WO2009064421A1 - Histone deacetylase inhibitors as skin lightening agents - Google Patents

Abstract

The present invention is directed to methods of inhibiting unwanted skin pigmentation, comprising locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase.

Description

HISTONE DEACETYLASE INHIBITORS AS SKIN LIGHTENING AGENTS

BACKGROUND OF THE INVENTION

Melanoma incidence continues to rise at an alarming rate while effective systemic therapies remain very limited. Microphthalmia associated transcription factor (MITF) is required for development melanocytes and is an amplified oncogene in a fraction of human melanomas. MITF also plays an oncogenic role in human clear cell sarcomas, which typically exhibit melanoma-like features. While MITF is in principal an attractive lineage-selective drug target for the malignancies, it is not known to contain intrinsic catalytic activity capable of direct small molecule inhibition. An alternative drug-targeting strategy is to identify and interfere with lineage-restricted mechanisms required for its expression. Histone deacetylase (HDAC) inhibitors (HDACi) have been shown to produce anti-cancer effects by inducing growth arrest and apoptosis or repressing angiogenesis. Prior to the invention it was not known that HDACi can also inhibit MITF.

SUMMARY OF THE INVENTION

According to the invention methods are provided of inhibiting unwanted skin pigmentation, by locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase. In one aspect of the invention the the histone deacetylase inhibitor is at least 8, 5, 4, 3, or 2-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase. In certain aspects of the invention the class III histone deacetylase inhibitor is sirtinol.

In one aspect of the invention methods are provided of inhibiting unwanted skin pigmentation, by repeatedly locally administering to the area of skin of the subject an effective amount of the histone deacetylase inhibitor to maintain an inhibited amount of skin pigmentation, wherein the histone deacetylase inhibitor is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase. In one aspect of the invention the the histone deacetylase inhibitor is at least 8, 5, 4, 3, or 2-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase.

In one aspect of the invention methods are provided of inhibiting unwanted skin pigmentation, by locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected based on its inability to induce p53.

In one aspect of the invention methods are provided of inhibiting unwanted skin pigmentation, by locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the effective amount of the histone deacetylase inhibitor is insufficent to induce p53.

In certain embodiments of the invention the unwanted skin pigmentation is hyperpigmentation associated with a condition selected from acanthosis nigricans, Addison's disease, biliary cirrhosis, cafe au lait spots, ectopic ACTH syndrome, eosinophilia-myalgia syndrome, ephelides (freckles), folate deficiency, hemochromatosis, junctional and compound nevi, lentigo, malabsorption, Nelson's syndrome, pellagra, pigmented actinic keratosis, pigmented keratinocyte tumors, POEMS syndrome, porphyria cutanea tarda, post-inflammatory hyperpigmentation, scleroderma, seborrheic keratosis, vitamin Bi₂ deficiency, and Whipple's disease.

In certain aspects of the invention, locally administering is topically administering.

In certain aspects of the invention the area of skin is free of dermal malignancy.

In one aspect of the invention methods are provided of inhibiting unwanted skin pigmentation, by locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor in combination with an effective amount of skin lightening agent, wherein the skin lightening agent is not retinoid.

In one aspect of the invention the skin lightening agent is leptomycin B.

In one aspect of the invention the skin lightening agent is a tyrosinase inhibitor. In certain embodiments of the invention the tyrosinase inhibitor is hydroquinone, kojic acid, kojic acid dipalmitate, arbutin, magnesium ascorbyl phosphate, or calcium D- pantetheine-S-sulfonate.

In one aspect of the invention methods are provided of maintainng an inhibited amount of skin pigmentation by repeatedly locally administering to the area of skin of the subject an effective amount of the histone deacetylase inhibitor in combination with an effective amount of skin lightening agent, wherein the skin lightening agent is not retinol, to maintain an inhibited amount of skin pigmentation.

In certain aspects of the invention the histone deacetylase inhibitor is selected from a short-chain fatty acid, a hydroxamic acid, an epoxyketone-containing cyclic tetrapeptide, a cyclic tetrapeptide, a benzamide, and Depudecin. In a certain embodiment of the invention the histone deacetylase inhibitor is a short-chain fatty acid selected from a butyrate, a phenylbutyrate, and a valproate. In a certain embodiment of the invention the short-chain fatty acid is sodium butyrate.

