some embodiments, 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 - 1 3 4). 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 fraises operated at different rotation speeds to remove the epidermis and dermis to differing skin depths levels. Adult human skin treated with DA completely re- epithelializes in 5-7 days with minor redness lasting up to a few weeks. Dermabrasion may be carried out using any technique known in the art or as described herein, e.g. , in the examples of Sections 25, 26, and 28-36 (see, e.g. , Sections 35 and 36 for protocols for dermabrasion in human subjects), infra. For example, as described in the examples of Sections 25, 26, 35, and 36, dermabrasion may be carried out using standard DA with aluminum oxide crystals using the Aseptico Econo-Denmabrader, Advance Microderm DX system, or M2-T system; standard DA with Bell Hand Engine with diamond fraize; etc.

[00294] For example, in some embodiments, DA is carried out using an abrasive wheel. In some embodiments, DA with an abrasive wheel is used in order to achieve pinpoint bleeding. In other embodiments, DA may be carried out using an abrasive wheel to achieve larger globules of bleeding and frayed collagen. In other embodiments, non-powered devices such as abrasive cloths can also be used to achieve the DA, with the optional achievement of the same endpoint(s).

[00295] In some embodiments, DA is accomplished using a device typically used for microdermabrasion. For example, in such DA protocols, 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"'). In some embodiments, the microdermabrasion device is used under sterile conditions. In some embodiments, 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. In other embodiments, DA is achieved by using a device for

microdermabrasion to the point where treatment is stopped upon the observation of larger globules of bleeding and frayed collagen, which signals the removal of the stratum corneum and epidermis into the papillary and reticular dermis. In some embodiments, 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.

[00296] In some embodiments, 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. In some such embodiments, 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.

[00297] In some embodiments, 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

embodiments, integumental perturbation disrupts the papillary dermis. In some

embodiments, integumental perturbation removes the papillary dermis. In some

embodiments, 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.

[00298] In some embodiments, integumental perturbation by one or more of the aforementioned methods is to a skin depth of 60 μιη. In some embodiments, integumental perturbation is to a skin depth of 60- 100 μπι. In some embodiments, integumental perturbation is to a skin depth of 100 μηι. In some embodiments, integumental perturbation is to a skin depth of 150 pm. In some embodiments, integumental perturbation is to a skin depth of 100-500 μιη. In some embodiments, integumental perturbation is to a skin depth of less than 500 μηι. In some embodiments, integumental perturbation is to a skin depth of 500- 1000 μηι. In some embodiments, 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.

Discussion

[00299] The pulse or intermittent lithium treatments described herein potentiate the formation of new hair follicles. Integumental perturbation produces in the affected skin tissue an increase in the number of hair follicle stem cells and in the plasticity of hair follicle cells, such that resident hair follicles may be reprogrammed. Accordingly, and without being bound by any theory for how the invention works, integumental perturbation in combination with a pulse or intermittent lithium treatment provides an environment for the formation of a large number of follicles with desired properties. [00300] New hair follicles originate from Hair Follicle Stem Cells (FSCs), oligopotent cells whose progeny can differentiate into the highly differentiated specialized cells of the hair follicle (see Amoh Y. et al. Human hair follicle pluripotent stem (hfPS) cells promote regeneration of peripheral-nerve injury: an advantageous alternative to ES and iPS cells. J Cell Biochem, 2009, 107: 1016-1020; and Amoh Y, et al. Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells. Proc Natl Acad Sci U S A. 2004 Sep

7; 101 (36): 13291-5. Epub 2004 Aug 26).

[00301] 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. 2009; (ii) the skin ("tissue derived follicle stem cells" or "TDFSC") (see, e.g. , Ito M, 2007, Nature 447:316-320); (iii) bone marrow ("bone marrow derived follicle stem cells" or "BMDFSC") (see, e.g. , Fathke et al., 2004, Stem Cells 22:812-822; and Rovo et al., 2005, Exp Hematol. 33:909-91 1); and/or (iv) from mesenchymal stem cells such as adipocyte stem cells.

[00302] FSCs generate new hair follicles that preserve the type of hair follicle that is typical for each location of skin or scalp. For example, FSCs from the coronal scalp of a male with MPHL typically generate atrophic follicles with vellus or club hairs. In contrast, 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.

[00303] However, if external signals are provided that interfere with this "default" program, the 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. For example, FSCs from the coronal scalp of a male with MPHL, under the influence of estrogen, can generate follicles with terminal hair that are not subject to involution in response to DHT. Such follicles have characteristics usually associated with: (i) pre-alopecia follicles in the coronal scalp; (ii) female-type follicles on the coronal scalp; or (iii) occipital scalp type follicles. Alternatively, by antagonizing estrogen or testosterone, the assumption of the default hair pattern in a particular skin area may be prevented. For example, a female's unwanted moustache hair may be reduced by perturbing the skin of the upper lip and administering a testosterone antagonist.

[00304J Thus, 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. In some embodiments, the third treatment (e.g. , estrogen or testosterone modulator, such as those described in Poulos & Mirmirani, 2005, Expert Opin. Investig. Drugs 14: 177-184 (incorporated herein by reference) is administered simultaneously with integumentai perturbation. In some embodiments, the third treatment is administered after integumentai 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 integumentai perturbation. In one embodiment, the third treatment is administered at the time of integumentai 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. In some such embodiments, the third treatment is administered at the time of integumentai perturbation and up to some time after scab falls off , for example, between 5 - 14 days following integumentai perturbation. In some embodiments, 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 integumentai perturbation, lithium administration, and the third treatment is preferably monitored and adjusted so that optimal results are achieved.

[00305] The following sections contain non-limiting examples of combination treatments that are specific for, respectively, (i) enhancing hair growth and/or treating conditions associated with hair loss and (ii) removal of unwanted hair and/or treating conditions associated with excessive hair growth. The treatments described in these sections may optionally be combined with the aforementioned treatments and/or with one another.

5.3.4.1 COMBINATION TREATMENTS FOR ENHANCING HAIR GROWTH

[00306] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a treatment that enhances hair growth and/or treats a disease or condition associated with excessive hair loss. Any treatment that enhances hair growth and/or treats a disease or condition associated with excessive hair loss that is known in the art or yet to be developed is contemplated for use in such combination treatments.

[00307] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more antiandrogens, such as. e.g., finasteride (e.g., marketed as Propecia or Proscar), fluconazole, spironolactone, flutamide, diazoxide, 17-alpha-hydroxyprogesterone, 1 1-alpha-hydroxyprogesterone, ketoconazole, RU58841 , dutasteride (marketed as Avodart), fluridil, or QLT-7704, an antiandrogen oligonucleotide, or others described in Poulos & Mirmirani. 2005, Expert Opin. Investig. Drugs 14: 177-1 84, the contents of which is incorporated herein by reference.

Commonly used dosage forms of finasteride that may be used in such combination therapies are, for example, oral finasteride at 1 mg/day. See, e.g. , Physicians ' Desk Reference, 2009, 63rd ed., Montvale, NJ: Physicians' Desk Reference Inc., entries for Propecia® and Proscar® at pages 2095-2099 and 2102-2106, respectively, which are incorporated herein by reference in their entireties. The regular dosages may be increased or decreased as directed by the physician. For example, a lower dosage may be used over a shorter duration owing to the synergistic effect of the combination with the intermittent lithium treatment or a pulse lithium treatment.

[00308] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more channel openers, such as, e.g., minoxidil (e.g., marketed as Rogaine or Regaine), diazoxide, or phenytoin.

Commonly used dosage forms of minoxidil that may be used in such combination therapies are topical solutions comprising 2% minoxidil or 5% minoxidil, for example, topical minoxidil foam 5%. The regular dosages may be increased or decreased as directed by the physician. For example, a lower dosage may be used over a shorter duration owing to the sy nergistic effect of the combination with the intermittent lithium treatment or a pulse lithium treatment.

[00309] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with an antiandrogen (e.g., finasteride, 5-alpha reduction inhibitors) and a channel opener (e.g. , minoxidil).

[00310J In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more antiandrogens, such as ketoconazole, herbs (such as, e.g. , saw palmetto, glycine soja, Panax ginseng, Castanea Sativa, Arnica Montana, Hedera Helix Geranium Maculatum), triamcinolone acetonide (e.g., suspension of 2.5 to 5 mg/ml for injection), a topical irritant (e.g. , anthralin) or sensitizer (e.g. , squaric acid dibutyl ester [SADBE] or diphenyl cyclopropenone [DPCPJ),

clomipramine, unsaturated fatty acids (e.g., gamma linolenic acid), thickeners (such as, e.g., carbomer, glycol distearate, cetearyl alcohol), caffeine or coffee, a hair loss concealer, bimatoprost (trade name: Latisse), niacin, nicotinate esters and salts, adenosine, methionine, or CaCl2. In some embodiments, the combination therapy comprises intermittent lithium treatment or a pulse lithium treatment in combination with nitroxide spin labels (e.g., TEMPO and TEMPOL). See United States Patent 5,714,482. which is incorporated herein by reference.

[00311 J In some embodiments, the combination treatment comprising intermittent lithium treatment or a pulse lithium treatment to enhance hair growth in a female subject is not finasteride or ketoconazole. In some embodiments, the combination therapy comprising intermittent lithium treatment or a pulse lithium treatment to enhance hair growth in a pregnant, female subject is not finasteride or ketoconazole.

[00312] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a copper peptide(s), preferably applied topically, or another compound with superoxide dismutation activity. In some embodiments, the combination therapy comprises intermittent lithium treatment or a pulse lithium treatment in combination with an agent that increases nitric oxide production (e.g. , arginine. citrulline, nitroglycerin, amyl nitrite, or sildenafil (Viagra)). In preferred embodiments, such compounds are administered further in combination with a catalase or catalase mimetic, or other antioxidant or free radical scavenger.

[00313] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more agents 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; 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.

[00314] In some embodiments, 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. In some embodiments, 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 intlammation in the site where hair growth is desired helps to recruit stem cells to the tissues that drive the formation of new follicles.

[00315] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with an antiapoptotic compound. In one embodiment, the antiapoptotic compound is not a Wnt or a Wnt agonist.

[00316] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more of stem cell therapy, hair cloning, hair transplantation, scalp massage, a skin graft, hair plugs, or any surgical procedure aimed at hair restoration.

[00317J In some embodiments, 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. For example, the Hairmax Lasercomb or the Leimo laser are non-limiting examples of devices that can be used to stimulate growth of hair and can be used in combination with the methods described herein.

[00318] In certain embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for enhancing hair growth prevents, delays, or reverses scalp hair loss in MPHL and/or diffuse hair thinning associated with aging. In some embodiments, intermittent lithium treatment or a pulse lithium treatment, alone or in combination with the aforementioned methods for enhancing hair growth, synchronizes hair follicle cells in the cell cycle. In a specific embodiment, 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). In another

embodiment, 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). In another embodiment, 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). In another embodiment, the lithium treatment arrests hair follicle stem 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 drugsfwhich leads to anagen follicles, and an increased number of follicles).

[00319] In some embodiments, intermittent lithium treatment or a pulse lithium treatment, alone or in combination with the aforementioned methods for enhancing hair growth, synchronizes hair follicle cells in the Follicle Cycle. In one such embodiment, the treatment regimen induces follicles to enter anagen. In another embodiment, the treatment regimen prevents follicles from entering catagen. In one embodiment, the treatment regimen induces follicles in telogen to enter exogen. or induces follicles in exogen to enter anagen.

[00320] In certain embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for enhancing hair growth improves the effectiveness of these methods, making the treatment more effective, efficient, cost-effective, and/or user friendly. For example, fewer treatments may be required. In certain

embodiments, one of the previously described hair growth enhancement treatments on its own is not cosmetically satisfactory, the benefits are too short-lived, or the hair that results from the treatment is vcllus hair, or other thin or patchy hair, or has inadequate pigmentation. When one of these treatments is combined with intermittent lithium treatment or a pulse lithium treatment, the hair that results may be more cosmetically satisfactory, longer lasting, terminal hair or head hair (depending on the type of hair intended as opposed to vellus hair, and/or thicker, more uniform, and properly pigmented hair. In certain embodiments, more than one hair will emerge from each follicle, leading to the appearance of thicker hair.

[00321] In certain embodiments, one of the aforementioned treatments to enhance hair growth is administered following integumental perturbation (as described herein) and lithium treatment. In one exemplary embodiment, integumental perturbation is followed by lithium treatment, which is then followed by one of the aforementioned treatments to enhance hair growth. In another exemplary embodiment, integumental perturbation accompanies lithium treatment, which is then followed by one of the aforementioned treatments to enhance hair growth. In another embodiment, integumental perturbation is prior to lithium treatment administered together with one of the aforementioned treatments to enhance hair growth.

[00322] In addition to the examples in Sections 6-13, 24, 25. 32, 35, and 36, the following embodiment is illustrative of the methods described herein. Integumental perturbation is achieved by either treatment with a fractional Erbium- YAG laser to epidermal or dermal depth, a fractional C02 laser to epidermal or dermal depth, or dermabrasion as described herein. This is followed by the stimulation of follicle formation by a pulse lithium treatment. Then, the follicles (or Follicle Stem Cells) are reprogrammcd, e.g., a miniaturizing male temporal scalp follicle (or Follicle Stem Cell) is changed to a non-miniaturizing female-type temporal scalp follicle (or Follicle Stem Cell) using one or more of the following drugs: estrogen, finasteride, dutasteride (Avodart™). Alternatively, the follicle type can be reprogrammed, e.g. , a miniaturizing male temporal scalp follicle can be changed to a non- miniaturizing male occipital scalp-type follicle, using a drug such as valproate.) Then, terminal hair growth is stimulated by the application of low energy light (using, e.g. , LaserMax or IPL) or minoxidil.

[00323] In certain embodiments, enhancement of hair growth is accomplished by a combination of integumental perturbation, as described herein, and lithium treatment without one of the aforementioned treatments for enhancing hair growth. In some embodiments, the combination of integumental perturbation and lithium treatment of an area of skin that already contains hair-producing follicles (preferably, terminal hair) increases production of hair in that area of skin. In some embodiments, the combination of integumental perturbation and lithium treatment is administered to skin that has been damaged and which no longer contains follicles. In such embodiments, the combination of integumental perturbation and lithium treatment may restore follicle production in that area of skin. In one such embodiment, an area of skin containing a wound that has not healed correctly, such as a scar (e.g., a keloid scar), is administered a combination treatment of integumental perturbation and lithium in order to restore hair follicles and/or growth to that area of skin.

[0032 J In certain embodiments, enhancement of hair growth is accomplished by lithium treatment alone. In some embodiments, lithium treatment of an area of skin that already contains hair-producing follicles increases production of hair in that area of skin. In some embodiments, the lithium treatment is administered to skin that has been damaged and which no longer contains follicles. In such embodiments, the lithium treatment may restore follicle production in that area of skin. In one such embodiment, 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. These effects may be accomplished by modulating the dosage of lithium.

[00325] 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 multiple shafts per pore were produced by the combination and not by either alone. 5.3.4.2 COMBINATION TREATMENTS FOR INHIBITING HAIR GROWTH OR REMOVAL OF UNWANTED HAIR

[00326] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more techniques of depilation (removal of the part of the hair above the surface of the skin) or epilation (removal of the entire hair, including the part below the skin) of a part of the skin affected by unwanted hair. Intermittent lithium treatment or a pulse lithium treatment can be used in combination with any form of epilation or depilation known in the art. Any treatment that inhibits the growth of unwanted hair, removes unwanted hair and/or treats a disease or condition associated with unwanted hair that is known in the art or yet to be developed is contemplated for use in such combination treatments. For example, see U.S. Patent No. 6,050,990, issued April 18, 2000, which is incorporated herein by reference in its entirety.

[00327] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more prescription drugs such as, e.g., eflornithine (trade name: Vaniqa, which is formulated as eflornithine hydrochloride, 13.9%) or another ornithine decarboxylase inhibitor, and/or 5-fluorouracil (5- FU, Efudex 5% cream). In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with epidermal growth factor ( EGF) or a mimetic thereof.

[00328] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more polyamine derivatives and/or analogs including pharmaceutically acceptable salts and solvates thereof; inhibitors of ornithine decarboxylase, such as difluoryl methyl ornithine (DFMO),

methylglyoxalbisguanylhydrazone (MGBG), hydrozino ornithine (HAVA), and mixtures thereof; N-acetyl cysteines ( AC); neutralized salts of a non-hydroxy C2-C40 dicarboxylic acids, preferably malonate salts; and mixtures thereof. See, e.g.. International Patent

Application Publication No. WO 2005/120451 , published December 22, 2005, which is incorporated herein by reference in its entirety.

[00329] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with Stryphnodendron adstringens (Martius) Coville bark extract, which suppresses terminal hair. See Vicente et al., 2009. J Eur Acad Dermatol Venereol 23:410-414. In another embodiment, the combination therapy comprises the use of fennel. See Javidnia et al., 2003, Phytomedicine 10:455-458. [00330] In some embodiments, the combination treatment for hair removal comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more antiandrogen drugs, such as cyproterone acetate, ketoconazole, or spironolactone (the latter marketed under the trade names Aldactone, Novo-Spiroton, Aldactazide, Spiractin,

Spirotone, Verospiron or Berlactone). In one embodiment, such therapy is for use in female subjects affected by idiopathic hirsutism. In some embodiments, such anti-androgen combination therapy is not for use in female subjects predisposed to, at risk for, or suffering from, cancer.

[00331] In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a pro-apoptotic compound or a cytotoxic agent (such as, e.g. , those described in U.S. Patent No. 6,050,990, which is incorporated herein by reference). In one embodiment, the pro-apoptotic compound is not a Wnt antagonist.

[00332] In some embodiments, the combination treatment for hair removal comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more of hair bleaching, shaving, waxing, sugaring, threading, plucking, use of an abrasive material, laser, electrolysis or electrology, use of an epilation device (e.g., commercially available devices such as Emjoi®, Epilady®, Duet, Legend, and EpiGirl, While Philips Norelco's Satinelle Epilator, and similar products by Panasonic, Braun, and Norelco), use of friction, exfoliation, burning, intense pulsed light ("IPL"; e.g., Flashlamp or F.piLight), use of a mechanical device, or use of a chemical depilatory such as thioglycolic acid (e.g., Nair®), use of turmeric optionally with other ingredients like besan powder and milk, or enzymes such as the Epiladerm-Complex.

[00333] In certain embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for hair removal or inhibiting hair growth improves the effectiveness of these methods, making the treatment more effective, efficient, cost-effective, and/or user friendly. For example, fewer treatments may be required. In certain embodiments, one of the previously described hair removal or growth inhibition treatments on its own is not cosmetically satisfactory, the benefits are too short-lived, or the hair that remains or grows back after the treatment is too pigmented, thick or course. When one of these treatments is combined with intermittent lithium treatment or a pulse lithium treatment, the results may be more cosmetically satisfactory, longer lasting, and the hair that remains or grows back are fewer in number, thinner (e.g., vellus hair only), less pigmented, and/or shorter. In certain embodiments, a subject previously had one or more hair follicles with numerous hairs emerging from it, and after the combination therapy, only one hair, preferably a vellus hair, emerges from each follicle, leading to the appearance of thinner hair.

[00334] In particular embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for hair removal or inhibiting hair growth prevents, delays, or reverses terminal hair growth on female axilla, female face, female legs, male ears, male nose, or male back.

[00335] In particular embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for hair removal or inhibiting hair growth synchronizes hair follicle cells in the Cell Cycle. In one such embodiment, the lithium treatment arrests hair follicle cells in G2/M phase, which synchronizes them; the lithium treatment is removed; and then their entry into the cell cycle and mitotic division is stimulated with other drugs that expose their DNA and renders them susceptible to cytotoxic drugs or apoptosis-inducing drugs that cause them to enter catagen and involute. In another embodiment, the lithium treatment arrests hair follicle cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed; and then their entry into the cell cycle and mitotic division is stimulated with other drugs, which exposes their DNA and renders them susceptible to cytotoxic drugs or apoptosis-inducing drugs, causing them to enter catagen and involute. In another embodiment, the lithium treatment arrests hair follicle stem cells in G2/M phase, which synchronizes them; the lithium treatment is removed; and then their entiy into the cell cycle and mitotic division is stimulated with other drugs which exposes their DNA and renders them susceptible to cytotoxic drugs or apoptosis-inducing drugs, causing the stem cells to enter catagen and involute. In another embodiment, the lithium treatment arrests hair follicle stem cells in late prophase or metaphase, which synchronizes them; the lithium treatment is removed and then their entry into the cell cycle and mitotic division is stimulated with other drugs which expose their DNA and renders them susceptible to cytotoxic drugs or apoptosis-inducing drugs, and thus they enter catagen and involute.

[00336] In particular embodiments, intermittent lithium treatment or a pulse lithium treatment in combination with the aforementioned methods for hair removal or inhibiting hair growth synchronizes hair follicles in the Follicle Cycle. For example, the lithium treatment may induce follicles to leave anagen, enter catagen and involute, or enter exogen. The lithium treatment may inhibit follicles from entering anagen, or maintain follicles in telogen, or maintain follicles in exogen. [00337] In certain embodiments, one of the aforementioned treatments to inhibit hair growth or remove unwanted hair is administered following integumental perturbation (as described herein) and lithium treatment. In one exemplary embodiment, integumental perturbation is followed by lithium treatment, which is then followed by one of the aforementioned treatments to inhibit hair growth or remove unwanted hair. In another exemplary embodiment, integumental perturbation accompanies lithium treatment, which is then followed by one of the aforementioned treatments to inhibit hair growth or remove unwanted hair. In another embodiment, integumental perturbation is prior to lithium treatment administered together with one of the aforementioned treatments to inhibit hair growth or remove unwanted hair.

[00338] In certain embodiments, inhibition of hair growth or removal of unwanted hair is accomplished by a combination of integumental perturbation, as described herein, and lithium treatment w ithout one of the aforementioned treatments for inhibition of hair growth or removal of unwanted hair. In some embodiments, the combination of integumental perturbation and lithium treatment of an area of skin that already contains follicles that do not produce hair or that produce only vellus hair further reduces hair in that area of skin.

[00339] In certain embodiments, inhibition of hair growth or removal of unwanted hair is accomplished by lithium treatment alone. In some embodiments, lithium treatment of an area of skin that already contains follicles that do not produce hair or that produce only vellus hair further reduces hair in that area of skin. These effects may be accomplished by modulating the dosage of lithium.

5.3.4.3 REGIMENS FOR COMBINATION

TREATMENTS

[00340] For any of the combination treatments described above, in specific embodiments, 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.

[00341] In one embodiment, 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. [00342J In another embodiment, the intermittent lithium treatment or a pulse lithium treatment and other treatment are administered at exactly the same time.

[00343] In yet another embodiment, 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. In another embodiment, 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. In one embodiment, the intermittent lithium treatment or a pulse lithium treatment and the other treatment are administered by different routes of

administration. In an alternate embodiment, 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. When administered simultaneously, 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.

[00344) In various embodiments, 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, 1 1 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart. In other embodiments, 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. In some embodiments, 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.

[00345] In one embodiment, 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.

[00346] In certain embodiments, 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. Not only does such cycling treatment have the advantages described herein (attributable, at least in part, to the synchronization of the hair and/or Follicle Cycle), 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. In such embodiments, 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. In certain embodiments, 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.

[00347] As described in the examples in Sections 6 to 23 and 36, infra, in some embodiments, subjects discontinue their current treatment (e.g., topical minoxidil, finasteride, eflornithine), and the lithium compound is applied for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with the lithium compound is discontinued and treatment with the current treatment is re-started. In some embodiments, the subject is treated with 10 cycles of the protocol: alternating topical lithium (e.g., for 1 week) with the other treatment (e.g. , for 3 weeks).

[00348) For embodiments in which the lithium treatment accompanies hair transplantation (e.g., follicular unit extraction), an area of scalp that was pre-treated with lithium and, e.g., minoxidil or finasteride, is used as a source for transplanted follicles. After hair follicle implantation, 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 followed by treatment with, e.g. , minoxidil or finasteride for three weeks.

[00349J Other regimens for combination treatments for use in the methods described herein include those described in Sections 5.3.1 - 5.3.3 supra.

5.4 INDICATIONS AND TREATMENT REGIMENS FOR ALOPECIA

[00350] Human hair patterning consists of gender specific changes that occur over the life of subjects and vary in degree between individuals and more generally between humans of different racial and ethnic backgrounds. Before puberty, males and females have similar patterns of scalp hair and the rest of their bodies are covered with largely invisible vellus hair. The forearms and legs grow thin, fine terminal hair gradually even before puberty. In males and females puberty is associated with terminal hair growth in the axilla, and anogenital regions. During puberty, both males and females grow terminal hair over forearms and legs, but males have quantitatively more growth in these regions. Males after puberty grow terminal hair over the moustache/beard, chest, and back regions. Later, males manifest varying degrees of loss of terminal hair on the scalp (vertex/corona and frontal/

temporal/parietal regions) in a process called "MPHL alopecia." Both males and females with genetic susceptibilities manifest diffuse scalp terminal hair thinning that is called "'androgenetic alopecia." Males manifest various degrees of terminal hair growth on ears, in the nose (nares) and of eyebrows. After menopause, females manifest moustache/beard hair growth. Both males and females manifest diffuse scalp hair thinning with age. Both females and males with genetic susceptibility manifest hair color changes with age.

[00351] The sex hormones, androgens and estrogens, play important roles in much of human hair patterning. A high ratio of androgen to estrogen activity drives both the process of transforming vellus hair to terminal hair (in puberty, e.g. axilla and anogenital regions; male moustache and beard) and in transforming terminal hair (and large terminal follicles) to vellus hair (and miniaturized follicles) in MPHL alopecia. Sex hormones are synthesized by the skin locally where they exert intracrine or paracrine actions. (Reviewed in, Zouboulis CC, Chen VVC. Thornton MJ, Qin , Rosenfield, 2007, "Sexual hormones in human skin" Horm Metab Res 39:85-95) The local level and activity of each sex steroid depends upon the activity of androgen- and estrogen-synthesizing enzymes and of appropriate androgen- and estrogen receptors in specific cell types. Androgens are regulated by five major enzymes and changes in the expression of isoenzymes; or changes in the expression of androgen receptor and androgen receptor transactivating factors regulate hair patterning. Estrogens are produced in sebaceous glands which express cytochrome P450 aromatase ("aromatase") that converts androgen to 17-beta-estradiol (E2). Estrogens act on Estrogen Receptor alpha (ERalpha) and Estrogen Receptor alpha (ERbeta) in human skin are expressed in site specific localizations. In addition to regulating hair patterning and growth, androgens have effects in sebaceous gland growth and differentiation, epidermal barrier homeostasis and wound healing; and estrogens regulate skin aging, pigmentation, hair growth, sebum production and skin cancer. (Ohnemus et al., 2006, Endocr Rev. 27(6):677-706, "The hair follicle as an estrogen target and source.")

Table 1. Types of Human Hair Follicles Producing Terminal Hair

Pubic (anogenital) - puberty driven, not androgen or estrogen specific, curly intermediate length

Armpit (axillary) - puberty driven, not androgen or estrogen specific, curly intermediate length

Scalp - throughout life, thins with aging

Vertex - induced by androgens in males to miniaturize and involute

Crown- induced by androgens in genetically susceptible males to miniaturize and involute

Temples- induced by androgens in genetically susceptible males to miniaturize and involute

Beard/Moustache- induced by androgens to at puberty, suppressed by estrogens, curly intermediate length

Chest/back- induced by androgens to at puberty, suppressed by estrogens, short length

Eyebrow - throughout life, thickens/grows with aging in men, straight short length Nose surface - on the surface of the nose; thickens/grows with aging in males, short length

Nose/nostrils (nares) - in the lumen; increases at puberty, thickens/grows with aging in males, short length

Ear (auricle) - appears with aging in males, short length

Leg- induced by androgens in males at puberty, suppressed by estrogens but not as completely as beard, short length

Arm- induced by androgens in males & females at puberty, suppressed by estrogens but not as completely as beard, short length

Eyelid/eyelashes - throughout life, single units

[00352] The location of specialized human hair follicles allow us to introduce a classification (Table 1 and Figure 1) of those follicles that produce terminal hair which highlights the distinctive features including type of hair produced (length, curl);

appearance/disappearance during life and regulation by sex steroids. This classification makes several assumptions in grouping together as similar types certain male and female follicles that may not be justified by future study. For example, except for scalp hair, humans have only small amounts of visible hair until puberty, when specialized hair follicles in the pubic (anogenital) and armpit (axillary) regions begin producing terminal hair. Some believe that pubic and axillary specialized follicles change their activity in response to only androgens in both males and females and that the hair follicles in the female pubic and axillary regions are extremely sensitive to androgens. The classification system is useful to show the variety and unique characteristics of specialized hair follicles in humans that can be modified using the methods described herein.

5.4.1 PATIENT POPULATIONS FOR ALOPECIA

AND HAIR GROWTH

[00353| A candidate subject for intermittent lithium treatment (i.e., alternating lithium treatment with "vacationed iday" periods) or a pulse lithium treatment for promoting hair growth is any subject suffering from hair loss, hair thinning, balding, or who has or has had a disease or condition associated therewith, or who wishes to enhance the growth or thickness of hair.

