WO2008066707A2 - Targeting prohormone convertase enzymes to suppress pomc-msh processing to induce skin lightening - Google Patents

Abstract

Methods and compositions useful for reducing skin pigmentation and hyperpigmentation are provided. The pigmentation or hyperpigmentation reduced according to the methods of the invention can arise independent of a UV-induced tanning response. Also provided are methods for identifying a candidate skin lightening agent. The methods and compositions of the invention are useful for treatment of a number of conditions, including age spots (also variously known as liver spots, sun spots, solar lentigo, and lentigines).

Description

TARGETING PROHORMONE CONVERT ASE ENZYMES TO SUPPRESS POMC-MSH PROCESSING TO INDUCE SKIN LIGHTENING

BACKGROUND OF THE INVENTION Ultraviolet (UV) radiation represents a definitive risk factor for skin cancer, in particular in combination with certain underlying genetic traits such as red hair and fair skin [1, 2]. Pigmentation of the skin results from the synthesis of melanin in the pigment-producing cells, the melanocytes, followed by distribution and transport of pigment granules to neighboring keratinocytes. It is commonly believed that melanin is crucial for absorption of free radicals generated within the cytoplasm by UV radiation, and in direct shielding from UV and visible light radiation [3-5]. Molecular and genetic data indicate that variations in the coding region of the melanocortin-1 -receptor (MClR) play an important role in tanning and pigmentation in humans [6]. MClR is expressed in melanocytes and is activated by its ligand alpha-Melanocyte Stimulating Hormone (α-MSH). This pro-pigmentation hormone is produced and secreted by both keratinocytes and melanocytes in the skin following UV radiation [7, 8].

The gene encoding α-MSH is proopiomelanocortin (POMC), a multi-component precursor for α-MSH (melanotropic), adrenocorticotropic hormone (ACTH; adrenocorticotropic), as well as the opioid peptide β-endorphin. Normal synthesis of α-MSH and ACTH is an important determinant of constitutive human pigmentation and the cutaneous response to UV radiation [8-10].

POMC was primarily identified in the pituitary gland (hypophysis), but the production of POMC and POMC-derived peptides is now known to be not confined to this organ. In humans, circulating levels of α-MSH and ACTH are low. Several independent reports have demonstrated synthesis of α-MSH and ACTH by epidermal keratinocytes and melanocytes [7, 12-17], and the cutaneous α-MSH content showed little change after hypophysectomy [18]. In addition, prior to the invention the mechanism underlying UV-mediated expression of α-MSH was not known.

SUMMARY OF THE INVENTION

It has now been surprisingly discovered according to the instant invention that UV-mediated induction of POMC and α-MSH in the skin, and thus skin pigmentation, is directly mediated by the tumor suppressor protein p53. In addition, it has also now been surprisingly discovered according to the instant invention that induction of POMC and α-MSH in the skin, and thus skin pigmentation, can be induced by the tumor suppressor protein p53 even in the absence of UV radiation. It has now further been unexpectedly discovered that treatment of unwanted skin pigmentation in a living subject, including in particular hyperpigmentation arising from causes other than and/or in addition to UV radiation, can be effected using one or more inhibitors of enzymes involved in the processing of POMC to yield α-MSH. Such enzymes specifically include, but are not limited to, prohomone convertase 1 (PCl) and prohomone convertase 2 (PC2). The invention in one aspect is a method for reducing or preventing skin pigmentation. The method according to this aspect of the invention includes the step of locally administering to a target {e.g., pigmented) skin an effective amount of an inhibitor of an enzyme involved in cleavage of proopiomelanocortin (POMC) to alpha melanocyte stimulating hormone (α-MSH), to reduce or prevent pigmentation of the skin. In one embodiment the locally administering is topically administering.

In one embodiment according to this aspect of the invention the enzyme is selected from PCl and PC2. In one embodiment the enzyme is PCl . In one embodiment the enzyme is PC2.

In one embodiment the inhibitor of the enzyme is an inhibitor of enzymatic activity by a functional form of the enzyme. In one embodiment the inhibitor of the enzyme is an inhibitor of expression of a functional form of the enzyme.

Further according to this aspect of the invention in one embodiment the skin pigmentation is hyperpigmentation associated with a condition selected from acanthosis nigricans, Addison's disease, age spots (also variously known as liver spots, sun spots, solar lentigo, and solar and simple lentigines), basal cell carcinomas, biliary cirrhosis, cafe au lait spots (which may be associated with neurofibromatosis or Albright's syndrome), ectopic ACTH syndrome, eosinophilia-myalgia syndrome, ephelides (freckles), folate deficiency, hemochromatosis, junctional and compound nevi, lentigo, malabsorption, melanosis secondary to metastatic melanoma, Nelson's syndrome, pellagra, pigmented actinic keratosis, pigmented keratinocyte tumors, POEMS syndrome, porphyria cutanea tarda, post-inflammatory hyperpigmentation, scleroderma, seborrheic keratosis, vitamin B₁₂ deficiency, and Whipple's disease. Other treatable diseases, conditions, or disorders include those that can be characterized by discolorations of the skin or hair such as, for example, hyperpigmentation caused by inflammation or from diseases such as melasma/chloasma and postinflammatory hyperpigmentation. Certain drugs are associated with the development of hyperpigmentation. These drugs include 5-fluorouracil (5-FU), busulfan, cyclophosphamide, and ACTH. Each of these lists is not meant to be limiting. In one embodiment the condition is specifically associated with induction of melanin production. In one embodiment the condition associated with hyperpigmentation is age spots (also variously known as liver spots, sun spots, solar lentigo, and lentigines). The invention in one aspect is a method of identifying a candidate skin lightening agent. The method according to this aspect of the invention includes the steps of contacting, under defined conditions, a test cell (e.g., a keratinocyte, melanocyte, fibroblast, etc.) with (a) an agent that induces proopiomelanocortin (POMC) expression, and (b) a test agent; measuring an amount of alpha melanocyte stimulating hormone (α-MSH) expressed by the contacted test cell (e.g., keratinocyte); and determining the test agent is a candidate skin lightening agent when the amount of α-MSH expressed by the contacted test cell (e.g., keratinocyte) is reduced compared to an amount of α-MSH expressed by a control test cell (e.g., a control keratinocyte) contacted, under the defined conditions, with the agent that induces POMC expression (but without the test agent). In one embodiment the test agent is a peptide at least six amino acid residues long.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. These and other aspects and embodiments of the invention are described in further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS The figures are illustrative only and are not required for enablement of the invention disclosed herein.

FIG. IA is a bar graph (left) and a set of western blots (right) depicting the induction of POMC mRNA (left, normalized to GAPDH) and p53, POMC, and α- tubulin (loading control) proteins (right) in human primary keratinocytes at indicated times following UV exposure. (-) refers to no UV and fold induction of POMC is calculated relative to untreated cells.

FIG. IB is a bar graph (left) and a set of western blots (right) depicting the induction of POMC mRNA (left, normalized to GAPDH) and p53, POMC, and α- tubulin (loading control) proteins (right) in mouse PAM212 keratinocytes at indicated times following UV exposure. (-) refers to no UV and fold induction of POMC is calculated relative to untreated cells.

FIG. 1C is a bar graph (left) and a set of western blots (right) depicting the induction of POMC mRNA (left, normalized to GAPDH) and p53, POMC, and α- tubulin (loading control) proteins (right) in mouse PAM212 keratinocytes either transfected (+) or not (-) with empty pcDNA vector or HA-p53 plasmid, as indicated.

FIG. 2A is a bar graph depicting the induction of POMC mRNA in PAM212 cells (filled bars) and the lack of induction of POMC RNA in PAM212 cells transfected with dominant-negative p53 (PAMDD, open bars) at indicated times following UV exposure. (-) refers to no UV and fold induction of POMC is calculated relative to untreated cells. FIG. 2B is a bar graph depicting the induction of POMC mRNA in primary keratinocytes isolated from wild-type mice (wtp53, filled bars) and the lack of induction of POMC RNA in primary keratinocytes isolated from p53-/- mice (p53-null, open bars) at indicated times following UV exposure. (-) refers to no UV and fold induction of POMC is calculated relative to untreated cells. FIG. 3 A (top) is a schematic representation of human POMC locus, indicating location of highly conserved p53-binding consensus sequence in the promoter region, and (bottom) various luciferase reporter constructs and corresponding fold induction of luciferase activity by UV, in transiently transfected PAM212 cells.

FIG. 3B is a bar graph depicting fold induction of luciferase activity in unirradiated PAM212 cells transfected with plasmid 5 (including p53 binding site, filled bars) or plasmid 2 (without p53 binding site, open bars), either alone or in combination with either control pcDNA3 vector or p53 vector.

FIG. 3 C is a bar graph depicting fold induction of luciferase activity in PAM212 cells transfected with plasmid 5 (including p53 binding site, filled bars), and lack of induction of luciferase activity in PAMDD cells transfected with plasmid 5 (open bars), at indicated times following UV exposure.

FIG. 3D is an image of a gel depicting p53 electrophoretic mobility shift assay (EMSA) in nuclear extracts isolated from PAM212 cells at indicated times following UV exposure.

FIG. 3E is a pair of images of stained agarose gels depicting association of p53 with POMC promoter by chromatin immunoprecipitation (ChIP), in human primary keratinocytes (top) and mouse PAM212 cells.

FIG. 4A is a pair of photographic images depicting ear pigmentation differences among UV irradiated p53-/- (UV, -/-), UV irradiated p53 wild-type (UV, +/+), and unirradiated p53 wild-type control (+/+) mice. FIG. 4B is a set of four photomicrographic images depicting melanin content in ear sections from mice in FIG. 4A.

FIG. 4C is a set of four photomicrographic images depicting POMC protein expression in ear sections from mice in FIG. 4A.

FIG. 4D is a bar graph depicting POMC mRNA expression in epidermis from UV irradiated and un-irradiated p53-null mice (p53-/-, open bars) and wild-type mice (p53+/+, filled bars) as measured by real-time RT-PCR.

FIG. 5 is a panel of 12 photomicrographic images depicting immunohistochemical staining of p53, α-MSH, and MITF in human foreskin at indicated times following UV irradiation. Arrows indicate the first time point when the staining marker is positive.

FIG. 6 A is a bar graph (top) and a set of western blots (bottom) depicting the induction of POMC mRNA (top, normalized to GAPDH) and p53, POMC, and α-tubulin (loading control) proteins (bottom) in mouse PAM212 keratinocytes incubated for indicated amounts of time in the presence of 25 μM etoposide. (-) refers to no etoposide and fold induction of POMC is calculated relative to untreated cells.

FIG. 6B is a photographic image depicting ear pigmentation differences among 5-FU-treated p53-/- (UV, -/-), 5-FU-treated p53 wild-type (5-FU, wt), and untreated ρ53 wild-type control (wt) mice.

FIG. 6C is a set of four photomicrographic images depicting melanin content in ear sections from mice in FIG. 6B.

DETAILED DESCRIPTION

The tumor suppressor protein p53 [20, 21] is a transcription factor which plays a pivotal role in the cellular response to genotoxic stress such as UV radiation and chemically induced DNA damage [22, 23]. It has been shown to directly activate transcription of numerous genes such as those regulating cell cycle progression, apoptotic cellular pathways, and others [24]. Loss of function of p53 leads to aberrant cell-growth and survival responses and as such, its dysregulation plays an integral part in the genesis of human cancer.

In the skin, p53 function is critical for tissue integrity following UV irradiation. p53-/- mice exhibit an enormously enhanced propensity to develop tumors following UVB by week 16, while none of the comparably treated p53+/+ mice developed skin tumors after 17 wk [25]. UV can induce "signature" mutations in the p53 gene, almost exclusively dipyrimidine C to T substitutions including CC to TT frame shift mutations, which are rarely seen in non-cutaneous tumors [26, 27]. These mutations were found in the skin of UV-irradiated mice months before tumor development [26]. Conversely, mutations in p53 are absent from most melanomas [28]. In addition to the above activities, p53 has been shown to be essential for the "sunburning" response [29], as demonstrated by the absence of apoptotic keratinocytes following UV irradiation of p53-/- mice. This important discovery provided a striking example of the pivotal role p53 has in regulation of keratinocyte apoptosis in the context of a naturally occurring environmental exposure. Collectively, these observations are consistent with the discovery, according to the instant invention, that p53 also participates in regulation of the pigmentation response to UV. The gene for POMC encodes a polypeptide hormone precursor that undergoes extensive, tissue-specific, post-translational processing via cleavage by subtilisin-like enzymes known as prohormone convertases (PCs). The classical motif for processing by PCs is KRj or RRJ,. There are eight potential cleavage sites within the polypeptide precursor and, depending on tissue type and the available convertases, processing may yield as many as ten biologically active peptides involved in diverse cellular functions. The encoded protein is synthesized mainly in corticotroph cells of the anterior pituitary where four cleavage sites are used; adrenocorticotropin, essential for normal steroidogenesis and the maintenance of normal adrenal weight, and lipotropin beta are the major end products. In other tissues, including the hypothalamus, placenta, and epithelium, all cleavage sites may be used, giving rise to peptides with roles in pain and energy homeostasis, melanocyte stimulation, and immune modulation. These include several distinct melanotropins, lipotropins, and endorphins that are contained within the adrenocorticotropin and β-lipotropin peptides. In humans the mature prohormone POMC is a 241 amino acid (aa) long, ca. 30 kDa protein first described in the pituitary gland (hypophysis). An amino acid sequence for human POMC is available as GenBank accession no. NP_001030333, the entire content of which is incorporated herein by reference. In the anterior pituitary, POMC is cleaved to yield ACTH and β-lipotropin. Further processing of ACTH in the pituitary middle lobe yields α-MSH (13 aa; also known as melanocortin; SYSMEHFRWGKPV (SEQ ID NO: I)). Other cleavage products derived from POMC in nonpituitary tissues include α-endorphin, β-endorphin, γ-endorphin, γ-lipotropin, β-MSH, γ-MSH, and corticotropin-like intermediate peptide (CLIP). Zhou A et al. (1999) J Biol Chem 274:20745-8. Melanocytes in epidermis express melanocortin 1 receptor (MClR) pigmentation and are one of the primary targets for α-MSH. At least a majority of α-MSH found in skin originates outside of the pituitary, because cutaneous α-MSH content showed little change following hypophysectomy in rats, and because in humans circulating levels of α-MSH are low. Schauer and co-workers then reported that human normal keratinocytes express POMC as well as α-MSH and ACTH. Schauer E et al. (1994) J Clin Invest 93:2258-62. It is now known that a variety of stimuli, including UV irradiation, stimulate epidermal keratinocytes to produce α-MSH, and that α-MSH, acting through MClR in a cAMP-dependent pathway, in turn stimulates melanocytes to produce melanin. Melanin released by melanocytes is taken up by keratinocytes, resulting in skin pigmentation.