In a certain embodiment of the invention the histone deacetylase inhibitor is a hydroxamic acid selected from a trichostatin, SAHA, oxamflatin, ABHA, Scriptaid, Pyroxamide, LBH589, and Propenamide. In a certain embodiment the hydroxamic acid is Trichostatin A (TSA). In a certain embodiment the hydroxamic acid is SAHA. In a certain embodiment the hydroxamic acid is LBH589.

In a certain embodiment the histone deacetylase inhibitor is an epoxyketone- containing cyclic tetrapeptide selected from trapoxin, HC-toxin, Chlamydocin, Diheteropeptin, WF-3161, CyI-I, and Cyl-2.

In a certain embodiment the histone deacetylase inhibitor is a cyclic tetrapeptide selected from FR901228, Apicidin, and a cyclic-hydroxamic-acid-containing peptide (CHAP).

In a certain embodiment the histone deacetylase inhibitor is a benzamide selected from MS-275 (MS-27-275) and CI-994.

In a certain embodiment of the invention the histone deacetylase inhibitor is Depudecin.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

Figure 1 is a panel of bar graphs and Westrern blots that depicting that HDACi drugs repress cell growth as well as M-MITF and SOXlO levels in melanoma cell lines. A, UACC62, UACC257, and primary human melanocytes (l°mel) were treated with HDAC inhibitors, sodium butylate (NaB) (shadow bars) and trichostatin A (TSA) (dotted bars) for 48 h. Relative growth rate of NaB- or TSA-treated cells was normalized by PBS- or ethanol (EtOH) -treated cells (filled bars), respectively. Data are mean ± S.D. of at least three independent experiments. B. Western blotting analysis of whole-cell lysates prepared from UACC62, UACC257, MeWo, or SK-MEL-5 human melanoma cells and l°mel treated with PBS, NaB, EtOH, or TSA for 24 h (Upper panel) or from UACC62 human melanoma cells treated with PBS, NaB, EtOH, TSA, DMSO, SAHA, or LBH589 for 24 h (lower panel). Lysates were immunoblotted with antibodies against MITF, SOXlO, acetylated histone 3 (Ace-H3), or α-tubulin. The arrows show the bands of M- MITF protein. Asterisk indicates A-MITF protein. C. Whole-cell lysates were prepared from DTC-I, SU-CCS-I, or CCS292 human clear cell sarcoma cells treated with PBS, NaB, EtOH, TSA for 24 h. Other conditions are represented as indicated in Fig. IB.

Figure 2 is a Western blot showing that overexpression of SOXlO can rescue the repression of M-MITF. Western blotting analysis of whole-cell lysates prepared from UACC62 overexpressing GFP, SOXlO, or SOXIO-mut cells. Arrowheads show the ectopic expressing SOXlO or SOXIO-mut. Other conditions are represented as indicated in Fig. IB.

Figure 3 is a panel of bar graphs, graphs and agarose gel depicting that HDACi repress transcription of the SOXlO gene. A. Quantitative reverse transcription PCR analysis of total RNA isolated from UACC62 (left panel) and 501mel (right panel) treated with 1 μM of TSA for indicated times. In each case, SOXlO (filled bars) or M- MITF (shadow bars) mRNA levels were normalized to glyceraldehyde-3 -phosphate dehydrogenase and performed in triplicate. Data are mean ± S.D. of at least three independent experiments. B. mRNA stability of SOXlO (left panel) and M-MITF (right panel) are shown. UACC62 cells were treated with Actinomycin D and after 15 min (0 h) ethanol (EtOH) (dotted line) or TSA (solid line) were added. Total RNA was isolated at indicated times. Other conditions are represented as indicated in Fig. 3A. C. Chromatin immunoprecipitations were performed from UACC62 cells treated with PBS, NaB, EtOH, and TSA. Protein-chromatin cross-linked complexes immunoprecipitated with either no antibody (no Ab) (lane 6-9), RNA polymerase II antibody (α-Pol-II) (lane 10- 13), or control antibody (control Ab) (lane 14-17), and input genomic DNA (lane 2-5) were run with DNA markers (M, lane 1 and 18) on a 2.0% agarose gel and stained with ethidium bromide. DNA segments show SOXlO exonl region (upper panel) and β-ac tin exon 3 region (lower panel).