[00354] The subject may be any subject, preferably a human subject, including male, female, intermediate/ambiguous (e.g. , XO), and transsexual subjects. In certain

embodiments, the subject is a human adolescent. In certain embodiments, the subject is undergoing puberty. In certain embodiments, 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 elderly. In certain embodiments, 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. In some embodiments, the subject is a male 20 to 50 years old. In some embodiments, the subject is a male 20 to 60 years old. In some embodiments, the subject is a male 30 to 60 years old. In some embodiments, the subject is a male 40 to 60 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.

[00355] In one embodiment, the intermittent lithium treatment or a pulse lithium treatment is delivered to an area in which hair growth is desired, for example, the scalp or face (e.g. , the eyebrow, eyelashes, upper lip, lower lip, chin, cheeks, beard area, or mustache area) or another part of the body, such as, e.g. , the chest, abdomen, arms, armpits (site of axillary hair), legs, or genitals. In some embodiments, hair restoration to a wounded or scarred part of the skin is desired. In one embodiment, the scar is caused by surgery, such as a face lift, skin graft, or hair transplant.

[00356] The subject may have a disease or disorder of balding or hair loss (including hair thinning), such as forms of nonscarring (noncicatricial) alopecia, such as androgenetic alopecia (AGA), including MPHL or FPHL (e.g. , thinning of the hair, i.e., diffuse hair loss in the frontal/parietal scalp), or any other form of hair loss caused by androgens, toxic alopecia, alopecia areata (including alopecia universalis), scarring (cicatricial) alopecia, pathologic alopecia (caused by, e.g. , medication, trauma stress, autoimmune diseases, malnutrition, or endocrine dysfunction), trichotillomania, a form of hypotrichosis, such as congenital hypotrichosis, or lichen planopilaris, or any other condition of hair loss or balding known in the art or described infra.

[00357] In some embodiments, the subject has hair loss caused by a genetic or hereditary disease or disorder, such as androgenetic alopecia.

[00358] In some embodiments, the subject has hair loss caused by anagen effluvium, such as occurs during chemotherapy (with, e.g., 5-fluorouracil, methotrexate, cyclophosphamide, vincristine). In addition to chemotherapy drugs, Anagen effluvium can be caused by other toxins, radiation exposure (such as radiation overdose), endocrine diseases, trauma, pressure, and certain diseases, such as alopecia areata (an autoimmune disease that attacks anagen follicles.)

[00359] In some embodiments, the subject has hair loss caused by telogen effluvium.

Telogen effluvium is caused frequently by drugs like lithium and other drugs like valproic acid and carbamazepinc. In addition to psychiatric drugs, telogen effluvium can be induced by childbirth, traction, febrile illnesses, surgery, stress, or poor nutrition. (See, Mercke et al., 2000, Ann. Clin. Psych. 12:35-42). [00360] In some embodiments, the subject has hair loss caused by or associated with medication, such as chemotherapy (e.g. , anti-cancer therapy or cytotoxic drugs), thallium compounds, vitamins (e.g. , vitamin A), retinoids, anti-viral therapy, or psychological therapy, radiation (e.g. , such as the banding pattern of scalp hair loss that may be caused by radiation overdose), trauma, endocrine dysfunction, surgery, physical trauma, x-ray atrophy, burning or other injury or wound, stress, aging, an autoimmune disease or disorder, malnutrition, an infection (such as, e.g., a fungal, viral, or bacterial infection, including chronic deep bacterial or fungal infections), dermatitis, psoriasis, eczema, pregnancy, allergy, a severe illness (e.g. , scarlet fever), myxedema, hypopituitarism, early syphilis, discoid lupus erythematosus, cutaneous lupus erythematosus, lichen planus, deep factitial ulcer, granuloma (e.g. , sarcoidosis, syphilitic gummas, TB), inflamed tinea capitis (kerion, favus), a slow-growing tumor of the scalp or other skin tumor, or any other disease or disorder associated with or that causes balding or hair loss known in the art or described infra.

[00361] In some embodiments, the subject has hair thinning, or "shock loss," or a bald patch caused by prior use as a source of tissue or follicles for hair transplantation or follicular unit transplantation.

[00362] In some embodiments, a candidate subject is a subject who wishes to enhance hair growth, for example, to have more hair, faster-growing hair, longer hair, and/or thicker hair. In some embodiments, the candidate is a subject who wishes to increase hair pigmentation. In some embodiments, the subject is not affected by a condition of excessive hair loss.

5.4.2 ANDROGENET C ALOPECIA

[00363] Both males and females develop diffuse hair loss in the frontal/parietal scalp called "thinning," which begins between 12 and 40 years of age. Perhaps more than males, females notice (and complain of) diffuse hair thinning progressively in middle age more than males, perhaps because diffuse alopecia is more noticeable and problematic for females because they do not suffer from MPHL and retain the frontal hairline. In females, thinning is known as "'Female Pattern Hair Loss (FPHL)" and is caused or exacerbated by androgens. (Price VH, 2003, J Investig Dermatol Symp Proc. 8( l ):24-7, Androgenetic alopecia in women).

[00364J Minoxidil is FDA approved for both males and females. Finasteride, while not approved for females, does provide a benefit. See, PDR entry for Propecia. which is incorporated herein by reference. Intermittent lithium treatment or a pulse lithium treatment can be used in combination with minoxidil or other channel openers, finasteride, dutasteride, flutamide, or other antiandrogens, laser therapy or other mode of photo-stimulation of hair follicles, dermabrasion, hair transplantation or other surgical treatment for treatment, or any other treatment provided in Section 5.3 supra. These treatments can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.4.3 MALE PATTERN HAIR LOSS (MPHL)

[00365] After puberty, males begin to lose the scalp hair over the vertex, crown and frontal/parietal areas in a relatively characteristic pattern that is a continuum (described by Hamilton Norwood scale). The loss of scalp hair in men is called MPHL and is known to be a process driven by the androgen, dihydrotestosterone (DHT), which can be inhibited and to some extent reversed by finasteride which inhibits the conversion of testosterone to DHT. Minoxidil can also delay or reverse MPHL.

[00366] Minoxidil and finasteride treatment are more effective at delaying the progression of MPHL than in reversing it. These agents are generally not effective a year or more after hair loss has occurred, consistent with some kind of terminal senescence or involution of the follicle (as will be discussed below). These treatments can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

[00367] Most drugs for hair loss aim to retain the existing hair follicles in their active cycling states, or to rejuvenate telogen hair follicles to actively cycling anagen states.

However, a treatment that encourages the growth of "new" hair follicles, combined with one that retains the hair follicles in their actively cycling states offer significant value to the individual who is balding. Moreover, since intermittent lithium treatment or a pulse lithium treatment synchronizes the hair/Follicle Cycle, a treatment intended to enhance hair growth or remove hair will be more effective, efficient, cost-effective, and user friendly. For example, fewer treatments may be required. The hair that results may be more cosmetically satisfactory, longer lasting, more terminal hair and/or thicker, more uniform, and properly pigmented hair.

5.4.4 AGING

[00368] Aging of humans results in programmed hair patterning, Diffuse hair loss, including thinning of the occipital scalp occurs in aging. [00369J It is believed that hair loss in postmenopausal women is related to the loss of estrogens (and/or a decrease in the estrogen/androgen ratio). Accordingly, in some embodiments, the combination treatments disclosed herein for age-related hair loss comprise a combination of intermittent lithium treatment or pulse lithium treatment and estrogen replacement therapy or androgen inhibition therapy.

[00370] Aging also results in change of follicle cycle control. In males, eyebrows grow longer and nares hair grow longer suggesting that the lengths of telogen and anagen are no longer regulated as closely. In other words, with aging there is a loss of the function of suppressing terminal hair growth. We propose that these changes are due to repopulation of follicles with new follicles that have been seeded by follicle stem cells (FSCs) and educated by nurse cells. Over time, however, an accumulation of errors in nurse cell education occurs, and the number of stem cells derived from bone marrowr decreases. While nurse cells also educate tissue stem cells, they are essential for BMSC. However, the timing of follicle cycle programming has not been transmitted with fidelity, so the lengths are not typical of the original follicles.

[00371] Intermittent lithium treatment or pulse lithium treatment may be used in combination with any of the treatments described in Section 5.3 in order to treat age-related hair loss. These treatments can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.4.5 HAIR COLOR CHANGES

[00372] Hair color changes in both males and females becoming progressively grayer (mixture of gray hair; white hair and black hair) and whiter. Color change is patterned, since scalp hair changes earlier than body beard hair or body hair. Beard hair may also change color in a pattern that follow a moustache line, before ultimately turning uniformly gray (typically a mixture of white and black hair). This is due to decreased melanin content in the hair shaft (supplied by melanocytes associated with hair follicles).

[00373] Using the treatments described herein can result in rejuvenated follicles that do not follow the hair pattern types and recruit/attract melanocytes and as a result produce colored hair. These treatments can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment. 5.4.6 FACTORS THAT REGULATE SEX HORMONE

SENSITIVITY OF HAIR FOLLICLE CELLS

[00374J Cytokines 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-betal 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- betal that inhibited KC growth (Inui et al, 2003, J Investig Dermatol Symp Proc. 8:69-71).

[00375] In certain embodiments, adjuvants and/or other stimulators of local cytokines are used in conjunction with the intermittent lithium treatment or pulse lithium treatment.

Without being bound by any theory, 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 follicle cell cycle and recruit new FSCs to follicles.

[00376] Melatonin is a protein hormone secreted by the pineal gland 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). These treatments can be administered during the lithium treatment "holidays". Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.4.7 TREATMENTS FOR DELAYING OR

REVERSING HUMAN HAIR PATTERNING

[00377] Given the regulation of human hair patterning by sex steroids, it is believed that humans evolved hair patterning to provide social signals in interactions such as mating and dominance. However, current fashion motivates many men to prevent, delay or reverse male MPHL. Minoxidil (an antihypertensive drug that opens the K+ channel) and antiandrogens, such as finasteride, dutasteride or ketoconazole, are reasonably effective in stimulating the growth of vellus and miniaturized hair in certain MPHL conditions. Finasteride is not approved for use in females. However, patient dissatisfaction with statistically significant, but cosmetically insignificant increase in hair counts contribute to poor compliance and unsatisfactory outcomes. Minoxidil use is further complicated by the fact that it is messy, leaves a residue, and requires daily application. In addition, the side effects from persistent finasteride or minoxidil treatment - such as sexual dysfunction - are another reason why subjects may benefit from a reduced treatment duration or treatment at a lower dose as may be enabled by the combination treatments described herein. Importantly, both minoxidil and finasteride are effective only for as long as it is taken; the hair gained or maintained is lost within 6-12 months of ceasing therapy. See, e.g., Rossi, ed., 2004, Australian Medicines Handbook. Adelaide: Australian Medicines Handbook. Even when effective, these drugs do no create hair follicles of the kind that were there before balding, and the resultant hair follicles are smaller and less dense.

[00378] Women also suffer from hair thinning and hair loss due to a variety of factors; for example, certain conditions, such as, e.g., polycystic ovary, result in male-pattern facial and body hair on females, which motivates them to remove or reduce hair. Many women also desire the prevention, delay or reversal of "female-pattern baldness," which may result from a variety of factors, for example, the aging process.

[00379] Similarly, current fashion motivates many women to remove facial, axillary and leg hair ("unwanted hair') by shaving, laser, depilation, plucking, wax, electrolysis, and medications such as Vaniqa (eflornithine hydrochloride 13.9%) (as well as others).

Competitive advantages motivate swimmers, body builders, and certain other athletes to remove body hair, and current fashion motivates many men to reduce or eliminate back hair and hair from other parts of the body such as nose hair, ear hair, and eyebrow hair.

[00380] These treatments can be administered during the lithium treatment "holidays.'* Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

[00381] Androgen receptor inhibitors are also useful for stimulating scalp hair growth and inhibiting beard and moustache hair (Hu LY, et al., 2007, Bioorg Med Chem Lett. 2007 17:5983-5988). These treatments can be administered during the lithium treatment

"holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.5 INDICATIONS AND TREATMENT REGIMENS FOR UNWANTED HAIR

[00382] A candidate subject for intermittent lithium treatment (i.e., alternating lithium treatment with "vacation/holiday" periods) or a pulse lithium treatment for inhibiting hair growth or removing hair is any subject who has or has had a condition, disease or disorder associated with excess hair or unwanted hair, or who wishes to inhibit the growth of hair or remove hair. In some embodiments, permanent hair removal is desired.

[00383] The subject may be any subject, preferably a human subject, including male, female, and transsexual subjects. In certain embodiments, the subject is a human adolescent. In certain embodiments, the subject is undergoing puberty. In certain embodiments, 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 elderly. In certain embodiments, 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. 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 breastfeeding.

[00384] In some embodiments, the intermittent lithium treatment or a pulse lithium treatment is delivered to an area of unwanted hair, for example, the head (e.g., the eyebrow, nose and nares, upper lip, lower lip, chin, cheeks, ears, or forehead) or another part of the body, such as, e.g., the chest, breast (e.g., the nipples), abdomen, neck, back, arms, armpits (site of axillary hair), legs, hands, feet, buttocks, or genitals. In some embodiments, hair removal from a wounded or scarred area of the skin is desired. In some embodiments, hair removal from darkly pigmented skin or a darkly pigmented area of the skin, such as a mole, freckle, or the genital area, is desired.

[00385J In particular embodiments, subjects who are candidates for intermittent lithium treatment or a pulse lithium treatment include those afflicted with hypertrichosis (excess hair not localized to the androgen-dependent areas of the skin), including generalized congenital hypertrichosis (congenital hypertrichosis lanuginosa), acquired generalized hypertrichosis (acquired hypertrichosis lanuginosa), patterned acquired hypertrichosis, localized congenital hypertrichosis, localized acquired hypertrichosis, paradoxical hypertrichosis, and "werewolf syndrome," pili multigemini, excess hair in androgen-dependent areas of the skin, idiopathic hirsutism, post-menopausal facial hair, axillary hair, back hair, ear hair, or any other disease, disorder, or form of unwanted hair or excessive hair as discussed infra and/or known in the art. 1

[00386J In some embodiments, the subject has excess hair caused by a genetic or hereditary disease or disorder. In some embodiments, the subject has excess hair growth caused by or associated with medication, such as chemotherapy (e.g., anti-cancer therapy, anti-viral therapy, psychological therapy), steroid therapy (e.g., systemic androgenic steroid therapy or corticosteroid therapy), antihypertensive drugs (e.g., minoxidil), or cyclosporine, radiation, trauma, endocrine dysfunction (such as, e.g., adrenal virilism, basophilic adenoma of the pituitary, masculinizing ovarian tumors, Stein-Leventhal syndrome), porphyria cutanea tarda, surgery, burning or other wound, stress, aging, an autoimmune disease or disorder, malnutrition (e.g., lanugo), an infection (such as, e.g. , a fungal, viral, or bacterial infection), dermatitis, psoriasis, eczema, pregnancy, menopause, allergy, or any other form of excessive hair growth known in the art. Any other disease or disorder associated with unwanted hair or excessive hair known in the art is also contemplated.

[00387] In some embodiments, a candidate subject is any subject who wishes to remove or prevent excess hair or unwanted hair, or who wishes to inhibit the growth of hair or remove hair, for example, to have less hair, slower-growing hair, shorter hair, and/or thinner hair. In some embodiments, the candidate is a subject who wishes to decrease hair pigmentation. In some embodiments, the subject is not affected by a condition or disease or disorder associated with excessive hair.

5.5.1 REGIONS OF INCREASED TERMINAL HAIR

GROWTH IN ADULT MALES

[00388] In addition to MPHL and post-puberty male pattern body hair growth, males also manifest varying degrees of growth of new or increased terminal hair in middle age (over 35 years). Males begin to grow terminal hair on the ears, particularly around the auricular canal; and have increased hair growth (length and density) in the nares, and increased hair growth of eyebrows (hair length and curl.) In some cases, males develop terminal hairs on the skin covering the central prominence of the nose.

[00389] The mechanisms of these transformations have not been studied carefully, but appear to be androgen driven since they are uncommon in females and rnore prominent in certain races/ethnic backgrounds (for example Scottish and Welsh men). However, some females experience the growth of ear hair as a side-effect of topical minoxidil treatment. [00390] Any combination treatment described in Section 5.3 may be used to reduce unwanted terminal hair in adult males. In some embodiments, the combination treatment comprises intermittent lithium treatment or a pulse lithium treatment in combination with a drug such as, e.g. , eflornithine (trade name: Vaniqa, which is formulated as eflornithine hydrochloride, 13.9%) or 5-fluorouracil (e.g. , 5-FU, Efudex 5% cream). In some embodiments, the combination treatment for hair removal comprises intermittent lithium treatment or a pulse lithium treatment in combination with one or more antiandrogen drugs. A combination treatment for hair removal may also comprise intermittent lithium treatment or a pulse lithium treatment with one or more of hair bleaching, shaving, waxing, sugaring, threading, plucking, use of an abrasive material, laser, electrolysis or electrology, use of an epilation device, use of friction, exfoliation, burning, intense pulsed light ("IPL"; e.g. , Flashlamp or EpiLight), use of a mechanical device (e.g. , epilators such as Epilady, Emjoi, etc.), or use of a chemical depilatory (e.g. , Nair ®).

[00391] These treatments for use in combination with lithium treatment can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.5.2 REGIONS OF INCREASED TERMINAL HAIR

GROWTH IN ADULT FEMALES

[00392] After menopause, females frequently experience increased hair growth to varying degrees on the face in the moustache/beard pattern. In females generally, estrogens suppress moustache/beard hair. In addition, women treated with androgens or with certain medical conditions retain the potential to develop beard and moustache hair. Estrogens (particularly estrogen-progesterone oral birth control pills) are believed to inhibit hair growth primarily by suppressing ovarian androgen production, however action on skin estrogen receptors may also play a role in these activities. Cyproterone acetate and spironolactone and other anti- androgens (e.g. ketoconazole) are effective as anti-androgens in reducing unwanted hair on females (e.g. idiopathic hirsutism), although there is wide variability in individual responses.

[00393] Vaniqa (eflornithine hydrochloride 13.9%) and other ornithine decarboxylase inhibitors, polyamine derivatives, as well as 5-fluorouracil (5-FU, Efudex 5% cream) and certain antiandrogens inhibit hair growth. These drugs (described in Section 5.3 supra) and the other drugs described in Section 5.3 can be used with, or as an adjunct to laser hair removal or to electrolysis, depilatory creams, plucking and waxing. Other products that suppress terminal hair have been described: (a) Vicente RA, et al, 2009, J Eur Acad Dermatol Venereol. 23(4):410-4, "'Double-blind, randomized, placebo-controlled trial of a cream containing the Stryphnodendron adstringens (Martius) Coville bark extract for suppressing terminal hair growth"; (b) Javidnia et al., 2003, Phytomedicine 10(6-7):455-8.

"Antihirsutism activity of Fennel (fruits of Foeniculum vulgare) extract. A double-blind placebo controlled study." These treatments can be administered during the lithium treatment "holidays." Alternatively, these treatments can be administered prior to or subsequent to a pulse lithium treatment.

5.6 METHODS FOR EVALUATING TREATMENT

5.6.1 ANIMAL MODELS

[00394] Human skin and hair have features that are relatively unique among terrestrial mammals. First, the great majority of human skin appears hairless to the naked eye, while the vast majority of other terrestrial mammals are essentially covered with visible hair. Second, visible human hair appears and disappears in patterns that have spatial and temporal components. Third, the patterns of visible human hair are distinct in typical male and females (exhibit gender dimorphism). Accordingly, it is evident that relative to other mammals, humans have distinct hair patterning and humans have correspondingly distinct molecular, cellular and tissue mechanisms that regulate hair growth and that control human hair, patterning. Modulating human hair growth and loss requires considerations that are unique to humans and for which other animals are insufficient models.

[00395] It should be noted that certain non-human primates share features of hair patterning with humans, but not to the degree or extent. Old World Apes (gorillas and chimpanzees) have areas of skin that lack visible hair; on the face surrounding the eyes, nose and mouth; on ears; and the plantar surfaces of hands and feet. In addition, Rhesus Macaque has patterned alopecia in males and females. Gorillas have hair patterning with respect to color on dominant males: i.e., the "Silverback." While certain of these mechanisms share similarities to humans, the extent and degree of hair patterning in human remains relatively unique.

5.6.1.1 HUMAN SKIN XENOGRAFT MODELS

[00396] Preliminary evidence of hair follicle neogenesis has been demonstrated in human skin (obtained from the hair line during a face lift procedure) grafted onto the back of an immunodcficicnt SCID mouse. Such human skin xenograft models arc useful for testing the safety and efficacy of the intermittent lithium treatments or a pulse lithium treatment described herein, as well as the combination treatments described in Section 5.3 supra.

Although any method for producing human skin xenografts known in the art may be used, an exemplar model is described in the example of Section 26 below.

[00397] Success of treatment aimed at improving hair growth can be measured by:

• increased terminal hair formation

• follicle synchronization so that the overall hair density appears to be greater compared to previous asynchronous hair growth

• increased anagen or decreased telogen

• follicle regeneration

• increased proliferation of dermal papilla

• increased recruitment or proliferation of stem cells to the follicle

• generation of new hair follicles

• increased diameter of existing or new hair (increased thickness of hair shafts)

• increased number of hair follicles at a more mature stage of development

• increased hair weight.

[00398] 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 5.3. Preferably, a human skin xenograft model is used. For example, 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 described in the examples of Sections 7, 9, 1 1 , 13, and 24-36 below. A synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for restoring or enhancing hair growth (described in, e.g., Section 5.3) 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).

[00399] In an alternative embodiment, the intermittent lithium treatment or a pulse lithium treatment is used in combination treatments (e.g. , described in Section 5.3 supra) to reduce unwanted hair growth. Success of such treatments can be measured using an animal model, e.g., the human xenograft mouse model described herein, by:

• decreased terminal hair formation • follicle synchronization so that synergies are achieved when the hair growth retardant is sequentially applied

• decreased anagen or increased telogen

• inhibition of follicle regeneration.

• decreased hair weight, thickness, or density

• decreased number of hair follicles at a mature stage of hair follicle development.

[00400] An additive or a synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for removing unwanted hair (described in, e.g..

Section 5.3) 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).

5.6.1.2 OTHER ANIMAL MODELS

[00401] 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 examples in Section 25 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.

5-6.2 METHODS FOR EVALUATING TREATMENT IN HUMANS

[00402] The safety and efficacy of the intermittent lithium treatments or pulse lithium treatment described herein may also be measured in human subjects according to methods known in the art. See, e.g., International Patent Application Publication No. WO

2005/084621 , published September 15, 2005, the contents of which is incorporated by reference herein in its entirety. For example, an intermittent lithium treatment or pulse lithium treatment may be administered in combination with minoxidil, finasteride, laser therapy, or a dermabrasion procedure for integumental perturbation to improve hair growth, as described in the Examples of Sections 6-13, 24, 25, 35, and 36 below.

[00403] Success of treatment aimed at improving hair growth can be measured by: • increased terminal hair formation (e.g., measuring new hair growth as an increased number of fibers in an affected area of the skin, or increased thickness (e.g. , diameter) or length of hair fibers)

• follicle synchronization so that the overall hair density appears to be greater compared to previous asynchronous hair growth (e.g., measured examination of a biopsy)

• increased anagen or decreased telogen (e.g., measured examination of a biopsy)

• increased follicle regeneration (e.g., measured examination of a biopsy)

• increased proliferation of dermal papilla (e.g., measured examination of a biopsy)

• increased recruitment or proliferation of stem cells to the follicle (e.g., measured examination of a biopsy)

• generation of new hair follicles (e.g., measured examination of a biopsy or by confocal microscope, by assessing number of hair follicles and/or by assessing morphological development stages of hair follicles compared to baseline or a negative control )

[00404] In some embodiments, the intermittent lithium treatment or pulse lithium treatment increases hair count by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, the intermittent lithium treatment or pulse lithium treatment increases terminal hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, the intermittent lithium treatment or pulse lithium treatment increases hair thickness by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. Such an improvement in hair count, terminal hair, or hair thickness 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.

[00405J A synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for restoring or enhancing hair growth (described in, e.g.. Section 5.3) 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).

[00406] In an alternative embodiment, the intermittent lithium treatment or a pulse lithium treatment is used in combination treatments (e.g. , described in Section 5.3 supra) to reduce unwanted hair growth. Any appropriate method for testing the safety and efficacy of such treatments may be used, for example, as described in the examples of Sections 14-23 below (see also sections on safety in the clinical protocol in Section 36). Success of such treatments can be measured by:

• decreased terminal hair formation (e.g. , measuring hair growth as a decreased number of fibers in an affected area of the skin, or decreased thickness (e.g. , diameter) or length of hair fibers)

• follicle synchronization so that synergies are achieved when the hair growth retardant is sequentially applied

• decreased anagen or increased telogen

• inhibition of follicle regeneration.

[00407] In some embodiments, the intermittent lithium treatment or pulse lithium treatment decreases hair count by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. In some embodiments, the intermittent lithium treatment or pulse lithium treatment decreases terminal hair by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75%o or more, or by 100% or more. In some embodiments, the intermittent lithium treatment or pulse lithium treatment decreases hair thickness by 5% or more, by 10% or more, by 15% or more, by 20% or more, by 25% or more, by 30% or more, by 40% or more, by 50% or more, by 75% or more, or by 100% or more. Such an improvement in hair count, terminal hair, or hair thickness may be measured after 1 day, 2 days, 3 days, 5 days, 1 week, 2 weeks, 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.

[00408] A synergistic effect of an intermittent lithium treatment or pulse lithium treatment on another treatment for removing unwanted hair (described in, e.g. , Section 5.3) 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).

-I l l - 5.6.3 IN VITRO MODELS

[00409] Skin expltmt model. The efficacy of the intermittent lithium treatments or pulse lithium treatment described herein may be tested using skin explants, for example, prepared from skin biopsies or other surgical procedures. See, e.g., Ballanger et al., supra.

6. EXAMPLE 1: INTERMITTENT TREATMENT OF HAMILTON-NORWOOD TYPE VI MALE-PATTERN ALOPECIA USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL MINOXIDIL

[00410] A male human subject, 30 years old, with Hamilton-Norwood type VI male- pattern alopecia presents complaining of continued hair loss despite treatment with topical minoxidil foam 5%, oral finasteride 1 mg/day. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to affected area of the scalp for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one wreek, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and he is evaluated after three weeks.

[00411] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers; and the patient's subjective evaluation of hair growth. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc.); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00412] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate (1 week) with minoxidil foam treatment (3 weeks in which finasteride treatment is continued). Response to therapy is measured by the methods described above.

7. EXAMPLE 2: INTERMITTENT TREATMENT OF HAMILTON-NORWOOD TYPE VI MALE-PATTERN ALOPECIA USING LITHIUM GLUC NATE DEVICE ALTERNATING WITH TOPICAL MINOXIDIL

[00413] A Caucasian male human subject, 30 years old, with Fitzgerald Type II skin (wherein Type I is the lightest and Type VI is the darkest (see Wcllcr et al, 2008, Clinical Dermatology, 4th ed., Maiden, MA: Blackwell Publishing, pp. 268)), with Hamilton- Norwood type VI male-pattern alopecia presents complaining of continued hair loss despite treatment with topical minoxidil foam 5%, oral finasteride 1 mg/day. The bald and transitional areas of the subject's scalp are prepared by shaving and then treated with a fractional and non-ablative Erbium- YAG laser with an emission at 1540-1 550 nm (set to 50- 70 J/cm2, treatment level of 8- 10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel ( Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to treated area of the scalp for one week. After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and he is evaluated after three weeks.

[00414] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers; and the patient's subjective evaluation of hair growth. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00415] The subject is optionally treated with 10 more cycles, e.g., to increase hair density, for example: 1 week of topical lithium gluconate followed by 3 weeks of minoxidil foam treatment, with optional laser treatment. Response to therapy is measured by the methods described above.

8. EXAMPLE 3: INTERMITTENT PRE-TREATMENT OF "DONOR" SCALP AREAS WITH LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL MINOXIDIL IN PREPARATION FOR A HAIR TRANSPLANT PROCEDURE IN A PATIENT WITH HAMILTON-NORWOOD TYPE VII MALE-PATTERN ALOPECIA

[00416] A male human subject, 30 years old, with Hamilton-Norwood type VII male- pattern alopecia presents complaining of continued hair loss despite treatment with topical minoxidil foam 5%, oral finasteride 1 mg/day.

8.1 TREATMENT A

[00417] Pre-treatment: In preparation for a hair transplant procedure, the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to the to the occipital and deep temporal areas area of the scalp from which follicles will be harvested for transplant for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and he is evaluated after three weeks.

[00418] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers; and the patient's subjective evaluation of hair growth. The treated area of affected scalp is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00419] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks). Response to therapy is measured by the methods described above.