It has now been discovered, according to the instant invention, that in addition to and separate from UV radiation, p53 directly induces keratinocytes to produce POMC and α-MSH, resulting in skin pigmentation.

As mentioned above, processing of POMC in the pituitary gland is reported to involve PCs. Two PCs expressed in the neuroendocrine system, including the pituitary, are prohomone convertase 1 (PCl, also known as PC3 or PC1/PC3) and prohormone convertase 2 (PC2). PCl belongs to a family of Ca²⁺-dependent, subtilisin-like serine endoproteases, the prohormone convertases (PCs), that are structurally related to the yeast prohormone processing enzyme kexin (Kex2). To date, seven mammalian members of this family have been identified and characterized, including the endoproteinases furin (subtilisin-like proprotein convertase 1; SPCl), PACE4 (SPC4), and the PCs PC2 (SPC2), PC 1 / PC3 (SPC3), PC4 (SPC5), PC5 / PC6 (SPC6), and PC7 / PC8 / LPC (SPC7).

Although some cell types express only one or the other of PCl and PC2, many neuroendocrine cells express both PCl and PC2. The substrate specificities of PCl and PC2 have been studied with purified enzymes, in cell culture, and in mice with disruption of the gene encoding PC2 (conventional PCl knockouts are lethal). Based on these studies it has been reported that PCl and PC2 are able to cleave many of the same substrates, although there are some sites cleaved preferentially by each. For example, PC2 has been reported to cleave Lys-Lys (KK) sites in POMC (necessary to produce α-MSH, CLIP, and β-endorphin), whereas PCl has negligible activity toward these sites in POMC but cleaves KK sites in proenkaphalin. Zhou A et al. (1993) J Biol Chem

268:1763-9; Breslin MB et al. (1993) J Biol Chem 268:27084-93; Zhou A et al. (1994) J Biol Chem 269:17440-7; Zhou A et al. (1999) J Biol Chem 274:20745-8. PCl is the predominant convertase in the anterior pituitary, where its action results in the release of intact ACTH and β-endorphin. In the pituitary middle lobe both PCl and PC2 are expressed, resulting in the further processing of ACTH to α-MSH.

The functions of mammalian PCl and PC2 have been further clarified through the generation, by homologous recombination, of mice deficient in one or the other of these enzymes. PC2-null animals have fasting hypoglycemia due to a lack of production of mature glucagon from the pancreatic α cell, and PCl -null mice, while sharing some elements of the phenotype reported in humans, uniquely have severely reduced linear growth attributed to failure of growth hormone releasing hormone production (Furuta et al., Proc. Natl. Acad. Sci. U.S.A. 94: 6646-6651, 1997; Zhu et al., Proc. Natl. Acad. Sci. U.S.A. 99: 10293-10298, 2002; O'Rahilly et al., N. Engl. J. Med. 23: 1386-1390, 1995). It is as yet unclear whether these enzymes have an identical range of actions in humans and to what extent dysfunction of these enzymes can contribute to human metabolic and endocrine disease. In addition, the absence of obesity in the PCl knock-out mouse is surprising, given the severe abnormalities of POMC processing and the importance of POMC- derived peptides in the control of energy balance, evidenced by the obesity of POMC- null mice. See Jackson et al., J. Clin. Invest. 112: 1550-1560, 2003; Jackson et al., Nature Genet. 16: 303-306, 1997. In human, PCl deficiency results in childhood obesity, hypogonadism, hypoadrenalism, postprandial hypoglycemia, hypocortisolemia, and evidence of impaired processing of ACTH and insulin precursors, and small intestinal dysfunction. The persistent production of hormones such as ACTH and GLP-I in the absence of any functional PCl suggests that other similar enzymes can partially compensate for lack of this convertase in humans (Jackson et al., J. Clin. Invest. 112(10): 1550-60, 2003).

Nucleotide and amino acid sequences of PCl and PC2 are known and are available, for example, as GenBank accession nos. NM_000439 (human PCl cDNA); NP_000430 (human PCl protein); NM_002594 (human PC2 cDNA); NP_002585 (human PC2 protein); NM_013628 (mouse PCl cDNA); NP_038656 (mouse PCl protein); NM_008792 (mouse PC2 cDNA); and NP_032818 (mouse PC2 protein). The entire content of each of these references is incorporated herein by reference. Purified recombinant forms of PCl and PC2 have been described. See, for example, Apletalina E et al. (1998) J Biol Chem 273:26589-95 and Johanning K et al. (1998) J Biol Chem 273:22672-80. Specific inhibitors of PCl and PC2 have been described. PCl is selectively inhibited by the 26 kDa neuroendocrine protein proSAAS and, more particularly, by fragments of proSAAS including the decapeptide VLGALLRVKR (Kj ~9 nM; SEQ ID NO: 2) and the hexapeptide LLRVKR within the C-terminal portion of pro SAAS. Zhou A et al. (1994) J Biol Chem 269:17440-7; Fricker LD et al. (2000) JNeurosci 20:639-48; Basak A et al. (2001) J Biol Chem 276:32720-8; Cameron A et al. (2000) FEBS Letts 473:135-8.

PC2 is selectively inhibited by the 21 kDa neuroendocrine protein 7B2 and, more particularly, by the 31-aa C-terminal peptide of 7B2. Lindberg I et al. (1995) Biochemistry 34:5486-93. More recently, Zhu et al. reported that the first 18 aa of the 31-aa C-terminal peptide of 7B2 is equally as effective an inhibitor of PC2 as the 31-aa C-terminal peptide, each with a Kj of -270 nM. Zhu X et al. (1996) Proc Natl Acad Sci USA 93:4919-24.

In addition to the foregoing inhibitors, in a study of a library of approximately 52 million hexapeptides Apletalina and co workers identified inhibitors of both PCl and PC2. Apletalina E et al. (1998) J Biol Chem 273:26589-95. Based on this study the most potent single dual inhibitor was identified as LLRVKR, with Kj values of 3.2 nM (for PCl) and 360 nM (for PC2). Additional examples of particularly potent hexapeptide inhibitors identified in this study include the following (Ibid):

Small molecule PCl / PC2 inhibitors, such as boronic acid or chloromethyl ketone derivatives of peptides that are otherwise substrates for PCl or PC2, may also be used in the instant invention. For example, the -COOH terminal of the tetrapeptide RVKR (or the other small peptides listed above) may be replaced with a functional group, such as a boronic acid or chloromethyl ketone that is able to form a covalent adduct with the active-site histidine or serine in the enzyme.

Merely to illustrate, the PC1/PC2 inhibitor can be represented in the general forumula:

wherein

Ri, R₂ and R₄ each independently represent -(CH₂)_n-NH-C(=NH)-NH₂ or -(CH₂)_n-NH₂;

R₃ represent a lower alkyl or a hydroxy 1 substituted lower alkyl;

Rp, independently for each occurrence, represents H or an amino-protecting group;

W represents a functional group which reacts with an active site residue of the targeted PCl or PC2 protease, as for example, -CN, -CH=NR₅,

- BS-X_{1 f} - BP-X_{1 f} -B O

R₅ represents H, an alkyl, an alkenyl, an alkynyl, -C(Xi)(X₂)X₃, -(CH₂)_m-R₆, -(CH₂VOH, -(CH₂)_n-O-alkyl, -(CH₂)_n-O-alkenyl, -(CH₂)_n-O-alkynyl, -(CH₂VO- (CH₂)_m-R₆, -(CHz)_n-SH, -(CH₂)_n-S-alkyl, -(CH₂)_n-S-alkenyl, -(CH₂)_n-S-alkynyl, - (CH₂)_n-S-(CH₂)_m-R₆, -C(O)C(O)NH₂, -C(O)C(O)OR₇;

Rg represents, for each occurrence, a substituted or unsubstituted aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle;

R₇ represents, for each occurrence, hydrogen, or a substituted or unsubstituted alkyl, alkenyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, or heterocycle; and

Y_] and Y₂ can independently or together be OH, or a group capable of being hydrolyzed in vivo to a hydroxyl group, including cyclic derivatives where Yi and Y₂ are connected via a ring having from 5 to 8 atoms in the ring structure (such as pinacol or the like),

R₅₀ represents O or S;

R₅] represents N₃, SH₂, NH₂, NO₂ or -0-R'₇; R₅₂ represents hydrogen, a lower alkyl, an amine, -0-R₇, or a pharmaceutically acceptable salt, or R₅₁ and R₅₂ taken together with the phosphorous atom to which they are attached complete a heterocyclic ring having from 5 to 8 atoms in the ring structure

Xj represents a halogen;

X2 and X3 each represent a hydrogen or a halogen;

m, independently for each occurrence, is zero or an integer in the range of 1 to 8; and

n, independently for each occurrence, is an integer in the range of 1 to 8.

In certain preferred embodiments, W represents

In certain preferred embodiments, R₅ is a hydrogen, or a halogentated lower alkyl.

In certain preferred embodiments, Xi is a fluorine, and X₂ and X₃, if halogens, are also fluorine.

These and other PCl and PC2 inhibitor compounds will find use in the practice of the instant invention.

The invention in one aspect is a method for reducing or preventing skin pigmentation. In one embodiment the skin pigmentation to be reduced or prevented according to the method of the invention can be a normal amount of pigmentation. For example, the method can be used if a subject desires to reduce or prevent pigmentation of at least a region of skin for cosmetic reasons. In one embodiment the subject may have a condition that results in hypopigmentation of one or more regions of the skin, or localized hypomelanosis, for example in vitiligo. Affected regions of skin have reduced or absent pigmentation, and these regions can be strikingly different from adjacent unaffected regions of skin with normal and full amounts of pigmentation. By reducing the degree of pigmentation or preventing the normal degree of pigmentation in unaffected skin, the contrast in pigmentation between affected skin and unaffected regions of skin can be reduced. In one aspect the invention provides a method for treating a condition associated with hyperpigmentation. As used herein, the terms "treat" and "treating" refer to reducing at least one sign or symptom of a disease or condition in a subject having or at risk of developing such disease or condition. In one embodiment the subject has the disease or condition. In one embodiment "treat" and "treating" refer to curing a disease or condition in a subject having such disease or condition. As used herein, the term "subject" refers to a living vertebrate. In one embodiment a subject is a mammal. In one embodiment a subject is a human.

Conditions associated with hyperpigmentation are conditions characterized at least in part by the presence of a greater-than-desired amount of endogenous skin pigmentation affecting at least a region of the skin of a subject. In one embodiment a condition associated with hyperpigmentation is a condition characterized at least in part by the presence of a greater-than-normal amount of endogenous skin pigmentation affecting at least a region of the skin of a subject. A greater-than-normal amount of endogenous skin pigmentation refers to an amount of pigmentation that is objectively greater than that amount of pigmentation present either (a) in another region of skin of the subject, including but not limited to an average amount of pigmentation of the skin of the subject, or (b) in the same region of skin of the subject at an earlier time, e.g., prior to development of the hyperpigmentation. In different embodiments the hyperpigmentation can accompany or be a manifestation of either a malignant or non-malignant (i.e., benign) condition. Endogenous skin pigmentation refers to skin pigmentation that is generated by cells in the skin, and it is to be distinguished, for example, from skin pigmentation arising from dye injected into the skin, e.g., tatooing, or other forms of exogenous skin pigmentation. Examples of conditions associated with hyperpigmentation, in addition to tanning, include acanthosis nigricans, Addison's disease, age spots (also variously known as age / liver spots, sun spots, solar lentigo, and solar and simple lentigines), basal cell carcinomas, biliary cirrhosis, cafe au lait spots (which may be associated with neurofibromatosis or Albright's syndrome), ectopic ACTH syndrome, eosinophilia- myalgia syndrome, ephelides (freckles), folate deficiency, hemochromatosis, junctional and compound nevi, lentigo, malabsorption, melanosis secondary to metastatic melanoma, Nelson's syndrome, pellagra, pigmented actinic keratosis, pigmented keratinocyte tumors, POEMS syndrome, porphyria cutanea tarda, post-inflammatory hyperpigmentation, scleroderma, seborrheic keratosis, vitamin B₁₂ deficiency, and Whipple's disease. Other treatable diseases, conditions, or disorders include those that can be characterized by discolorations of the skin or hair such as, for example, hyperpigmentation caused by inflammation or from diseases such as melasma/chloasma and postinflammatory hyperpigmentation. Certain drugs are associated with the development of hyperpigmentation. These drugs include 5-fluorouracil (5-FU), busulfan, cyclophosphamide, and ACTH. Each of these lists is not meant to be limiting. The subject compounds may ultimately reduce melanin levels in the skin by inhibiting POMC synthesis / processing, α-MSH production, and ultimately the production of melanin, whether the melanin is produced constitutively or in response to ultraviolet radiation, such as sun exposure. Thus, some of the active compounds in the present invention can be used to reduce skin melanin content in non-pathological states so as to induce a lighter skin tone, as desired by the user, or to prevent melanin accumulation in the skin that has been exposed to ultraviolet radiation. These compounds can also be used in combination with skin peeling agents, including glycolic acid or trichloroacetic acid face peels, to lighten skin tone and to prevent repigmentation.