Figure 4 is a panel of graphs that depict SOXlO enhancer-element suppression by HDACi. A. Effects of HDAC inhibitors on the human SOXlO enhancer. Relative luciferase activity in transient transfection assays is shown for UACC62, 501mel, and HeLa cells. Luciferase activity was normalized by internal control activity (pRL-CMV). Relative luciferase activity of pGL3-TK+enhancer R (filled bars) is shown as the ratio to the normalized luciferase activity obtained with pGL3-TK (open bars) in each control, respectively. Data are mean ± S. D. of at least three independent experiments. B. Functional analysis of the SOXlO enhancer in SOXlO promoter. Left panel shows the reporter plasmids used. The open arrows indicate the SOXlO enhancer. X shows point mutation of the TCF/LEF/SOX family/SRY-binding sites. Relative luciferase activity in transient transfection assays of UACC62 is shown to the right. Luciferase activity was normalized to cotransfected renilla luciferase activity. Relative luciferase activity is shown as the ratio to the normalized luciferase activity obtained with the SOXlO promoter without enhancer. Data are mean ± S.D. of at least three independent experiments.

Figure 5 is a panel of photographs and bar graphs showing HDACi affect on cell growth/survival— rescue by ectopic SOXlO. UACC62 (-), GFP-, SOXlO-, or SOXlO- mut-overexpressing cells were incubated with TSA (10 nM) for 24 h. After TSA treatments, the cells were cultured in medium without TSA for 7 days. Upper panel shows representative photographs of colony- forming assays (on plastic). Lower panel shows relative survival rate of TSA-treated cells (dotted bars), normalized to EtOH- treated control cells (filled bars). Data are mean ± S.D. of at least three independent experiments. The relative survival rate with * in UACC62/SOX10 cells is significantly higher than the value obtained in UACC62/GFP cells, p < 0.01.

Figure 6 is a Western blot showing the effect of Sirtinol on p53 and alpha-tubulin in PAM212 keratinocytes.

Figures 7-12 further illustrate the effects of HDAC inhibitors on pigmentation.

DETAILED DESCRIPTION OF THE INVENTION

It has now been discovered that selective histone deacetylase (HDAC) targeting can be used to downregulate pigmentation. This invention is based at least in part on the discovery that certain HDAC inhibitors upregulate p53 in keratinocytes. It has been shown that upregulation of p53 stimulates skin pigmentation. Therefore induction of p53 would work against goal of the de-pigmentation by HDAC inhibitor drugs.

It has now been discovered that multiple histone deacetylases (HDAC)-inhibitor drugs potently suppress MITF expression in melanoma and clear cell sarcoma cells. This suppression is seen to occur via inhibition of SOXlO expression. SOXlO is a neural crest-restricted transcriptional regulator of the MITF promoter, whose germline mutation in man and multiple species produces Waardenburg Syndrome, as does mutation of MITF. Via suppression of SOXlO and MITF, certain HDAC inhibitor drugs are thus candidates to play therapeutic roles in inhibiting unwanted skin and/or hair pigmentation, alone or in combination with other skin lightening agents. In addition, via suppression of SOXlO and MITF, certain HDAC inhibitor drugs are candidates to play therapeutic roles in targeting human malignancies dependent upon these lineage specific transcription factors. In a prefferd embodiment the HDAC inhibitor drugs are HDAC inhibitors that are at least 10-fold more effective in inhibiting a class I or class II HDACs in comparison to a class III HDACs. In a certain embodiment of the invention the HDAC class III inhibitor is sirtinol, nicotinamide, or splitomicin.

The protein family of mammalian histone deacetylases (HDACs) can be divided into three subclasses (Gray and Ekstrδm, 2001). HDACs 1,2, 3, and 8 which are homologues of the yeast RPD3 protein constitute class I. HDACs 4, 5, 6,7, 9, and 10 are related to the yeast Hda 1 protein and form class II. Recently, several mammalian homologues of the yeast Sir2 protein have been identified forming a third class of deacetylases which are NAD dependent. All of these HDACs appear to exist in the cell as subunits of a plethora of multiprotein complexes. In particular, class I and II HDACs have been shown to interact with transcriptional corepressors mSin3, N-CoR and SMRT which serve as bridging factors required for-the recruitment of HDACs to transcription factors. The HDAC and the many known HDAC inhibitors are further described in WO 2005/000289, WO2005/092283, and WO 2007/038459 and references cited therein which are hereby incorporated in their entirety.