[00420] Post-transplantation: The area of scalp that was pre-treated with lithium and minoxidil is used as a source for the transplanted follicles. After hair follicle implantation, treatment with lithium gluconate is initiated for one week, and then discontinued and followed by treatment with topical minoxidil foam for three weeks.

[00421] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers; and the patient's subjective evaluation of hair growth. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc.); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00422] The subject is treated with 10 cycles of the protocol : alternating topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks).

8.2 TREATMENT B

[00423] The subject is provided a treatment in which the bald crown scalp receives in order: (i) Erbium- YAG laser; (ii) lithium; and (iii) estrogen during the phase where follicle stem cells are reorganizing and reforming hair follicles. The estrogen treatment reprograms the follicle stem cells to alter their sensitivity/response to androgens. In effect, this could be described as rendering follicle stem cells either (a) '"female-type" with respect to crown scalp follicle cells or (b) "occipital scalp type" in terms of their lack of response to androgens by involution. 9. EXAMPLE 4: INTERMITTENT PRE-TREATMENT OF "DONOR" SCALP AREAS USING LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL MINOXIDIL IN PREPARATION FOR A HAIR TRANSPLANT PROCEDURE IN A PATIENT WITH HAMILTON-NORWOOD TYPE VII MALE-PATTERN ALOPECIA

[00424] A Caucasian male human subject, 30 years old, with Fitzgerald Type II skin (wherein Type I is the lightest and Type VI is the darkest (see Weller et al. , 2008, Clinical Dermatology, 4th ed., Maiden, MA: Blackwell Publishing, pp. 268)), with Hamilton- Norwood type VII male-pattern alopecia presents complaining of continued hair loss despite treatment with topical minoxidil foam 5%, oral finasteride 1 mg/day.

[00425] Pre-treatment: In preparation for a hair transplant procedure, 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/cm2, treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to the to the occipital and deep temporal areas area of the scalp from which follicles will be harvest for transplant for one week. After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and he is evaluated after three weeks.

[00426] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00427] The subject is optionally treated with 10 more cycles, e.g., to increase hair density, for example: topical lithium gluconate (1 week) with minoxidil foam treatment (3 weeks), with optional laser treatment. Response to therapy is measured by the methods described above.

[00428] Post-transplantation: The area of scalp that was treated with lithium and minoxidil is used as a source for the transplanted follicles. After hair follicle implantation, treatment with lithium gluconate is initiated for one week, and then discontinued and followed by treatment with topical minoxidil foam for three weeks.

[00429] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00430] The subject is treated with 1 0 cycles of the protocol: alternating Erbium- YAG laser treatment followed by topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks).

10. EXAMPLE 5: INTERMITTENT TREATMENT OF A POSTMENOPAUSAL PATIENT WITH DIFFUSE THINNING OF SCALP HAIR USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL MINOXIDIL

[00431] A female human subject, 65 years old, complains of diffuse thinning of scalp hair growth after menopause. She describes continued hair loss despite treatment with topical minoxidil foam 2% and oral finasteride 1 mg/day. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to affected area of the scalp for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and she is evaluated after three weeks.

[00432] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen. etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00433] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( I week) with minoxidil foam treatment (3 weeks). Response to therapy is measured by the methods described above.

11. EXAMPLE 6: INTERMITTENT TREATMENT OF A POSTMENOPAUSAL PATIENT WITH DIFFUSE THINNING OF SCALP HAIR USING A LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL MINOXIDIL

]00434] A Caucasian female human subject Fitzgerald Type II skin, 65 years old, complains of diffuse thinning of scalp hair growth after menopause. She describes continued hair loss despite treatment with topical minoxidil foam 2% and oral finasteride 1 mg/day. 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/cirf , treatment level of 8- 10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to the treated area of the scalp for one week. After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and she is evaluated after three weeks.

[004351 Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division: follicles growing new hair fibers, follicles with no hair fibers.

[00436] The subject is optionally treated with 10 more cycles, e.g. , to increase hair density, for example: topical lithium gluconate (1 week) followed by minoxidil foam treatment (3 weeks), with optional laser treatment. Response to therapy is measured by the methods described above.

12. EXAMPLE 7: INTERMITTENT PRE-TREATMENT OF "DONOR" SCALP AREAS OF A POST-MENOPAUSAL PATIENT WITH DIFFUSE

THINNING OF SCALP HAIR USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL MINOXIDIL IN PREPARATION FOR A HAIR TRANSPLANT PROCEDURE

[00437] A female human subject, 65 years old, with Fitzgerald Type II skin complains of diffuse thinning of scalp hair growth after menopause. She describes continued hair loss despite treatment with topical minoxidil 2% foam and oral finasteride 1 mg/day.

[00438J Pre-treatment: In preparation for a hair transplant procedure, the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to the occipital and deep temporal areas area of the scalp from which follicles will be harvested for transplant for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and she is evaluated after three weeks.

[00439] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00440] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks). Response to therapy is measured by the methods described above.

[00441] Post-transplantation: The area of scalp that was pre-treated with lithium and minoxidil is used as a source for the transplanted follicles. After hair follicle implantation, treatment with lithium gluconate is initiated for one week, and then discontinued and followed by treatment with topical minoxidil foam for three weeks.

[00442] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00443[ The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks).

13. EXAMPLE 8: INTERMITTENT PRE-TREATMENT OF "DONOR" SCALP AREAS OF A POST-MENOPAUSAL PATIENT WITH DIFFUSE THINNING OF SCALP HAIR USING A LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL MINOXIDIL IN PREPARATION FOR A HAIR TRANSPLANT PROCEDURE

[00444] A Caucasian female human subject (with Fitzgerald Type II skin, wherein Type I is the lightest and Type VI is the darkest), 65 years old, complains of diffuse thinning of scalp hair growth after menopause. She describes continued hair loss despite treatment with topical minoxidil 2% foam and oral finasteride 1 mg/day.

[00445] Pre-treatment: In preparation for a hair transplant procedure, 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% treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical minoxidil and to apply the Lithium gluconate 8% gel to the occipital and deep temporal areas area of the scalp from which follicles will be harvested for transplant for one week (to synchronize the hair follicle cells in G2/M phase arrest ). After one week, treatment with lithium gluconate is discontinued and treatment with topical minoxidil foam is re-started and she is evaluated after three weeks.

[004461 Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00447] The subject is optionally treated with 10 more cycles, e.g. , to increase hair density, for example: alternating Erbium- YAG laser treatment plus topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks). Response to therapy is measured by the methods described above.

[00448] Post-transplantation: The area of scalp that was pre-treated with lithium and minoxidil is used as a source for the transplanted follicles. After hair follicle implantation, treatment with lithium gluconate is initiated for one week, and then discontinued and followed by treatment with topical minoxidil foam for three weeks, with or without laser treatment.

[00449] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of scalp); thickness of fibers; length of hair fibers. The treated area of affected scalp is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division: follicles growing new hair fibers, follicles with no hair fibers.

[00450] The subject is optionally treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with minoxidil foam treatment (3 weeks), with or without laser treatment.

14. EXAMPLE 9: INTERMITTENT TREATMENT OF POST MENOPAUSAL FACIAL HAIR USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL EFLORNITHINE

[00451] A female human subject, 50 years old, complains of moustache hair growth after menopause. Vaniqa® (eflornithine hydrochloride 13.9%) is being applied without much relief. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflornithine and to apply the Lithium gluconate 8% gel to affected area of the face for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical eflomithine is re-started and she is evaluated after three weeks.

[00452] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of facial skin); thickness of fibers; length of hair fibers; and the patient's subjective evaluation of hair growth inhibition. The treated area of affected face is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc.); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00453] The subject is optionally treated with 10 more cycles, e.g., to decrease hair density, for example: alternating topical lithium gluconate (1 week) with eflomithine foam treatment (3 weeks). Response to therapy is measured by the methods described above.

15. EXAMPLE 10: INTERMITTENT TREATMENT OF POST MENOPAUSAL FACIAL HAIR USING LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL EFLORNITHINE

[00454] A Caucasian female human subject (with Fitzgerald Type II skin, wherein Type I is the lightest and Type VI is the darkest), 50 years old, complains of moustache hair growth after menopause. Vaniqa® (eflomithine hydrochloride 13.9%) is being applied without much relief. 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/cm2, treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflomithine and to apply the Lithium gluconate 8% gel to the treated area of the face for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical eflomithine is re-started and she is evaluated after three weeks.

[00455] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of facial skin); thickness of fibers; length of hair fibers. The treated area of affected face is biopsied and studied for distribution of follicles in various stages of Follicle Cycle ( anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers. [00456] The subject is optionally treated with 10 more cycles, e.g.. to decrease hair density, for example: topical lithium gluconate ( 1 week) followed by with etlomithine foam treatment ( 3 weeks), with or without laser treatment. Response to therapy is measured by the methods described above.

16. EXAMPLE 11: INTERMITTENT TREATMENT OF POST MENOPAUSAL FACIAL HAIR USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL 5-FLUOROURACTL

[00457] A female human subject, 50 years old, complains of moustache hair growth after menopause. Vaniqa (eflornithine hydrochloride 13.9%) is being applied without much relief. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflornithine and to apply the Lithium gluconate 8% gel to affected area of the face for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical 5-fluorouracil (5-FU, Efudex 5% cream) is started and she is evaluated after three weeks.

[00458] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of facial skin); thickness of fibers; length of hair fibers. The treated area of affected face is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00459] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with 5-FU cream treatment (3 weeks). Response to therapy is measured by the methods described above.

17. EXAMPLE 12: INTERMITTENT TREATMENT OF POST MENOPAUSAL FACIAL HAIR USING LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL 5-FLUOROURACIL

[00460] A Caucasian female human subject (with Fitzgerald Type II skin, wherein Type I is the lightest and Type VI is the darkest), 50 years old, complains of moustache hair growth after menopause. Vaniqa (eflornithine hydrochloride 13.9%) is being applied without much relief. 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/cm2, treatment level of 8- 10. and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflornithine and to apply the Lithium gluconate 8% gel to the treated area of the face for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical 5-fluorouracil (5-FU, Efudex 5% cream) is started and she is evaluated after three weeks.

[00461J Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of facial skin); thickness of fibers; length of hair fibers. The treated area of affected face is biopsied and studied for new follicle growth, bifurcating follicles undergoing follicle division; follicles growing new hair fibers.

[00462] The subject is optionally treated with 10 more cycles, e.g. , to decrease hair density, for example: topical lithium gluconate ( 1 week) followed by 5-FU cream treatment (3 weeks), with or without laser treatment. Response to therapy is measured by the methods described above.

18. EXAMPLE 13: INTERMITTENT TREATMENT OF AXILLARY HAIR

USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL EFLORN1THINE

[00463] A female human subject, 30 years old, complains of axillary hair growth and irritation from using razors to shave her armpits. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) ) and instructed to discontinue topical eflornithine and to apply the Lithium gluconate 8% gel to the affected area of the axilla for one week (to synchronize the hair follicle cells in G2/ phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical Vaniqa (eflornithine hydrochloride 13.9%) is started and she is evaluated after three weeks.

[00464] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of axillary skin); thickness of fibers; length of hair fibers. The treated area of affected axillary skin is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2. M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00465] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with eflornithine foam treatment (3 weeks). Response to therapy is measured by the methods described above. 1 . EXAMPLE 14: INTERMITTENT TREATMENT OF AXILLARY HAIR USING LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL EFLORNITHINE

[00466] A Caucasian female human subject (with Fitzgerald Type 11 skin, wherein Type 1 is the lightest and Type VI is the darkest), 30 years old, complains of axillary hair growth and irritation from using razors to shave her armpits. The subject is administered a fractional and non-ablative laser therapy using an Erbium- Y AG laser with an emission at 1540- 1550 nm (set to 50-70 J/cm2, treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflomithine and to apply the Lithium gluconate 8% gel to the treated area of the axilla for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical Vaniqa (eflomithine hydrochloride 13.9%) is started and she is evaluated after three weeks.

[00467] Response to therapy is detem ined by measuring new hair growth (increased number of fibers in an affected area of axillary skin); thickness of fibers; length of hair fibers. The treated area of affected axillary skin is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00468J The subject is optionally treated with 10 more cycles, e.g. , to decrease hair density, for example: topical lithium gluconate (T week) followed by eflomithine foam treatment (3 weeks), with or without laser treatment. Response to therapy is measured by the methods described above.

20. EXAMPLE 15: INTERMITTENT TREATMENT OF BACK HAIR

USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL EFLORNITHINE

[00469J A male human subject, 30 years old, complains of excess back hair growth.

Repeated waxings have had short term effects and are painful. The subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed apply it to affected area of the skin for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical Vaniqa (eflomithine hydrochloride 13.9%) is started and he is evaluated after three weeks. [00470) Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of back skin); thickness of fibers; length of hair fibers. The treated area of affected back skin is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc. ); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00471] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate ( 1 week) with eflornithine foam treatment (3 weeks). Response to therapy is measured by the methods described above.

21. EXAMPLE 16: INTERMITTENT TREATMENT OF BACK HAIR

USING LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL EFLORNITHINE

[00472] A Caucasian male human subject (with Fitzgerald Type II skin, wherein Type I is the lightest and Type VI is the darkest), 30 years old, complains of excess back hair growth. Repeated waxings have had short term effects and are painful. 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/cm2, treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed apply it to the treated area of the skin for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical Vaniqa (eflornithine hydrochloride 13.9%) is started and he is evaluated after three weeks.

[00473] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of back skin); thickness of fibers; length of hair libers. The treated area of affected back skin is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair libers, follicles with no hair fibers.

[00474] The subject is optionally treated with 10 more cycles, e.g. , to decrease hair density, for example: topical lithium gluconate (1 week) followed by eflornithine foam treatment (3 weeks), with or without laser treatment. Response to therapy is measured by the methods described above. 22. EXAMPLE 17: INTERMITTENT TREATMENT OF EAR HAIR

USING LITHIUM GLUCONATE GEL ALTERNATING WITH TOPICAL EFLORNITHINE

[00475] A male human subject, 55 years old, complains of ear (auricle) hair growth. The subject is provided with a topical preparation of Lithium gluconate 8% ge! (Lithioderm 8% gel) and instructed discontinue topical eilornithine and to apply the Lithium gluconate 8% gel to affected area of the ear for one week (to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical eilornithine is re-started and he is evaluated after three weeks.

[00476] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of ear ); thickness of fibers; length of hair fibers. The treated area of affected ear is biopsied and studied for distribution of follicles in various stages of Follicle Cycle (anagen, catagen, etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ): new follicle growth, bifurcating follicles; follicles undergoing follicle division; follicles growing new hair fibers, follicles with no hair fibers.

[00477] The subject is treated with 10 cycles of the protocol: alternating topical lithium gluconate (1 week) with eilornithine foam treatment (3 weeks). Response to therapy is measured by the methods described above.

23. EXAMPLE 18: INTERMITTENT TREATMENT OF EAR HAIR USING

LITHIUM GLUCONATE DEVICE ALTERNATING WITH TOPICAL EFLORNITHINE

[00478] A Caucasian male human subject (with Fitzgerald Type II skin, wherein Type I is the lightest and Type VI is the darkest), 55 years old, complains of ear (auricle) hair growth. 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 , treatment level of 8-10, and 8 passes) and the subject is provided with a topical preparation of Lithium gluconate 8% gel (Lithioderm 8% gel) and instructed to discontinue topical eflomithine and to apply the Lithium gluconate 8% gel to the treated area of the ear for one week ( to synchronize the hair follicle cells in G2/M phase arrest). After one week, treatment with lithium gluconate is discontinued and treatment with topical eilornithine is re-started and he is evaluated after three weeks.

[00479] Response to therapy is determined by measuring new hair growth (increased number of fibers in an affected area of ear); thickness of fibers; length of hair fibers. The treated area of affected ear is biopsied and studied for distribution of follicles in various stages of Follicle Cycle fanagen, catagen. etc.); distribution of follicle cells in various stages of cell cycle (e.g. G2, M, etc. ); new follicle growth, bifurcating follicles; follicles undergoing follicle division: follicles growing new hair fibers, follicles with no hair fibers.

[00480] The subject is optionally treated with 10 more cycles, e.g., to decrease hair density, for example: topical lithium gluconate ( 1 week) followed by eflornithine foam treatment (3 weeks), with or without laser treatment. Response to therapy is measured by the methods described above.

24. EXAMPLE 19: ABLATIVE LASER TREATMENT VARIATIONS

[00481] The treatments presented in the examples in Sections 7, 9, 1 1, 13, 15, 17, 19, 21 , and 23 may alternatively be accomplished by applying an ablative laser treatment in place of the non-ablative laser treatment. In such ablative laser treatments, the application of Lithium gluconate is sterile and, optionally, the treatment area is covered by a bandage. For example, ablative laser treatment may accomplished using an Erbium-YAG laser at 2940 nm or a C02 laser at 10,600 nm.

24.1 ULTRAPULSE CO? FRACTIONAL LASER

[00482] After shaving/clipping of the existing hair in the area to be treated, and followed by cleaning with antiseptic, 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? laser is used to disrupt the epidermis and dermis to approximately 100 to 500 μΜ 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.

24.2 ULTRAPULSE CO2 ABLATION LASER

[00483] After shaving/clipping of the existing hair in the area to be treated, and followed by cleaning with antiseptic, Lidocaine HCL 2% with Epinephrine 1 .100,000 are injected to anesthetize the surface of the area to be treated. An Ultrapulse C02 laser (ablative mode) is used to disrupt the epidermis and dermis to approximately 100 to 500 μΜ 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. The

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.

24.3 CANDELA SMOOTH PEEL FULL-ABLATION ERBIUM LASER

[00484] After shaving/clipping of the existing hair in the area to be treated, and followed by cleaning with antiseptic, 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 μΜ deep. The treated area is a 1.5 cm x 1.5 cm square.

25. EXAMPLE 20: DERMABRASION TREATMENT VARIATIONS

[00485] The treatments presented in the examples in foregoing Sections 7, 9, 1 1 , 13, 15, 17, 19, 21 , and 23 may alternatively be accomplished by applying the following

dermabrasion treatment in place of the laser treatment.

[00486] After shaving/clipping of the existing hair in the area to be treated, followed by cleaning with antiseptic, 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 μ , 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. In an alternative example, a Bell Hand dermabrasion device may be used.

[00487] Variations of this protocol are found in the example of Sections 28-32, which present mouse studies using dermabrasion, and the protocols for use in humans in the examples of Sections 35 and 36. In the mouse studies, dermabrasion was carried out using a microdermabrasion device. While dermabrasion in humans may also be carried out using a microdemiabrasion device, where sterile conditions are preferential, a dermabrasion device is preferably used.

26. EXAMPLE 21 : HUMAN SKIN XENOGRAFT ANIMAL MODEL FOR

ASSESSING OR CONFIRMING EFFICACY OF LITHIUM TREATMENT REGIMEN

[00488] 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 μΐ, 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, NDR1 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. After healing and successful "'take,1' prior to wounding, the human skin is analyzed using in vivo confocal microscopy, histology and/or photography to determine the ''contror or "pre-wounded" state of the skin graft. Prior to wounding, mice are anesthetized with ketamine (80

mg/kg)/xylazine (20 mg/kg) delivered i.p. in a volume of < 100 μΐ, and monitored by toe pinch to determine the surgical plane of anesthesia. The epidermis of the human skin is removed using a microdcrmabrasion device to dermabrade as described above. (Experiments with dermabrasion on ex vivo human abdominal skin have established the initial parameters for removal of epidermis, however some testing in 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 approximately 50%). The wounds are allowed scab and heal naturally. The mice are observed and photographed daily in order to monitor the formation of the scab and the timing of its detachment (scab detachment should occur within 2 weeks of wounding). As soon as the scab detaches, 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, in vivo confocal microscopy, histology and/or photography is performed daily (until the end of the experiment) following scab detachment in order to monitor hair follicle neogenesis (confocal microscopy is noninvasive but does require anesthesia). 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. 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 μΐ, 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. For the experimental design in which the human

( xenograft) skin is wounded, there are 3 treatment groups (lithium compositions, vehicle, no lithium composition or vehicle) with 2 different delivery methods (IP and topical). With 10 mice per group, this requires 50 mice (only 1 group of "no lithium composition or vehicle"). The most efficacious combination of lithium composition and mode of delivery is repeated in 3 more independent experiments (with only lithium composition-treated and vehicle groups), thus adding 60 more mice, giving a total of 1 10 mice. An identical experiment is carried out, but without dermabrasion wounding (epidermal removal), requiring an additional 1 1 mice. This yields a total of 220 mice for the wounding and lithium composition portion of this experiment. An additional 20 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.

27. EXAMPLE 22: MODULATION OF RELEASE PROFILES

OF LITHIUM IONS AS A FUNCTION OF FORMULATION COMPOSITION

[00489] 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.

27.1 TEST FORMULATIONS

[00490| The 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.

Table 2. Lithium Chloride Compositions

Table 2 (continued).

ID Code Formulation Identity Composition

Lithium Lithium Gluconate Gel, Lithium gluconate = 8%

gluconate 8% Glycerol = 10%

hydrogel Carbopol 980 = 1.5%

Methyl Paraben = 0.10%

Propyl Paraben-0.05%

NaOH = Q.S.P. to pH 6.8

Purified Water = Q.S.P. to 100

27.2 METHODS

[00491 J Skin Preparation. Percutaneous absorption was measured using the in vitro cadaver skin finite dose technique. Cryopreserved, split-thickness human cadaver trunk skin was obtained from a skin bank and stored in water-impermeable plastic bags at < -20° C until use. Prior to the experiment, skin was removed from the bag, thawed at room temperature for 20 minutes and then cut into sections large enough to fit on 0.81 cm2 Franz diffusion chambers. The deraial chamber was filled with phosphate buffered saline, pH 7.4 ± 0.1 , and the epidermal chamber (chimney) left open to ambient laboratory conditions. Skin samples were comprised of both full thickness (epidermis plus dermis) as well as dermal tissue. 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 R.PM and its skin surface temperature maintained at 32.0° ± 1.0 °C.

Franz Cell Testing Conditions;

Temperature: 32°C

Receptor Solution: PBS (Spectrum, USP grade).

[00492] Dosing and Sample Collection. All formulations were applied to the skin sections using a positive displacement pipette set to deliver 0.1 g. The dose was spread throughout the surface of the skin. At pre-selected time intervals after test formulation application, aliquots of the reservoir solution were removed and replaced with an equivalent amount of the fresh solution (phosphate buffered saline), and an aliquot taken for analysis. [00493] Analytical Methods. Quantification of Lithium Chloride was done using the Infinity (tm) Lithium test kit from Thermo Fisher (Middletown, Virginia). The concentration of lithium in each aliquot was calculated. A calibration curve was first generated and subsequently used to derive the concentration of lithium in each aliquot collected.

27.3 RESULTS AND DISCUSSION

[00494] The results for the percutaneous absorption of Lithium Chloride are shown in Figures 6-10. The water/oil (w/o) emulsion 28A displayed the lowest percent permeated through the dermis, with 60% released over a 12 hour time period. This emulsion was significantly different than the other prototypes, in that the drug was entrapped within lanolin alcohol lipid spheres and dispersed in a non-aqueous continuous medium (mineral oil). The emulsion 35 A' demonstrated relatively rapid permeation through the dermis, with 100% released in approximately 5 hours at 32 °C. The formulation BX is a neutral hydrogel, with its gel-like consistency produced by the presence of high molecular weight hydroxyethyl cellulose (HEC). The diffusion of lithium through the hy drogel and through the dermis is slower than 35 A', 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.

28. EXAMPLE 23: IN VIVO TIME-COURSE ASSESSMENT OF PERMEATION AND RESIDENCE TIME OF TOPICALLY ADMINISTERED LITHIUM IONS (AS A FUNCTION OF FORMULATION TYPE) THROUGH MOUSE SKIN TREATED WITH DERMABRASION AND FULL-THICKNESS EXCISION

[00495] 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.

[00496] Formulations selected in this experiment were : 35A\ 35BX and BV-001 -003A with their respective compositions as shown in Table 2 supra. 28.1 EXPERIMENTAL DESIGN

[00497] 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.

[00498] 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.

[00499] 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.

[00500] 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.

[00501] Skin and Plasma samples were taken every day at 2 hours, 4 hours and 8 hours post dosing to establish the skin permeation and residence time, correlated with lithium ion plasma concentrations in mM.

28.2 RESULTS AND DISCUSSION

[00502] The data (Figure 11) show that Li ions can be delivered through skin that has been perturbed by a standard method of integumental perturbation, such as dermabrasion. Significant levels of Li can be delivered through the dermis, with peak levels at

approximately 8 mM Li and trough levels at approximately 0.03 to 0.09 mM. Multiple dosing is preferred in order to achieve significant levels of Li in the skin. The pharmacokinetic profile shows that "pulsed" Li delivery can be accomplished.

[00503] 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.

[00504] These data demonstrate that Li ions can be delivered adequately through skin that has been perturbed by dermabrasion. In this dosing format, a once or twice daily

administration of lithium for a short period of time is envisioned. [00505] 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.

29. EXAMPLE 24: IN VIVO SKIN AND PLASMA DISTRIBUTION OF LI WITH SUBCUTANEOUSLY DELIVERED LITHIUM CHLORIDE IN A DOSE- ESCALATING FASHION IN C57/BLK MICE. WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS SKIN EXCISION

[00506] In this example, the skin and the corresponding plasma concentrations of Li ions were determined following subcutaneous administration of lithium chloride at increasing dose concentrations. T his protocol can also be adapted to determine follicular neogenesis as a function of increasing dose concentrations of lithium.

29.1 EXPERIMENTAL DESIGN

]00507] All lithium-containing formulations used lithium chloride dissolved in isotonic saline.

[00508] 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.

[00509 J Lithium treatment of the dermabraded mice started on the day of dermabrasion (= Day 1 ). DA mice received 42 mg/kg. 127 mg/kg, or 381 mg/kg subcutaneously, twice daily for 4 days, and one dose on the 5th day.

Table 3. DA

tt Animals /

FTE Group Grp Dose (mg/kg)

1 15 42 j

2 1 5 127

3 15 381 *

Wound + Vehicle

4 1 5 (saline)

5 1 Wound Only

No Wound, No

6 1 5 Treatment

injection.

[00510J Lithium treatment of FTE mice started the day of scab detachment (at day 10- 1 1 ). FTE mice received 64 mg/kg, 150 mg/kg, or 240 mg/kg subcutaneously, twice daily for 4 davs, and one dose on the 5th day. [00511 J At the 5 day, either one hour before intended dosing (for trough levels) or one hour after dosing ( for peak levels), the mice were sacrificed, and the entire wound area of skin was analyzed for Li concentration and blood was drawn and centriruged into red blood cells (RBC) and plasma. Then, at the 21 5t day, a section of skin was biopsied and analyzed for Li concentration, along with the corresponding plasma and RBC concentrations.

[00512] Mice that received FTE treatment as a mode of wounding were dosed on the day of scab detachment ( e.g. , on day 10- 1 5 post-wounding) with a single dose of lithium chloride for 5 days. At the 5th day of dosing, either one hour before intended dosing ( for trough levels) or one hour after dosing (for peak levels), the mice were sacrificed and the wound was biopsied and analyzed for Li concentration. Correspondingly, blood was drawn and centrifuged into RBC and plasma and assayed for lithium levels.

[00513] Lithium concentrations were measured by the validated bioanalytical ICP method provided below. LOQ for Li in the assay = 50 mM.

29.2 VALIDATED BIOANALYTICAL ICP METHOD FOR MEASURING SKIN LITHIUM CONCENTRATION

29.2.1 SCOPE AND APPLICATION

[00514] This method was developed to quantify lithium in murine skin, plasma and pellet of red blood cells (RBC). 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.

[00515] 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

[00516J Processing of plasma samples. A known amount of lithium working standard was added to measured volume of plasma. Concentrated nitric acid ( 1 mL) was added, and the mixture was heated in a microwave for approximately fifteen minutes. After digestion was complete, 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-μιη nylon filter, it was ready for injection into the 1CP/MS.

[00517] Processing of skin samples. Each skin sample was diced into smaller pieces. A known amount of lithium working standard was added to measured skin mass. Concentrated nitric acid (1 mL) was added and the mixture heated in a microwave for approximately fifteen minutes. After digestion was complete, digest was cooied and purified water was added to achieve a total volume of 50 mL. Once the solution was mixed and passed through a 0.45-μιη nylon filter, it was ready for injection into the ICP/MS.

[00518] Processing of pellet of red blood cells. To a v ial containing pellet of erythrocytes, 0.5 mL of 0.125% Igepal* CA-630 (dissolved in purified water) was added. 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- μηι nylon filter, it was ready for injection into the ICP/MS.

29.2.2 METHOD VALIDATION AND RESULTS

[00519] Prior to performing any quantitative assays on unknown test samples, method validation was performed to ensure that the methodology was sensitive, accurate, precise, and reproducible for the analyte of interest. The parameters to be characterized during method validation were system suitability. Lower Limit of Quantitation (LLOQ), precision, and accuracy (recovery).