In one embodiment the condition or hyperpigmentation is specifically associated with induction of melanin production. In one embodiment the condition associated with hyperpigmentation is age spots (also variously known as liver spots, sun spots, solar lentigo, and lentigines).

One of ordinary skill in the art will appreciate that the endpoint chosen in a particular case will vary according to the disease, condition, or disorder being treated, the outcome desired by the patient, subject, or treating physician, and other factors. Where the composition is being used to lighten skin color such as, for example, to reverse hyperpigmentation caused by, for example, inflammation or diseases such as melasma, or to lighten hair color, any one or a number of endpoints can be chosen. For example, endpoints can be defined subjectively such as, for example when the subject is simply "satisfied" with the results of the treatment. For pharmacological compositions, the endpoint can be determined by the patients or by the treating physician's satisfaction with the results of the treatment. Alternatively, endpoints can be defined objectively. For example, the patient's or subject's skin or hair in the treated area can be compared to a color chart. Treatment is terminated when the color of the skin or hair in the treated area is similar in appearance to a color on the chart. Alternatively, the reflectance of the treated skin or hair can be measured, and treatment can be terminated when the treated skin or hair attains a specified reflectance. In another method, the amount of melanin in the skin or hair can be measured.

A hyperpigmented region of skin can involve and refer to an area of skin from as small as about 1 mm² up to and including the entire surface of the skin. In certain common embodiments a hyperpigmented region of skin can involve and refer to an area of skin from about 1 cm to tens of cm . There can be a single hyperpigmented region or there can be more than one hyperpigmented region in a given subject. When there is more than one hyperpigmented region in a subject, the various hyperpigmented regions can be similar or dissimilar to one another in size, shape, and/or pigmentation.

The subject skin-lightening compounds / agents can be formulated alone or in combination with other agents. When provided in a topical formulation, agents can be co-formulated with emollients, emulsifiers, solvents, waxes, thickeners, film formers, humectants, preservatives, surfactants, perfumes, buffering agents, chelating agents, emulsion stabilizers, opacifying agents, pH adjusters, propellants, coloring agents, and the like. Such forms of the compositions can be formed into formulations, such as lotions, creams, gels, aerosols and sticks, in accordance with procedures well known in the art.

The compounds / agents of the present invention can be administered orally in solid or semi-solid dosage forms, such as hard or soft-gelatin capsules, tablets, or powders, or in liquid dosage forms, such as elixirs, syrups, or suspensions. The compounds can also be administered parenterally, in sterile liquid dosage forms. Since topical application is preferred, other dosage forms are possible including mousse or foams, patches, ointments, creams, gels, lotions, solutions, suppositories, or formulation for transdermal administration. Because in vivo use is contemplated, the composition is preferably of high purity and substantially free of potentially harmful contaminants, e.g., at least National Food grade, generally at least analytical grade, and preferably at least pharmaceutical grade. To the extent that a given compound must be synthesized prior to use, such synthesis or subsequent purification shall preferably result in a product that is substantially free of any potentially contaminating toxic agent that may have been used during the synthesis or purification process.

Thus the method according to this aspect of the invention includes the step of locally administering to pigmented skin an effective amount of an inhibitor of an enzyme involved in cleavage of POMC to α-MSH, to reduce pigmentation of the skin. In one embodiment the locally administering is topically administering.

Certain inhibitors of PCl and PC2, enzymes involved in cleavage of POMC to α-MSH, are described above. These include peptides that act as competitive inhibitors of the enzymatic action of either or both of PCl and PC2. Peptides and other inhibitors of enzymatic action of PCl and PC2 can be formulated for local administration to skin, for example either for local injection or for topical administration. The formulation optionally can include one or more agents useful for promoting uptake of active agent by keratinocytes.

In addition to peptide inhibitors of PCl and PC2, inhibitors of PCl in one embodiment can refer to agents that specifically interfere with the expression of functional PCl, and similarly inhibitors of PC2 in one embodiment can refer to agents that specifically interfere with the expression of functional PC2. Inhibitors of expression specifically include compositions useful for RNA interference (RNAi) affecting PCl and PC2. Beinfeld and coworkers, for example, recently reported successful in vitro inhibition of PC 1 expression in, and thereby reduction of cholecystokinin (CCK) secretion by, At-T20 cells, using two different methods of RNAi. Beinfeld MC et al. (2006) Peptides 27:905-10. Inhibitors of expression can alternatively or in addition include compositions useful for antisense inhibition of PCl and PC2. Nucleic acid molecules and other inhibitors of expression of PCl and PC2 can be formulated for local administration to skin, for example either for local injection or for topical administration. The formulation optionally can include one or more agents useful for promoting uptake of active agent by keratinocytes.

Additional agents useful for inhibiting PCl and/or PC2 enzyme expression and/or function also can be formulated for local administration to skin, for example either for local injection or for topical administration. The formulation optionally can include one or more agents useful for promoting uptake of active agent by keratinocytes. As noted above, the invention embraces antisense oligonucleotides that selectively bind to nucleic acid molecules encoding a PCl or PC2 prohormone convertase to decrease expression and activity of these proteins.

As used herein, the term "antisense oligonucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA. The antisense molecules are designed so as to interfere with transcription or translation of a target gene upon hybridization with the target gene or transcript.

Antisense oligonucleotides that selectively bind to a nucleic acid molecule encoding PCl or PC2 are particularly preferred. Those skilled in the art will recognize that the exact length of the antisense oligonucleotide and its degree of complementarity with its target will depend upon the specific target selected, including the sequence of the target and the particular bases which comprise that sequence.

It is preferred that the antisense oligonucleotide be constructed and arranged so as to bind selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions. Based upon the nucleotide sequences of nucleic acid molecules encoding PCl and PC2, (e.g., GenBank Accession Nos. listed above) or upon allelic or homologous genomic and/or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention. In order to be sufficiently selective and potent for inhibition, such antisense oligonucleotides should comprise at least about 10 and, more preferably, at least about 15 consecutive bases which are complementary to the target, although in certain cases modified oligonucleotides as short as 7 bases in length have been used successfully as antisense oligonucleotides. See Wagner RW (1995) Nat Med 1 :1116-18. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases. Although oligonucleotides may be chosen which are antisense to any region of the gene or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N-terminal or 5' upstream sites such as translation initiation, transcription initiation, or promoter sites. In addition, 3 '-untranslated regions may be targeted by antisense oligonucleotides. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs. In addition, the antisense is targeted, preferably, to sites in which mRNA secondary structure is not expected (see, e.g., Sainio K et al. (1994) Cell MoI Neurobiol 14:439-57) and at which proteins are not expected to bind.

In one set of embodiments, the antisense oligonucleotides of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These oligonucleotides may be prepared by art-recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance (or at least do not interfere with) their stability or targeting or which otherwise enhance (or at least do not interfere with) their therapeutic effectiveness.

The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acid molecules has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters, and peptides.

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxy 1 group at the 3' position and other than a phosphate group at the 5' position. Thus modified oligonucleotides may include a 2'-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose.

The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acid molecules encoding a prohomone convertase, together with pharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of a pharmaceutical composition. Such a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art. For use by injection, the compositions should be sterile and contain a therapeutically effective amount of the antisense oligonucleotides in a unit of weight or volume suitable for administration to a subject.

The methods of the invention also encompass use of isolated short RNA that directs the sequence-specific degradation of a target mRNA, e.g., a PCl or PC2 prohormone convertase mRNA, through a process known as RNA interference (RNAi). RNA interference is known to occur in a wide variety of organisms, including embryos of mammals and other vertebrates. It has been demonstrated that double-stranded RNA (dsRNA) is processed to RNA segments 21-23 nucleotides (nt) in length, and furthermore, that they mediate RNA interference in the absence of longer dsRNA. Thus, these 21-23 nt fragments are sequence-specific mediators of RNA degradation and are referred to herein as short interfering RNA (siRNA) or RNAi. Methods of the invention encompass the use of these fragments (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) to enable the targeting of PCl or PC2 mRNAs for degradation in mammalian cells useful in the therapeutic applications discussed herein.

Methods for design of the RNAs that mediate RNAi and methods for transfection of the RNAs into cells and animals are well known in the art, including some that are readily commercially available. See, for example, Verma NK et al (2004) J Clin Pharm Ther 28(5):395-404; Mello CC et al. (2004) Nature 431 :338-42; Dykxhoorn DM et al. (2003) Nat Rev MoI Cell Biol 4(6):457-67; Proligo (Hamburg, Germany); Dharmacon Research (Lafayette, CO, USA); Pierce Chemical (part of Perbio Science, Rockford, IL, USA); Glen Research (Sterling, VA, USA); ChemGenes (Ashland, MA, USA); and Cruachem (Glasgow, UK). The RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Most conveniently, siRNAs are obtained from commercial RNA oligonucleotide synthesis suppliers. In general, RNAs are not too difficult to synthesize and are readily provided in a quality suitable for RNAi. A typical 0.2 μmole-scale RNA synthesis provides about 1 milligram of RNA, which is sufficient for 1000 transfection experiments using a 24-well tissue culture plate format.

The prohormone convertase cDNA-specific siRNA is designed preferably by selecting a sequence that is not within 50-100 bp of the start codon and the termination codon, avoids intron regions, avoids stretches of 4 or more bases such as AAAA, CCCC, avoids regions with GC content <30% or >60%, avoids repeats and low complexity sequence, and avoids single nucleotide polymorphism sites. The prohormone convertase siRNA may be designed by a search for a 23-nt sequence motif AA(N 19). If no suitable sequence is found, then a 23-nt sequence motif NA(N21) may be used with conversion of the 3' end of the sense strand siRNA to dTdT. Alternatively, the prohormone convertase siRNA can be designed by a search for NAR(N 17) YNN. The target sequence may have a GC content of around 50%. The siRNA targeted sequence may be further evaluated using a BLAST homology search to avoid off-target effects on other genes or sequences. Negative controls can be designed by scrambling targeted siRNA sequences. The control RNA preferably has the same length and nucleotide composition as the siRNA but has at least 4-5 bases mismatched to the siRNA. The siRNA molecules can comprise a 3' hydroxyl group. The siRNA molecules can be single-stranded or double- stranded, wherein such double-stranded molecules can be blunt ended or comprise overhanging ends (e.g., 5' and/or 3') from about 1 to about 6 nucleotides in length (e.g., pyrimidine nucleotides, purine nucleotides). In order to further enhance the stability of the siRNA, the 3' overhangs can be stabilized against degradation. The RNA can be stabilized by including purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine-uridine dinucleotide 3' overhangs by 2'-deoxythymidine is tolerated and does not affect the efficiency of RNAi. The absence of a 2' hydroxyl significantly enhances the nuclease resistance of the overhang in tissue culture medium. The siRNA molecules used in the methods of the present invention can be obtained using a number of techniques known to those of skill in the art. For example, the siRNA can be chemically synthesized or recombinantly produced using methods known in the art. Such methods are described in U.S. Published Patent Application Nos. US2002-0086356A1 and US2003-0206884A1 that are incorporated herein by reference in their entirety.

The methods described herein are used to identify or obtain RNA molecules that are useful as sequence-specific mediators of PCl and PC2 prohormone convertase mRNA degradation and, thus, for inhibiting PCl and PC2 prohormone convertase activity. Expression of PCl and PC2 prohormone convertase can be inhibited in humans in order to prevent the protein from being translated and thus to inhibit the processing of POMC to yield α-MSH.

Any RNA can be used in the methods of the present invention, provided that it has sufficient homology to the target gene to mediate RNAi. The RNA for use in the present invention can correspond to the entire prohormone convertase gene or a portion thereof. There is no upper limit on the length of the RNA that can be used. For example, the RNA can range from about 21 base pairs (bp) of the gene to the full length of the gene or more. In one embodiment, the RNA used in the methods of the present invention is about 1000 bp in length. In another embodiment, the RNA is about 500 bp in length. In yet another embodiment, the RNA is about 22 bp in length. In certain embodiments the RNA is 21 to 23 nucleotides in length.

In the description that follows, the term "active agent" shall refer to an inhibitor of PCl or PC2 prohormone convertase activity and/or expression, as described above. Active agents can optionally be combined with one or more other therapeutic agents. The active agent and other therapeutic agent(s) may be administered simultaneously or sequentially. When the active agents and other therapeutic agent(s) are administered simultaneously, they can be administered in the same or separate formulations, but they are administered at the same time. The active agent and the other therapeutic agent(s) are administered sequentially when the administration of the active agent is temporally separated from the administration of the other therapeutic agent(s). The separation in time between the administration of these compounds may be a matter of minutes or it may be longer. Other therapeutic agents include but are not limited to anti-cancer therapy and tyrosinase inhibitors hydroquinone, kojic acid, kojic acid dipalmitate, arbutin, magnesium ascorbyl phosphate, and calcium D-pantetheine-S- sulfonate.

The active agents may be administered in conjunction with an anti-cancer therapy. Anti-cancer therapies include cancer medicaments, radiation and surgical procedures. As used herein, a "cancer medicament" refers to an agent which is administered to a subject for the purpose of treating a cancer. As used herein, "treating cancer" includes preventing the development of a cancer, reducing the symptoms of cancer, and/or inhibiting the growth of an established cancer. In other aspects, the cancer medicament is administered to a subject at risk of developing a cancer for the purpose of reducing the risk of developing the cancer. Various types of medicaments for the treatment of cancer are described herein. For the purpose of this specification, cancer medicaments are classified as chemotherapeutic agents, immunotherapeutic agents, cancer vaccines, hormone therapy, and biological response modifiers. The chemotherapeutic agent may be selected from the group consisting of methotrexate, vincristine, adriamycin, cisplatin, non-sugar containing chloroethylnitrosoureas, 5-fluorouracil (5-FU), mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin, carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyl transferase inhibitor, faresyl transferase inhibitor, MMP, MTA/L Y231514, L Y264618/Lometexol, Glamolec, CI-994, TNP-470, Hycarntin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone, Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340, AG3433, Incel/VX-710, VX-853, ZDOlOl, ISI641, ODN 698, TA 2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f, Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin, Metastron/strontium derivative,

Temodal/Temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine, Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid, SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609 (754)/RAS oncogene inhibitor, BMS-182751 /oral platinum, UFT(TegafurΛJracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer, Campto/Levamisole,

Camptosar/Irinotecan, Tumodex/Ralitrexed, Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/Fludarabine, Pharmarubicin/Epirubicin, DepoCyt, ZDl 839, LU 79553/Bis-Naphtalimide, LU 103793/Dolastain, Caelyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD 0473/Anormed, YM 116, Iodine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide, Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD 9331,

Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog, nitrosoureas, alkylating agents such as melphalan and cyclophosphamide, Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustine phosphate sodium, Etoposide (VP 16-213), Floxuridine, Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p'-DDD), Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG), Pentostatin (2'deoxycoformycin), Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate, but it is not so limited.