The structure of chromatin plays a central role in biological processes requiring access to DNA. Chromatin consists of basic histones and other structural proteins which participate in control of gene transcriptional (1-3). Histone proteins are post- transcriptionally modified in multiple ways; including acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, providing a framework for controlling chromatin processes (4). Two particular groups of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) are intimately associated with transcriptional regulation through regulating the acetylation status of histones as well as a variety of nonhistone proteins (5).

Recently, HDAC inhibitors (HDACi) have been shown to exhibit anti-cancer effects, and suberoylanilide hydroxamic acid (SAHA) has been FDA approved for treatment of cutaneous T cell lymphoma (6-9). Cancer-related consequences of HDACi drugs include growth arrest and apoptosis as well as repression of angiogenesis (10). The effect of HDACi on growth arrest and apoptosis has been also shown in melanoma cells, but the mechanism has been poorly understood (11).

Microphthalmia-associated transcription factor (MITF) is a basic- helix/loop/helix-leucine-zipper transcription factor which plays an essential, lineage specific role in melanocyte development (12-14). The MTTF gene has a complex- promoter organization, giving at least nine distinct isoforms sharing exons 2 to 9. Some of these isoforms exhibit tissue-specific expression such as M-MITF, which is specific to melanocytes. (15, 16). The melanocyte-specifϊc promoter is located most proximal to the common downstream exons and is known as the M-promoter (17). As a transcription factor MITF regulates multiple genes related to melanin synthesis, control of apoptosis, or cell cycle progression (18-20). Transcription factors implicated in the regulation of the M-MITF promoter include paired box gene 3 (PAX3), cAMP-responsive element binding protein (CREB), SRY (sex-determining region Y)-box 10 (SOXlO), lymphoid enhancer-binding factor 1 (LEFl) (and/or TCFl/3/4), one cut domain 2 (ONECUT-2) and MITF itself (21-25). Appropriate regulation of MITF is required for cell growth/survival in melanocytes and recent work has implicated this gene as an oncogene due to its amplification in a fraction of human melanomas (26). Human Clear Cell Sarcomas typically contain a chromosomal translocation which produces the EWS-ATFl fusion protein (27-29). This chimeric oncoprotein directly triggers dysregulated expression of MITF, which in turn appears to play a vital role within this tumor (30). Clear Cell Sarcoma cells were also found to express SOXlO, which is required for MITF expression, as it is in melanocytes (30).

SOXlO belongs to a family of transcription factors related by homology to the high-mobility group (HMG) box region of the the testis-determining factor SR Y (31). The transcription factor SOXlO is necessary for the development of melanocytes and the enteric nervous system (32). SOXlO transactivates several gene promoters, including MITF, EDNRB, and c-RET (33-40). Germline heterozygous mutation of SOXlO produces Waardenburg Syndrome type 4 (melanocyte defects and megacolon) (for review see 41). Similarly, germline heterozygous mutation of MITF is responsible for Waardenburg Syndrome type 2a (melanocyte defects without megacolon), reflecting the epistatic relationship among these transcription factors. Although several targets of SOXlO have been identified, little is known regarding the mechanism(s) regulating expression of SOXlO (42, 43).

It has now been discovered that multiple HDAC inhibitor drugs, sodium butyrate (NaB), Trichostatin A (TSA), SAHA, and LBH589, repress MITF expression in melanoma and clear cell sarcoma cells. This repression is mediated by suppression of the SOXlO promoter. The resulting data suggest that in addition to MITF, SOXlO plays an important role in melanoma growth/survival and HDACi may represent a plausible small molecular approach to targeting suppression of these factors in melanoma and clear cell sarcoma. The invention in one aspect is a method for inhibiting, 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.

In one embodiment the area of skin that is treated is free of dermal malignancy. Dermal malignancy includes melanomas and any other type of skin cancer and cancerous skin lesions.

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, sun spots, solar lentigo, and solar and simple lentigines), 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, 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 MITF expression via inhibition of SOXlO expression, 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.

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 cm² to tens of cm². 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, emulsifϊers, 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.

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 HDAC inhibitor to reduce pigmentation of the skin. In one embodiment the locally administering is topically administering.

Certain HDAC inhibitors are described herein. HDAC inhibitors 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 the description that follows, the term "active agent" shall refer to a HDAC inhibitor or a skin lightening agent, as described herein.