[00520] System Suitability. The method assay was shown to work with sample matrices collected from many animals. Biological samples were obtained from at least six different mice that experimentally were not exposed to lithium, and replicates of these control samples were individually spiked with known amounts of the lithium-standard. Samples were processed for analysis as described above. In the absence of any interference, the amount of lithium measured with the analytical assay was expected to be identical to the spiked amount. This was the case, as shown in Table 4 for each of the three murine matrices: plasma, skin and pellet of red blood cells. Percent Recovery, which is the ratio of amount measured to amount spiked, was near 100% for all three matrices, varying between 98 - 1 10%. Matrix variability between replicate samples had a %CV value of less than 3%, which met acceptance criterion. (Acceptance Criterion: %CV < 15.0)

[00521] This included running six replicates of the mid range standard of the calibration curve. The Standard Deviation (SD) and Coefficient of Variation (CV) for the plasma, skin and RBCs were calculated. All three matrices met the criterion for %CV; for plasma it was 2.78, for skin it was 1.54 and for RBCs it was 1.18. The results are presented in Table 4. Table 4.

System S.lt.bilHy

[00522] Specificity. This test was performed to demonstrate that none of the components in test samples, either originally present or added to them during processing, contained significant amounts of test analyte. Because Igepal* CA-630 solution was used for the processing of RBC pellets, its lithium content was also tested. No interference was found in either water or Igepal-70 even when they were directly injected into the analytical instrument. The results in Table 5 indicate the level of lithium that was measured for blank unincurred matrix samples. These results include contribution from all solutions used in processing and indicate that lithium content was about 0.050, 0.034 and 0.035 μg/L for control plasma, skin and RBC pellets, respectively. (Acceptance Criterion: No significant interference from water and matrix blanks)

[00523] Water, plasma, skin, RBCs and Igepal® CA-630 blanks were run to show the specificity of the system. The baseline noise of lithium in plasma, skin, and pellet of red blood cells was 0.050, 0.034, and 0.035. The results are presented in Table 5. Table 5.

.Specificity

[00524| Linearity. Calibration curves for lithium assay are shown in Figure 12 for the various matrices runs. Each of these curves was obtained by spiking purified water with various amounts of lithium. The calibration curve was linear over the range of 0.05-50 μg/L when run with all matrices. The linearity of the curve was based on the coefficient of correlation associated with the scatter of the data points around the calculated straight line. In all cases, the r2 value for the coefficient of correlation exceeded 0.99.(Acceptance Criterion: The coefficient of correlation for each standard will have an r2 = 0.99 or greater)

[00525] The coefficient of correlation for the calibration curve was greater than 0.99

(Figure 12).

[00526] Precision at Lower Limit of Quantitation (LLOQ). LLOQ was measured at a concentration of 0.1 The lowest point in the calibration curve was not selected due to the noise level of the blank matrices. Six replicate unincurred blank matrix samples were spiked with lithium. Each of the six replicates was injected three times into the IPC. The results are presented in Table 6. In terms of %CV, precision for all six replicates was less than 9%, for all matrices. (Acceptance Criterion %CV for six replicates will not exceed 20.0)

[00527] This was calculated from the results of six replicates of a calibration standard at 0.1 ^ig/L of the calibration curve. The %CV for plasma was 5.83, for skin 8.83 and for RBC 8.46. The results are presented in Table 6. Table 6.

Lower UmU «f Quantitation

[00528] Accuracy. Accuracy was measured as % recover of measured analyte when blank matrix samples were spiked with known amounts of lithium. This test differs from that described in the section entitled '"System Suitability" in using fewer replicate samples, making no attempt to insure replicate matrices have originated from separate animals and in extending the measurement to three concentrations, which correspond to the low, mid, and upper end of the calibration range. Such spiked samples were also prepared and run routinely with unknown test samples— and so are called QC samples. They were the basis for demonstrating assay accuracy every time unknown samples were run. Table 7 presents the results obtained during validation. Except near LLOQ, percent recovery of measured analyte was found to be within 10% of its known amount. (Acceptance Criterion: Recovery of QC samples will be 80-120% for plasma and 70-130% for skin or RBCs)

[00529] Accuracy or recovery was measured with QC standards that were run in triplicate. Concentration of the three QC samples corresponded to the low, mid, and upper points of the calibration range. The recoveries for the plasma standards were from 87.2- 109%, for skin standards 70.6- 102% and for RBC 104- 123%. The results are presented in Table 7.

|00530] Precision. 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.

(Acceptance Criterion: %CV will not exceed 15.0.) Table 7.

Table 8.

Precision [005311 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. (00532) Intermediate Precision. The procedure used for intermediate precision was essentially the same as for the section entitled "Precision," but in this case the QC sample was prepared on a different day. Specifically, the plasma, pellet of red blood cells and skin were collected from the same mouse species as used for preclinical testing of the Sponsor's lithium-bascd drug. Results are presented in Table 9. In terms of %CV, precision was less than 1 .16%. (Acceptance Criterion: %CV will not exceed 15.0.)

Table 9.

Intermediate frecuton

[00533] Intermediate precision measurement was conducted according to the procedure in the section entitled "Precision," using a freshly prepared QC sample from the plasma, skin and the RBCs of the mice. The plasma, skin and the RBCs were from the same species of mice which were used for the preclinical mouse experiments described herein. The %CV for plasma was 1 .03, for skin 1.15 and for pellet of RBCs 0.530.

29.2.3 CALCULATIONS

Recovery

[00534] % Recovery - Calculated Concentration x 100

True Concentration

Coefficient of Correlation

[00535] r =∑¾zY where z is the standard score

n-1 Standard Deviation

Ϋ [individual data (X) - Mean Value (X)]2

[005361 Standard Deviation (SD)

n - i

% Coefficient of Variation

[00537] % CV = SD x 100

Mean

29.2.4 CONCLUSION

[00538] In this study, a method for the quantification of lithium in murine plasma, skin and red blood cells (RBC) was developed and validated.

[00539] The coefficient of correlation exceeded 0.99 for each of the three sample matrices over the calibration range from 0.1 g/L to 50 μg L. Plasma, skin, and RBC blank samples were spiked with known quantities of lithium to make standards. Standards for each matrix were prepared separately for each replicate of each parameter tested. The method was validated using 0.1 μσ/L as the lower limit of quantitation (LLOQ). All criteria for the System Suitability, LLOQ, Accuracy, Precision, and Intermediate Precision were met. It is noted that for LLOQ, all three matrices were measured with 0.1 ^ig/ standard instead of 0.05

(which was the lowest point in the calibration curve) due to matrix interference. This did not affect the outcome of the study.

29.3 RESULTS AND DISCUSSION

[00540] In the DA experiments, the 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 peak, ranged from 0.00035 mM-0.0029 mM (Figures 13 and 14). Li concentrations in blood, at peak, ranged from 0.2 mM-3.4 mM (Figures 14, 15A and 15B).

[00541] When lithium is administered subcutaneously in the FTE experiment, as for the DA experiment, the Li concentrations in skin and blood increased in a dose-related fashion. Like for the DA experiments, little Li ion extraction into RBC (on average, regardless of the dosage, RBC had 0.004-0.005 mM Li) and skin (Figure 16) was observed. Most of the Li was in the plasma (Figure 17). Li concentrations in skin at peak ( 1 hour post dosing, i.e., on day 5 (wherein day 1 = day of scab detachment and tlrst day of dosing)) were 0.00022-0.0015 mM (see Figures 16 and 18). Li concentrations in skin at trough (24 h post dosing on day 5) were 0.0001 -0.0009 mM Li. However, Li concentrations in blood at peak ( 1 hr post dosing on day 5) were between 0.695 mM - 1.059 mM Li (see Figures 17 and 18), and trough concentrations in blood were 0.02-0.09 mM Li. Animals injected with > 300 mg/kg had toxicity effects.

[00542] Thus, 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 15B). 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.

30. EXAMPLE 25: IN VIVO DISTRIBUTION OF LI WITH ONCE DAILY TOPICALLY ADMINISTERED LITHIUM FORMULATIONS IN C57 BLK MICE, WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS EXCISION

[00543] 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 l lydrogel (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.

Table 10

Formulation # 1 (Lithium Gluconate Formulation # 2 (Lithium Chloride

Hydrogel, "*HC73(F; also referred to Hydrogel, "BV-001 -003A"; also

herein as "lithium gluconate") referred to herein as "lithium chloride")

Lithium Gluconate, 8% Lithium Chloride, 8%

CarboPol 980 CarboPol 980

Glycerol Glycerol

Methyl Paraben Methyl Paraben

Propyl Paraben Propyl Paraben

Distilled Water Distilled Water

Sodium hydroxide (for pH adjustment) Sodium hydroxide (for pH adjustment)

Formula Weight: 202 g/'mol Formula Weight : 42.39 g/'mol

30.1 EXPERIMENTAL DESIGN

[00544] 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). Each day, animals were sacrificed at 1 h post-dose (for measurement of peak levels), 4-6 h post-dose, and 22-24 h post-dose (for measurement of trough levels) (i.e., tissue samples (skin and blood) were taken at t=l , 4-6h, 24h, 25h, 28h, 48h, 49h, 52h, 72h, 73h, 76h, 96h). See Table 1 1 below for a schematic of the experimental design. Table 11

3rd Dose 4th Dose

[00545) Each wound was dosed with a fonnulation 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.

[00546] Lithium ion concentrations were measured by ICP/MS/MS, using the validated method provided in Section 29.2 supra.

30.2 RESULTS AND DISCUSSION

[00547] The data show that Li ions are delivered into skin that has been perturbed by DA or FTE and into the blood when administered topically (see top and bottom graphs, respectively, of Figures 11 and 19, respectively). Lithium Chloride hydrogel and Lithium Gluconate hydrogel have similar pharmacokinetic profiles (compare light gray and dark gray curves, respectively). Significant levels of Li were delivered through the dermis, with initial peak levels in skin at approximately 8 mM Li and initial trough levels at approximately 0.3 to 0.09 mM (see top graphs of Figures 1 1 and 19). The pharmacokinetic profile shows that "pulsed" Li delivery can be accomplished, i.e., there is not a sustained release profile and repeated dosings are required to maintain peak or near-peak levels.

[00548] Lithium blood levels (see bottom graphs of Figures 11 and 19) were generally an order of magnitude lower than in skin, possibly because the formulation used - in which the Li ions are complexed with CarboPol 980 to form a polymer - enhances its residence in the skin, in contrast to Li ions in. for example, saline, which is expected to be highly water soluble. [00549] These data demonstrate that with topical administration of lithium chloride hydrogel or lithium gluconate hydrogel, Li ions are delivered through skin that has been perturbed by DA or FTE, but enter the blood at a much lower level. Delivery occurs in a pulsatile fashion.

31. EXAMPLE 26: IN VIVO DISTRIBUTION OF LI WITH TWICE DAILY TOPICALLY ADMINISTERED LITHIUM FORMULATIONS IN C57/BLK MICE, WITH THEIR SKIN TREATED WITH DERMABRASION OR FULL THICKNESS EXCISION AND CORRELATION TO HAIR FOLLICLE NEOGENESIS

[00550] The purpose of the experiment in this example was to evaluate the absorption of Li ions into the skin and blood compartments as a function of dosage (% Li) and dosing frequency in an in vivo mouse model developed for follicle neogenesis, with twice/day topical administration of two lithium formulations, a Lithium Chloride Hydrogel and a Lithium Gluconate Hydrogel (8%) (also referred to as "lithium gluconate, 8%") (and see Table 10 above), as well as a 1 % Lithium Gluconate Hydrogel (also referred to as "'lithium gluconate, and a 16% Lithium Gluconate Hydrogel (also referred to as "lithium gluconate, 16%"). This example provides an assessment of the rate of permeation and residence time of lithium ions provided in the formulations in conjunction with in vivo mouse experiments to assess hair follicle neogenesis. This experiment shows that topically administered lithium can induce formation of new hair follicles. It is postulated that lithium induces differentiation of stem cells into neogenic hair follicles.

31 1 EXPERIMENTAL DESIGN, RESULTS, AND DISCUSSION

[00551] 24 C57/BL 6 mice were enrolled in each group. There were 3 groups in total. The mice were dermabraded as discussed above and dosing was started at day 1, immediately after debriding the mouse skin with dermabrasion, and continued twice daily to day 4. Scab formation on the wound occurs approximately at day 1 , and thus the formulations are delivered prior to scabbing of the wounds. Each day, animals were sacrificed at 1 h post-dose (for measurement of peak levels), 4-6 h post-dose, and 22-24 h post-dose (for measurement of trough levels). See Table 12 below for a schematic of the experimental design.

[00552] 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. Table 12

[00553] Lithium ion concentrations were measured by ICP/MS/MS, using the validated method provided in Section 29.2 supra.

|00554) The data show that topical administration of Lithium Gluconate hydrogel delivers Li in a dose-correlated fashion into skin that has been perturbed by DA and, to a much lesser extent, into the blood (see Figure 20). Significant levels of Li were delivered through the dermis, and the trough concentrations were significantly higher with the twice daily dose than with once daily dosing. The pharmacokinetic profile shows that "pulsed" Li delivery is still maintained with twice daily dosing, i.e. , there is not a sustained release profile and repeated dosings are required to maintain peak or near-peak levels.

32. EXAMPLE 27: MOUSE MODEL OF LITHIUM TREATMENT FOLLOWING INTEGUMENTAL PERTURBATION USING FULL THICKNESS EXCISION OR DERMABRASION

(00555J The example in this section provides exemplary protocols for integumental perturbation by full thickness excision (FTE) and dermabrasion in mice and the results of studies conducted using these protocols that demonstrate the efficacy of combination therapies comprising lithium and integumental perturbation in inducing hair growth.

[00556] Specifically, this example provides protocols using four different combinations of lithium and integumental perturbation to induce hair follicle neogenesis: 1 ) full thickness excision + subcutaneouslv delivered lithium, 2) full thickness excision + topically delivered lithium, 3) dermabrasion + subcutaneously delivered lithium, and 4) dermabrasion + topically delivered lithium. 32.1 FULL THICKNESS EXCISION PROTOCOL

[00557] 1. Twelve ( 12)-day old C57BL6/J mice pups are used. They are fed high fat food from the day they arrive to the day of surgery (10 days).

[00558] 2. When the mice are 21 days old, full thickness excision (FTE) surgery is carried out.

[00559] 3. Mothers, domes, and high fat food is removed from the cages. Food is replaced with normal food.

[00560] 4. All pups for the experiment are placed into a large container to randomize.

[00561] 5. The pups are weighed one at a time. If under 7 g, they are placed into a separate "runts'' cage and FTE is not performed on these mice. If weight is made, mice are injected with calculated buprenorphine ("BUP"; 0.05 mg/kg). A stock BUP solution may be used that works out to 0.009 mg/ml, so 50 μΐ per 9 g mouse is injected. Place 6 mice per cage until all mice are weighed and given buprenorphine. During the administration of the anesthesia and for the duration of the time that the mice were anesthetized, the cages are placed on heating pads which are set to low heat.

[00562] a. ALTERNATE: If working with many mice (50+), it may be preferable to stagger the BUP dosing in order to avoid the effect of the analgesic wearing off before surgery. It is preferable to stay as close as possible to BUP administration at 1 -2 hours pre- surgery.

[00563] 6. One hour after giving the analgesic, the mice from one cage are injected with ketamine (70 mg/kg) / xylazine (8 mg/kg). Again, a stock that works out to 50 μΐ per 9 g mouse may be used.

[00564] a. ALTERNATE: Giving an additional 10 μΐ KX above the calculated dose (based on weight) seems to put the mice out to toe pinch quicker without increased fatality. However, it is only suggested to do this if few mice are not fully going out to toe pinch.

[00565] 7. Once mice are anesthetized, the proper number is car-punched, their weight recorded, their back hair shaved with clippers, and a 1 .5 cm x 1.5 cm box is marked on the rear dorsum.

[00566] 8. Eye ointment is applied to the mice, to keep their eyes from drying out during their immobilization, and the cages are pre- warmed on low-setting heating pads.

[00567] 10. The surgery site on the rear dorsum is sprayed/wiped with alcohol (70% ethanol) to prevent infections. [00568] 1 1 . A full thickness excision (FTE) ( 1.5 cm2) along the marked lines is cut out from the skin (cut inside the vertical lines and directly on the horizontal lines) using a pair of blunt-tip scissors and curved-tip forceps.

[00569] a. ALTERNATE: If letting mice live past day-5 post scab detachment, India ink should be used to mark the corners and sides of the wound (8 "dots").

]00570] 12. Finished mice are placed back in the pre-warmed cages and the cages are left on the pads until all mice awake. Eye ointment is reapplied if/when necessary.

[00571] 13. Each cage is supplied with a dish of wet food (moistened regular chow), regular dry chow, water, a dish of flavored JELL-O®, and a water bottle with flavored Prang (bio-serv.com, F2351-S; also avail. From Fisher) mixed in to aid in hydration. Saline is administered to mice that exhibit signs of pain, dehydration, malnourishment, or stress. Optionally, all cages can be provided with flavored Prang for the duration of the experiment in addition to their water supply. The mice may also be given flavored JELL-O® for the 2 days post surgery (JELL-O® when introduced in earlier experiments for rehydration purposes had a positive effect - mice eagerly ate the JELL-O® and looked healthier on days following the procedure).

[00572] 14. The mice are monitored and weighed over the next two days, as well as 2-3 times per week thereafter. The FTE wounds are allowed to heal by secondary intention. Dose mice AM and PM (approximately 10 AM and 5 PM) with BLIP on day 1 after FTE. Dose AM and if necessary in PM on day 2. Replace JELL-O® or water as needed.

[00573] 15. Mice are monitored daily for scab detachment (occurs 1 1 - 18 days post FTE).

[00574] 16. On the day of scab detachment, mice are placed into treatment groups:

[00575] 16a. For topical lithium experiment, animals are assigned to groups as follows: the 1 st mouse to lose its scab is placed in Lithium gluconate hydrogel (also abbreviated in this section as "lithium gluconate'"), 8%; the 2nd - lithium gluconate, 16%; the 3rd - Lithium Chloride hydrogel (also abbreviated in this section as "lithium chloride"), 8%; the 4th - lithium gluconate 1 %; the 5th - Placebo; and the 6th - No Treatment. This order is repeated as scabs detach until all mice are distributed evenly into treatment groups. After all mice are allotted to treatment groups, the sample sizes arc as follows: No treatment with mock handling (mock handling = Pick up and handle mice as if being dosed. An empty capillary tube is used to mock spread out drug onto their back under the bandage. This is done to ensure all mice undergo the same stresses during dosing): No treatment (n=18); Placebo (n=18); Lithium Chloride 8% (n= 19); lithium gluconate 1% (n= 19); lithium gluconate 8% (n=19); lithium gluconate 16% (n=19) (numbers correspond to the mice in the actual experiment summarized in Section 32.3, in which 1 12 mice remained alive at the time of scab detachment).

[00576] For topical lithium experiment, bandages (to prevent mice from licking off the drug) are applied as follows: On the day of scab detachment, the mice are administered 75% of a normal dose of ketamine-xvlazine according to weight. While the mice are anesthetized, the posterior dorsal, posterior lateral, and posterior ventral sides are shaved and treated with Nair. Care is taken to ensure that Nair did not cover the wounded area. Mice are then wrapped in Tegaderm & Telfa pad bandages. The Tegaderm is tightly wrapped around the mouse with the Telfa pad fixed over the wound / treatment area. The first treatment is administered once the bandages are in place. The mice are placed back into their cages before the anesthesia wears off. If at any point during the treatment period the mice escape from their bandage, the bandage is replaced. Mice are checked for bandages at every dosing interval.

[00577] 16 b. For subcutaneous lithium experiment, animals are assigned to groups as follows: On the day of scab detachment, mice are placed into five treatment groups: Saline (n=15), LiCl 64 mg/kg (n=15), LiCl 150 mg/kg (n=15), LiCl 240 mg/kg (n=15), No treatment (n=15) (numbers correspond to the mice in the actual experiment summarized in Section 32.3). Note that there are no bandages applied for the subcutaneous lithium experiment.

[00578] 17 a. For the topical lithium experiment, dosing is as follows: topical lithium doses ( 100 ul per 20 g mouse) are administered twice per day (for each mouse respectively), with the morning dose at ~ 1 1 AM and the afternoon dose at ~ 5 PM. The day of scab detachment is denoted as Scab Detachment Day 1 (SDl). Mice in treatment groups receive an AM and PM dose on SDl , SD2, SD3, and SD4 (8 total doses). Drummond wiretrols and accompanying ΙΟΟμΙ capillary tubes are used to dispense 1 ΟΟμΙ onto the wound site of each mouse. The tube is then used to spread out the drug to encompass the entire wound. One capillary tube is used per mouse. Drug treatment vials are replaced when necessary.

[00579] 17 b. For the subcutaneous lithium experiment, dosing is as follows: mice are dosed once their scab detaches (SDl = Scab Detachment - 1 ). Doses are given twice per day (am and pm) subcutaneously ( 100 μΐ for a 20 gram mouse).

[00580J 18. All mice are harvested on SD5, including mice used for determination of "peak" and "trough" lithium levels (see below). The mice are not treated on their respective harvest day (SD5), unless that mouse is used for a peak dosage sample, i.e. , selected as a peak value point for testing lithium compound levels in blood and skin. Some mice from each group were selected for evaluation of lithium levels in the skin and blood. Per group, three mice are used for "peak'" levels and three mice for '"trough" levels. Mice selected for the peak levels have blood drawn I hour post AM dosing on SD5 (9 total doses). Mice selected for "trough" levels have blood drawn 18-24 hours post SD4 PM dose (i.e., prior to harvest without treatment). On day 5 post scab detachment, approximately 0.5 ml of blood is collected per mouse, using the cheek lancet technique, and the mice are sacrificed. Wound skin is then analyzed by confocal microscopy and collected as described below.

[005811 19. Processing of mice for data collection: In preparation for in vivo scanning laser microscopy, the hair surrounding the healed wound is trimmed to reveal the wound surface. The surface is imaged with a confocal microscope; this includes 1 -3 Vivablocks (8 mm x 8 mm) at depths between 40-100pm, and 1 -3 Vivastacks (500 uM x 500 uM ) of areas with many neogenic hair follicles (NHF's) or interesting features. The mice are sacrificed, and the entire wound is excised (excluding normal surrounding tissue as much as possible), and tissue allocated in a procedure that lasts ~ 3-5 minutes per mouse after the time of death. Following excision, the wound is bisected. Half of the wound is placed on a section of a 3x5 inch index card and stored in cold 4% Paraformaldehyde for histology. The other half is frozen on dry ice for biochemical assays, including lithium levels. The frozen samples are placed on dry ice for the duration of the harvest period and then transferred to a -80° Celsius freezer for long term storage. The histology samples are taken out of the 4% Para- Formaldehyde solution following overnight fixation and moved to 30% sucrose/1 X PBS. Within 24 hours the samples are taken from the sucrose solution and dabbed dry, then embedded in OCT. The OCT is frozen in a slurry of crushed dry ice and 2-O-niethyl-butane. Cryosections are generated for histology. In the actual experiment the results of which are summarized in Section 32.3, for quantification of histology, approximately 10 slides were generated from the cut edge (midline) of the sample, and the first slide was stained with hematoxylin and eosin and was used for quantification. In the minority of cases, the tissue sections on the first slide were damaged, which then necessitated use of the next slide in the sequence. Only samples in which the wound was clearly demarcated from the adjacent normal were used for quantification of neogenic hair follicles.

[00582J Section 32.3 provides the results of an experiment in which the above FTE protocol was used.

32.2 DERMABRASION PROTOCOL

[00583| In the following protocol, a microdermabrasion device is used to perform dermabrasion. [00584| 1. 7 week old C57BL6/J female mice are obtained. The mice are allowed to acclimate for 2 weeks + 5 days before the day of surgery. Mice are 9 weeks +- 5 days old on the day of procedure.

[00585] 2. Mice are weighed and treated with buprenorphine ("BUP"; single IP injection. 0.05 mg/kg, 50 μΐ per 20 g mouse using a dosing solution of 0.02 mg/ml) 60 minutes before the procedure, on the day following dermabrasion (two doses, ~ 8 hours apart), and as needed, the second day after dermabrasion.

[00586] 3. After one hour has passed mice are re- weighed (because they are not ear punched before ketamine/xylazine dose), anesthetized with ketamine (80 mg/kg) / xylazine (8 mg/kg), and ear punched for identification. Mice are given an eye ointment to keep their eyes from drying out during their immobilization.

[00587] 4. Once the mice have ceased being mobile both the left and right sides of the dorsal rear back skin are clipped.

[00588] a. Nair® is applied for 1 minute to the right and left flank, the hair wiped off with a wet paper towel, and dried with paper towel.

[00589] 5. The mice are dermabraded once they do not react to a toe-pinch.

[00590] a. The microdermabrasion device settings (Advanced Microderm, DX model) are set to max vacuum, large tip, and max mixture.

[00591] b. aluminum oxide crystals are used for dermabrasion.

[00592] c. 10 passes on the right dorsal rear flank are carried out (each pass is a single movement from cranial to caudal direction; skin is held taught).

[00593] d. The left side of the body is not dermabraded.

[00594] 6. After dermabrasion, the 4 corners and the midpoints along the edge of the wound are tattooed with an injection of India ink (using a tuberculin syringe); 8 total tattoo marks made.

[00595] 7. Prior to waking up, the mice are bandaged as in the FTE protocol (only topical lithium experiment is bandaged), and dosing initiated (day 1 ) as follows:

[00596] A. For the topical lithium experiment: 1) Lithium gluconate hydrogel (also referred to in this section as "lithium gluconate") 1 % (n=15); 2) lithium gluconate 8% (n=15); 3) lithium chloride hydrogel (also referred to in this section as ''lithium chloride") 8% (n= 15); 4) lithium gluconate 16% (n=T 5); 5) placebo (n=15); and 6) no treatment (with mock handling)(n=15) (described in Section 32.3).

[00597] B. For the subcutaneous lithium experiment: lithium chloride 42 mg/kg (n=15), lithium chloride 127 mg/kg (n=15), lithium chloride 381 mg/kg (n=15), lithium chloride 600 mg/kg (n= 19). Saline (n=T 5), Wound-No Treatment (n=T 5), and No Wound-No Treatment (n=5). (No Wound-No Treatment is anesthetized and depilated with Nair, but not dermabraded) (for the experiment described in Section 32.3).

[00598] 8. The mice are then placed back into their respective cages which were pre- warmed on low-heat heating pads prior to surgery and kept on heating pads (under cage) until they wake up.

[00599] 9. Upon waking up (in the afternoon), the mice are treated again with the specified treatment, according to the label on their cage. Twice-a-day dosing continued until day 4 post-MDA / day 5 total

[00600] 10. During the dosing period, weights and observations of mice are recorded daily. The time of deliver)' of dose is recorded. For the subcutaneous lithium experiment, water consumption is monitored, as well as each cage provided with a salt water source (450 mM NaCl).

[00601 ] 1 1. Tissue is harvested from 1 cage (2-5 mice) of each group on day 5 post-DA for i) bioassay of target, ii) assay for concentration of compound in skin, and iii) histology.

[00602) a. This is done by resecting the skin from the right side and observing the ink spots from the dermal side of the skin. The wound area is cut out and divided into three pieces for assays as indicated above.

[00603] 12. Blood samples are also collected into potassium-EDTA vacutainers (2 ml capacity, lavender cap) from these same animals via cheek puncture lancets. The blood is centrifuged (maximum speed in non-refrigerated microfuge), the supernatant (plasma) is removed to a separate microfuge tube, labeled, and then frozen (along with the RBC cell pellet) for storage and shipment. Blood is taken within 1 -2 hours of the last dose to detenmine peak levels of lithium compound in skin, or alternatively prior to harvest without dosing if trough levels are desired.

[00604] 13. Remaining animals (~10 per group) are allowed to survive until 21 -23 days post dermabrasion at which time they were clipped and razored (disposable razor) on both the wound and non-wounded skin. A full thickness excision of skin from both treatment sites is excised. The skin samples are separately placed on 3x5 note cards and the surface of the excised skin (wound - right side; non-wounded - left side) analyzed by confocal microscopy in order to quantify the thickness of the hair shafts, density of hair shafts, the density of hair pores, and the number of shafts per pore.

[00605] 14. Following confocal microscopy, the skin is divided into three pieces as above. [00606] a. For biochemical assays, samples are immediately placed in Eppendorf tubes and frozen on dry ice, after which they are transferred to -80 °C freezer.

|00607] b. For histology, a piece of tissue is immersed in 4% PFA/PBS and stored at 4 °C until processing for paraffin histology, or for cryoscctioning, then changed to 30% sucrose/PBS (after overnight fixation) for between 12-24 hours. Samples are then embedded in optimal cutting temperature (OCT) and stored at -80 °C.

[00608] Section 32.3 provides the results of an experiment in which the above DA protocol was used.