The immunotherapeutic agent may be selected from the group consisting of Ributaxin, Herceptin, Quadramet, Panorex, IDEC- Y2B8, BEC2, C225, Oncolym, SMART M195, ATRAGEN, Ovarex, Bexxar, LDP-03, ior t6, MDX-210, MDX-11, MDX-22, OV103, 3622W94, anti-VEGF, Zenapax, MDX-220, MDX-447, MELIMMUNE-2, MELIMMUNE-I, CEACIDE, Pretarget, NovoMAb-G2, TNT, Gliomab-H, GNI-250, EMD-72000, LymphoCide, CMA 676, Monopharm-C, 4B5, ior egf.r3, ior c5, BABS, anti-FLK-2, MDX-260, ANA Ab, SMART IDlO Ab, SMART ABL 364 Ab and ImmuRAIT-CEA, but it is not so limited.

The cancer vaccine may be selected from the group consisting of EGF, Anti- idiotypic cancer vaccines, Gp75 antigen, GMK melanoma vaccine, MGV ganglioside conjugate vaccine, Her2/neu, Ovarex, M-Vax, O-Vax, L-V ax, STn-KHL theratope, BLP25 (MUC-I), liposomal idiotypic vaccine, Melacine, peptide antigen vaccines, toxin/antigen vaccines, MVA-based vaccine, PACIS, BCG vacine, TA-HPV, TA-CIN, DISC-virus and ImmuCyst/TheraCys, but it is not so limited. The term effective amount refers to the amount necessary or sufficient to realize a desired biologic effect. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular active agent being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular active agent and/or other therapeutic agent(s) without necessitating undue experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day may be contemplated to achieve appropriate systemic levels of compounds. Appropriate system levels can be determined by, for example, measurement of the patient's peak or sustained plasma level of the drug. "Dose" and "dosage" are used interchangeably herein.

Generally, daily topical doses of active compounds will be from about 10 nanograms (ng)/cm² per day to 10 milligrams (mg)/cm² per day. It is expected that topical doses in the range of 500 ng/cm² to 5 mg/cm², in one or several administrations per day, will yield the desired results. Dosage may be adjusted appropriately to achieve desired drug levels, depending upon the mode of administration. For example, it is expected that dosing for local administration by direct injection would be from one order to several orders of magnitude lower per day than for topical administation. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate levels of active agent.

For any compound described herein the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for active agents which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan. The formulations of the invention are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

For use in therapy, an effective amount of the active agent can be administered to a subject by any mode that delivers the active agent to the desired site or surface. Administering the pharmaceutical composition of the present invention may be accomplished by any means known to the skilled artisan. Preferred routes of administration include but arc not limited to local injection and topical.

The compounds, when it is desirable to deliver them locally, may be formulated for parenteral administration by injection, e.g., by bolus injection. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

In addition to the formulations described previously, the compounds may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Particularly suited for topical administration are pharmaceutical compositions comprising the active agent formulated as granules, powders, emulsions, suspensions, creams, lotions, drops or other suitable preparations disclosed herein, in whose preparation excipients and additives and/or auxiliaries such as solubilizers are customarily used as described herein.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems.

For a brief review of methods for drug delivery, see Langer (1990) Science 249:1527-

1533, which is incorporated herein by reference.

The active agents and optionally other therapeutic(s) may be administered per se

(neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p- toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group. Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004- 0.02% w/v).

The pharmaceutical compositions of the invention contain an effective amount of active agent and optionally other therapeutic agent(s) included in a pharmaceutically- acceptable carrier. The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The therapeutic agent(s), including specifically but not limited to the active agent, may be provided in particles. Particles as used herein means nano or microparticles (or in some instances larger) which can consist in whole or in part of the active agent or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the active agent in a solution or in a semi-solid state. The particles may be of virtually any shape. Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate). The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug there from. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described herein. Several delivery systems, such as the ones described in detail below, may also be used to deliver the subject compounds / agents. It is well known that the skin is an effective barrier to penetration to many chemical agents. The epidermis of the skin has an exterior layer of dead cells called the stratum corneum which is tightly compacted and oily and which provides an effective barrier against gaseous, solid or liquid chemical agents, whether used alone or in water or oil solutions. If an agent penetrates the stratum corneum, it can readily pass through the basal layer of the epidermis and into the dermis. If the agent is harmful, e.g., a toxic chemical, penetration of the stratum corneum is an event to be prevented.

Although the effectiveness of the stratum corneum as a barrier provides great protection, it can also frustrate efforts to apply beneficial agents directly to local areas of the body. The inability of physiologically active agents to penetrate the stratum corneum has resulted in a great deal of research on penetration-enhancing agents for the skin. See for example U.S. Pat. Nos. 3,989,815; 3,989,816; 3,991,203; 4,122,170; 4,316,893; 4,405,616; 4,415,563; 4,423,040; 4,424,210; and 4,444,762.

That being said, various delivery systems suitable for use in the present invention are known to those of skill in the art and can be used to deliver effective amounts of the subject agents to decrease pigmentation. In general, any formulation that can penetrate the skin barrier (stratum corneum) so that the subject agent can contact keratinocytes and/or melanocytes in the skin is preferred. For example, encapsulation in liposomes, or microcapsules are examples of delivery systems that can be used. In addition, the compositions of the invention may be formulated in various solvents, gels, creams, lotions or solutions to facilitate simple application to the skin and/or hair follicles. Aerosolized compositions, comprising a suspension of very fine particles of a solid or droplets of a liquid in a gaseous medium, may also be utilized to deliver effective amounts of the subject agent. The suspension is stored under high pressure and released in the form of a fine spray or foam and can be applied directly to the skin or hair.

Liposomes

In certain embodiments, a liposome preparation can be used. The liposome preparation can be comprised of any liposome which penetrates the stratum corneum and fuses with the cell membrane of keratinocytes / melanocytes, resulting in delivery of the contents of the liposome into the cell. Liposomes can be prepared by methods well- known to those of skill in the art. For example, liposomes such as those described in U.S. Pat. No. 5,077,211; U.S. Pat. No. 4,621,103; U.S. Pat. No. 4,880,635 or U.S. Pat. No. 5,147,652 can be used. See also Yarosh, D., et al., J. Invest. Dermatol., 103(4): 461-468 (1994) or Caplen, N. J., et al. Nature Med, 1(1): 39-46 (1995).

The liposomes can specifically target the appropriate cells (e.g., epidermal keratinocytes / melanocytes). In a preferred embodiment of the invention, the liposomal composition is applied directly to the skin or hair of a mammal, in the area where decreased pigmentation is desired.

Lotions and creams according to the present invention generally comprise a solution carrier system and one or more emollients. Lotions typically comprise from about 1% to about 20%, preferably from about 5% to about 20%, of emollient; from about 50% to about 90%, preferably from about 60% to about 80%, water; and a pharmaceutically effective amount of an agent described herein.

Liposomes (lipid vesicles) may also prove useful as a solvent for the subject agent, or as a means of encapsulating the subject agent, or as a means of complexing with the subject agents. Liposomes are aqueous compartments enclosed by a lipid bilayer. They are produced by techniques well known to those skilled in the art. For example, liposomes can be produced by suspending a suitable lipid, such as phosphatidyl choline, in an aqueous medium. This mixture is then sonicated to give a dispersion of closed vesicles that are quite uniform in size. Among the useful liposomes are stratum corneum lipid liposomes formed from epidermal ceramides, cholesterol, palmitic acid and cholesterol sulfate, such as described in Abraham et al, 1999. Journal Invest Derma, 259-262.

Many lipids are believed suitable for use in making the liposomes, many of which are commercially available, e.g. Liposome Kit is available from Sigma Chemical Company, St. Louis, Missouri under catalog number L-4262. Liposome Kit L-4262 contains Lalpha-phosphatidylcholine (egg yolk), dicetyl phosphate and cholesterol. It is a negatively charged lipsome mixture, another suitable negatively charged liposome mixture available from Sigma Chemcial Company is L-4012 which contains L- alphaphosphatidylcho line, dicetyl phosphate and cholesterol. Suitable positively charged liposome mixtures available from Sigma Chemical Company contains L-alpha- phosphatidylcholine, stearylamine and cholesterol (catalog numbers L-4137 and L- 3887). Categories of lipids in suitable liposomes are phospholipids, glycosphingolipids, ceramides, cholesterol sulfate and neutral lipids. Various combinations of these lipids are found in neonatal mouse, pig and human stratum granulosurn and stratum corneum. Other categories of lipids which can be used to make the liposomes are straight chain fatty acids, glycerol esters, glycerides, phosphoglycerides, sphingolipids, waxes, terpenes and steroids. Specific preferred lipids suitable for use are phosphatidyl choline, dicetyl phosphate and cholesterol.

The liposomes may simply be used as the solvent for the subject agents ~ i.e., after the liposomes are produced and isolated the subject agents are added to the liposomes.

The subject agents may also be encapsulated in (or trapped in) the compartment portion of the liposome. This can be done by adding an aqueous solution of the subject agents to a suitable lipid and mixing {e.g., sonicating) to produce the liposomes containing the subject agents. To make the aqueous solution of the subject agents, it may be desirable, as discussed above, to add additional water soluble components {e.g. alcohols, acetone, and the like) to increase the solubility of the subject agents in the aqueous solution or to help maintain the subject agents in the aqueous solution. The subject agents may also be added directly to a suitable lipid and mixed therewith so that there is a blend of the subject agents and lipid. Then when an aqueous solution is added to this blend and sonicated to produce the liposomes, the subject agents will be in the lipid layer of the liposome and not the compartment of the liposome.

The liposome (as solvent) and the subject agent composition or the liposomes (MC Activator in compartment or lipid layer) can then be combined with a suitable topical vehicle, e.g. a lotion, gel or cream vehicle. The lipid mixture which forms the liposome can be any of the conventional mixtures available or discussed in the literature which are pharmaceutically and cosmetically acceptable.

Preferred lipid mixtures contain a phosphatidyl choline, dicetyl phosphate and cholesterol. The lipid mixtures which form the liposomes are commercially available in a solvent such as ethanol or chloroform. A typical mixture contains on a weight basis, seven parts phosphatidylcholine, 2 parts dicetyl phosphate and one part cholesterol. Although topical or oral delivery would seem the most practical, for some human subjects who are extremely light sensitive due to treatment with various prescription medicines (e.g. tetracycline) or who are afflicted with certain medical conditions (e.g. burn patients) or genetic disorders (e.g. xeroderma pigmentosum), it is conceivably advantageous to deliver the composition systemically by means of intravenous, subcutaneous or intramuscular routes.

Organogels

In one embodiment, the subject agent is a composition for diffusional transdermal delivery of medication to a patient, which comprises the subject agent that it may be applied topically and conform to and adhere to the patient's skin for a period of time sufficient for a significant portion of the medication to be delivered transdermally to the patient. The basic composition of this embodiment is a mixture of an organogel, a solubilized the subject agent and a carrier combined with a drug release agent. Penetration enhancement is provided by the organogel and by the release agent. In the exemplary process, an organogel can be formed from lecithin and isopropyl palmitate. These two materials are thoroughly blended and mixed until a substantially uniform gel structure forms. The organogel, which is the base for the cream composition, can be formed at the time that the composition is to be formulated. The drug or medication is solubilized with a solvent, such as water, alcohol or other appropriate solvent, again by mixing in a known manner. When it is desired to start formation of the actual composition, the solubilized agent is mixed thoroughly into the organogel matrix, again by conventional mixing techniques. The technique used will of course be such that the organogels structure is not broken down. Finally, a carrier, such as water or alcohol, and a drug release agent, such as a polyoxymer, are blended. The carrier/release agent mixture can be made up in large lots and stored under refrigerator until needed, at which time an appropriate quantity can be taken for and the remainder retained in refrigerated storage. The carrier/release agent mixture is then mixed with the drug/organogel mixture to produce the final "cream" composition.

Considering first the organogel, the blend of the two components will typically be in the range of from about 25% to 75% (by weight) of the lecithin component, the remainder being the fatty acid ester component. The "lecithin component" may be lecithin, any comparable fatty acid phospholipid emulsifying agent, such as fatty acids and their esters, cholesterol, tri-glycerides, gelatin, acacia, soybean oil, rapeseed oil, cottonseed oil, waxes or egg yolk, or any other material which acts in the same manner as lecithin.