32.3 SUMMARY OF EXPERIMENTAL DESIGN AND RESULTS

[00609] Based on results from the experiments described in the examples of Sections 22 to 31. an experiment to assess hair follicle neogenesis in mice was undertaken using the protocols described in Section 32.1 and 32.2 supra, as follows: One set of mice was enrolled for DA with 8% lithium chloride hydrogel, placebo and sham (untreated) (as described in Table 7). A second set of mice was enrolled for FTE (as described in Table 13). Dosing for the FTE groups was started at approximately day 10 - 15 post wounding, when the scab detached from the wound (this is referred to herein as "Day 1 "; each mouse was monitored for scab detachment and when this occurred, this was set as "Day 1 " of treatment), and continued twice daily to day 4 post-scab detachment. Dosing for DA starts on the day of the DA procedure.

Table 13.

[00610] Samples were taken from the mice administered topical and subcutaneous lithium at day 5 (skin and blood) to assess skin and blood concentrations at peak and trough. For the dermabrasion experiment, 5 of 15 mice are sacrificed to take samples at day 5 post DA. For the FTE experiment, all mice are sacrificed at day 5 post scab detachment (first day of treatment), some of which are used for measuring lithium levels in blood and skin. The results for the topical experiment are shown in Table 14. The blood levels at "peak" were at least 9-fold lower than the corresponding skin levels.

Table 14.

FTE / Skin (values in mM Li)

Peak Stdev Trough Stdev

Placebo 0.00 0.00 0.00 0.00

Lithium Gluconate. 1 % 0.92 0.93 0.15 0.21

Lithium Gluconate, 8% 2.67 2.55 0.70 0.12

Lithium Gluconate, 16% 1 1 .20 0.77 2.65 2.41

Lithium Chloride, 8% 3.77 2.29 0.25 0.15

FTE / Blood (values in mM Li)

Peak Stdev Trough Stdev

Placebo 0.00 0.00 0.00 0.000

Lithium Gluconate, 1 % 0.04 0.04 0.03 0.043

Lithium Gluconate, 8% 0.30 0.24 0.29 0.38

Lithium Gluconate, 16% 0.19 0.17 0.18 0.13

Lithium Chloride. 8% 0.30 0.045 0.12 0.015

DA / Skin (values in mM Li)

Peak Stdev Trough Stdev

Placebo 0.00 0.00 0.00 0.00

Lithium Gluconate. 1% 0.73 0.47 0.01 0.01

Lithium Gluconate, 8% 3.08 1.43 0.28 0.42

Lithium Gluconate, 16% 8.26 2.31 0.75 0.57

Lithium Chloride, 8% 1.12 0.30 0.22 0.06

DA / Blood (values in mM Li)

Peak Stdev Trough Stdev

Placebo 0.00 0.000 0.00 0.000

Lithium Gluconate, 1 % 0.03 0.02 0.01 0.004

Lithium Gluconate, 8% 0.29 0.07 0.09 0.02

Lithium Gluconate, 16% 0.91 0.14 0.1 1 0.02

Lithium Chloride. 8% 0.27 0.022 0. 10 0.035

[00611] Parts of the same tissue samples shown in Table 14 were also used to assess hair growth.

32.3.1 FTE RESULTS

[00612] In the FTE study using topically administered lithium, quantification of confocal images showed that, compared to placebo, treatments containing 8% lithium (lithium gluconate 8% or lithium chloride 8%) had higher mean and median numbers of germs, germ densities within the germ forming region (GFR), and germ densities within the total wound area (TWA). The 8% lithium chloride treatment also had higher mean and median GFR areas and percent coverages of TWA by GFR, while the 8% lithium gluconate treatment had higher mean, but not median, GFR areas and percent coverages of TWA by GFR. Although results for the lowest strength lithium treatment (1% lithium gluconate) were essentially

indistinguishable and in some cases slightly less favorable than those of the placebo group, there was not an overall dose response because the results for lithium gluconate 16% were less favorable that those for the two 8% strengths. Results from the group of mice that did not receive any treatment were generally superior to placebo, 1 % lithium gluconate, and occasionally rivaled those from the 8% and 16% strength treatments.

[00613] As shown in Figures 21-23, in the FTE experiment using topically administered lithium, relative to placebo and lithium gluconate 1 % and 16%, lithium chloride 8% and lithium gluconate 8% promoted increased differentiation of neogenic hair follicles as detected histologically. Compared to placebo, the lithium chloride 8% treatment was statistically significantly superior with respect to both the total number of neogenic hair follicles and the proportion of mature stages (Stages > 5). The lithium gluconate 8% treatment also appeared to be superior to placebo, but the differences did not reach statistical significance after adjusting for multiple comparisons. The lithium gluconate 16% treatment had somewhat better results than placebo, but none of the comparisons approached statistical significance. The results for the lithium gluconate 1% group were similar to those for placebo.

[00614] It is noted that mice from all groups treated with topical lithium had

approximately the same weight gain profile throughout the entire experiment (see Figure 24), indicating that topical lithium (at all concentrations) did not have any negative effects, such as systemic effects that negatively impact overall health of the mice, and then which may indirectly affect hair growth.

[00615] Insignificant effects were seen in the experiments in which lithium was delivered subcutaneously, despite achieving maximal tolerated doses in the mice. It is postulated that lithium administered subcutaneously at non-toxic dosages did not achieve high enough concentrations in the skin to effect hair follicle neogenesis.

32.3.2 DA RESULTS

[00616] In the dermabrasion experiment (using dermabrasion + topical lithium), it was intended to analyze four variables using confocal imaging: hair shaft density, hair pore density, number of hair shafts per pore, and hair shaft diameter. Since the number of pores and hair shafts was the same, the density analysis was done on hair shafts. No formal histological analysis was made in this experiment as hair follicle density was unchanged among all treatment groups.

[00617] Compared to placebo, the mean hair shaft diameter was statistically significantly larger (16%) for mice receiving lithium gluconate 8% (see Figures 25 and 26). The lithium chloride 8% and lithium gluconate 16% treatments also had higher mean hair shaft diameters, but unlike the lithium gluconate 8% groups, the differences did not reach statistical significance. With respect to hair shaft density, there were no statistically significant differences between placebo and any of the lithium-containing treatments. Results from the group of mice that did not receive any treatment were comparable to those of the placebo group.

[00618] The histological hallmark of male and female pattern hair loss is miniaturization of hair follicles with a progressive transformation of terminal hair follicles into vellus-like follicles: terminal hair follicles have a shaft diameter of greater than 0.06 mm, whereas vellus-like follicles are defined as hairs with a hair shaft diameter of 0.03 mm or less and are thinner than the hair's inner root sheath (Dinh and Sinclair, 2007). The findings of an increase in hair shaft diameter in the 8% and 16% lithium groups and the observation histologically that the hair follicles appeared to disassemble and later reassemble during wound healing is suggestive that hair follicle neogenesis occurs also following more superficial wounding in association with topical application of lithium.

[00619] It is noted that mice from all groups treated with topical lithium had

approximately the same weight gain profile throughout the entire experiment (data not shown), indicating that topical lithium (at all concentrations) did not have any negative effects, such as systemic effects that negatively impact overall health of the mice, and then which may indirectly affect hair growth.

[00620] Insignificant effects were seen in the experiments in which lithium was delivered subcutaneous ly, despite achieving maximal tolerated doses in the mice. It is postulated that lithium administered subcutaneously at non-toxic dosages did not achieve high enough concentrations in the skin to effect hair follicle neogenesis.

32.3.3 SUMMARY

[00621] Topical delivery of lithium achieved positive effects on hair follicle neogenesis in both the full thickness excision and dermabrasion models. Both the 8% lithium formulations (lithium chloride and lithium gluconate) used were found to improve growth of hair in the mouse skin. Compared to placebo, the animals treated with either topical formulation of 8% lithium had a higher percentage of neogenic hair follicles at a more mature stage of development (see Figures 21 to 23), increased thickness of hair shafts (see Figures 25 and 26), an increased number of neogenic hair follicles in the wound site (Table 15 and Figures 27 to 31), and normal patterning of hair follicles (as determined by density of neogenic hair follicles: see Table 15 and Figures 32 and 33).

Table 15.

[00622] Specifically, for the FTE + topical lithium experiment, in addition to an increased number of neogenic hair follicles observed histologically, twice-daily lithium 8% treatments increased the maturation of neogenic hair follicles (the numerical difference from the lithium chloride 8% group achieved statistical significance). For the dermabrasion + topical lithium experiment, twice-daily lithium 8% treatments increased the shaft diameters of hair follicles formed after dermabrasion (the numerical difference from the lithium gluconate 8% group achieved statistical significance). The effect of lithium 8% for hair follicle neogenesis and in increasing hair shaft diameters in these experiments was greater than that associated with the lithium 1% and 16% concentrations. Although 16% lithium gluconate administration resulted in significant delivery of Li to the skin, new hair follicles were not observed in mice treated with this concentration.

[00623] In summary, twice-daily topical application of lithium 8% induces hair follicle neogenesis following integumental perturbation by either full thickness excision or dermabrasion. The results of these experiments provide evidence of efficacy of pulsatile treatment of lithium in combination with integumental perturbation.

33. EXAMPLE 28: COMPARISON OF RELEASE RATES OF COMPLEXED AND NON-COMPLEXED LITHIUM CHLORIDE SOLUTIONS

[00624] One approach to slowing down release of Li ions from a matrix is to "complex" or bind the positively charged ions to a negatively charged bioadhesive polymer. This experiment presents a correlation of release rate to the extent of binding of Lithium to the anionic polymer. The anionic polymer was selected to be partially crosslinked polyacrylic acid, or Carbopol 980, with ionizable carboxylic acid groups with a pKa ~5-6.

33.1 EXPERIMENTAL DESIGN

[00625] Solutions of lithium chloride were prepared to 1. 5 and 8% in saline. These solutions are hypothesized to have instant release into the medium. Solutions of lithium chloride in 1% Carbopol were prepared in strengths 0.5%, 1%, 4%, 8%, 10% and 20% w/w. To prepare these solutions, lithium chloride solutions were added to a solution of Carbopol 980, dissolved previously with distilled water and pi I to 7. The shear flow rheology of each of these formulations was tested in a cone-plate Brookfield Viscometer at temperature of 37 °C. In vitro release experiments were carried out using dialysis cassettes immersed in phosphate buffered saline at pH 7.4, with slow stirring.

33.2 RESULTS

[00626] Viscosity. The shear flow viscosity of lithium chloride was very low, as in water (< 10 cp). The viscosity of placebo Carbopol 980 at a 1% solids content was 3800 cp at 37 °C. The viscosity dropped as higher concentrations of Lithium Chloride were added to the Carbopol due to complexation and neutralization of the negative charge. The solution precipitated at 20% w/w Lithium Chloride, demonstrating the complexation had been completed and the resulting complex had exceed the theta point. The viscosity of Lithium Chloride as a function of concentration was as follows: Placebo : 3800 cp > 0.5% Li Carbopol : 2800 cp > 1% Li Carbopol : 2000 cp > 4% Li Carbopol : 1500 cp > 8% Li Carbopol : 1015 cp > 10% Li Carbopol : 430 cp > 20% Li Carbopol : precipitate.

[00627] Release Rates. The release rate of complexed Li was slowest for the precipitate and highest for uncomplexed Lithium Chloride in Saline. Since the concentration of Carbopol was identical in all of the complexation solutions, this suggests that complexed Li can be utilized as a technique to retard release of the highly water soluble Li ion from a matrix.

[00628] At pH > pKa, aqueous solutions of CarboPol increase dramatically in viscosity, due to repulsion of ionized carboxylates. Positively charged Li ions readily bind to the anionic polymer. Thus, the release kinetics of Li+ from a gel of CarboPol is due to its de- binding kinetics. The release of Li from this matrix is slower than its release from a matrix such as saline. 34. EXAMPLE 29: MICROENCAPSULATION OF LITHIUM

GLUCONATE IN BIODEGRADABLE POLY (D,L-LACTIDE-CO- GLYCOLIDE PLG) MICROSPHERES

[00629] Lithium gluconate was encapsulated in PLG microspheres for the purpose of developing a sustained release Li Gluconate formulation that can be embedded into the dermis.

34.1 EXPERIMENTAL DETAILS

[00630] Lithium Gluconate was dissolved in water at a concentration of 8% and complexed with Carbopol 980. This is referred to as the "complexed Lithium Gluconate solution." Separately, Lithium Gluconate was dissolved in water at a concentration of 8%.

[00631] 1 ml of the Lithium Gluconate complex was added to 4 ml of 28 mg/ml PLG solution in methylene chloride and homogenized for 50 s at 7500 RPM. The resulting milky white dispersion was immediately poured into a aqueous solution containing 1% PVA and homogenized for another 1 minute at 7500 RPM. The dispersion was then poured into a large excess of 0.5% PVA and stirred for two hours at ambient temperature to evaporate the methylene chloride. The spheres were then washed three times with water and lyophilized to produce a free-flowing powder.

[00632] The encapsulation efficiency of complexed Lithium Gluconate was measured by a colorimetric Li assay.

[00633] In another experiment, uncomplexed Li was encapsulated and encapsulation efficiency measured by a colorimetric Li assay.

34.2 RESULTS AND DISCUSSION

[00634] The encapsulation efficiency of Li when using complexed Li was approximately 55%. In contrast, the encapsulation efficiency of Li using uncomplexed Li Gluconate was 7.74%. Spheres obtained by this method were approximately 1 -4 microns in diameter, as shown in Figures 34 and 35.

[00635] This experiment demonstrates that encapsulation efficiency in microspheres can be enhanced with complexation of Li with an anionic polymer, prior to encapsulating in the PLG matrix. 35. EXAMPLE 30: CLINICAL EVALUATION OF NEOFOLLIC LA

DEVELOPMENT RESULTING FROM APPLICATION OF LITHIUM GLUCONATE IN COMBINATION WITH DERMABRASION WITH OPTIONAL REPROGRAMMING

[00636] The following example provides a protocol for demonstrating the importance of timing of lithium gluconate treatment for the optimization of follicular neogenesis after epidermal disruption. In this protocol, patients are treated with a pulse lithium of 8% lithium gluconate (topical gel) in combination with dermabrasion, optionally also in combination with an additional treatment to enhance hair growth. As controls, 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. |00637] Although any patient population may be treated, preferred patients are Caucasian males 20-50 years of age, have androgenetic alopecia with the presence of a transition zone defined as an area possessing both terminal and miniaturized hairs, have a Fitzpatrick skin type 1-4 (higher Fitzpatrick skin type ratings are not preferred due to the increased risk of keloid formation and hypopigmentation in these subjects). 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. 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.

[00638] Dermabrasion using alumina particles is performed on Day 0. Dermabrasion is performed to a depth of approximately 100 μΜ, 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 scalp skin corresponding to transitional areas (or advancing margin) of balding in the vertex region. 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. In the event that limited follicle neogenesis is observed on day 14, another biopsy may be performed on day 17. Subjects scheduled for day 1 1 biopsies for whom the scab in the wound detaches before day 8, will have the biopsy rescheduled for 3 days afterwards. Conversely, subjects for whom the scab has not detached by day 10 will have the biopsy visit rescheduled for 3 days after the scab detaches. It is expected that the scab will detach around days 6-10. Subjects return on day 46-50 for follow up photography of the treated area.

[00639] 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 in two sites: one site corresponding to the area of greatest balding on the vertex and one site corresponding to a transitional area (or advancing margin) of balding in the vertex region.

35.1 DETERMINATION OF TREATMENT SITES

[00640] Two sites on each subject's scalp are identified for treatment, both corresponding to transitional areas (or advancing margins) of balding in the vertex region. Some patients may be treated in a site of greatest balding on the vertex region.

35.2 SCANNING LASER CONFOCAL MICROSCOPY

[00641] Scanning laser confocal microscopy (CM) is a non-invasive imaging technique that uses laser light to visualize the skin in vivo. Progress of treatment, starting at day 0, may be monitored using CM alone or in combination with photography. This fluid immersion microscope requires oil / water immersion to measure changes in the index of refraction within the tissue as detected by the reflected laser light. Real-time noninvasive confocal infrared imaging of the epidermis, papillary dermis, and superficial reticular dermis to a maximum depth of 350 μηι is possible with resolution comparable to conventional light microscopy. Skin can be imaged in its native state without the fixing, sectioning, and staining necessary for histology. As such, dynamic processes can be noninvasively observed over an extended period of time. As a research tool, RCM has been reported to facilitate the in vivo assessment of several pigmented (Grimes, 2005) and nonpigmented skin lesions. See Curiel- Lewandrowski, et al., Use of in vivo confocal microscopy in malignant melanoma: an aid in diagnosis and assessment of surgical and nonsurgical therapeutic approaches. Arch Dermatol 140 (2004), pp. 1 127-1 132; Gerger et al., Diagnostic applicability of in vivo confocal laser scanning microscopy in melanocytic skin tumors, J Invest Dermatol 124 (2005), pp. 493- 498; Swindells et al. Reflectance confocal microscopy may differentiate acute allergic and irritant contact dermatitis in vivo, J Am Acad Dermatol 50 ( 2004), pp. 220-228; Aghassi, et al., Time-sequence histologic imaging of laser-treated cherry angiomas with in vivo confocal microscopy, J Am Acad Dermatol 43 (2000), pp. 37- 1 : and Gonzalez et al. Non-invasive (real-time) imaging of histologic margins of a proliferative skin lesion. In Vivo. J Invest Dermatol 1998 1 1 1 : 538-539. This technique has been successfully used to image hair follicle neogenesis in mice and should have similar utility in people. Incorporating CM imaging into this protocol will help determine the optimal biopsy day for a subject.

[00642] Confocal microscopy is performed using a device (Vivascope; Lucid, Inc.) used for human studies. Subjects are positioned in an inclined or sitting position to allow visualization of the treated area of the scalp. Each subject remains still in the imaging position for a minimum of 15 minutes of imaging per subject. A medical grade adhesive secures the fluid immersion ring to the surface of the skin. The ring remains attached throughout the imaging session and a new ring is applied on each test site. CM is performed on baseline Day 0, 7, and 14 ( 1 1 and 17 if punch biopsy was not taken on Day 14).

35.3 DERMABRASION

[00643] 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 μΜ, which includes removal 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. Each dermabraded area is approximately a 1.5 cm x 1.5 cm square. Suitable dermabrasion devices are the ASEPTICO ECONO-DERMABRADER from

Tiemann and Company, the DX system from Advanced Microderm (see, e.g. ,

http://www.advancedmicroderm.com'products/tech_specs.html), or the M2-T system from Genesis Biosystems. 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.

[00644] 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.

[00645] Following the procedure 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. Do 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.

[00646] The treated are may itch as the new skin grows and may be slightly swollen, sensitive, and bright pink for several weeks after dermabrasion.

[00647] The following measures are taken to prevent any complications.

[00648] · Inform your doctor of any yellow crusting or scabs - this may be the start of an infection.

[00649] · Swelling and redness should subside after a few days to a month. Persistent redness of an area could be the sign of a scar forming so contact your doctor immediately.

[00650] · No swimming is permitted for the first 7 days following dermabrasion.

[00651] · To avoid pigmentation, once the new skin is healed, keep out of the sun and apply a broad spectrum sunscreen daily for at least 3 months after microdermabrasion. Even the sun through window-glass can promote unwanted pigmentation.

35.4 PUNCH BIOPSY

[00652] 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 Ethalon sutures. Vaseline and band-aid are applied. Tissue samples are stored in formalin for histological analysis. 35.5 PRIMARY ENDPOINTS

[00653] Histologic analysis of hair follicle neogenesis following dermabrasion. The null hypothesis is that no (0) neogenic follicles will form, since that is the current dogma in humans. A positive response to treatment is characterized as the appearance of 3 or more neogenic follicles in a 4 mm punch biopsy.

[00654] Among the factors to be evaluated when determining success of treatment arc: crusting/scabbing; comedones; infection; pigmentary changes (e.g. , absent, hypopigmentation (mild, moderate or severe), or hyperpigmentation (mild, moderate or severe)); scarring; re- epithelialization; or presence of hair follicles by gross observation.

35.6 SECONDARY ENDPOINTS

[00655] 1) To determine which day after epithelial disruption is follicle neogenesis most active.

[00656] 2) To quantify the number and morphological developmental stage of follicles in each biopsy.

[00657] 3) Clinical characteristics of dermabraded areas.

35.7 RESULTS

[00658] An exploratory clinical study without lithium, i.e. , to determine the effect of dermabrasion on formation of new hair follicles, was carried out in accordance with the protocol described above. In particular, the goal of the exploratory clinical study was to examine whether removal of epidermis and some papillary dermis could induce hair follicle neogenesis in human scalp.

[00659] General morphological parameters were assessed from sections stained by hematoxylin and eosin (H&E). The presence of elastin fibers was determined using the Luna stain. Immunohistochemistry was performed using antibodies against BerEP4 (a marker of embryonic hair follicles), and i67 (a marker of cell proliferation). Alkaline phosphatase (AP) histochemistry was used to identify the dermal papilla of the HF. Digital camera images were used to record the visible aspects of the wound and the healing process.

[00660] Thirteen of the 15 enrolled subjects had skin biopsies in each of their two dermabraded sites. Two of these 13 subjects ( 15%) had neogenic-like hair follicles present in both their left and right sites, for a total of four positive biopsies out of 26 adequate biopsies ( 15%). [00661] Ten subjects had biopsies of the control sites. No neogenic-like hair follicles were present where there had been no dermabrasion.

36. EXAMPLE 31 : CLINICAL EVALUATION OF LITHIUM GLUCONATE AND INTEGUMENTAL PERTURBATION IN HAIR FOLLICLE REGROVVTH

f 00662 J This example provides a clinical protocol to examine the use of topically applied lithium to promote hair follicle neogenesis in male subjects with androgenetic alopecia in association with integumental perturbation. The two methods that will be used include the more superficial disruption with dermabrasion and the deeper (extending to the level of the subcutaneous fat) full thickness skin excision using punch biopsy.

[00663] Based on preclinical data (see, e.g. , the example of Section 32, supra) and the exploratory human data (see the example of Section 35), the clinical hypothesis underpinning the primary and secondary objectives of this protocol is that topical lithium will increase the number of neogenic-like hair follicles in skin that has undergone integumental perturbation (also referred to in this section as "controlled perturbation") to place it into a regenerative state. The controlled perturbation can be more superficial (e.g. with a dermabrasion affecting mostly the epidermis) or can be more profound (e.g. when a punch biopsy affects both the epidermis and the deeper dermis). It is also possible that this type of stimulation may lead to macroscopic evidence of hair growth.

36.1 PROTOCOL DRUG

[00664] This protocol uses lithium gluconate 8% gel (Lithioderm). it is formulated as Lithium gluconate 8.00 g/100 g; 100 g of gel contains 274.80 mg of lithium ion. Excipients present in the Lithioderm formulation are Carbomer (Carbopol 980), glycerol, sodium hydroxide, methylparaben, propylparaben, and purified water.

[00665] Lithioderm is marketed in France, approved for local (topical) treatment of facial seborrheic dermatitis in adults (redness and flaking), and granted reimbursement status by the Agence francaise de securite sanitaire des produits de sante (AFSAPPS) in 2002. See also http://www.labcatal.eom/produits31d.htm#rcpl

[00666] Lithioderm is recommended for use in a symptomatic treatment of seborrheic dermatitis. For seborrheic dermatitis, Lithioderm is administered in 2 applications per day, 1 morning and 1 evening for 2 months. The gel is applied on the entire face in a thin layer on clean, dry skin, massaging gently until penetration of the product. Measures of systemic transition in vivo and a model in vitro suggest a generally low absorption of lithium after topical application of Lithioderm Gel.

[00667] Usage of Lithioderm should be discontinued or avoided if there is hypersensitivity to any constituents, and the product should not be administered concomitantly with lithium orally and / or topically. In the absence of clinical data with lithium used topically, and although the doses used are low, as a precaution, it is preferable not to administer the drug during the first trimester of pregnancy. Administration of lithium at doses used in psychiatry may cause fetal malformation involving mainly the heart. The use of this product is possible during lactation; however, Lithioderm gel should not be applied on the breasts. Lithioderm gel is generally well tolerated. Rare cases of burning or increase of erythema have been reported, most often transient. Due to the presence of methyl parabens and propyl, there is a risk of contact dermatitis, and in exceptional cases, immediate reaction with urticaria and bronchospasm.

[00668] Lithium succinate 8% formulated in a petrolatum-based ointment (Efadermin) is available on the market in Germany also for the treatment of seborrheic dermatitis.

Lithioderm instead of Efadermin is used in the current protocol; however, the protocol may also be tested with Efadermin.

[00669] The topical safety of these products, including irritation and sensitization of the formulations is well-established. The safety profile is characterized as well tolerated, with side effects of rare cases of burning or increase of erythema, which are usually transient. In a Good Laboratory Practice (GLP)-compliant acute dermal irritation toxicity study (Waller, 2009), application of Lithioderm 0.1 mL or placebo gel (vehicle alone) twice-daily for 7 days to New Zealand white rabbits (10M, l OF) was not irritating to the skin of the rabbit, and did not adversely affect the healing of dermabraded skin. The systemic adverse drug reactions (ADRs) associated with oral lithium therapy (used to treat acute mania and for the prophylaxis of recurrent affective disorders) have not been reported with topical application of lithium (SmPC Lithioderm; SmPC Efadermin), due to the much smaller doses and minimal systemic exposure associated with topical administration.

[00670] Thus the use of Lithioderm in this protocol poses no new safety concerns. The exposure to Li+ after the intended application of Lithioderm (target 0.1 mL to 3 sites twice- daily, with a range based on varying deliverable droplet size of 0.068 mL to 0.219 mL) is anticipated to be limited, consistent with what has been reported for its use in seborrheic dermatitis (Dreno et al, 2002). In this protocol, at the maximum dose that is envisaged, even assuming all of the lithium applied was absorbed, the total maximum lithium gluconate exposure is 1 10.4 tng per day ( 1.38 g [0.23 x 6] x 80 mg/g). In terms of Li+ absorbed, this translates to a maximum exposure of 3.79 mg of Li+ per day. This level of exposure is anticipated to be similar to the exposure that occurs when patients use an 8% lithium product to treat seborrheic dermatitis over their cheeks, nose, and forehead (the SmPC for Lithioderm states to apply to the entire face in a thin layer twice-daily [morning and evening] for 2 months).

[00671] Oral lithium (as the carbonate salt) is administered in significantly higher doses. Acute mania can be treated with 1200-2400 mg/day in divided doses in hospitalized patients, targeting a serum concentration between 0.9 and 1.1 mM (Baldessarini and Tarazi, 2006). For outpatients for treatment and for long-term use to prevent recurrent manic-depressive illness, desired serum Li+ levels are 0.6 to 1 .25 mM, which is usually achieved with 900 mg to 1 ,500 mg of lithium carbonate per day in divided doses. In these patients treated with oral lithium carbonate, mild to moderate adverse reactions may begin to occur at Li+ serum concentrations of >1.5 mM.

[00672] In patients with seborrheic dermatitis treated with lithium gluconate 8% gel (Lithioderm®), the serum lithium levels at Day 28 and Day 56 of treatment were reported to be 4.48 micrograms/L (0.00064 mM Li+) and 4.15 micrograms/L (0.00060 mM Li+), respectively, compared to a value of 2.76 micrograms/L (0.00040 mM Li+) at baseline (prior to dosing) (Dreno & Moyse, 2002). In the placebo group the corresponding values remained unchanged at 2.6 micrograms/L (0.00037 mM Li+). This indicates minimal systemic exposure to Li+ through skin compromised with seborrheic dermatitis, and as the authors note, the increase to 4.4 micrograms/L remains far from the toxic level of 9000

micrograms/L (1.297 mM Li+), and very low compared with patients treated by systemic lithium. In the present protocol, if the systemic lithium exposure from topical administration of Lithioderm is comparable to that in the treatment of seborrheic dermatitis, serum concentrations would be approximately 900-fold below the lower limit of the therapeutic range for the use of oral lithium carbonate for acute mania (0.6- 1.0 mM), with a safety margin in excess of 2,000 relative to the concentrations usually associated with toxic symptoms ( 1.5 mM).

36.1.1 LITHIODERM TREATMENT REGIMEN

[00673| Lithium treatment regimens combined with the 2 different types of integumental perturbation are used in this protocol. [00674| In this protocol, on Day 0 the first integumental perturbation is with dermabrasion (DA); Lithioderm (also referred to in this section as "drug" or "product") is applied topically immediately following DA and then continued twice daily through the second dose on Day 1 1.

[00675] On Day 14, the second type of integumental perturbation, i.e. , a full thickness excision (FTE) biopsy (also referred to as "punch biopsy"), extending to the level of the subcutaneous fat, is performed. The drug will have been discontinued after the final dose on Day 1 1 (2 days prior to this procedure). Application of drug begins again on Day 17, and continues twice daily through to the end of Day 34, with a final dose on Day 35. See the intervention schedule below.

Intervention Schedule l v l I I- 111111 i 11 i -1-

• bi piy ifTE - :jf>pr- . ! I -:io>o - ,Ι

• IO t RE '.cab :!vt i ,n-.or,l

- appro , 'irne t ioj

[00676] The first biopsy on Day 14 is a full thickness excision that is being combined with drug (Lithioderm or placebo) to evaluate if this different integumental perturbation combined with pharmacologic modulation by lithium induces follicular neogenesis. A time-point that is three days post-biopsy (Day 17) is selected for the beginning of drug dosing. Nineteen days of treatment post biopsy is chosen.

[00677] Because the SmPC of Lithioderm indicates that its topical application is generally well tolerated when the drug is applied twice daily for 2 months in the treatment for seborrheic dermatitis, it is postulated that the increased possibility of catching cells that are receptive (before the inductive window for follicular neogenesis may be closed) outweighs the risk of dosing for a few extra days.

36.1.2 RISKS, DISCOMFORTS AND POTENTIAL BENEFITS

Pharmacological intervention

[00678] The preclinical studies and human studies presented herein show that stimulation of follicular neogenesis may represent a successful therapeutic intervention in the treatment of androgenic alopecia. Based on these data, topical (dermal) lithium application (e.g. , lithium gluconate 8% gel) is expected to enhance hair follicle neogenesis.

[00679] Data from a primary pharmacodynamic study in mice following full thickness excision (FTE) (see. e.g., the examples of Sections 28-30 and 32 supra) suggest that twice- daily (bid) topical application of lithium-containing gel (Lithioderm) at a concentration of 8% might enhance wound-induced hair follicle neogenesis following FTE. Following dermabrasion, twice-daily topical application of Lithioderm gel 8% resulted in a statistically significant increase in hair shaft diameter compared to placebo suggesting that hair follicle neogenesis may occur also following more superficial wounding in association with topical application of lithium (see example of Section 32). Thus, current preclinical experimental evidence suggest that topically applied lithium may promote follicular neogenesis and finally hair growth.

[00680] Lithioderm® 8% gel is designated for the treatment of seborrheic dermatitis. The active ingredient is lithium gluconate (8 g lithium gluconate/100 g gel: 80 mg lithium gluconate/1 g gel). According to the SmPC for Lithioderm®, 100 g of gel contains 274.80 mg of lithium element (or litihium ions) (2.748 mg/g gel). Lithioderm is approved in France for the treatment of seborrheic dermatitis. In Germany and Switzerland, there is another approved lithium-containing preparation (Efaderm®) on the market for the same indication. It contains 8% lithium succinate and 0.05% zinc sulfate as active ingredients.

[00681] In general, due to the extremely low plasma levels of Li+, adverse

pharmacodynamic interactions of clinical relevance are not expected. No topical drug-drug interactions have been specified in the prescribing information (SmPC Lithioderm, SmPC Efadermin). No published data have been identified to suggest drug-drug-interactions between topical lithium gluconate 8% gel and other topically applied products. There is no evidence of mutagenicity or carcinogenicity. At normal therapeutic oral doses, it is unlikely that lithium would be teratogenic. However, due to lack of suitable clinical studies, its use may not be suitable in the first trimester of pregnancy. Similarly, due to lack of suitable clinical experience, it may be advisable that patients avoid excessive exposure to sunlight during the treatment with lithium gluconate 8% gel.

[00682J The local tolerance of lithium-containing gel is good. Lithium per se is non- sensitizing. However, excipients in the gel formulation (Lithioderm) may cause allergic reactions. It is noted that the oral use of lithium is well established worldwide since the 19th century for the treatment of bipolar disorders. A variety of oral formulations and lithium salts, i.e. carbonate, sulfate, citrate, succinate, hydrogen aspartate, have yielded an extensive body of data on the safe use of this drug in humans. The same has been shown with dermally applied lithium in the treatment of seborrheic erythema using much smaller doses than oral lithium and is to be expected in the treatment of patients with alopecia.

[00683J Therefore, the drug is well characterized in other indications. The formulation lithium gluconate, 8% gel has proven excellent clinical efficacy and safety in patients with seborrheic dermatitis in two clinical studies reported and during daily practice and as demonstrated in the SmPCs of the products approved (e.g. France, Germany and

Switzerland).

[00684] The use of lithium gluconate 8% gel in the treatment of androgenic alopecia does not pose new safety concerns. Lithium gluconate 8% gel is used in this protocol at dosages recommended for the treatment of seborrheic dermatitis. The following will be applied (see also Table 16 below): one droplet to 3 sites bid, whereby the deliverable droplet size varies from 0.068 mL to 0.21 mL. At the maximum dose assuming the highest weight of a droplet (0.219 mL that corresponds to 0.230 g, taking a density of 1.05 g/mL gel product into account), the lithium exposure by lithium gluconate 8% gel is expected to be 3.79 mg Li +/day (0.230 g [weight of one droplet] x 3 sites bid [x 2] = 1.38 g = 3.79 mg lithium element [or lithium ion]). Taking an average body weight of 50 kg into account, the human daily lithium exposure amounts to 0.0758 mg/kg (2.80 mg/m2). On the other hand, it should be noted that the estimated daily dietary lithium intake varies from 0.65 to 3.1 mg (0.013 - 0.062 mg kg) (Schrauzer, 2002).

Table 16. Explanation of Gel Droplet Volume and Weights, Lithium Gluconate Weight Range Delivered, Lithium Element Weight Range Delivered*

Cat Category Details

#

5 Total weight range of gel 432 mg to 1380 mg of active or placebo gel

administered to site in a single day

Lowest = 72 mg x 2 x 3 = 432 mg

(either active or placebo, applied per

day on three sites, for a twice daily Highest = 230 mg x 2 x 3 = 1380 mg

application)

6 Total weight range of lithium 34.56 mg to 110.4 mg of Lithium Gluconate

gluconate administered in a single

Lowest = 0.432 g x 80 mg lithium gluconate/g gel = day (API present in active gel, applied

34.56 mg of lithium gluconate

per day on three sites, for a twice daily

application) Highest = 1.38 g x 80 mg lithium gluconate/g gel =

110.4 mg of lithium gluconate

7 Total weight range of Lithium 1.19 mg to 3.79 mg of Lithium element Lowest = element (administered in a single (0.432 g gel x 274.80 mg Lithium elemental OOg gel = day (in the active gel, applied per day 1.19 mg Li element

on three sites, for a twice daily

Highest = (1.38 g gel x 274.80 mg Lithium element) application)

)÷100g gel = 3.79 mg Lithium element

* Three tubes of Lithium Gluconate, 8% gel and 3 tubes of Placebo were used to determine the weight of each droplet of approximately 0.1 mL of the active and placebo gels (n=50 droplets). All other values presented in this document are calculated values. As per SmPC of "LITHIODERM 8%, gel" the qualitative and quantitative composition is 8.00 g of Lithium gluconate per 100 g of active gel. This translates to 8,000 mg per 100 g of gel or 80 mg per 1 g of gel. As per SmPC of

"LITHIODER 8%, gel" 100 g gel contains 274.80 mg of Lithium base. Active gel is the

LITHIODERM product with all incorporated active and excipients. Similarly, the placebo gel is the placebo with all incorporated excipients. Lithium Gluconate is the active pharmaceutical ingredient in the active gel. Lithium Element is the lithium (which can be in the form of lithium ions) contained in the active gel.

[00685] Oral lithium (as the carbonate salt) is administered in significantly higher doses (up to 1500 mg/day [corresponding to 282 mg Li+, 5.64 mg/kg, 208.6 mg/m2, respectively], depending on the indication, with dose adjustment to maintain the desired therapeutic plasma levels). Although oral bioavailability can vary between formulations, Li+ is generally absorbed readily and almost completely from the gastrointestinal tract following oral ingestion, and the toxicity is related to the serum concentration of Li+. In patients suffering from seborrheic dermatitis, the highest serum lithium level detected was 4.48

(0.00064 mM) during a long-term treatment (28-56 days) with Lithioderm (Dreno and

Moyse, 2002). In comparison to Li+ levels obtained by oral dosing (0.6 mM), the lithium concentration observed after repeated dermal application is approximately 900 times lower (0.6/0.00064 mM). (see Table 17). Table 17.

Daily lithium load mg/day mg/kg/day mg/nr/day blood level

( in a 50 kg (mM)

person)

by Lithioderm*' 3.79 0.0758 2.80 0.00064

Entire tube of Lithioderm 82.44 1.65 60.93 0.014

by oral dosing up to 282 up tu 5.64 208.6 0.6*

Ratio oTaliderm&\iamM i∞e 74 74 74 938

Ratio oralJdeTmidmiiK tlix

3.42 3.42 3.42 42.86

dietary intake** 0.65 - 3.1 0.013 - 0.062 0.48 - 2.29

* Considered as the lowest therapeutically effective serum concentration (Jope, 2003)

** Schrauzer, 2002

[00686] Conversion of absolute animal doses ( 0.55 mg/mouse) into body weight-related doses (27.5 - 55 mg/kg, assuming a mouse weight range of 10-20 grams) shows that even high topical exposures to lithium does not result in toxicologically relevant plasma levels in mice.

[00687] In the theoretical case, if someone accidentally smeared the entire contents of the Lithioderm tube (i.e. 82.44 mg of Lithium element [or lithium ions] in 30 grams of gel) on skin, the dermal lithium dose in a 50 kg person would be 1.65 mg Li/kg (82.44 mg Li/50 kg), which is 21.77 times more than the maximum intended exposure of 0.0758 mg Li/kg (3.79 mg Li/50 kg). Correspondingly, the estimated serum level would then translate to 0.014 mM Li+, which is approximately 42 times less than the oral dose (Table 17). Therefore, dermal (topical) lithium has a broader safety margin than orally administered lithium and can be used safely.

Physical Interventions

[00688] Dermabrasion. Dermabrasion is a procedure used to remove the superficial layers of the skin by a rotating diamond wheel or bnish attached to a hand-piece. The pressure of the hand-piece, the speed of movement of the hand-piece, and the number of passes per area of the skin determines the depth of skin removed by the procedure.

Dermabrasion has been used to treat photo-damage skin, scars, and acne scars (Grimes, 2005). In skilled hands dermabrasion generates a reproducible, precisely controlled abrasion of the stratum corneum and epidermis and superficial dermis.

[00689] During the dermabrasion procedure, subjects are provided with protective coverings to protect their eyes from debris. Subjects wearing contact lenses are asked to remove them.

[00690] Subjects who receive dermabrasion may experience an allergic reaction to the anesthetic (used to modulate pain during the procedure), mild pain, scab formation and crusting. In epidermal disruption procedures, small particles from the dermabrader may also remain in the dermis and could cause discoloration, a tattoo or an allergic reaction.

Occasional adverse effects include infection, hypo or hyper-pigmentation and scarring.

[00691] Punch Biopsy. Subjects undergo 4 mm punch biopsies on Day 14, and 5 mm or 6 mm punch biopsies on Day 168, using standard biopsy techniques. Follow-up visits are made on Days 35 and 182 to document healing of the punch biopsies and to remove sutures from the Day 68 biopsy.

[00692] The punch biopsy 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) is injected to anesthetize each of three sites that are to be biopsied. 4 mm, 5 mm or 6 mm punch biopsies are performed. The 4 mm Day 14 biopsy is allowed to heal by secondary intention. The Day 168 biopsy site is closed with 2 4.0 Ethilon sutures. Tissue samples may be stored in buffered 4% paraformaldehyde for review by a pathologist.

[00693] Potential rare side effects of punch biopsy include infection, reaction to the anesthetic, bleeding, poor wound healing, mild pain, nerve damage and pigment change. The punch biopsy that heals by secondary intention carries a risk of leaving a small scar. The second punch biopsy (in the same area of the first punch biopsy) closed with a suture(s) is expected to remove such a scar and heal with an acceptable cosmetic result. It is noted that a published study concluded that 4 mm biopsy sites allowed to heal by second intention (which in this protocol are the first biopsies and are scheduled on Day 14) appear at least as good as biopsy sites closed primarily with suture (Christenson et al, 2005). In the published study, for the larger 8 mm punch biopsies, the subjects preferred the primary closure with suturing. The second punch biopsy in this protocol, which takes place on Day 168, is 5 mm: at the discretion of the clinician and if this is in accordance with the subject's wish, the second biopsy may be 6 mm, or an elliptical biopsy may be performed (by hand, or with an excisor) in order to assure scar removal that might follow the first biopsy, or to ensure removal of any photography tattoos. This protocol specifies that the biopsies on Day 168 are to undergo primary closure with suturing.

[00694| Wound Coverings for Dermabrasion and Punch Biopsies. In the human study provided in Section 35 supra, no dressing was employed alter dermabrasion of the scalp in subjects with androgenetic alopecia. This procedure was generally well tolerated and resulted in follicular neogenesis is some subjects. For the first 4 mm punch biopsy in the current protocol, although coverings should be avoided if possible, a simple gauze dressing after this procedure on Day 14 can be applied and left on overnight to absorb oozing.

[00695] Photography, Hair Dye and Tattoos. As possible controls for efficacy, macrophotographs may be taken of three sites for each subject at baseline (Day - 14) and at Days 84 and 168. High quality photographs are allow for accurate hair count, unit area density, and hair shaft thickness analysis. Two types of macrophotographs may be obtained for each of three target areas: 1) Macrophotograph without contact plate to document skin surface for 4 mm punch biopsy scar evaluation; 2) Macrophotograph with contact plate for hair analysis.

[00696] To optimize photography identification of hair shafts and hair shaft thickness, each of the target areas may be stained with commercially available hair dye (Revlon Roux Lash and Brow Tint in Black). A small dot tattoo (black indelible ink) may be placed in the center of each of the three target areas. The size of these tattoos is similar to this period character: The tattoos may serve as a reference point to exactly identify the same sites for photography on Day- 14 and Days 84 and 168 to facilitate the drawing of accurate comparisons. The three sites may be photographed without and with the contact plate on the EpiFlash (Nikon camera fitted with a Canfield EpiFlash). This methodology, including the dot tattoos, is what was employed in the Phase 2 and Phase 3 clinical trials that formed the basis of regulatory approval for finasteride for the treatment of androgenetic alopecia in men (Kaufman et al, 1998; Leyden et al, 1999).

36.2 OBJECTIVES

36.2.1 PRIMARY OBJECTIVE

[00697] The primary objective of this clinical protocol is to induce an increased number of neogenic-like hair follicles with controlled cutaneous perturbation using dermabrasion plus topical lithium gluconate 8% gel, compared to treatment with dermabrasion plus topical placebo gel at Day 168. 36.2.2 SECONDARY OBJECTIVE

[00698] The secondary objective of the clinical protocol is to increase the number of hair follicles with controlled cutaneous perturbation using a 4 mm punch biopsy plus topical lithium gluconate 8% gel. compared to treatment with a 4 mm punch biopsy plus topical placebo gel at Day 168.

36.2.3 EXPLORATORY OBJECTIVES

[00699) Exploratory objectives of the protocol are:

[00700] (i) To count the number of photographically detected hairs induced by treatment with controlled cutaneous perturbation plus the topical application of lithium gluconate 8% gel compared to treatment with controlled cutaneous perturbation plus the topical application of placebo gel (at Day 84 and Day 168, versus Day -14);

[00701 ] (ii) To measure the shaft thickness of photographically detected hairs induced by treatment with controlled cutaneous perturbation plus the topical application of lithium gluconate 8% gel compared to treatment with controlled cutaneous perturbation plus the topical application of placebo gel (at Day 84 and Day 168, versus Day - 14);

[00702] Each of the main endpoints of the protocol is based on assessment of histological and biological markers in biopsy specimens.

[00703] A further objective is to further characterize the safety and tolerability of topical lithium gluconate 8% gel compared to topical placebo gel in the setting of controlled cutaneous perturbation and in the setting of no cutaneous perturbation by dermabrasion.

[00704| Another objective is to obtain preliminary P data on the assumed approximate peak and trough systemic exposure to serum Li+ following controlled cutaneous perturbation using dermabrasion and a single-dose of topical drug on Day 0, and on the approximate peak and trough systemic exposure to serum Li+ following punch biopsy (Day 14) and then multiple dosing (Day 17 through Day 35) on Day 35.

36.3 PROTOCOL SUMMARY

[00705J Subject receive lithium gluconate 8%, gel (Lithioderm) or placebo ( a vehicle control with a composition the same as Lithioderm minus the active substance) to be applied topically twice-daily (morning and evening, at least 8 hours apart) at an approximate dose of volume of 0.1 mL applied twice daily to three sites, for a total intended volume of 0.6 mL. Due to droplet size variability, this translates to an actual total volume range of 0.41 to 1.32 mL. [00706] In treatment period 1 , application of drug continues until the end of Day 1 1 . The sites are biopsied at Day 14 (4 mm punch), and the wounds allowed to heal by secondary intention. Application of drug continues in treatment period 2, from Day 17 until Day 35. Subjects may be asked to return on Day 84 and Day 168 for repeat photography sessions. Monthly safety follow-up phone calls may be performed on Days 1 12 and 140. On Day 168, the initial punch biopsy sites is re-biopsied and sutured (5 mm, or 6 mm at the discretion of the practitioner and if in accordance with the subject's wish in order to assure scar removal, that might follow the first biopsy, and any photography tattoo removal). Subjects should be seen on Day 182, when suture removal is planned. For the purposes of this protocol, the end of the treatment period is defined as the last clinical visit of the last subject.

[00707] In order to ensure procedural practicalities, four subjects are treated initially (as per protocol) and must reach Day 14 (and completed the first biopsy) without operationally significant incident before any subsequent subject is treated.

36.4 DISCUSSION OF PROTOCOL DESIGN, INCLUDING THE CHOICE OF CONTROL GROUPS

[00708] A placebo treatment group (vehicle only) may serve as a control group to show what an agent-free therapy contributes in terms of efficacy and safety.

|00709] A non-dermabraded scalp site may serve as a control to show the effect of dermabrasion on the number of neogenic-like hair follicles and serve as a benchmark for the local safety and tolerability of the drug (lithium gluconate 8% gel or placebo gel [vehicle only]).

36.5 PATIENT POPULATION

36.5.1 INCLUSION CRITERIA

[00710] For a controlled clinical study, preferred subjects are Caucasian male subjects aged between 20 and 65 years of age who have, optionally,

[00711 J (i) androgenetic alopecia with the presence of a vertex transition zone defined as an area possessing both normal and miniaturized hairs, Hamilton Norwood type 3V, 4, 5, 5A or 5V (see Figure 5) with a vertex area large enough to accommodate all 3 treatment sites; and

[00712| (ii) a Fitzpatrick skin type 1-4 (higher Fitzpatrick skin type ratings may preferably be excluded due to the increased risk of keloid formation and hypopigmentation in these subjects); [00713] Subjects must be willing and able to comply with the procedures and should have no foreseeable reason to prevent completion of the clinical protocol.

36.5.2 EXCLUSION CRITERIA

[00714] Subjects who fulfill one or more of the criteria listed below may be excluded:

[00715] General exclusions:

[00716] · participation in another clinical trial within the 30 days directly preceding this clinical protocol, earlier participation in this clinical protocol, or simultaneous participation in a clinical trial;

[00717] · suspicion of drug and/or alcohol abuse;

[00718] · a clinical history suggestive of intolerance, allergies or idiosyncrasies to the drug(s) or the ingredients of the product(s), or to agents that may be used in any of the protocol's procedures;

[00719] · a psychiatric condition that might limit participation and/or that lead to the assumption that the ability to completely understand the consequences of consent is missing;

[00720] · any disease or circumstances on account of which the subject should not participate in the protocol, including any clinically significant medical condition that may interfere with the interpretation of the results, including an uncontrolled chronic disease. For example, subjects with a history of clinically significant diseases e.g. cardiovascular, hematological, endocrine, hepatic, renal, immunodeficiency disorder, coagulation abnormalities, or malignancy may be excluded.

[00721] Specific exclusions:

[00722] · history of keloids or hypertrophic scarring,

[00723] · history of poor wound healing;

[00724] · history of diabetes mellitus;

[00725] · history of coagulopathy/bleeding diathesis;

[00726] · history of hypersensitivity to lidocaine/epinephrine or to the components of the product;

[00727] · immune compromise or undergoing therapy to treat an immune disorder;

[00728] · serum creatinine, blood urea nitrogen (BUN) or fasting blood glucose above the upper limits of normal (ULN), clinically significant abnormalities in liver function tests (3 times the upper limit of normal); gamma glutamyl transpeptidase (GGT), alkaline

phosphatase, aspartate aminotransferase (AST) or alanine aminotransferase (ALT); or the presence of proteinuria (greater than a trace on urine dipstick); results to be confirmed by Day - 14. All these values are based on the ULN for the laboratory performing the safety labs;

[007291 · clinically significant abnormalities in vital signs or ECG results, in the opinion of the practitioner;

[00730] · HIV, hepatitis B, hepatitis C positivity (negative status for each must be confirmed by Day - 14);

[00731] · diagnosis of any active skin condition that would interfere with the clinical procedures or evaluations described herein;

[00732] · sunburned skin;

[00733] · previous hair transplant surgery;

[00734] · current or recent use (within preceding 6 months) of isoretinoin

(ROACCUTANE);

[00735] · current or recent use (within preceding 6 months) of minoxidil (ROGAI E);

[00736] · unwillingness to abstain from the personal use of hair dye from the start of screening through and including the last clinic visit for the protocol;

[00737] · started, stopped, changed dose or changed regimen of 5 alpha reductase inhibitors, such as finasteride (PROPECIA), within the preceding 12 months;

[00738] · current or recent use (within preceding 30 days) of hormone therapy, corticosteroids (except inhaled steroids and topical steroids to non-scalp areas) or other immunomodulators;

[00739] · current or recent use (within preceding 30 days) of any lithium-containing product;

[00740] · current or recent use (within preceding 14 days) of an anticoagulant or antiplatelet agent, including acetylsalicylic acid.

36.6 TREATMENTS ADMINISTERED

[00741] In this protocol, four treatments are described:

lithium gluconate 8% gel + dermabrasion;

placebo gel + dermabrasion;

• lithium gluconate 8% gel + no dermabrasion;

• placebo gel + no dermabrasion.

[00742] T e outcome of the more superficial perturbation (dermabrasion) in conjunction with drug (primary objective; see Section 36.2.1) is evaluated at Day 14. The outcome of deeper perturbation (secondary objective, see Section 36.2.2) is determined from the repeat punch biopsies (extending to the level of the subcutaneous fat) to be made at Day 168. For the secondary objective there are two treatment groups:

• lithium gluconate 8% gel + punch biopsy;

placebo gel + punch biopsy.

[00743] Treatment (with Lithium gluconate 8% gel or matching placebo, i.e. vehicle - collectively referred to herein as "product") comprises two modalities: (i) Drug (lithium gluconate 8% gel or placebo [vehicle]); and (ii) Controlled cutaneous perturbation.

[00744] Drug treatment (treatment period 1 ) starts on Day 0 (immediately after dermabrasion) and continues until the end of Day 1 1 , 2 days prior to punch biopsy 1. Drug treatment (treatment period 2) begins again on Day 17 (3 days after the first biopsy) and continues to Day 35.

[00745] The dose is an approximate volume of 0.1 mL, applied twice daily to three sites, for a total intended volume of 0.6 mL. Due to droplet size variability, this translates to an actual total volume range of 0.41 to 1.32 mL. A dose of 0.1 mL corresponds to a droplet the size of a small pea. The product is applied with a disposable finger stall "Fingerlinge"' and gently applied to cover the area of treatment. If a dose is missed, it should not be applied later; the second dose shall remain as intended (ca. 0.1 mL).

[00746] On Days 0 through Day 2 at least the first of the two intended daily doses of drug are applied in the clinic to each of the three sites. After Day 2, if the subject demonstrates the capability to apply the drug correctly, he may do this at home (except where indicated [e.g., prior to, and including, Day 35] when that application will be under supervision in the clinic). However, if at any time it appears that the subject is not applying the product as prescribed, then both daily applications should be in the clinic.

[00747] Drug is applied twice a day, morning and evening, at least 8 hours apart (although on Days 0 and 17, start of treatment periods 1 and 2, drug may be applied 5 hours apart if required, or down to 3 hours if absolutely necessary, to facilitate completion of required assessments).

[00748] Two types of cutaneous perturbation are used.

• Day 0: Dermabrasion using a hand-held dermabrader, to an estimated depth of approximately 100 microns to two of the three scalp sites. One of three treatment sites in each subject may remain unabraded as a control.

Day 14: full-thickness 4 mm punch-biopsy (extending to the level of the subcutaneous fat) to each site. This 4 mm punch may be used for two purposes:

o create the deeper perturbation modality o yield histology data for the combination of dermabrasion / drug

treatment

[00749] The procedures for dermabrasion and the two sets of punch biopsies are described below.

36.6.1 DERMABRASION

[00750] · Shave/clip the existing hair in the area to be treated;

[00751] · Thoroughly clean the areas to be dermabraded with isopropyl alcohol as an antiseptic cleansing agent,

[00752] · Inject lidocaine 1 I L 2% with epinephrine 1 :200,000 (0.5 mL to 2 mL /site) to anesthetize the surface of the areas to be treated (each dcrmabraded area is to be a 1 .5 cm x 1.5 cm square);

[00753] · Important: Allow the lidocaine plus epinephrine anesthetic 20 minutes to take effect - this should be timed to ensure that a full 20 minutes has elapsed after the injection and the procedure is performed

[00754] · If subject w¾ars contact lenses, they should be removed

[00755] · Protect subject's eyes with shields

[00756] · Perform standard dermabrasion (using a hand-held dermabrader with standard grit diamond fraize) to a depth of approximately 100 microns, that includes removal of 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);

[00757] · The dermabraded areas may be lightly patted clean with saline moistened 4x4 gauze pads.

[00758] To achieve hemostasis:

[00759] · Apply direct pressure using a gauze pad with finger pressure to control any bleeding (as the wounds are only partial thickness; it is likely that this will be effective: if not follow the procedure as for first punch biopsy: see below).

General information on dermabrasion:

[00760] The procedure begins with shaving/clipping of the existing hair in the areas to be treated followed by a thorough cleaning of the area to be treated with antiseptic cleansing agent.

[00761] The clinician should choose 3 areas with a small number of very small hairs to be the treatment sites. A numbing solution is injected to numb the surface that is being treated. Wait 20 minutes for the numbing solution to take full effect. Remove contact lenses, if worn, and wear a shield to protect eyes. The clinician then carefully maneuvers the dermabrasion tool over the three areas, one at a time, to carefully remove layers of skin until he/she reaches the desired level. The dermabraded areas may be wiped gently with sterile salt solution or sterile water and gauze to remove debris. If there is any bleeding the clinician presses on the sites with a gauze pad, until it stops. Fach procedure should only take a few minutes.

[00762] Following the procedures the treated skin will be red, swollen and tender. The wounds are cared for until new skin starts to grow; this usually takes 7- 10 days. The scab is allowed to come off completely by itself.

36.6.2 FIRST PUNCH BIOPSY PROCEDURE

[00763J Note: This procedure uses a 4 mm punch.

[00764] · Thoroughly clean the areas to be biopsied with isopropyl alcohol as an antiseptic cleansing agent;

[00765] · Select the biopsy site.

[00766] · Inject lidocaine HCL 2% and epinephrine 1 :200,000 (0.5 cc to 2 mL/site) into the dermis to raise an intradermal wheal appropriate to the size of the intended biopsy and causing an orange peel appearance to the skin;

[00767] · Important: Allow the lidocaine plus epinephrine anesthetic 20 minutes to take effect - this should be timed to ensure that a full 20 minutes has elapsed after the injection and the biopsy is performed;

[00768] The actual biopsy procedure itself is as described by Alguire and Mathes, 1998:

[00769] · Stabilizing the skin with the thumb and forefinger, stretching it slightly perpendicular to the normal skin tension lines to produce an oval rather than a round wound, place the punch perpendicular to the skin and apply firm and constant downward pressure with a circular motion;

[00770] · When the punch reaches the subcutaneous fat (indicated when a "give"' is felt), remove the punch and apply downward finger pressure to pop up the core;

[00771] · Completely elevate the core with gentle use of forceps or needle tip, and excise it at the base with small tissue scissors.

[00772] To achieve hemostasis,

[00773] · Surgeon applies timed continuous pressure for 10 minutes using a gauze pad with finger pressure directly over the biopsy site and repeats for a further 10 minutes or longer if necessary. Because the effects of chemical agents and electro-cautery on follicular neogenesis are unknown, pressure is the preferred method for hemostasis. [00774] · If still bleeding, surgeon performs light electro-cautery.

General Information on First Punch Biopsy

[00775] The procedure begins with a thorough cleaning of the areas to be treated, with antiseptic cleansing agent.

[00776] A numbing solution is injected to numb the surfaces that are being treated. Wait 20 minutes for the numbing solution to take full effect. The clinician uses a 4 mm biopsy tool. The clinician carefully maneuvers the punch biopsy tool over the 3 areas to carefully remove layers of skin until he/she reaches the desired level. Each procedure should only take a few minutes. The clinician will press on the sites with a gauze pad for 10 minutes to stop the bleeding, and for an extra 10 minutes, or longer, if needed. Although stopping the bleeding by pressure will be tried first and is preferred, if any of the sites are still bleeding, light electro-cautery may be used.