The other component is an organic solvent/emollient, particularly including fatty acid esters, of which the esters of the saturated alkyl acids are preferred. A preferred solvent/emollient is isopropyl palmitate or isopropyl myristate. However, there are numerous compounds available which exist in liquid form at ambient temperatures and will function in a manner equivalent to the fatty acid esters. These are all quite well known and include, but are not limited to, the following:

Ethanol

Propylene glycol

Water

Sodium oleate Leucinic acid

Oleic acid

Capric acid

Sodium caprate

Laurie acid Sodium laurate

Neodecanoic acid

Dodecylamine

Cetyl lactate

Myristyl lactate Lauryl lactate

Methyl laurate

Phenyl ethanol

Hexamthhylene lauramide

Urea and derivatives Dodecyl n,n-dimethylamino acetate

Hydroxyethyl lactamide

Phyophatidylcholine

Sefsol-318 (a medium chain glyceride) Isopropyl myristate

Isopropyl palmitate

Surfactants (including): polyoxyethylene (10) lauryl ether diethyleneglycol lauryl ether Laurocapram (azone)

Acetonitrile

1-decanol

2-pyrrolidone

N-methylpyrrolidone N-ethyl-1-pyrrolidone

1 -methyl-2-pyrrolidone

1 -lauryl-2-pyrrolidone

Sucrose monooleate

Dimethylsulfoxide Decylmethylsulfoxide

Acetone

Polyethylene glycol (100-400 mw)

Dimethylacetamide

Dimethylformamide Dimethylisosorbide

Sodium bicarbonate

Various C₇ to C₁₆ alkanes

Mentane Menthone Menthol Terpinene D-terpinene Dipentene N-nonalol Limonene Ethoxy diglycol

This combination of the phospholipid emulsifying agent and the fatty acid or fatty acid ester or equivalent thereof forms an organogel. For the example, the organogel can be a lecithin organogel, which is both isotropic and thermally reversible. At temperatures greater than about 4O⁰C the organogel will become a liquid and its viscosity will be greatly reduced. Water can be also be added to control the viscosity of the organogel. The organogel serves as one of the penetration enhancers in the cream, and acts on the stratum corneum of the skin to promote interaction between the phospholipids of the cream and the phospholipids of the skin. This causes a disruption in the normal regular arrangement of layers in lipids in the stratum corneum so that openings are created which then allow the drug to pass more easily through the skin. The organogel will be compatible with a wide variety of lipophilic, hydrophilic and amphoteric drugs and medications.

Using the above-described lecithin organogel and its components as an example, the properties needed for inclusion of a subject agent will be evident to those skilled in the art. The various compounds, polymers, etc. comprising the organogel, the solubilized drug and the carrier/polyoxymer components must all be compatible with each other, so that chemical reactions do not occur which would adversely affect the efficacy or safety of the cream composition; they must be mutually soluble so that they can be mixed and blended to a uniform consistency; they must be such that the resulting cream composition has a viscosity under ambient conditions which is low enough to allow it to be applied easily and smoothly to the skin, but not so low that the cream acts as at least in part like a liquid and cannot be retained on the skin where it is applied; they must not be toxic, irritating or otherwise harmful to the patient; they must be sufficiently stable that the overall composition will have a reasonable shelf life and service life; and, as a practical matter, they must be available at reasonable cost. The subject agent to be administered may need to be solubilized in a solvent to enable it be blended properly with the organogel and the carrier/release agent. Typical solvents for such use include water, the low molecular weight alcohols and other low molecular weight organic solvents. Solvents such as water, methanol, ethanol and the like are preferred. The purpose of solubilizing is to enable the subject agent to become properly dispersed in the final cream. It is possible that a few drugs or medications might themselves be sufficiently soluble in the cream that a solvent, and therefore a separate solubilizing step, would not be needed. For the purpose of this description, therefore, the term "solubilized" drug or medication shall be considered to include those drugs or medications which can be dispersed or dissolved into the cream with or without the presence of a separate solvent. Usually the amount each of medication and solvent which will be present, based on the entire composition, will be in the range of up to <1% to 20%, with the preferred concentration of each being about 10%. The concentrations of both need not be identical.

Humectants The compositions of this invention may also be formed by combining the subject agent with effective amounts of water and a humectant. These compositions are predominantly water with enough humectant added to form a cosolvent mixture that will dissolve the subject agent.

The humectant will generally be present in amounts of about 1 to about 7% by weight of the total composition with about 4 to about 5% being preferred. The balance of the composition is water such that the total amount of ingredients (water, humectant, and the subject agent equals 100% by weight. Thus, such compositions may contain water in amounts of about 91 to about 98.95% by weight of the total compositions- with about 91 to about 98.9% being suitable. Humectants well known in the art may be used. Examples of humectants include propylene glycol, sorbitol, and glycerin. Other suitable humectants may include fructose, glucose, glutamic acid, honey, maltitol, methyl gluceth-10, methyl gluceth-20, sodium lactate, sucrose, and the like.

Non-ionic surfactants

Moreover, the inclusion of the non-ionic surfactant in the composition of this J invention produces a more uniform skin tan rather than spotty tans produced by using tanning compositions which do not contain such non-ionic surfactants.

The non-ionic surfactant which is particularly well suited in the practice of this invention is polyoxyethylene 4 lauryl ether which is available from ICI Americas, Inc., Wilmington, Delaware, and is sold under the trade name BRIJ 30. This surfactant is also0 referred to as laureth-4, which is its CTFA (Cosmetic Toiletry and Frangrance Association) adopted name. Other non-ionic surfactants of this type which can be used in this invention include polyoxyethylene 4 lauryl ether containing 0.01% butylated hydroxy anisole (BHA) and 0.005% citric acid as preservatives. This surfactant is also available from ICI Americas, Inc. and is also known by its CTFA adopted name of5 Laureth-4 and sold under the trade name BRIJ 30 SP. Still other non-ionic surfactants which are suitable in the compositions of this invention are: polyoxyethylene 23 lauryl ether, known by its CTFA adopted name of Laureth-23 (trade name BRIJ 35); polyoxyethylene 23 lauryl ether containing 0.01% BHA and 0.005% citric acid, known by its CTFA adopted name of Laureth-23 (trade name BRIJ 35 SP); polyoxyethylene 20 cetyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA adopted name of Ceteth-2 (trade name BRIJ 52); polyoxyethylene 10 cetyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA and adopted name of Ceteth-10 (trade name BRIJ 56); polyoxyethylene 20 cetyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA adopted name of Ceteth-20 (trade name BRIJ 58); polyoxyethylene5 2 stearyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA name of Steareth-2 (trade name BRIJ 72); polyoxyethylene 10 stearyl ether with 0.001% BHA and 0.005% citric acid, known by its CTFA name of Steareth-10 (trade name BRIJ 76); polyoxyethylene-2 oleyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA name of Oleth-2 (trade name BRIJ 92); polyoxyethylene-2 oleyl ether (low color0 and odor) with 0.01% BHA and 0.005% citric acid, known by its CTFA name of Oleth-2 (trade name BRIJ 93); polyoxyethylene 10 oleyl ether with 0.01% BHA and 0.005% citric acid, known by its CTFA name of Oleth-10 (trade name BRIJ 96) and polyoxyethylene 10 Oleth ether (low color and odor) with 0.01% BHA and 0.005% citric acid, known by its CTFA name of Oleth- 10 (trade name BRIJ 97).

The aforementioned non-ionic surfactants may be generally referred to as polyoxyethylene alkyl ethers and may be used alone or in admixture with one another. Another type of non-ionic surfactants which may be used in the present invention is polyoxyethylene 20 sorbitan monolaurate, known by its CTFA name of Polysorbate- 20 (trade name TWEEN 20) and polyoxyethylene 4 sorbitan monolaurate, known by its CTFA name of Polysorbitan-21 (trade name TWEEN 21), and other such polyoxyethylene derivatives of sorbitan fatty acid esters. Other types of non-ionic surfactants which may be used in the composition of this invention are sorbitan fatty acid esters which include sorbitan monolaurate, known by its CTFA adopted name of Sorbitan Laurate (trade name ARLACEL 20); sorbitan monopalmitate, known by its CTFA adopted name of Sorbitan Palmitate (trade name ARLACEL 40); sorbitan monostearate, known by its CTFA adopted name of Sorbitan Stearate (trade name ARLACEL 60); sorbitan monooleate, known by its CTFA adopted name of Sorbitan Oleate (trade name ARLACEL 80); sorbitan sesquioleate, known by its CTFA adopted name of Sorbitan Sesquioleate (available under the trade names ARLACEL 83 and ARLACEL C); sorbitan trioleate, known by its CTFA adopted name of Sorbitan Trioleate (trade name ARLACEL 85); glycerol monstearate and polyoxyethylene stearate, known by its CTFA adopted name of Glycerl Stearate and PEG-100 Stearate (trade name ARLACEL 165); and glycerol monoleate diluted with propylene glycol and containing 0.02% BHA and 0.01% citric acid added as preservatives, known by its CTFA adopted name of Glycerl Oleate and Propylene Glycol (trade name ARLACEL 186).

Bioadhesives

Absorption of the subject agents and contact with melanocytes may be further improved by the use of bioadhesive polymers. In some embodiments, bioadhesive polymers may be included in the formulations of the invention to improve transport and retention of drug microparticles and nanoparticles. In general terms, adhesion of polymers to epithelial tissues may be achieved by (i) physical or mechanical bonds, (ii) primary or covalent chemical bonds, and/or (iii) secondary chemical bonds (e.g., ionic). Secondary chemical bonds, contributing to bioadhesive properties, include dispersive interactions {e.g., Van der Waals interactions) and stronger specific interactions, which include hydrogen bonds. The hydrophilic functional groups responsible for forming hydrogen bonds are the hydroxyl (-OH) and the carboxylic acid groups (-C00H).

As used herein "bioadhesion" generally refers to the ability of a material to adhere to a biological surface, such as skin or hair, for an extended period of time. Bioadhesion requires contact between a bioadhesive material and a surface {e.g., tissue and/or cells). Thus the amount of bioadhesive force is affected by both the nature of the bioadhesive material, such as a polymer, and the nature of the surrounding medium. Suitable polymers include polylactic acid (2 kDa MW, types SE and HM), polystyrene, poly(bis carboxy phenoxy propane-co-sebacic anhydride) (20:80) (poly (CCP:SA)), alginate (freshly prepared); and poly(fumaric anhydride-co-sebacic anhydride (20:80) (p(FA:SA)), types A (containing sudan red dye) and B (undyed). Other high-adhesion polymers include p(FA:SA) (50:50) and non-water-soluble polyacrylates and polyacrylamides. Suitable polymers that are bioadhesive include soluble and insoluble, nonbiodegradable and biodegradable polymers. These can be hydrogels or thermoplastics, homopolymers, copolymers or blends, natural or synthetic.

Two classes of polymers that may be useful bioadhesive properties are hydrophilic polymers and hydrogels. In the large class of hydrophilic polymers, those containing carboxylic groups {e.g., poly(acrylic acid)) exhibit the best bioadhesive properties, and therefore polymers with the highest concentrations of carboxylic groups should be the materials of choice for bioadhesion on soft tissues. Among polymers known to provide good results are sodium alginate, carboxymethylcellulose, hydroxymethylcellulose and methylcellulose. Some of these materials are water-soluble, while others are hydrogels.

Rapidly bioerodible polymers such as poly(lactide-co-glycolide), polyanhydrides, and polyorthoesters, having carboxylic groups exposed on the external surface as their smooth surface as they erode, are also excellent bioadhesive polymers.

Representative natural polymers include proteins, such as zein, modified zein, casein, gelatin, gluten, serum albumin, or collagen, and polysaccharides, such as cellulose, dextrans, polyhyaluronic acid, polymers of acrylic and methacrylic esters and alginic acid. Representative synthetic polymers include polyphosphazines, poly(vinyl alcohols), polyamides, polycarbonates, polyalkylenes, polyacrylamides, polyalkylene glycols (e.g., polyethylene glycol (PEG)), polyalkylene oxides, polyalkylene terephthalates, polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyvinylpyrrolidone (PVP), polyglycolides, polysiloxanes, polyurethanes and copolymers thereof. Representative synthetically modified natural polymers include alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, and nitrocelluloses.

Specific polymers include, but are not limited to, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose (HPMC), hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxymethyl cellulose, cellulose triacetate, cellulose sulfate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), polyφutyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly (ethylene terephthalate), polyvinyl acetate), polyvinyl chloride, polystyrene, polyvinyl pyrrolidone, polyvinylphenol, poly(butic acid), poly(valeric acid), poly(lactide-co- caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, blends and copolymers thereof. These polymers can be obtained from sources such as Sigma Chemical Co., St.

Louis, MO., Polysciences, Warrenton, PA, Aldrich, Milwaukee, WI, Fluka, Ronkonkoma, NY, and BioRad, Richmond, CA or synthesized from monomers obtained from these suppliers using standard techniques.

Polyanhydrides are an example of a mucoadhesive polymer. Suitable polyanhydrides include polyadipic anhydride, polyfumaric anhydride, polysebacic anhydride, polymaleic anhydride, polymalic anhydride, polyphthalic anhydride, polyisophthalic anhydride, polyaspartic anhydride, polyterephthalic anhydride, polyisophthalic anhydride, poly carboxyphenoxypropane anhydride and copolymers with other polyanhydrides at different mole ratios.

Chitosan

Microencapsulation can be particularly useful to deliver the subject agents that might otherwise cause local irritation. Various commercial microcapsules and nanocapsules are available which differ in the type of polymers used to make the capsule wall such as Hallcrest Microcapsules (gelatin, gum arabic), Coletica Thalaspheres (collagen), Lipotec Millicapsules (alginic acid, agar), Induchem Unispheres (lactose, microcrystalline cellulose, hydroxypropyl-methylcellulose), Kobo Glycospheres 5 (modified starch, fatty acid esters, phospholipids) and Softspheres (modified agar).

Chitosan is a natural, biodegradable cationic polysaccharide that can be used for topical formulation of the subject agents. It is derived by deacetylating chitin, a natural material extracted from fungi, the exoskeletons of shellfish and from algae and has previously been described as a promoter of wound healing [Balassa, U.S. Pat. No.

W 3,632,754 (1972); Balassa, U.S. Pat. No. 3,911,116 (1975)]. Chitosan comprises a family of polymers with a high percentage of glucosamine (normally 70-99%) and N-acetylated glucosamine (1-30%) forming a linear saccharide chain of molecular weight from 10,000 up to about 1,000,000 Dalton. Chitosan, through its cationic glucosamine groups, interacts with anionic proteins such as keratin in the skin conferring some bioadhesive

/5 characteristics. In addition, when not deacetylated, the acetamino groups of chitosan are an interesting target for hydrophobic interactions and contribute to some degree to its bioadhesive characteristics [(Muzzarelli et al., In: Chitin and Chitinases Jolles P and Muzzarelli RAA (eds), Birkhauser Verlag Publ., Basel, Switzerland, pp.251-264 (1999)].