[00777] A light sterile gauze pad and tape dressing will be put over each site. This will need to come off after the first night.

[00778] Following the procedure the areas will be red, swollen and tender. Wounds are cared for until new skin starts to grow; this usually takes 7-10 days. The scab is allowed to come off completely by itself.

36.6.3 SECOND PUNCH BIOPSY PROCEDURE

[00779] Note: This procedure is intended to use a 5 mm punch: at the discretion of the clinician and if this in accordance with the subject's wish the second biopsy may be 6 mm in order to assure scar removal that might follow the first biopsy, and removal of any photography tattoo. Alternatively, to ensure full scar removal, an elliptical biopsy may be performed (by hand, or with an excisor).

[00780] As for the first punch biopsy:

[00781 ] · Thoroughly clean the areas to be biopsied with isopropyl alcohol as an antiseptic cleansing agent;

[00782] · Inject lidocaine HCL 2% and epinephrine 1 :200,000 into the dermis to raise an intradermal wheal appropriate to the size of the intended biopsy and causing an orange peel appearance to the skin;

[00783] · Important: Allow the lidocaine plus epinephrine anesthetic 20 minutes to take effect - this should be timed to ensure that a full 20 minutes has elapsed after the injection and the biopsy is performed;

[00784] The actual biopsy procedure itself is as described by Alguire and Mathes, 1 98: [00785] * Stabilizing the skin with the thumb and forefinger, stretching it slightly perpendicular to the normal skin tension lines to produce an oval rather than a round wound, place the punch perpendicular to the skin and apply firm and constant downward pressure w ith a circular motion;

[00786] · When the punch reaches the subcutaneous fat (indicated when a ''give is felt), remove the punch and apply downward finger pressure to pop up the core ;

[00787] · Completely elevate the core with gentle use of forceps or needle tip, and excise it at the base with small tissue scissors.

[00788] To achieve hemostasis:

[00789] · Insert sutures to eliminate dead space and achieve good closure of skin;

[00790] · If wound continues to bleed surgeon applies continuous pressure for 5 minutes using a gauze pad with finger pressure directly over the biopsy site;

[00791] · If still bleeding continue to apply pressure. Sutures may be removed to check that there is no arteriolar bleeding point (in which case reinsert sutures using a larger needle and apply a pressure bandage);

[00792] · Check wound in 20 minutes and if still oozing apply pressure bandage and arrange next day follow up.

[00793] Wash around the treatment areas and also the hair, if required, with sterile water or saline to ensure that the subject's scalp and hair are clean. Further washing with sterile water or saline will be performed the following day, if required, or if requested by the subject

[00794] Note: the second punch biopsy is scheduled to be 5 mm: at the discretion of the investigator and if in accordance with the subject's wish, the second biopsy may be 6 mm in order to assure scar removal that might follow the first biopsy.

General Information on second punch biopsy

[00795] The procedure begins with a thorough cleaning the area to be treated with antiseptic cleansing agent.

[00796] A numbing solution is injected to numb the surface that is being treated. Wait 20 minutes for the numbing solution to take full effect. The clinician uses a 5 mm biopsy tool, although if the scar from the first biopsy is larger than expected and the patient wishes the whole scar to be removed, a 6 mm punch may be used. This punch biopsy should also remove any photography tattoo, if appropriate to do so. The clinician carefully maneuvers the punch biopsy tool over the 3 areas to carefully remove layers of skin until he/she reaches the desired level. He will then stitch the wound. Each procedure should only take a few minutes. [00797] If a site is bleeding, the clinician will press on it with a gauze pad for 5 minutes to stop the bleeding. If a site is still bleeding after this time, pressure may be applied for longer. Also the stitches may be removed to check that there is no reason for the bleeding, and the site can then be re-stitched if required using a different needle, and a pressure bandage can be applied for an extra 10 minutes if needed. The wound will be checked after 20 minutes and if still oozing a pressure bandage will be put on and you will be seen the next day for follow up. A sterile wound covering is applied.

[00798] Following the procedure the area may be red, swollen and tender. Healing of the wound usually takes 7-10 days. Stitches are removed in about 2 weeks.

36.6.4 CONTROLLED CUTANEOUS PERTURBATION

[00799] Dermabrasion (Day 0): For a control, one of the subject's three treatment sites is left unabraded.

[00800] Punch biopsy: All subjects receive punch biopsies of the two dermabraded and the one non-dermabraded site on two occasions (Day 14 and Day 168), extending to the level of the subcutaneous fat.

36.7 SELECTION OF DOSE

36.7.1 PRODUCT

[00801] The dose selected (for subjects assigned to lithium gluconate 8% gel) is in accordance with the approved use of Lithioderm 8% gel for the treatment of seborrheic dermatitis [SmPC derm] to cover the areas intended in this protocol (3 x 1.5 cm x 1 .5 cm). The SmPC for derm describes application morning and evening on the entire face in a thin layer, but in this protocol application is to be only to the three specific (1.5 cm x 1.5 cm) sites.

36.8 SELECTION OF TIMING OF DOSES AND PROCEDURAL

INTERVENTIONS

36.8.1 DRUG

f 00802] Drug is applied immediately following Dermabrasion (Day 0). and is allowed to "wash out" for 2 days (Day 12- Day 14) prior to the Day 14 biopsy. Drug treatment is restarted 3 days post-biopsy (Day 17) and continues through Day 35.

36.8.2 CONTROLLED CUTANEOUS PERTURBATION [00803] This protocol provides controlled cutaneous disruption by a dermabrasion in the first period followed by a full thickness excision by a 4 mm punch biopsy in the second period. After an initial dermabrasion and a regimen of topically applied lithium, drug is washed out for an appropriate period before the 4 mm punch biopsy. The 4 mm punch provides tissue to analyze the potential efficacy of lithium in combination with dennabrasion, the more superficial disruption. It also creates the deeper perturbation that can be tested with lithium for induction of follicular neogenesis.

[00804] The second punch biopsy at Day 168 provides tissue to analyze the potential efficacy of lithium in combination with the deeper, full thickness excision from the first punch biopsy. At Day 168, any hair follicles that are evident on histological or clinical examination would be by definition new, since the second biopsy is over the site of the first biopsy, which should have removed all pre-existing hair elements.

36.8.3 PHARMACOKINETIC SAMPLING

[00805] Blood is analyzed to assess levels of lithium in serum. Because exposure is anticipated to be very low (Dreno & Moyse, 2002), samples are drawn 1 hour after the application of drug.

[00806] It is expected that the systemic exposure to Li+ (serum levels) will be maximal shortly after the scalp perturbation procedures, which remove the stratum corneum and epidermis, exposing the dermal vasculature in the papillary dermis. Since the surface areas of the exposed vasculature is greater with the dermabrasion ( 1.5 cm x 1.5 cm square) than that of the punch biopsy punch (0.4 cm in diameter), samples are taken at baseline before dermabrasion (Day 0 as a baseline) and 1 hour after drug administration on dermabraded skin (Day 0).

[00807] This protocol also permits examination of the levels of lithium in the serum after multiple topical applications. Because the uninterrupted duration of twice-daily dosing with lithium during the second period of this protocol after the Day 14 punch biopsy (19 days) is longer than during the first period after the Day 0 dennabrasion ( 12 days), samples are taken just prior to and 1 hour after the morning dose on Day 35, which is the last day of multiple dosing in the second period of the protocol.

36.8.4 NOT PERMITTED CONCOMITANT TREATMENT

[00808] It is preferable that the following medications are not permitted prior to the protocol, nor are they permitted concomitantly: [00809] · isoretinoin (ROACCUTANE), or within the preceding 6 months;

[00810] · minoxidil (ROGAINE), or within the preceding 6 months;

[00811] · hormone therapy, or within the preceding 30 days;

[00812] · corticosteroids, or within the preceding 30 days, except inhaled steroids or topical steroids to non-scalp areas;

[00813] · other immunomodulators, or within the preceding 30 days;

[00814] · any lithium-containing product (topical or oral), or within the preceding 30 days;

[00815] · any anticoagulant or antiplatelet agent (including acetylsalicylic acid), or within the preceding 14 days.

[00816] In addition, subjects who have started or stopped 5 alpha reductase inhibitors, such as finasteride (PROPECIA), within the preceding 12 months are to be excluded.

[00817] During the protocol period, subjects should not use hair dye, or alter their hairstyle.

[00818] In the human study provided in the example of Section 35 supra, no dressing was employed after dermabrasion of the scalp in subjects with androgenetic alopecia. This procedure was generally well tolerated and resulted in follicular neogenesis is some subjects. For the first 4 mm punch biopsy in the current protocol, although coverings should be avoided if possible, a simple gauze dressing after this procedure on Day 14 can be applied and left on overnight to absorb oozing.

[00819] There are no published data to suggest drug-drug-interactions between topical application of lithium gluconate 8% gel and other topically applied products; however, to avoid any confounding of assessment of effect, compounds such as sunscreens, moisturizers and other compounds (such as aluminum chloride) should not be used until complete re- epithelialization has occurred and the skin appears clinically normal.

36.8.5 PERMITTED CONCOMITANT TREATMENT

[00820] The following concomitant medications are permitted:

[00821] · inhaled corticosteroids;

[00822] · topical to non-scalp areas;

[00823] · 5 alpha reductase inhibitors, such as finasteride (PROPECIA), provided that this has been maintained at constant dose and dosing regimen within the preceding 12 months, and the subject intends to continue on this stable regimen throughout the duration of the present treatment. [008241 Paracetamol and/or codeine-containing preparations may be taken to control any pain or discomfort following the procedures as analgesics such as aspirin and nonsteroidal anti-inflammatory agents may have an adverse effect on hemostasis.

[00825] After the dermabrasion and biopsy procedures, ideally the wound sites are left completely unperturbed by the subject, and scalp and hair not be washed at all for the first 3 days, although the clinician may offer gentle irrigation with sterile water or saline during clinic visits just after the Day 0 dermabrasion and Day 14 biopsy. After the second punch biopsy (Day 168), which is sutured, after the first 3 days the subject may remove the bandage and cleanse the wound gently with soap and water, pat dry with a clean towel, apply a thin film of topical antibiotic, and apply a new bandage until the next clinic visit (Days 171- 182). For the next 5 days (Days 3 through 7) after each of the three procedures, only water should be used to clean the hair. After day 7 the surrounding hair may be washed with a small amount of standard shampoo (a mild baby shampoo, which will be provided). So as not to mistakenly remove any of the applied test medication, hair washing should be done just before drug is about to be applied again, allowing time for the hair and scalp to dry.

[00826] On Day 35, after the last dose of drug, the subject's scalp is examined to decide if it may be covered with makeup while subjects wait for the biopsy and stitches at the end of the clinical protocol period (which are intended to remove the tattoo, if subject wishes, and make any scar smaller).

[00827] From Day 0 (dermabrasion day) to the end of the clinical protocol period (Day 182), a light hat should be worn by the subject when going outside to avoid pigmentation. During the 2 periods of drug application, a light hat that does not remove or smear the product should be used.

36.9 TREATMENT COMPLIANCE

[00828] On Day 0 through Day 2, at least one of the two applications of the drug

(alternatively referred to herein as "product") should be at the clinician's site (e.g., physician's office). After Day 2, if the subject demonstrates the capability to apply the product correctly, the subject is provided with instructions for dosing and may apply product at home (except on attendance for any assessment visit [prior to, and including, Day 35] when that application will be under supervision in the clinic).

36.10 VARIABLES AND PROCEDURES

36.10.1 EFFICACY VARIABLES Primary Efficacy Variable

[00829] The primary efficacy variable is the number of neogenic-like hair follicles induced by treatment with controlled cutaneous perturbation using dermabrasion plus topical lithium gluconate 8% gel, compared to treatment with dermabrasion plus topical placebo gel.

[00830] The primary efficacy variable is evaluated using a 4 mm skin punch biopsy taken 14 days after the dermabrasion and topical treatment (Day 14).

[00831] As hair follicle development is a continuum and based on both histological and biochemical markers, at this time-point neogenic-like hair follicles are identified by fulfilling various criteria that include the following attributes: shorter length than vellus and/or vellus- like hair follicles, lack of a connection with a pre-existing pilosebaceous unit, lack of a pore at the skin surface, lack of a well-differentiated sebaceous gland, lack of a hair shaft, lack of an elastin-negative "streamer" or "dermal channel," and positive staining for 4'-6-Diamidino- 2-phenylindole (DAPI), alkaline phosphatase, BerEP4, and Ki67.

Secondary Efficacy Variable

[00832] The secondary efficacy variable is the number of hair follicles induced by treatment with controlled cutaneous perturbation using a 4 mm punch biopsy plus topical lithium gluconate 8% gel, compared to treatment with a 4 mm punch biopsy plus topical placebo gel.

[00833] This variable is evaluated approximately 5.5 months after the first biopsy (on Day 168), by biopsy, at the site of the first biopsy which was allowed to heal by secondary intention.

[00834| If any hair follicles are present it is assumed that these must derive from neogenic- like hair follicles, since the original hair follicles were removed by the first punch biopsy.

36.10.2 SAFETY VARIABLES

[00835] Safety and tolerability of lithium gluconate 8% gel applied topically and epidermal disruption by dermabrasion and punch biopsies is assessed by targeted evaluation of the treated scalp sites (using data collected for. e.g., local tolerability, wound healing and Vancouver Scar Scales [see below]) and, as is typical for clinical trials, the reporting of adverse events (AEs). Visits on Days 1, 2, 7, 12, 15, 17, 22 and 182 are optionally safety visits to assess the scalp sites (and using AE reporting if indicated). [00836 J The Vancouver Scar Scale (VSS) has 4 separate domains: pigmentation (graded 0 = normal, to 2 = hyperpigmentation), vascularity (graded 0 = normal, to 3 = purple), pliability (graded 0 = normal, to 5 = contracture) and height (graded 0 = normal, 3 = 5 mm):

PIGMENTATION

0. Normal;

1 . Hypopigmented;

2. Hyperpigmentation;

VASCULARITY

0. Normal: resembles the color over the rest of the body area;

1. Pink;

2. Red;

3. Purple.

PLIABILITY

0. Normal

1. Supple: flexible with minimal resistance;

2. Yielding: giving way to pressure;

3. Firm: inflexible, not easily moved, resistant to manual pressure

4. Banding: rope-like tissue that blanches with extension of scar;

5. Contracture: permanent shortening of scar, producing deformity or distortion. HEIGHT

0. Normal: flat;

1. < 2 mm;

2. 2-5 mm;

3. > 5 mm.

[00837] In addition, liver and renal function, fasting blood glucose and urinalysis may be performed at screening and on Days 182. A physical examination may be performed at screening and Day 182. Vital signs and ECG may be performed at screening. On days 0, 14, and 168, when dermabrasion and punch biopsies are performed, respectively, vital signs should be repeated.

36.10.3 EXPLORATORY EFFICACY VARIABLES

[00838] Exploratory efficacy variables are:

[00839] i) the difference in the number of hairs captured by photography on Day 84 and Day 168 versus Day - 14; [00840] ii) the difference in hair shaft thickness captured by photography on Day 84 and Day 168 versus Day - 14;

[00841 J iii) the histological characteristics in a second skin punch biopsy on Day 168;

[008421 Pharmacokinetics (P ): P sampling is done at baseline (Day 0) before

dermabrasion and 1 hour after drug administration to dermabraded skin (Day 0), and before and 1 hour after drug administration on Day 35..

36.11 MEASUREMENTS AND PROCEDURES

[00843] A flow chart summarizing the protocol is presented below.

Schematic of Protocol Period and Events

Photo Photo Photo

Screening Dermabrasion 1 Biopsy 2nd Biopsy

[00844] In this protocol, the intended total treatment period with dmg is 31 days (Days 0- 1 1 and Days 17-35 ).

]00845] The treatment periods are as follows:

[00846] 1. Screening (Day -28 to Day -0);

[00847] 2. Optional photography 1 , including optional tattoo placement, and use of hair dye (Day -14);

[00848] 3. Dermabrasion and first day of application of drug (Day 0):

[00849] 4. First treatment period (Day 0 - Day 1 1 ), to include optional safety checks of treatment sites on days 1 , 2, 7 and 12. Clinic visit on Day 12 (no treatment at this visit);

|00850] 5. First punch biopsy (Day 14), followed by optional safety checks of treatment sites on days 15, 17 and 22;

[00851] 6. Second treatment period (Day 1 7 - Day 35);

[00852] 7. Follow-up period 1 after first biopsy (T O weeks), leading up to optional photography 2 (including use of hair dye) (Day 84); [00853] 8. Follow-up period 2 (3 months), optional safety follow-up on Days 1 12 and 140, leading up to punch biopsy 2 and optional photography 3, Day 168 (including use of hair dye);

[008541 9. Follow-up period 3 (2 weeks); completion of clinical protocol (Day 182).

36.12 PROCEDURES AND MEASUREMENTS DURING THE TREATMENT PERIODS

36.12.1 BASELINE VISIT (DAY DAY OF

DERMABRASION

[00855] Before any procedures take place, the following must be carried out:

|00856] · Confirm continuing consent of patient;

|00857] · Document concomitant medications;

[00858] · Examine scalp, including visibility of the tattoo (if used as a control)

[00859] Thereafter, it is recommended to:

[00860] · Check concomitant medications since last visit;

[00861] · Monitor treatment sites on scalp and any AEs;

[00862] · Take pre-dose blood sample for baseline measurement of serum Li+;

[00863] · Perform vital signs (blood pressure, pulse [both recorded after subject seated for at least 5 minutes], respiration rate and body temperature).

[00864] · Perform dermabrasion procedure;

[00865] · Gently clean site and surrounding hair by irrigating with sterile water or saline;

]00866] · Apply drug at at least the first of the two intended daily doses of drug post- dermabrasion in the clinic to each of the three sites (two dermabraded. one non- dermabraded). The second application on this day may be done in the clinic or subject under supervision (or by subject at home, if considered capable);

[00867] · On Day 0 drug may be applied 5 hours apart if required, or down to 3 hours if necessary to facilitate completion of all assessments. However, inform the subject that dosing should be morning and evening, at least 8 hours apart on all other days in treatment period 1. Provide disposable finger stalls "Fingerlinge," at least 2 per treatment day;

[00868] · Take blood sample for measurement of serum Li+, 1 hour post dose;

[00869] · With site and hair having been cleaned after the procedure, encourage the subject to follow proper wound care procedures, to keep the sites and scalp dry and not to disturb the dermabraded sites; offer that the dermabrasion sites and surrounding hair will be inspected and if need be cleaned by the clinic staff on the next day's visit;

[00870] The sequential procedure for subsequent days is shown in the following sections.

36.12.2 DAYS 1 AND 2 (1 AND 2 DAYS RESPECTIVELY AFTER DAY 0), DAY 7 (± I)

(00871 ] · For all visits to the clinician, it is recommended to check concomitant medications since last visit; monitor AEs; Inspect the wound sites (checking for scab loss) and hair. Remind the subject that no wound covering is to be used at any time; encourage the subject to keep the sites and scalp dry and not to disturb the dermabraded sites; offer to clean surrounding hair and skin outside the dermabraded sites if desired by the patient.

[00872] · Apply at least one dose of product in clinic (by staff or subject under supervision).

36.12.3 DAY 12 (- 1/+21

[00873] This visit may be conducted on Day 1 1 (if the subject still applies both doses required for Day 1 1 ) or up to Day 14 (if the subject is contacted by telephone and reminded to stop dosing after the evening of Day 1 1 ). All efforts must be made to ensure that the number of dosing days will remain the same for all subjects.

[00874] · If any photography tattoo has faded such that treatment sites would be difficult to find, re-apply a new tattoo directly on top of the previous faded dot (at most, just one reapplication will be required);

36.12.4 DAY 14 (-11 + 2): DAY OF FIRST PUNCH BIOPSY

[00875] There must be a minimum of 1.5 days between ending treatment period I on Day 1 1 and biopsy on Day 14.

[00876] · Perform vital signs (blood pressure, pulse [both recorded after subject seated for at least 5 minutes], respiration rate and body temperature).

[00877] · Perform 4 mm punch biopsies. If a tattoo is used, for each site, an area just centripetal (toward the center of the head) from the tattoo is selected. Biopsy very close to the tattoo, taking care not to remove it.

[00878] · Irrigate around the treatment areas and also the hair, if required, with sterile water or saline to ensure that the subject's scalp and hair are clean;

[00879] · Apply simple gauze dressing, to be left on overnight; 36.12.5 DAYS 15 (± 1. 1 DAY AFTER DAY 14) AND 17 (- 1/ +3)

[00880] Day 15 takes place the day after biopsy. The time between biopsy (Day 14) and the start of treatment period 2 (Day 17) must be a minimum of 1 .5 days.

[00881] · Day 15 only: ensure dressing removed;

[00882] · Day 17 only: drug is applied. This may be by clinic staff or subject under supervision. On Day 17 drug may be applied 5 hours apart if required, or down to 3 hours if necessary to facilitate completion of all assessments. However, confirm to the subject that dosing should be morning and evening, at least 8 hours apart on all other days in treatment period 1 ;

[00883] · Day 17 only: advise subject to stop medication after the evening application on Day 34; remind subject not to cover wound at night;

36.12.6 DAY 22 (± 1): FOLLOW-UP

36.12.7 DAY 35 (-1/ +3 DAYS): END OF APPLICATION OF DRUG

[00884] Treatment Period 2 must be 19 days long, with a single dose applied in the clinic on Day 35

[00885] · Check compliance with procedures (application of drug and no wound covering);

[00886] · Take blood draw for serum lithium;

[00887] · Apply last dose of medication;

[00888] · If the photography tattoo has faded such that treatment sites would be difficult to find, re-apply a new tattoo directly on top of the previous faded dot (at most, just one reapplication will be required);

36.12.8 DAY 84 (± 3 DAYS): DAY OF PHOTOGRAPH

[00889] · If the photography tattoo has faded such that treatment sites would be difficult to find, re-apply a new tattoo directly on top of the previous faded dot (at most, just one reapplication will be required);

[00890| · Locate the 3 treatment sites. Shave hair within treatment sites to length of 1 mm (each treatment site to be an area of 1.5 cm x 1.5 cm) and take the first photograph without the contact plate. [008 1 j · Complete Vancouver Scar Scale for each treatment site in the scalp. For each site, this is the area just centripetal (toward the center of the head) from the tattoo (if used) where the Day 14 first punch biopsy was done and where a scar may be.

[00892] · Stain the hair with the supplied hair dye and again using the tattoo (if used) as a reference point perform any remaining photography.

36.12.9 DAYS 112 AND 140 (± 3 DAYS): FOLLOW UP

36.12.10 DAY 168 (± 3 DAYS): DAY OF SECOND PUNCH BIOPSY

[00893] · Perform vital signs (blood pressure, pulse [both recorded after subject seated for at least 5 minutes], respiration rate and body temperature).

[00894] · If using tattoo, use it to locate the treatment sites on the scalp. Shave hair within treatment sites to length of 1 mm (each treatment site to be an area of 1 .5 cm x 1.5 cm) and take a photograph (if desired for a control). Stain the hair with the supplied hair dye and perform the remaining photography;

[00895] · Complete Vancouver Scar Scale, to be assessed separately for each of the treatment sites. If a tattoo is used, for each site, this is the area just centripetal (toward the center of the head) from the tattoo where the Day 14 first punch biopsy was done and where a scar may be.

[00896] · Perform punch biopsies at each of the three treatment sites (the procedure is as for the first punch biopsy; however as wound closure will be achieved through suturing this overrides the procedure to achieve hemostasis). These are planned to be 5 mm biopsies but may be 6 mm, at clinician's discretion and in accordance with the wishes of the subject, to ensure complete scar excision and removal of any photography tattoos;

[00897] · Irrigate around the treatment areas and also the hair, if required, with sterile water or saline to ensure that the subject's scalp and hair are clean;

[00898] · Remind subject to fast from the evening before their Day 182 visit;

[00899] · Schedule appointment for Day 182 visit (unless biopsy wound is still oozing after 20 minutes and a pressure bandage has been applied. In this case arrange follow up on

Day 169).

36.12.11 DAY 182 (± 3 DAYS): FOLLOW UP

[00900] · Repeat blood sampling for renal and hepatic function (serum creatinine,

[BUN], GOT, alkaline phosphatase, AST and ALT) and fasting blood glucose; [00901 ] · Perform urine dipstick for the presence of proteinuria (greater than a trace on urine dipstick);

[00902] · Repeat physical examination of major body systems, including general appearance, cardiovascular, respiratory, gastrointestinal, haemato-lymphatic. brief neurological, musculoskeletal and skin;

[00903] · Remove sutures (wound healing assumed);

36.12.12 END OF PROTOCOL

[00904] The planned end-of this protocol is Day 182 (± 3 days).

36.12.13 APPROPRIATENESS OF MEASUREMENTS

[00905] The battery of measures that make up the primary, secondary (and exploratory) endpoints to examine the efficacy of lithium gluconate 8% gel following two modalities of controlled cutaneous perturbation are based on histological and biological markers and are outlined below:

Measurements of Primary Efficacy Variables

[00906] The primary efficacy variable, number of neogenic-like hair follicles, is evaluated using a 4 mm skin punch biopsy (extending to the level of the subcutaneous fat) which is taken 14 days after the dermabrasion and 12 days of topical treatment with drug (Day 14).

[00907] As hair follicle development is a continuum and based on both histological and biological markers, at this time-point neogenic-like hair follicles are identified by fulfilling various criteria that include the following attributes: shorter length than vellus and/or vellus- like hair follicles, lack of a connection with a pre-existing piloscbaceous unit, lack of a pore at the skin surface, lack of a well-differentiated sebaceous gland, lack of a hair shaft, lack of an elastin-negative "streamer" or "dermal channel", and positive staining for 4'-6-Diamidino- 2-phenylindole (DAPI), alkaline phosphatase, BerEP4, and Ki67.

[00908] At 14 days post dermabrasion, neogenic-like HFs have diameters of

approximately 80 μΜ. In order to generate tissue sections for staining, 4 mm punch biopsies are serially sectioned using a cryostat set at 8 μΜ per section (minimum thickness for cryosections). Sections are collected 2 per slide. Each slide can be used for a different staining assay.

[00909] If no sections are lost during sectioning/collection, an 80 μΜ wide structure will generate 10 sections, with 2 sections per slide, yielding approximately 5 slides, for approximately 5 assays. [00910] The following assays are done, each requiring approximately at least 1 slide:

• H and E (to asses morphological criteria)

Elastin stain (to look for the dermal channel)

Alkaline phosphatase stain (to look for the dermal papilla)

• BerEP4 - i67 double label IF (expression of these markers are an indication of follicle epithelium and proliferating cells, respectively)

[00911] One extra slide is available for other markers or if any of above would need to be redone.

Measurements of Secondary Efficacy Variables

[00912] The secondary efficacy variable, number of hair follicles at any stage of development in the healed tissue area of the second skin punch biopsy, are evaluated using a 5 mm (or 6 mm, if indicated) punch biopsy which will be obtained at approximately 5.5 months (Day 168), extending to the level of the subcutaneous fat, at the site of the first biopsy that was allowed to heal via secondary intention. The histological techniques that are used for analysis of the Day 168 biopsy are the same as those discussed above (Section

"Measurements of Primary Efficacy Variables"), for the Day 14 biopsy, although BerEP4, Ki67 and AP assays are only be performed if there are structures present that resemble early neogenic-like follicles.

Additional Exploratory Variables

[00913] Change in scalp hair count and hair shaft thickness may be evaluated with color macropho to graphs and computerized imaging (Canfield Scientific, Inc. Fairfield, NJ USA). Since the pathogenesis of androgenetic alopecia is characterized by miniaturization and dropout of follicles, effective treatments increase the number and thickness of hairs (Olsen 2003. Pierard-Franchimont et al., 1998). In this protocol, hair count and hair shaft thickness are measured at Day 84 and Day 168, and compared to Day -14 (baseline for photography). This type of photography, labeling, image analysis, and data-basing has been used to capture and evaluate endpoints in clinical trials that supported the marketing approval of treatments for androgenetic alopecia, in these studies, separation of active treatment from placebo in these photographically recorded hair growth parameters was observed after 6 months of treatment and beyond (Kaufman et al., 1998, Leyden et al., 1999).

Safety Variables

[00914] Safety and tolerability of lithium gluconate 8% gel applied topically and epidermal disruption by dermabrasion and punch biopsies may be assessed by targeted evaluation of the treated scalp sites and the reporting of adverse events (AEs). Visits on Days 1 , 2, 7, 12, 15, 17, 22 and 182 may be safety visits to assess the scalp sites. Adverse events may be reported at safety phone calls (or other follow-up) on Days 1 12 and 140.

[00915] In addition, the following clinical and laboratory safety parameters may be measured at screening and on Day 182: liver and renal function, fasting blood glucose and urinalysis. A physical examination may be performed at screening and Day 182 (last clinic visit). Vital signs and ECG arc performed at screening. Vital signs may be performed on days 0, 14, and 168, when dermabrasion and punch biopsies are performed.

36.13 EXPECTED ADVERSE EVENTS AND PRECAUTIONARY

MEASURES

[00916] Lithioderm is approved for the treatment of seborrheic dermatitis in

immunocompetent individuals. Expected side effects are rare cases of burning or increase of erythema, usually transient (AFSSAPS, 2002).

]00917] The following adverse events may occur when following the foregoing protocol: bleeding, pain, redness, swelling, exudation, crusting and infection. Every effort is intended to minimize these risks.

[00918] Paracetamol or codeine-containing compounds are to be used for analgesia, if required (concomitant use of aspirin is not permitted).

[00919] Other adverse events should be monitored and treated as indicated.

36.14 PHARMACOKINETIC AND BIOANALYTICAL METHODS

36.14.1 LABORATORY TESTS

[00920] Blood for measurement of serum Li+ is taken at Day 0 (before dermabrasion and 1 h after drug administration on dermabraded skin), and Day 35 (pre and 1 h post administration of drug).

Blood Volume

[00921] Each blood sample for the measurement of clinical laboratory tests requires 10 mL of blood (safety) and 8 mL (HIV, Hep B/C); and blood for Li+ measurements requires 3 mL. A total of 2 blood samples for safety clinical laboratory tests (screening and Day 182), 1 blood sample for HIV, Hep B/C (screening) and 4 blood samples for Li+ PK (2 samples on Day 0 and 2 samples on Day 35) are drawn throughout the protocol treatment course:

Safety Labs: 2 x 10 = 20 mL

HIV, Hep B/C: 1 x 8 = 8 mL Lithium P : 4 x 3 12 mL

Total: 40 mL.

36.14.2 PUNCH BIOPSIES

First Biopsy

[00922] The first skin punch biopsy (4 mm), which is taken 14 days after the dermabrasion and topical treatment (Day 14) will undergo histology to count the number of neogenic-like follicles.

[00923| As hair follicle development is based on both histological and biological markers, neogenic-like hair follicles are identified in the first biopsy by fulfilling various criteria that include the following attributes: shorter length than vellus and/or vellus-like hair follicles, lack of a connection with a pre-existing pilosebaceous unit, lack of a pore at the skin surface, lack of a well-differentiated sebaceous gland, lack of a hair shaft, lack of an elastin-negative "streamer" or "dermal channel," and positive staining for 4'-6-Diamidino-2-phenylindole (DAPI), alkaline phosphatase, BerEP4, and Ki67.

Second Biopsy

[00924] On Day 168 a second skin punch biopsy is performed over the 3 sites where there was a first punch biopsy on Day 14. In addition to providing samples for an analysis of efficacy, any scar tissue formed from the 4 mm punch biopsies on Day 14 is excised by this second biopsy on Day 168, which is then closed by sutures. At the discretion of the clinician and if in accordance with the subject's wish, the Day 168 punch is a 5 mm or 6 mm skin biopsy in order to assure scar removal. It is also intended that this second biopsy remove any photography tattoo if in accordance with the subject's wish. The sutures are removed at a follow-up 2 weeks later (Day 182).

[00925] This biopsy may undergo histology to count the number of hair follicles (as described for the first biopsy above) and for scar characteristics. However, during histology for hair follicles BerEP4, i67 and AP assays can only be performed if there are structures present that resemble early neogenic-like follicles.

36.14.3 PHOTOGRAPHY

[00926] Macrophotographs can be used to provide macroscopic evidence of hair growth. Color macrophotographs of the treatment sites are taken where the hair has been clipped and are centered with a dot tattoo to ensure reproducibility. Macrophotographs may be taken using an industry standard camera and ringflash with associated scanners and imaging software.

36.15 TARGET VARIABLES

36.15.1 PRIMARY EFFICACY VARIABLE

[00927] The number of neogenic-like hair follicles in each of the Day 14 biopsies, as determined by histological examination.

36.15.2 SECONDARY EFFICACY VARIABLES

[00928] The number of hair follicles in a second skin punch biopsy taken approximately 5.5 months after the first biopsy (Day 168) from the site of the Day 14 biopsy which was allowed to heal by secondary intention.

36.15.3 EXPLORATORY EFFICACY VARIABLES

• The difference in the number of hairs captured by photography on Day 84 and versus Day - 14;

• The difference in hair shaft thickness captured by photography on Day 84 and versus Day - 14;

• The histological characteristics in a second skin punch biopsy on Day 1 8.

36.15.4 SAFETY MEASUREMENTS

[00932] · Adverse events (AEs)

[00933] · Treatment site evaluations made at each visit (using local tolerability, wound healing and Vancouver Scar Scales).

[00934] · Liver and renal function, fasting blood glucose and urinaly sis are performed at screening and Day 182. A physical examination is also performed at screening and Day 182. Vital signs and ECG are performed at screening. On days 0, 14 and 168, when dermabrasion or punch biopsies are performed, vital signs are repeated

36.15.5 OTHER ASSESSMENTS

[00935] Serum lithium concentration at baseline before dermabrasion and 1 h after drug administration on dermabraded skin (Day 0), and prior to and 1 h after drug administration on Day 35.

36.16 INTENDED RESULTS (00936) Topical lithium is expected to increase the number of neogenic-like hair follicles in skin that has undergone controlled perturbation by dermabrasion compared to

dermabrasion plus topical placebo. This is formalized in the following hypotheses:

|00937] HO: Topical lithium does not promote hair follicle neogenesis

[00938] HI : Topical lithium promotes hair follicle neogenesis.

[00939] For the primary efficacy endpoint (number of neogenic-like hair follicles in Day 14 biopsy), these hypotheses are expressed mathematically as

[00940] HO: μΤ < μθ versus HI : μΤ > μθ

[00941] where μΤ and μ€ denote the true population mean numbers of neogenic-like hair follicles in subjects treated with the test treatment (twice daily topical application of 8% lithium gluconate gel to dermabraded scalp) and control treatment (twice daily topical application of placebo gel to dermabraded scalp), respectively.

[00942] The effect of topical lithium on hair follicle neogenesis is also examined by testing

[00943] HO: πΤ < nC versus HI : nT > nC

[00944] where πΤ and TIC denote the true proportions of subjects with at least one neogenic-like follicle in the populations of all subjects treated with the test treatment (twice- daily topical application of lithium gluconate 8% gel to dermabraded scalp) and control treatment (twice-daily topical application of placebo gel to dermabraded scalp), respectively.

36.17 STATISTICAL METHODS

36.17.1 PRIMARY ENDPOINT

[00945] In order to assess the efficacy of topical application of lithium gluconate 8% gel on hair follicle neogenesis in dermabraded sites, a generalized linear model (McCullagh & Nelder, 1989) is estimated for the mean number of neogenic-like hair follicles per Day 17 biopsy site. A Poisson regression may be used initially, but negative binomial regression or zero-inflation models (Lambert, 1992) may be used to better fit the data. Factors in the model will be the fixed effects of treatment and treatment site, and the random effects of subjects, and subjects' Hamilton-Norwood classification as a covariate. The one-sided null hypothesis HO: μΤ < μθ will be rejected in favor of the alternative H I : μΤ > μθ at the a = 1 5% level of statistical significance if the two-sided 70% confidence interval for μΤ - iC is entirely above zero. In addition, two-sided 95% confidence intervals will be constructed for the differences between each pair of treatments (including the non-abraded sites). [00946| The treatment groups are compared with respect to the proportions of subjects in each treatment group with at least one neogenic-like hair follicle in the Day 14 biopsy of either dermabraded site. The one-sided null hypothesis HO: πΤ < %C will be rejected in favor of the alternative H I : πΤ > 7rC at the a = 15% level of statistical significance if the two-sided 70% confidence intervals for πΤ - nC is entirely above zero. In addition, a two-sided 95% confidence interval will be constructed.

36.17.2 SECONDARY ENDPOINTS

[00947] The numbers of hair follicles induced in the Day 168 biopsies are analyzed analogously to the primary endpoint (numbers of neogenic-like follicles in the Day 14 biopsies). In addition, the differences in the numbers of hair shafts and shaft thickness are captured by photography on Days 84 and 168 versus Day - 14.

36.173 DETERMINATION OF SAMPLE SIZE

[00948| Since the nature of the distribution of the primary efficacy variable (number of neogenic-like hair follicles) is unknown, the sample size for a controlled study is ideally based on the number of subjects in each treatment group with at least one neogenic-like hair follicle which can be assumed to follow a binomial distribution. If this is the first clinical exploration of the effect of dermabrasion with pharmacological modulation on follicular neogenesis, the type I error rate (significance level) is set relatively high at a = 15%.

[00949] Ninety-two (92) subjects (i.e., 46 per treatment group) would provide 80% power to reject the null hypothesis 110: πΤ < nC in favor of the alternative H I : πΤ > nC at the a = 15% level of statistical significance if

[00950] · the probability of a subject having at least one neogenic-like hair follicle is TCC = 0.15 in the control group as estimated from the human study presented in Section 35 supra, and

[00951] · the probability of a subject having at least one neogenic-like hair follicle is π Τ = 0.30 in the test treatment group.

36.18 REFERENCES

[00952] AFSSAPS (Agence francaise de securite sanitaire des produits de sante); 2002. Available on-line via http://www.labcatal.com/produits31d.htm (Accessed 09-Jan-2010)

[00953] Alguire PC, Mathes BM ( 1998). Skin biopsy techniques for the internist, J Gen Intern Med 13 :46-54. |00954] Baldessarini RJM, Tarazi FI (2006). In: Goodman and Oilman's The

Pharmacological Basis of Therapeutics. Ed. Brunton LL: 1 1 th edition. New York, NY:

McGraw-Hill, Section III, Chapter 18: pp. 485-90.

[00955] Christenson LJ, Phillips P . Weaver AL, Otley CC (2005). Primary closure vs second-intention treatment of skin punch biopsy sites. Archives of Dermatology 141 : 1093- 1099.

[00956] Dinh QC, Sinclair R (2007). Female pattern hair loss: Current treatment concepts. Clinical Interventions in Aging 2(2) 189-199.

[00957] Dreno B & Moyse D (2002). Lithium gluconate in the treatment of seborrhoeic dermatitis: a multicenter, randomised, double-blind study versus placebo. Eur J Dermatol 12(6):549-552.

[00958] Grimes, 2005, Microdermabrasion. Dermatol Surg 31 : 1351 -1354.

[00959] Kaufman KD. Olsen ΕΛ, Whiting D, Savin R, DeVillez R, Bergfeld W, Price VH, Van Neste D, Roberts JL, Hordinsky M, Shapiro J, Binkowitz B, Gormley GJ. Finasteride in the treatment of men with androgenetic alopecia. Finasteride Male Pattern Hair Loss Study Group. J Am Acad Dermatol. 1998 Oct; 39(4 Pt 1 ) 39: 578-89.

[00960] Lambert D (1992). Zero-inflated Poisson Regression, with an application to defects in manufacturing. Technometrics 34, 1— 14.

[00961] Leyden J, Dunlap F, Miller B, Winters P, Lebwohl M, Hecker D, Kraus S, Baldwin H, Shalita A, Draelos Z, Markou M, Thiboutot D, Rapaport M, Kang S, Kelly T, Pariser D, Webster G, Hordinsky M, Rietschel R, Katz HI, Terranella L, Best S, Round E, Waldstreicher J. Finasteride in the treatment of men with frontal male pattern hair loss. J Am Acad Dermatol. 1999 Jun: 40(6 Pt 1 ): 40930-7.

[00962] McCullagh P, Nelder JA ( 1989). Generalized Linear Models. Chapman & Hall, London, 2nd edition.

[00963] Olsen EA. Current and novel methods for assessing efficacy of hair growth promoters in partem hair loss. J Amer Acad Dermatol 48:253-62, 2003.

[00964] Pierard-Franchimont C, De Doncker P, Cauwenbergh G, Pierard GE.

Ketoconazole shampoo: effect of long-term use in androgenic alopecia. Dermatology.

1998; 196 (4): 474-7.

[00965] Schrauzer GN (2002). Lithium: Occurrence, Dietary Intakes, Nutritional

Essentiality. Journal of the American College of Nutrition, Vol. 21, No. 1 , 14-21. [00966j Stough D, Stein K. Haber R, Parsley WM, Vogel JE. Whiting DA, Washenik (2005) Psychological Effect, Pathophysiology, and Management of Androgenetic Alopecia in Men. Mayo Clin Proc; 80( 10): 1316-1322.

[00967] Summary of Product Characteristics (SmPC), EFADERMIN. Available via Fachinformation http://wmv.louis-widmer.de/003 lwd_03030502_de.php?lang=de (Accessed August 2009; accessed April 30, 2010)

[00968] Summary of Product Characteristics (SmPC), LI 1 HIODERM, 8% gel. Available on-line at http://afssaps-prd.afssaps.fr/php/ecodex/rcp/R01 14584.htm (Accessed December 28, 2009; accessed April 30, 2010).

[00969] Waller DP. Acute dermal irritation toxicity study. Test Article Lithioderm 8%, Gluconate. Study No. 093-001. PreLabs LLC 2160 South First Avenue. Maywood, Illinois 60153, USA. Date of report Dec-2009.

[00970] All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[00971] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the illustrative embodiments and/or appended claims.

37. EXAMPLE: PREPARATION OF SYNTHETIC BIODEGRADABLE

PLA:PLG SCAFFOLDS AND MODULATION OF LI+ RELEASE BY

VARYING THE POLYMER COMPOSITION

[00972] The objective of this experiment was to develop prototypes of biodegradable scaffold patches that could be placed on integumentally perturbed tissue. The "scaffold" is a three-dimensional structure that can provide a high surface area for cell attachment. The cell signaling agent incorporated in the scaffold matrix is lithium gluconate, which in water ionizes into gluconate anions and Li+ ions. By varying the polymer composition of the scaffold matrix, the release properties of Li+ can be modulated from 3 days to 14 days. 37.1 PREPARATION AND CHARACTERIZATION OF BIODEGRADABLE DRUG LOADED MESH PATCHES

[00973] Solutions of lithium gluconate were prepared in distilled water at a concentration of 50 mg/ml. Poly(lactide-co-glycolide) (PLG), MW 12000 g/mole, poly(lactic acid) (PLA), MW 30.000 g/mole and blends thereof, were used to prepare fibrous scaffolds. The blends of polymers were 100/0 PLA/PLG, 50/50 PLA/PLG, 25/75 PLA PLG and 0/100 PLA/PLG, respectively. PLA and PLG were purchased from Purac, Inc.

[00974] Λ cotton candy machine (Gold Medal Floss, Cat# 3024) was set at a setting at 3 (there are five settings in total, ranging from temperatures of 40 °C to 200 °C).

[00975] 1 g of a blend of 100/0 PLA and 1 ml of the lithium gluconate solution was fed into the hopper, which resulted in fine fibers collecting (much like spider web) in the collection chamber. The fibers with incorporated drug were collected and pressed into patches of lg each, a low pressure Carver press. The patches were then punched out into 1 square inch squares.

[00976] A similar procedure was followed for the other blends of 50/50 PLA/PLG, 25/75 PLA PLG and 0/100 PLA PLG.

[00977] Scanning electron micrographs (SEM) (see Figure 36A and B)were taken of the patches. By SEM, the mesh size, or open-cell size was estimated to be around 100-200 microns. Estimated thickness of the fabricated patches was in the range of 500- 1000 microns.

[00978] The patches were placed into mesh buckets in dissolution baths containing phosphate buffered saline at 37 °C and pi I 7.4, to simulate physiological conditions. Aliquots of the dissolution media were retrieved at predetermined time-points and analyzed for Li+ content by flame-emission atomic adsorption spectroscopy (AA).

[00979] Figure 36C is a plot of Percent Cumulative Release of Li+ as a Function of Time in Days, overlaid with release profiles of four different blends.

37.2 RESULTS

[00980] SEM. Scanning electron micrographs of 100/0 PLA and 0/1 0 PLG is shown in Figure 36A and B. The micrographs demonstrated a tibrous texture.

[00981] Visual and F!exural Modulus. The pressed fiber patches were tested for flexural strength by a simple flex method of bending the patch between the thumb and the index finger. The patches could be bent, but they were brittle to the touch. Future patches should incorporate some plasticizing polymers such as PEGs, or silicones, to impart some flexibility to the patches. By SEM, the mesh size, or open-cell size was estimated to be around 100-200 microns. Estimated thickness of the fabricated patches was in the range of 500- 1000 microns. (00982) Release Rates. The release of Li+ could be modulated by varying the ratio of PEA to PLG. As a rule of thumb, the higher crystallinity of the poly(lactide) (PLA) slows down the release of Li+ from the matrix. The amorphous nature of poly (lactide-co-glycolide) (PLG) result in higher release rates of Li+. The approach of blending various ratios of PLA: PLG can be utilized effectively to modulate the release rate of Li+ from the matrix.

[00983] Biodegradability. The biodegradability of the patches can be tested in vitro, by incubation of pre-weighed patches in phosphate buffer saline. pH 7.4 at 37 °C. Over time, the patches are removed from the bath and dried in a vacuum oven maintained at 30 °C. The weight of the patches at T=0 and t=t provides biodegradation profile. Since the polymers degrade by hydrolysis and not by enzymolysis, the degradation buffer would not contain enzymes.

[00984] Bioadhesion. The bioadhesiveness of the drug-loaded patches can be assessed by placing the patch of wet tissue, inverting the tissue and measuring the rate at which the patch detaches from the tissue.

[00985] Cell Adhesion. The propensity of the drug-loaded patches to adhere to cells is measured by in-vitro culture of COS cells or keratinocytes in the presence of the scaffolds.

38. ILLUSTRATIVE EMBODIMENTS

[00986] The invention can be illustrated by the non-limiting, embodiments set forth in the following paragraphs.

1. A method for modulating hair growth in a human subject, comprising administering a pulse treatment or intermittent treatments of a lithium composition that delivers an effective amount of lithium ions to a human subject in need thereof.

2. The method of paragraph 1 in which the pulse treatment or intermittent treatment of the lithium composition is administered by topical administration to the skin.

3. The method of paragraph 1 in which the pulse treatment or intermittent treatment of the lithium composition is administered to the skin surface, transdermally, or intradermally. The method of paragraph 1 in which the pulse treatment or intermittent treatment of the lithium composition is administered by subcutaneous, parenteral or oral administration. The method of paragraph 1 in which the lithium composition comprises lithium gluconate, lithium carbonate, or lithium succinate. The method of paragraph 1 in which the lithium composition does not comprise lithium chloride. The method of paragraph 1 in which the lithium in the lithium composition is encapsulated in microspheres. The method of paragraph 7, wherein the lithium in the lithium composition is encapsulated in microspheres of sizes between 0.10 microns and 200 microns. The method of paragraph 8, wherein microspheres are between 0.20 microns and 50 microns. The method of paragraph 7, wherein the lithium in the lithium composition is encapsulated in liposomes of sizes between 10 nm and 50 microns. The method of paragraph 10, wherein the liposomes are between 500 nm and 20 microns. The method of paragraph 1 in which the lithium composition is administered as a cold liquid, which gels at a temperature of 32 °C - 37 °C. The method of paragraph 1 in which the lithium composition is administered as a liquid, which then hardens into a depot that delivers lithium over time. The method of paragraph 1 in which the lithium composition is administered as a hydrogel. The method of paragraph 1 in which the lithium composition comprises one or more excipients that complex to lithium. The method of paragraph 15, wherein the excipient comprises hyaluronic acid, polyacrylic acid or alginic acid. The method of paragraph I in which the lithium composition comprises one or more permeation enhancing agents or carriers that solubilize the lithium in skin. The method of paragraph 1 in which the lithium composition comprises propylene glycol, polyethylene glycol or ethanol. The method of paragraph 1 in which the pulse treatment is a single dose of the lithium composition administered over a period of 1 day to 1 month. The method of paragraph 1 in which the intermittent treatments are multiple courses of lithium treatments interrupted by lithium treatment holidays. The method of paragraph 20 in which a second treatment is administered during the lithium treatment holidays. The method of paragraph 21 in which the duration of the second treatment is similar to the duration of the lithium treatment. The method of paragraph 21 in which the duration of the second treatment is different from the duration of the lithium treatment. The method of paragraph 1 in which a second treatment is administered to the human subject receiving the lithium treatment. The method of paragraph 24 in which the duration of the second treatment is identical to the duration of the lithium treatment. The method of paragraph 24 in which the duration of the second treatment is different from the duration of the lithium treatment. The method of paragraph 24 in which the second treatment is administered concurrently with the lithium treatment. The method of paragraph 24 in which the second treatment is administered before the lithium treatment. The method of paragraph 24 in which the second treatment is administered after the lithium treatment. The method of paragraph 21 or 24 in which the second treatment is integumental perturbation. The method of paragraph 30 in which the integumental perturbation removes the epidermis partially or completely. The method of paragraph 30 in which the integumental perturbation removes all of the epidermis and part of the dermis. The method of paragraph 30 in which the integumental perturbation does not remove the epidermis. The method of paragraph 30 in which the integumental perturbation is accomplished by laser, light, heat, microneedle rollers, ultrasound, iontophoresis, electrophoresis, or radiation treatment, or a combination thereof. The method of paragraph 30 in which the integumental perturbation is accomplished by laser. The method of paragraph 35 wherein the integumental perturbation by laser is fractional and non-ablative. The method of paragraph 36, wherein the fractional, non-ablative integumental perturbation by laser is performed by use of an Erbium- YAG laser at 1500-1 90 nm. The method of paragraph 35 wherein the integumental perturbation by laser is fractional and ablative. The method of paragraph 38, wherein the fractional, ablative integumental perturbation results in fractional ablation of the skin at a depth between 100 microns and 4000 microns into the skin. The method of paragraph 38, wherein the fractional, ablative integumental perturbation results in fractional ablation of the skin at a depth approximating the depth of a full-thickness excision wound. The method of paragraph 38, wherein the fractional, ablative integumental perturbation results in fractional ablation of the skin over an area of 1.5 cm x 1 .5 cm to 15 cm x 15 cm. The method of paragraph 38, wherein the fractional, ablative integumental perturbation results in fractional ablation of the skin at a depth density of the micro- thermal zones of the fractional ablation approximates that of a full bulk ablation of the entire area of treatment. The method of paragraph 38, wherein the fractional, ablative integumental perturbation is by full bulk ablation, wherein the tissue of the entire area of treatment is ablated. The method of paragraph 43, wherein the fractional, ablative integumental perturbation by bulk ablation is over an area of 1.5 cm x 1.5 cm to 15 cm x 15 cm. The method of paragraph 43, wherein the fractional, ablative integumental perturbation by bulk ablation is accomplished at 10,600 nm using a carbon dioxide laser. The method of paragraph 43, wherein the fractional, ablative integumental perturbation by bulk ablation is accomplished at 2940 nm using a Erbium- YAG laser. The method of paragraph 35 wherein the integumental perturbation by laser is non- fractional and ablative. The method of paragraph 47, wherein the non- fractional, ablative integumental perturbation is by full bulk ablation, wherein the tissue of the entire area of treatment is ablated. The method of paragraph 47. wherein the non-fractional, ablative integumental perturbation by bulk ablation is over an area of 1.5 cm x 1.5 cm to 15 cm x 1 cm. The method of paragraph 47, wherein the non-fractional, ablative integumental perturbation by bulk ablation is accomplished at 10,600 nm using a carbon dioxide laser. The method of paragraph 47, wherein the non-fractional, ablative integumental perturbation by bulk ablation is accomplished at 2940 nm using a Erbium- YAG laser. The method of paragraph 30 in which the integumental perturbation is accomplished using a microneedle array. The method of paragraph 52, wherein the microneedle array is in the form of a roller or flat plate. The method of paragraph 52, wherein the microneedle array can disrupt a skin area of 1 .5 em x 1.5 cm to 15 cm x 15 cm. The method of paragraph 52, wherein the microneedle array can disrupt skin at a depth of 100 microns to 4000 microns. The method of paragraph 52, wherein the microneedle array has hollow needles. The method of paragraph 52, wherein the microneedle array top has a luer-lock fitting that can accommodate a syringe to deliver drug. The method of paragraph 57, wherein the volume of the syringe is 1 ml to 3 ml. The method of paragraph 30 in which the integumental perturbation is accomplished by injury. T he method of paragraph 59, wherein the mode of injury is mechanical. The method of paragraph 60, wherein the mechanical injury is accomplished by microdermabrasion. dermabrasion, tape stripping, full thickness excision of tissue. The method of paragraph 59, wherein the mode of injury is thermal injury. The method of paragraph 60, wherein the thermal injury is accomplished by laser. The method of paragraph 30 in which the integumental perturbation is accomplished by inducing inflammation. The method of paragraph 64 in which the method of inducing inflammation is by application of an adjuvant. 66. The method or" paragraph 65, wherein the adjuvant is selected from the group of sodium dodecyl sulfate, aluminum salts, monophosphoryl lipid A, and cetyl triammonium bromide (CTAB).

67. The method of paragraph 64 in which the method of inducing inflammation is injury to tissue.

68. The method of paragraph 64 in which the method of inducing inflammation is by application of an cytokine (e.g., IL- l beta).

69. The method of paragraph 64 in which the method of inducing inflammation is by application of an antigen (e.g. tetanus toxoid).

70. The method of paragraph 30 in which the method of integumental perturbation is accompanied by stem cell mobilization (i.e., G-CSF).

71. The method of paragraph 30 in which a third treatment is administered to the human subject receiving the lithium treatment and integumental perturbation.

72. The method of paragraph 71 in which the third treatment stimulates hair growth.

73. The method of paragraph 72 in which the third treatment is the administration of minoxidil.

74. The method of paragraph 72 in which the third treatment is the administration of a 5- α reductase inhibitor.

75. The method of paragraph 72 in which the third treatment is the administration of minoxidil and a 5-a reductase inhibitor.

76. The method of paragraph 74 in which the 5-a reductase inhibitor is finasteride.

77. The method of paragraph 75 in which the 5-a reductase inhibitor is finasteride.

78. The method of paragraph 72 in which the third treatment is the administration of bimatoprost.

79. The method of paragraph 72 in which the third treatment is the administration of an anti -senescence agent. 80. The method of paragraph 72 in which the third treatment is a surgical transplantation of hair follicles.

81. The method of paragraph 72 in which the stimulation of hair growth is on the scalp or face, chest, abdomen, arms, armpits, legs, or genitals, or a wounded or scarred area of the skin of the subject.

82. The method of paragraph 71 in which the third treatment inhibits hair growth.

83. The method of paragraph 82 in which the third treatment is the administration of eflornithine.

84. The method of paragraph 82 in which the third treatment is the administration of a cytotoxic drug.

85. The method of paragraph 82 in which the third treatment is the administration of eflornithine and a cytotoxic drug.

86. The method of paragraph 84 in which the cytotoxic drug is 5-fluorouracil.

87. The method of paragraph 85 in which the cytotoxic drug is 5-fluorouracil.

88. The method of paragraph 82 in which the inhibition of hair growth is on the head, chest, breast, abdomen, neck, back, arms, armpits, legs, hands, feet, buttocks, genitals, or a wounded or scarred area of the skin of the subject.

89. The method of paragraph 21 or 24 in which the second treatment stimulates hair growth.

90. The method of paragraph 89 in which the second treatment is by low intensity laser.

91. The method of paragraph 90 in which the low intensity laser is used either

concurrently or intermittently with lithium treatment.

92. The method of paragraph 90 in which the low intensity laser is the HairMax Laser comb.

93. The method of paragraph 89 in which the second treatment is the administration of minoxidil. 94. The method of paragraph 89 in which the second treatment is the administration of a 5- reductase inhibitor.

95. The method of paragraph 89 in which the second treatment is the administration of minoxidil and a 5-a reductase inhibitor.

96. The method of paragraph 94 in which the 5-a reductase inhibitor is finasteride.

97. The method of paragraph 95 in which the 5-a reductase inhibitor is finasteride.

98. The method of paragraph 89 in wrhich the second treatment is the administration of bimatoprost.

99. The method of paragraph 89 in which the second treatment is the administration of an anti-senescence agent.

100. The method of paragraph 89 in which the second treatment is a surgical

transplantation of hair follicles.

101. The method of paragraph 89 in which the stimulation of hair growth is on the scalp or face, chest, abdomen, arms, armpits, legs, or genitals, or a wounded or scarred area of the skin of the subject.

102. The method of paragraph 21 or 24 in which the second treatment inhibits hair

growth.

103. The method of paragraph 102 in which the second treatment is the administration of eflornithine.

104. The method of paragraph 102 in which the second treatment is the administration of a cytotoxic drug.

105. The method of paragraph 102 in which the second treatment is the administration of eflornithine and a cytotoxic drug.

1 6. The method of paragraph 104 in which the cytotoxic drug is 5-fluorouracil.

107. The method of paragraph 105 in which the cytotoxic drug is 5-fluorouracil. 108. The method of paragraph 102 in which the inhibition of hair growth is on the head, chest, breast, abdomen, neck, back, arms, armpits, legs, hands, feet, buttocks, genitals, or a wounded or scarred area of the skin of the subject.