In certain embodiments, a high viscosity chitosan is first mixed in the presence of

20 the subject agents dispersed in a suitable solvent to form a matrix, this matrix can then be precipitated under vigorous stirring conditions in the presence of anionic polymers and at higher pH values to form nano and micron size particles that can penetrate the stratum corneum or outer skin layer. This preparation of chitosan-based particles avoids the use of surfactants or emulsifiers which can cause skin irritation or other adverse reactions. 5 These chitosan formulations can provide such advantages as preferable tissue distribution of the drug, prolonged half life, controlled drug release and reduction of drug toxicity. In certain preferred embodiments, chitosan particles can be used for the topical delivery of water insoluble subject agents, where the sustained release of the drug is obtained by precipitating the chitosan/active agent matrix in the presence of anionic 0 polymers at pH conditions greater than 6.0 under vigorous stirring conditions. In addition, the chitosan microparticles disclosed in the present invention are able to act as delivery vehicles without leaving polymeric residues on the skin. The absence of residues may be due to the bioadhesiveness of chitosan to the skin surface as mentioned earlier which allows for greater penetration into the stratum corneum or the outer layer of the skin.

The term "high viscosity" chitosan refers to a chitosan biopolymer having an apparent viscosity of at least about 100 cps for 1% solutions in 1% acetic acid as measured using a Brookfield LVT viscometer at 25⁰C with appropriate spindle at 30 rpm. The viscosity of the chitosan solution can readily be determined by one of ordinary skill in the art, e.g., by the methods described in Li et al., Rheological Properties of aqueous suspensions of chitin crystallites. J Colloid Interface Sc 183:365-373, 1996. In addition, viscosity can be estimated according to Philipof s equation: V=(I +KC)⁸, where V is the viscosity in cps, K is a constant, C is the concentration expressed as a fraction (Form No. 198-1029-997GW, Dow Chemical Company). In certain embodiments, the high viscosity chitosan preferably has a viscosity greater than at least 100 cps, and more preferably greater than at least 500 cps. The term "dispersing agent" as used herein comprises any suitable solvent that will solubilize or suspend the water insoluble or slightly water soluble active agent but does not chemically react with either chitosan or the active substance. Examples include soybean oil, dibutyl hexanedioate, cocoglycerides, aliphatic or aromatic esters having 2- 30 carbon atoms (e.g. cococaprylate/caprate), coconut oil, olive oil, safflower oil, cotton seed oil, alkyl, aryl, or cyclic ethers having 2-30 carbon atoms, cycloaliphatic or aromatic hydrocarbons having 4-30 carbon atoms, alkyl or aryl halides having 1-30 carbon atoms.

The term "anionic polymer" refers to negatively charged polymers which can form a complex with chitosan such as poly(acrylic acid) and derivatives, xanthan gum, sodium alginate, gum arabic, carboxy methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, carrageenan, polyvinyl alcohol, sulfated glycosaminoglycans such as chondroitin sulfate and dermatan sulfate.

Other Ingredients

The subject agents can be formulated with sunscreening agents, such as UVA type, UVB type, or a combination of both. Generally, the sunscreening agents are used in amounts effective to provide the desired level of protection against UVA and/or UVB radiation. The sunscreening agents are used in amounts of, for example, about 2% to about 20% by weight of the total composition. Typical UVB type sunscreening agents include substituted para-aminobenzoates, alkyl esters of paramethoxycinnamate and certain esters of salicylic acid.

Typical UVA type sunscreening agents include certain benzophenones and dibenzoyl methanes.

Representative UVB type sunscreening agents include but are not limited to: (A) IDEA Methoxyinnamate (diethanolamine salt of p-methoxy hydro cinnamate), e.g., trade name BERNEL HYDRO from Bernel Chemical Co., Inc.; (B)Ethyl Dihydroxypropyl PABA (ethyl dihydroxypropyl p-aminobenzoate), e. g., trade name AMERSCREEN P from Amerchol Corp.; (C)Glyceryl PABA (glyceryl-p-aminobenzoate), e.g., trade name NIPA G.M.P.A. from NIPA Laboratories, Inc.; (D)Homosalate (Homomenthyl salicylate), e.g., trade name KEMESTER HMS from Humko Chemical; (E)Octocrylene, (2-ethylhexyl- 2-cyano-3,3diphenylacrylate), e.g., trade name UVINUL N-539 from BASF Chemical Co.; (F)Octyl Dimethyl PABA (Octyl dimethyl paminobenzoate, 2- ethylhexyl pdimethylaminobenzoate, Padimate 0), e.g., trade names AMERSCOL, ARLATONE UVB, and ESCALOL 507 from Amerchol Corp., ICI Americas, Inc., and Van Dyk, respectively; (G)Octyl Methoxycinnamate (2-ethylhexyl- pmethoxycinnamate), e.g., trade name PARSOL MCX from Bernel Chemical Co. Inc., or Givaudan Corp.; (H) Octyl Salicylate (2-ethylhexy salicylate), e. g., trade name SUNAROME WMO from Felton Worldwide, Inc.; (I)PABA (P-amino benzoic acid), e.g., trade name PABA from EM Industries, Inc. and National Starch & Chemical Corp., or trade name NIPA PABA from NIPA Laboratories Inc.; (J)2-Phenyl-benzimidazole-5- Sulphonic acid (Novantisol), e.g., trade name EUSOLEX 232 and NEO-HELIOP AN HYDRO from EM Industries, Inc. and Haarmann & Reimer Corp., respectively; (K)TEA Salicylate (triethanolamine salicylate), e.g., trade names SUNAROME W and SUNAROME G from Felton Worldwide, Inc.; (L)3-(4-methylbenzlidene)camphor or 3- (4methylbenzylidene)boran-2-one, e.g., trade name EUSOLEX 6300 from EM Industries, Inc.; and (M) Etocrylene (2-ethyl-2-cyano-3,3'di phenylacry late), e.g., trade name UVINUL N-35 from BASF Chemical Co. Representative UVA type sunscreening agents include but are not limited to: - (A)Benzophenone-3 (2-hydroxy-4- methoxybenzophenone), e.g., trade name SPECTRA-SORB UV-9 and UVINUL M-40 from American Cyanamid Co. and BASF Chemical Co., respectively; (B)Benzophenone-4 (sulisobenzone), e.g., trade name UVINUL MS-40 from BASF Chemical Co.; (C) Benzophenone-8 (dioxybenzone), e.g., trade name SPECTRA-SORB UV-24 from American Cyanamid Co.; (D)Menthyl Anthranilate (Menthyl-2- aminobenzoate), e.g., trade name SUNAROME UVA from Felton Worldwide, Inc.; (E)Benzophenone-l (2,4- dihydroxybenzophenone), e.g., trade name UVINUL 400 and UVASORB 2 OH from BASF Chemical Co. and TRI-K Industries, Inc., respectively; 4(F) Benzophenone-2 (2,2',4,4'-tetrahydroxy-benzohpenone), e.g., trade name UVINUL D-50 from BASF Chemical Co.; (G) Benzophenone-6 (2,2'-dihydroxy-4,4'dimethoxy- benz.ophenone), e.g., trade name UVINUL D-49 from BASF Chemical Co.; (H)Benzophenone-12 (octabenzone), e.g., trade name UVINOL 408 from BASF Chemical Co.; (1)4- isopropyl dibenzoyl methane (l-p-cumenyl3-phen.yipropane-l,3- dione), e.g. trade name EUSOLEX 8020 from EM Industries, Inc.; and (J)Butyl methyl dibenzoyl methane (4-t-butyl-4'methoxydibenzoyl methane), e.g. trade name PARSOL 1789 from Givaudan Corporation; Physical sunscreening agents may also be used. For example, red petrolatum in amounts of about 30 to about 99% by weight of the total co mposition, or titanium dioxide in amounts of about 2 to about 25% by weight of the total composition may be used. Talc, kaolin, chalk, and precipitated silica may also be used in effective amounts, e.g., about 1% to about 10% by weight of the total composition.

Additional sunscreening agents include lawsone (hydroxynaphthoquinone, ClOl- 1603, the coloring matter of henna leaves) with dihydroxy acetone.

Usually, when used, at least one UVB type and at least one UVA type sunscreening agent is used.

For example, at least one of the following UVB type sunscreening agents can be used: from about 1.5 to about 8.0% by weight of the total composition of octyl dimethyl PABA; octyl para-methoxycinnamate in amounts of about 1.5 to about 7.5% by weight of the total composition; homomenthyl salicylate in amounts of about 4.0 to about 15% by weight of the total composition; and octyl salicylate in amounts of about 3 to about 5% by weight of the total composition.

Also, for example, at least one of the following UVA type sunscreening agents can be used: benzophenone-3 in amounts of about 0.5 to about 6% by weight of the total composition; benzophenone-8 in amounts of about 0.5 to about 3% by weight of the total composition; and menthyl anthranilate in amounts of about 3.5 to about 5.0% by weight of the total composition. Using the ingredients disclosed above (e.g., emollients, emulsifiers, film formers, and the like), the riboflavin, riboflavin phosphate or mixtures thereof can be incorporated into formulations such as lotions, creams, gels mousses, waxed based sticks, aerosols, alcohol sticks and the like. These formulations are well known in the art, for example see Balsam, M.S., and Sagrin, E. (Editors) Cosmetic Science and Technology, Second Edition, Volumes 1 and 2, Wileylnterscience, a division of John Wiley & Sons, Inc., New York, copyright 1972; and Flick E. W., Cosmetic and Toiletry Formulations, Noyes Publications, 1984.

Emollients may be used in amounts which are effective to prevent or relieve dryness. Useful emollients may include: hydrocarbon oils and waxes; silicone oils; triglyceride esters; acetoglyceride esters; ethoxylated glyceride; alkyl esters; alkenyl esters; fatty acids; fatty alcohols; fatty alcohol ethers; ether-esters; lanolin and derivatives; polyhydric alcohols (polyols) and poly-ether derivatives; polyhydric alcohol

(polyol) esters; wax esters; beeswax derivatives; vegetable waxes; phospholipids; sterols; and amides.

Thus, for example, typical emollients include mineral oil, especially mineral oils having a viscosity in the range of 50 to 500 SUS, lanolin oil, mink oil, coconut oil, cocoa butter, olive oil, almond oil, macadamia nut oil, aloe extract, jojoba oil, safflower oil, corn oil, liquid lanolin, cottonseed oil, peanut oil, purcellin oil, perhydrosqualene (squalene), caster oil, polybutene, odorless mineral spirits, sweet almond oil, avocado oil, calophyllum oil, ricin oil, vitamin E acetate, olive oil, mineral spirits, cetearyl alcohol (mixture of fatty alcohols consisting predominantly of cetyl and stearyl alcohols), linolenic alcohol, oleyl alcohol, octyl dodecanol, the oil of cereal germs such as the oil of wheat germ cetearyl octanoate (ester of cetearyl alcohol and 2-ethylhexanoic acid), cetyl palmitate, diisopropyl adipate, isopropyl palmitate, octyl palmitate, isopropyl myristate, butyl myristate, glyceryl stearate, hexadecyl stearate, isocetyl stearate, octyl stearate, octylhydroxy stearate, propylene glycol stearate, butyl stearate, decyl oleate, glyceryl oleate, acetyl glycerides, the octanoates and benzoates of (C 12-Cl 5) alcohols, the octanoates and decanoates of alcohols and polyalcohols such as those of glycol and glycerol, and ricin-oleates of alcohols and poly alcohols such, as those of isopropyl adipate, hexyl laurate, octyl dodecanoate, dimethicone copolyol, dimethiconol, lanolin, lanolin alcohol, lanolin wax, hydrogenated lanolin, hydroxylated lanolin, acetylated lanolin, petrolatum, isopropyl lanolate, cetyl myristate, glyceryl myristate-, myristyl myristate, myristyl lactate, cetyl alcohol, isostearyl alcohol stearyl alcohol, and isocetyl lanolate, and the like.

Emulsifiers (emulsifying agents) may be used in amounts effective to provide uniform blending of ingredients of the composition. Useful emulsifiers may include anionics such as: fatty acid soaps, e.g., potassium stearate, sodium stearate, ammonium stearate, and triethanolamine stearate; polyol fatty acid monoesters containing fatty acid soaps, e. g., glycerol monostearate containing either potassium or sodium salt; sulfuric esters (sodium salts), e.g., sodium lauryl sulfate, and sodium cetyl sulfate; and polyol fatty acid monoesters containing sulfuric esters, e.g., glyceryl monostearate containing sodium lauryl- sulfate; Cationics such as : N(stearoyl colamino formylmethyl) pyridium chloride; N- soya-N-ethyl morpholinium ethosulfate; Alkyl dimethyl benzyl ammonium chloride; diisobutylphenoxytheoxyethyl dimethyl benzyl ammonium chloride; and cetyl pyridium chloride; Nonionics such as: polyoxy ethylene fatty alcohol ethers, e.g., polyoxyethylene lauryl alcohol; polyoxypropylene fatty alcohol ethers, e.g., propoxylated oleyl alcohol; polyoxyethylene fatty acid esters, e.g., polyoxyethylene stearate; polyoxyethylene sorbitan fatty acid esters, e.g., polyoxyethylene sorbitan monostearate; sorbitan fatty acid esters, e.g., sorbitan monostearate; polyoxyethylene glycol fatty acid esters, e.g., polyoxyethylene glycol monostearate; polyol fatty acid esters, e.g., glyceryl monostearate and propylene glycol monostearate; and ethoxylated lanolin derivatives, e.g., ethoxylated lanolins, ethoxylated lanolin alcohols and ethoxylated cholesterol.

Surfactants may also be used in the compositions of this invention. Suitable surfactants may include those generally grouped as cleansing agents, emulsifying agents, foam boosters, hydrotropes, solubilizing agents, suspending agents and nonsurfactants (facilitates the dispersion of solids in liquids).

The surfactants are usually classified as amphoteric, anionic, cationic and nonionic surfactants.

Amphoteric surfactants include acylamino acids and derivatives and N- alkylamino acids.

Anionic surfactants include: acylamino acids and salts, such as, acylglutarnates, acylpeptides, acylsarcosinates, and acyltaurates; carboxylic acids and salts, such as, alkanoic acids, ester carboxylic acids, and ether carboxylic acids; sulfonic acids and salts, such as, acyl isethionates, alkylaryl sulfonates, alkyl sulfonates, and sulfosuccinates; sulfuric acid esters, such as, alkyl ether sulfates and alkyl sulfates.

Cationic surfactants include: alkylamines, alkyl imidazolines, ethoxylated amines, and quaternaries (such as, alkylbenzyldimethylammonium salts, alkyl betaines, heterocyclic ammonium salts, and tetra alkylammonium salts).

Nonionic surfactants include: alcohols, such as primary alcohols containing 8 to 18 carbon atoms; alkanolamides such as alkanolamine derived amides and ethoxylated amides; amine oxides; esters such as ethoxylated carboxylic acids, ethoxylated glycerides, glycol esters and derivatives, monoglycerides, polyglyceryl esters, polyhydric alcohol esters and ethers, sorbitan/sorbitol esters, and triesters of phosphoric acid; and ethers such as ethoxylated alcohols, ethoxylated lanolin, ethoxylated polysiloxanes, and propoxylated polyoxyethylene ethers.

Suitable waxes which may prove useful include: animal waxes, such as beeswax, spermaceti, or wool wax (lanolin); plant waxes, such as carnauba or candelilla; mineral waxes, such as montan wax or ozokerite; and petroleum waxes, such as paraffin wax and miorocrystalline wax (a high molecular weight petroleum wax). Animal, plant, and some mineral waxes are primarily esters of a high molecular weight fatty alcohol with a high molecular weight fatty acid. For example, the hexadecanoic acid ester of tricontanol is commonly reported to be a major component of beeswax.

Suitable waxes which may be useful also include the synthetic waxes 'including polyethylene polyoxyethylene and hydrocarbon waxes derived from carbon monoxide and hydrogen.

Representative waxes also include: Peresin; cetyl esters; hydrogenated jojoba oil; hydrogenated jojoba wax; hydrogenated rice bran wax; Japan wax; jojoba butter; jojoba oil; jojoba wax; munk wax; montan acid wax; ouricury wax; rice bran wax; shellac wax; sufurized jojoba oil; synthetic beeswax; synthetic jojoba oils; trihydroxystearin; cetyl alcohol; stearyl alcohol; cocoa butter; fatty acids of lanolin; mono-, di- and triglycerides which are solid at 250°C, e.g., glyceyl tribehenate (a triester of behenic acid and glycerine) and C18-C36 acid triglyceride (a mixture of triesters of C18-C36 carboxylic acids and glycerine) available from Croda, Inc., New York, NY under the trade names- Syncrowax'HRC and Syncrowax HGL-C, respectively; fatty esters which are solid at 250⁰C; silicone waxes such as methyloctadecaneoxypolysiloxane and poly (dimethylsiloxy) stearoxysiloxane; stearyl mono- and diethanolamide; rosin and its derivatives such as the abietates of glycol and glycerol; hydrogenated oils solid at 25O⁰C; and sucroglycerides.

J Thickeners (viscosity control agents) which may be used in effective amounts in aqueous systems include: algin; carbomers such as carbomer 934, 934P, 940 and 941; cellulose gum; cetearyl alcohol, cocamide DEA, dextrin; gelatin; hydroxyethylcellulose; hydroxypropylcellulose; hydroxypropyl methylcellulose; magnesium aluminum silicate; myristyl alcohol; oat flour; oleamide DEA; oleyl alcohol; PEG-7M; PEG14M; PEG- W 9OM; stearamide DEA; Stearamide MEA; stearyl alcohol; tragacanth gum; wheat starch; xanthan gum; and the like.

Suitable film formers which may be used include: acrylamide/sodium acrylate copolymer; ammonium acrylates copolymer; Balsam Peru; cellulose gum; ethylene/maleic anhydride copolymer; hydroxyethylcellulose; hydroxypropylcellulose; 75 polyacrylamide; polyethylene; polyvinyl alcohol; pvm/MA copolymer (polyvinyl methylether/ maleic anhydride); PVP (polyvinylpyrrolidone); maleic anhydride copolymer such as PA- 18 available from Gulf Science and Techno logy; PVP/hexadecene copolymer such as Ganex V-216 available from GAF Corporation; acrylic/acrylate copolymer; and the like.

20 Generally, film formers can be used in amounts of about 0.1% to about 10% by weight of the total composition with about 1% to about 8% being preferred and about 0.1% to about 5% being most preferred.

Preservatives which may be used in effective amounts include: butylparaben; ethylparaben; imidazolidinyl urea; methylparaben; o-phenylphenol; propylparaben; 25 quaternium-14; quaternium-15; sodium dehydroacetate; zinc pyrithione; and the like.

The preservatives are used in amounts effective to prevent or retard microbial growth. Generally, the preservatives are used in amounts of about 0.1% to about 1% by weight of the total composition with about 0.1% to about 0.8% being preferred and about 0.1% to about 0.5% being most preferred. 0 Perfumes (fragrance components) and colorants (coloring agents) well known to those skilled in the art may be used in effective amounts to impart the desired fragrance and color to the compositions of this invention. The invention in one aspect is a method of identifying a candidate skin lightening agent. The method can be performed in vitro or in vivo. The method includes the step of contacting, under defined conditions, a test cell (e.g., keratinocyte) with an agent that induces proopiomelanocortin (POMC) expression. Contacting refers to any suitable physical contact between two entities. Defined conditions refers to any selected and preferably reproducible set of chemical, physical, and temporal parameters suitable for the purpose of carrying out the method. Such defined conditions generally include physiological conditions including temperature, pH, osmotic strength, pθ₂, concentration of glucose and other nutrients, and the like, as well as amount, frequency, and duration of contact with the contacted agent.

In a preferred embodiment, the test cell is a keratinocyte. A keratinocyte refers to a keratinocyte either in vitro, e.g., either in primary culture or as a cell line, or in vivo. Methods for primary culture of keratinocytes are known. See, for example, Marcelo CL et al. (1978) J Cell Biol 79(2 Pt l):356-70. Agents that induce POMC expression in keratinocytes specifically include, without limitation, UV radiation, p53, pyrimidine- pyrimidine (e.g., thymidine-thymidine) dinucleotide dimers (see Eller MS et al. (1996) Proc Natl Acad Sci USA 93:1087-92; Kichina J et al. (1996) Pigment Cell Res 9:85-91; Goukassian DA et al. (1999) J Invest Dermatol 112:25-31).

The method also includes the step of contacting the test cell (e.g., a keratinocyte, a melanocyte, a fibroblast, etc.) with a test agent. A test agent, as used herein, refers to any defined chemical entity or defined combination of such entities, including small organic molecules (up to 1.5 kDa) and isolated biomolecules such as peptides, proteins, nucleic acids, carbohydrate polymers, and lipids. Test agents can be provided as members of a library of such agents, including for example a library of compounds prepared using combinatorial chemistry according to methods well known in the art, that may include at least tens, hundreds, or thousands of compounds. Biomolecules include naturally occurring biomolecules as well as biomolecules artificially generated by human design.

The method further includes the step of measuring an amount of α-MSH expressed by the contacted test cell (e.g., keratinocyte). The measuring step in one embodiment involves direct measurement of α-MSH. This can be accomplished, for example, using standard methods in a suitable α-MSH-specific enzyme-linked immunosorbent assay (ELISA) to determine the amount of α-MSH secreted into (keratinocyte) cell culture medium in vitro. Alternatively or in addition, the step of measuring the amount of α-MSH can be performed indirectly, for example by measuring pigmentation of keratinocytes, particularly in vivo. The pigmentation can be measured using any suitable method, including histological examination and melanin quantification, as described in the examples below.

The method further includes the step of determining the test agent is a candidate skin lightening agent when the amount of α-MSH expressed by the contacted test cell (e.g., keratinocyte) is reduced compared to an amount (i.e., a control amount) of α-MSH expressed by a control test cell (e.g. , a control keratinocyte) contacted, under the defined conditions, with the agent that induces POMC expression. Comparison to a negative control (no test agent) can be made either concurrently with a given test measurement or with a suitable historical control. Test and control measurements are preferably made using the same type of assay, e.g., ELISA. In one embodiment the test amount of α-MSH is at least 5 percent less than a corresponding control amount of α-MSH. In various other embodiments the test amount of α-MSH is at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, at least 30 percent, at least 40 percent, or at least 50 percent less than a corresponding control amount of α-MSH. In one embodiment the test amount of α-MSH is at least 95 percent less than a corresponding control amount of α-MSH. In one embodiment the test amount of α-MSH is 100 percent less than a corresponding control amount of α-MSH.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES

Example 1

UV Treatment Leads to Upregulation of POMC and a-MSHIn Vitro Previous data had suggested that the POMC gene is upregulated at both protein and mRNA levels following UV irradiation of skin [7, 12-17]. Although RNA upregulation could occur through a variety of mechanisms, the proximal lkb promoter region of the POMC gene was examined, searching for consensus transcription factor binding elements which are conserved between human, rat, and mouse. Among the various consensus elements found, one was particularly noteworthy due to its known regulation by UV: p53. Primary human keratinocytes and the mouse keratinocyte line PAM212 were therefore examined following UV, for both POMC and p53 levels. Results of these experiments are shown in FIG. 1.

Primary keratinocytes and melanocytes were isolated and grown from normal human or mouse skin as described [83-85]. Briefly, human or mouse primary keratinocytes were cultured in Keratinocyte serum-free medium (SFM) (Invitrogen Corporation, USA). Cell cultures were studied in passage 2 after limited in vitro expansion from primary cultures. Melanocyte and fibroblast contamination was eliminated by differential trypsinization. Cells were grown to 40-60% confluence prior to use in irradiation experiments, in humidified incubators supplemented with 5% CO₂. The mouse keratinocyte cell line PAM212 was generously provided by Sr. Stuart Yuspa (NIH).

Keratinocytes were exposed to ultraviolet radiation in a Stratalinker UV chamber (Stratagene,Cedar Creek,TX) equipped with 15W 254 nm UVB bulbs (Germicidal lamp FGl 5T8 made in Japan) at a dose of 100J/m². After irradiation, cells were incubated in DMEM medium in humidified incubators supplemented with 5% CO₂ until time of assay.

RNA and protein were collected at time 0 and different time points after irradiation, as indicated. For quantitative RT-PCR, total RNA was converted into cDNA using Superscript™ HI reverse Transcriptase kit (Invitrogen). cDNA expression was quantified using QuantiTect Probe RT-PCR kits (Qiagen, Valencia, CA) and ICycler machine (BioRad, Hercules, CA). Gene-specific primer sets were as reported [13]. Taqman PCR reactions were done in triplicate for each sample and normalized to GAPDH. Western blotting was performed using the following antibodies: anti-p53: DO-7 (Calbiochem, OPO3L), CM-5 (Vector, VP-P56), ICl 2MAB (Cell Signaling, 2524) and Pab241 (Oncogene, AB-I); and anti-POMC (Pro sci, XW-7447 and Phoenix H-029- 30). Enzyme immunoassay was performed using the alpha-MSH EIA kit (Phoenix 5 Pharmaceuticals Inc., EK-043-01).

The 100 J/m² UVB dose administered in this experiment is equivalent to the Standard Erythema Dose (SED) [30, 31] commonly used as a measure of sunlight. As a reference, the ambient exposure on a clear summer day in Europe is approximately 30-40 SED. Also, an exposure dose of 4 SED would be expected to produce moderate W erythema on un-acclimated white skin, but minimal or no erythema on previously exposed (tanned) skin.

UV markedly induced expression of POMC mRNA and protein by 6 hours, and p53 induction was already maximal by 3 hours, consistent with its known stabilization by UV (FIG. IA and FIG. IB). At 24 h the levels of POMC protein were lower relative 15 to that found after 6h in keratinocytes (human as well as mouse), probably as a result of its proteolytic processing and secretion [7, 8]. Analysis of the corresponding culture media by ELISA demonstrated >30-fold induction of α-MSH secretion by keratinocytes after UV.

20 Example 2

POMC Is Induced By p53 Overexpression

To test whether POMC is a p53-responsive gene in keratinocytes, pcDNA-HA- p53 or empty vector was introduced into the PAM212 keratinocyte cell line and POMC expression was assessed by a real-time quantitative RT-PCR assay and immunoblotting 5 (FIG 1C). POMC expression was significantly induced in response to p53 at both mRNA and protein levels. The rapid induction of POMC following UV radiation of keratinocytes is consistent with the rapid, post-translational stabilization responsible for p53 upregulation following UV [32-34]. 0 Example 3

UV-Mediated Upregulation of POMC Requires p53 A synthetic dominant-negative p53 allele (p53DD [35]) was stably introduced into the PAM212 keratinocyte line, to yield PAM212/p53DD or, equivalently, PAMDD cells. UV-irradiated PAM212 or PAMDD cells were processed for RNA and protein isolation as described in Example 1. As shown in FIG. 2A, ectopic expression of p53DD was seen to abrogate induction of POMC mRNA and protein levels following UV exposure. Keratinocytes from wild-type and p53-null mice (littermates) were also studied. p53-nullizygous keratinocytes exhibited no measurable POMC mRNA upregulation following UV irradiation (FIG. 2B). Of note, western blotting demonstrated that basal POMC expression (prior to UV) was not significantly diminished in the absence of p53, suggesting that p53 is not globally required for POMC expression, but is essential for the UV -responsive induction of POMC in keratinocytes. This finding is corroborated by the obvious fact that p53-/- C57BL/6 mice have black fur.

Example 4

POMC Is a Direct Transcriptional Target ofp53

A potential p53 binding-site was identified in the POMC 5 '-flanking region, 300 bp upstream of the transcription initiation site in humans, with a similar site in the mouse promoter [36, 37]. A series of luciferase reporters was tested for UV responsiveness after transfection into PAM212 keratinocytes.

A fragment of the human POMC promoter (-680 to +1 relative to the transcription start site) and a series of unidirectional truncations from the 5' end of POMC (-580/+1, -480/+1, -280/+3, and -101/+1) were generated by PCR and were inserted into the PGL-3 basic vector (Promega) upstream of the luciferase reporter gene in 6-well plates (2 μg DNA/well) using Lipofectamine 2000 (GIBCO BRL) according to the manufacturer's instructions. Promoter constructs were co-transfected with the pRL- TK plasmids (Promega). 24 h after transfection, the cells were irradiated by UVB (100J/m², as described in Example 1) and 24 h later were lysed and assayed using Dual Luciferase reagents (Promega). Promoter activity was measured by Luciferase levels, normalized to the constitutively expressed Renilla.

Electrophoretic mobility shift assays (EMSA) were done using LightShift Chemiluminescent EMSA kit (Pierce Biotechnology Inc., Rockford, IL, USA) according to the manufacturer's instructions. For competition experiments, 5-, 15-, or 50^:fold excess unlabeled POMC oligo (wild-type 5'-BiO-AGGCAAGATGTGCCTTGCGCTC-B' (SEQ ID NO:3) or mutant 5'-CCCGAAGATGTGCCTTGGCAAA-S' (SEQ ID NO:4) in double-stranded configurations) was incubated with the extract for 10 min before the addition of labeled oligo and the incubation proceeded for an additional 20 min at room temperature. In supershift experiments, 1 μL of anti-p53 antibody (ABl, Oncogene) was subsequently added and incubated for an additional 15 min at room temperature.

Chromatin immunoprecipitation was performed as described [87, 88], using anti- p53 antibody (AbI, Oncogene), and control anti-IgG (Santa Cruz Biotechnology, Inc.). DNA released from precipitated complexes were amplified using primers for the p21 and actin promoters and for the POMC promoter region (from -160 to -64 of mouse POMC promoter and from -381 to -260 of human POMC promoter). Primers for actin and p21 were as reported [89, 90]. POMC promoter primers were as follows:

human, forward 5'-TGCGAACCAGGCAGATGCCA-S' (SEQ ID NO:5); human, reverse 5'-TTAGAACGGGCGGGAGGCTT-S' (SEQ ID NO:6); mouse, forward 5'-CAGATGCGCCTTGCGCTCAG-S' (SEQ ID NO:7); mouse, reverse 5'-ACCTTCCTGGCAGCGCTTC-S' (SEQ ID NO:8).

As shown in FIG. 3A and FIG. 3B, deletion mutants as well a site-specific mutation at the p53 consensus element abrogated UV-responsiveness of the POMC promoter. Furthermore, parallel transfections into PAM212 and PAM212/p53DD revealed that suppression of endogenous p53 is sufficient to abrogate the UV-induced reporter activity (FIG. 3C). Classical electrophoretic mobility shift assay (EMSA) demonstrated a UV-induced DNA binding activity that was supershifted by anti-p53 antibody and with sequence specificity for the p53 consensus probe (but not point mutant) in keratinocyte nuclear extracts (FIG. 3D). To determine whether p53 occupies the endogenous POMC promoter in cells, chromatin immunoprecipitation (ChIP) from UV-irradiated vs. un-irradiated mouse keratinocytes (PAM212) or human primary keratinocytes was used. p53 binding to the POMC promoter was detected following UV, whereas no association was detected in un-irradiated cells (FIG. 3E). Controls included ChIP of the p53 response element in the p21 promoter, and the actin promoter (negative control). These data suggest that p53 directly modulates transcriptional activity of the POMC promoter following UV irradiation.

Example 5 UV/p53 Induction of POMC Occurs Preferentially in Keratinocytes

To assess whether the induction of POMC by UV (via p53) occurs in non- keratinocytes, melanocytes, fibroblasts, and spleen cells were exposed to UV as described in Example 1. All three lineages displayed reproducible POMC induction, but the magnitude of the effect was significantly greater in keratinocytes (16-25 fold in keratinocytes vs. ~3-fold in non-keratinocytes). Using p53-/- primary melanocytes, it was found that even the modest (~3-fold) induction of POMC by UV in melanocytes appears to require p53.

Example 6 Deficient Tanning Response ofp53-/- Mice

To test the in vivo requirement of p53 for UV-induced pigmentation, age- matched wild-type and p53-null C57BL/6 mice were subjected to UV followed by evaluation of ears and tails, two locations containing epidermal melanocytes (furry regions lack epidermal melanocytes [38]. p53-deficient (-/-) mice were C57BL/6 TSG- p53^® Nl 2 purchased from Taconic Farms (Hudson NY, USA). These p53-deficient mice were originally generated by Donehower. et al [82].

Animals were exposed to ultraviolet irradiation (40 J/m² UVB, once a day, 5 days per week, for 10 weeks) in a custom-made lucite chamber (Plastic Design Corporation, Massachusetts) designed to allow freedom of movement while being irradiated. UV was delivered by a double bank of UVB lamps. UVA was filtered by chamber (Plastic

Design Corporation, Massachusetts) and UV emittance was measured with the use of a UV photometer (UV Products, Upland, CA) equipped with UVB measuring head. Skin samples were biopsied at indicated time points after UV exposure. Animals were either sacrificed by CO₂ or anesthetized with isoflurane anesthesia prior to ear sampling. Ear sections were immediately placed in 10% buffered formalin until paraffin embedding and sectioning (done by the rodent histopathology core service at Harvard Medical School). Hematoxylin/Eosin and Fontana-Masson staining were performed by the histopathology core. Immunohistochemistry was performed according to standard protocols with the following antibodies: anti-p53: DO-7 (Calbiochem, OPO3L), CM-5 (Vector, VP-P56); anti-POMC (Pro sci, XW-7447 and Phoenix H-029-30); and anti-Mitf (C5 or D5 [86]). As shown in FIG. 4A, visible tanning of ears and tails was observed in wild-type but not in p53-null mice. Interestingly, baseline pigmentation was not appreciably different in fur of p53 wild-type vs. null mice, but was reproducibly slightly lighter in epidermal tail skin of p53-nulls. Histologic analyses revealed absence of both POMC and melanin induction in UV irradiated p53-/- skin (FIG. 4B and FIG. 4C). POMC mRNA induction was also directly measured in skin of the same mice following UV radiation. As shown in FIG. 4D, significant POMC mRNA induction was observed following UV in p53 wild-type mice but was absent in p53-/- mice. Aside from α-MSH, another proteolytic cleavage product of POMC is the opioid receptor ligand β-endorphin, suggested to be a mediator of sunseeking behavior in man [39-41]. Expression of β- endorphin, like α-MSH, was induced by UV in a p53-dependent manner. These data indicate that p53 is essential for POMC induction in vivo following UV and establishes p53 as an integral molecule in the tanning response.

To asess whether similar events occur in the UV response of human skin, discarded normal human skin specimens were exposed to UV (in a manner similar to the keratinocytes in Example 1) and stained over a timecourse for p53, α-MSH peptide, and the melanocyte transcription factor MITF. Induction of MITF by MSH/MCIR/cAMP indicates activation of the pigmentation pathway [13, 42] and also identifies skin melanocytes at the basal epidermis. As shown in FIG. 5, p53 was rapidly induced in virtually every epidermal keratinocyte by 1 hour following UV exposure. MSH was induced a bit later (3-6 hours), again throughout the epidermal keratinocyte population. MITF was strongly induced at 6 hours, and was obseved to localize to the basal epidermal population, where it stains melanocyte nuclei (FIG. 5) as previously reported [43]. These results indicate a similar temporal induction of signaling components following UV irradiation of either mouse or human skin.

Example 7 Role ofp53 in Non-UV Induction of Pigmentation A role for p53 in the UV-pigment response is notable because p53 protein may be stabilized by various non-UV stresses, raising the possibility that it may participate in cutaneous pigmentation in a variety of non-UV associated settings. To test this, PAM212 keratinocytes were treated with the topoisomerase inhibitor etoposide, and induction of p53 and POMC were measured. As shown in FIG. 6A, both p53 and POMC were induced. A simple test of the possibility that p53 may participate in non- UV skin hyperpigmentation is the response to topical 5-fluorouracil (5-FU), a known inducer of p53 [44] and a drug which is used in multiple human dermatologic conditions, which has been described to induce hyperpigmentation as a side effect in a fraction of patients [45]. Three p53(+/+) and three knockout (-/-) mice were treated with 2% 5-FU, once a day, 5 d per week, for 3 wk. As shown in FIG. 6B and FIG. 6C, chronic exposure to topical 5-FU induced hyperpigmentation in p53 wild-type, but not p53-/-, mouse skin, demonstrating that non-UV triggers of p53 can also induce pigmentation. This result is consistent with previous reports that DNA damage (or its repair) can stimulate tanning [46, 47], and suggests that the mechanism involves p53-mediated mimicking of the UV-pigmentation response in keratinocytes.

Example 8

Inhibition of UV -Mediated POMC and a-MSH Induction in Vitro The experiments described in Example 1 are performed with the additional step of including at least one inhibitor of either expression or function of PCl and/or PC2 in the cell culture medium. The inhibitor or inhibitors are added over a range of concentrations from 1 nM to 1 mM. UV irradiation, RNA and protein analysis are performed as described in Example 1. Inhibitor present at an effective dose effectively reduces induction of POMC and α-MSH in vitro, compared to results described in Example 1.

Example 9

Decreased Tanning Response inp53 Wild-Type Mice Treated with Prohormone Convertase Inhibitor

The experiments using p53 wild-type mice described in Example 6 are performed with the additional step of topically administering at least one inhibitor of either expression or function of PCl and/or PC2 (at least 1 μg to each ear, 5 days a week, for 10 weeks) to the ears of mice on the same schedule they are exposed to UV irradiation. The inhibitor or inhibitors are administered over a range of doses from 1 μg to 1 mg per ear per treatment day. UV irradiation, skin sampling, histologic and immunohistologic examination, RNA and protein analysis are performed as described in Example 6.

Inhibitor administered at an effective dose effectively reduces tanning response in vivo, compared to results for p53 wild-type mice described in Example 6.

Example 10 Decreased Tanning Response in Human Skin Treated with Prohormone Convertase

Inhibitor

Experiments using human skin described in Example 6 are performed with the additional step of including at least one inhibitor of either expression or function of PCl and/or PC2 in the culture medium. The inhibitor or inhibitors are added over a range of concentrations from 1 nM to 1 mM. UV irradiation, RNA and protein analysis are performed as described in Example 6. Inhibitor present at an effective dose effectively reduces induction of p53, α-MSH, and MITF in vitro, compared to results described in Example 6.

Example 11

Inhibition of Non-UV Induction of Pigmentation In Vitro Experiments with PAM212 cells described in Example 7 are performed with the additional step of including at least one inhibitor of either expression or function of PCl and/or PC2 in the cell culture medium. The inhibitor or inhibitors are added over a range of concentrations from 1 nM to 1 mM. Treatment with etoposide and analysis of p53 and POMC are performed as described in Example 7. Inhibitor present at an effective dose effectively reduces induction of p53 and POMC in vitro, compared to results described in Example 7.

Example 12

Inhibition of Non-UV Induction of Pigmentation In Vivo Experiments with p53 wild-type mice described in Example 7 are performed with the additional step of topically administering at least one inhibitor of either expression or function of PCl and/or PC2 (at least 1 μg to each ear, 5 days a week, for 3 weeks) to the ears of mice on the same schedule they are exposed to 5-FU. The inhibitor or inhibitors J are administered over a range of doses from 1 μg to 1 mg per ear per treatment day. Tanning and melanin assessment are performed as described in Example 7. Inhibitor administered at an effective dose effectively reduces tanning and melanin content in vivo, compared to results described in Example 7. 0

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EQUIVALENTS

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

What is claimed is:

Claims

1. A method for reducing or preventing skin pigmentation, comprising locally administering to a target (e.g., pigmented) skin an effective amount of an inhibitor of an enzyme involved in cleavage of proopiomelanocortin (POMC) to alpha melanocyte stimulating hormone (α-MSH), to reduce or prevent pigmentation of the skin.

2. The method of claim 1 , wherein the skin pigmentation is hyperpigmentation associated with a condition selected from acanthosis nigricans, Addison's disease, age spots (also variously known as liver spots, sun spots, solar lentigo, and lentigines), basal cell carcinomas, biliary cirrhosis, cafe au lait spots (which may be associated with neurofibromatosis or Albright's syndrome), ectopic ACTH syndrome, eosinophilia- myalgia syndrome, ephelides (freckles), folate deficiency, hemochromatosis, junctional and compound nevi, lentigo, malabsorption, melanosis secondary to metastatic melanoma, Nelson's syndrome, pellagra, pigmented actinic keratosis, pigmented keratinocyte tumors, POEMS syndrome, porphyria cutanea tarda, post-inflammatory hyperpigmentation, scleroderma, seborrheic keratosis, vitamin B₁₂ deficiency, and Whipple's disease.

3. The method of claim 1 or claim 2, wherein the locally administering is topically administering.

4. The method according to any one of claims 1-3, wherein the enzyme is selected from PCl and PC2.

5. The method according to claim 4, wherein the enzyme is PCl .

6. The method according to claim 4, wherein the enzyme is PC2.

1. A method of identifying a candidate skin lightening agent, comprising contacting, under defined conditions, a test cell (e.g., keratinocyte) with

(a) an agent that induces proopiomelanocortin (POMC) expression, and (b) a test agent; measuring an amount of alpha melanocyte stimulating hormone (α-MSH) expressed by the contacted test cell (e.g., keratinocyte); and determining the test agent is a candidate skin lightening agent when the amount of α-MSH expressed by the contacted test cell (e.g., keratinocyte) is reduced compared to an amount of α-MSH expressed by a control test cell (e.g., control keratinocyte) contacted, under the defined conditions, with the agent that induces POMC expression.