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 tyrosinase inhibitors hydroquinone, kojic acid, kojic acid dipalmitate, arbutin, magnesium ascorbyl phosphate, and calcium D-pantetheine-S-sulfonate. In a preferred embodiment of the invention the HDAC inhibitor compound is not combined with retinoid.

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/cm to 5 mg/cm , 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 are not limited to local injection and topical use.

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.

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.

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.

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, lauric 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, l-methyl-2-pyrrolidone, l-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 40°C 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.

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.

Moreover, the inclusion of the non-ionic surfactant in the composition of this 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 also 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 of 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 2 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); polyoxyethylene 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 color 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).

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 (-COOH).

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 methyl cellulose. 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), poly(butyl 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), poly( vinyl 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.

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 (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. 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 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., hi: 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 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 emulsifϊers which can cause skin irritation or other adverse reactions. 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 polymers at pH conditions greater than 6.0 under vigorous stirring conditions, hi 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) , 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.

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.

Emulsifϊers (emulsifying agents) may be used in amounts effective to provide uniform blending of ingredients of the composition. Useful emulsifϊers 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 moφholinium ethosulfate; Alkyl dimethyl benzyl ammonium chloride; diisobutylphenoxytheoxyethyl dimethyl benzyl ammonium chloride; and cetyl pyridium chloride; Nonionics such as: polyoxyethylene 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 250°C; 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 250°C; and sucroglycerides.

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- 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; 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.

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; 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.

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 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 General Methods Cell cultures. UACC62 and UACC257 human melanoma cells were cultured in RPMI- 1640 medium (Mediatech Inc.) containing 10% fetal bovine serum (FBS) and penicillin/streptomycin/L-glutamine. MeWo and SK-MEL-5 human melanoma cells were cultured in DMEM medium (Mediatech Inc.) containing 10% FBS and penicillin/streptomycin/L-glutamine. 501mel human melanoma cells were cultured in Ham's F-IO medium (Mediatech Inc.) containing 10% FBS and penicillin/streptomycin/L-glutamine. Primary human melanocytes between passages 2 and 5 were derived from neonatal foreskins and grown in TIVA media (Ham's FlO (Mediatech Inc.), 7% fetal bovine serum, penicillin/streptomycin/glutamine (Invitrogen), I x IO^-4 M 3 -isobutyl-1 -methyl xanthine (IBMX; Sigma), 50 ng ml^"1 12-O-tetradecanoyl phorbol-13-acetate (TPA; Sigma), 1 μM Na₃VO₄, and 1 x 10^'3 M N⁶,2'-O- dibutyryladenosine 3:5-cyclic monophosphate (dbcAMP; Sigma)). The stable UACC62 transformant cells containing pLNCX-GFP, pLNCX-SOXIO, and pLNCX-SOXIO-mut were selected with G418 (500 μg/ml) (Mediatech, Inc), and referred to as UACC62/GFP (control), UACC62/SOX10, UACC62/SOX10-mut. These stable transformed lines were grown in RPMI- 1640 medium containing 10% FBS, 500 μg/ml G418, and penicillin/streptomycin/L-glutamine. The cells were treated with sodium butyrate (NaB) (2.5 mM) (Sigma-Aldrich, St. Louis, MO, USA), Trichostatin A (TSA) (1 μM) (Sigma- Aldrich), suberoylanilide hydroxamic acid (SAHA) (10 μM), or LBH589 (10 μM) (Novartis) for various times as indicated.

Vector generation. pLNCX-GFP, pLNCX-SOXIO, and pLNCX-SOXIO-mut were constructed from pLNCX retroviral vector (Clontech). To construct the luciferase reporter containing TK promoter (pGL3-TK), the TK promoter (Bglll/Hindlll fragment) was subcloned from pRL-TK and inserted into pGL3 -basic. The human SOXlO promoter region, the SOXlO enhancer sequences, and the mutated SOXlO enhancer sequences were generated by polymerase chain reaction (PCR) from the BAC clone, RP5-1039K5 (BACPAC Resource Center, Children's Hospital Oakland Research Institute, Oakland, California). The primers used were as follows: SOXlO promoter region: 5'-ggg gta ccc art tec cac ccc eta cac ccc ttt gga c-3' (sense) (SEQ ID NO: 1) and 5'-gaa gat eta gga agt gga aaa ccg tgt ccc aag gtg-3' (antisense) (SEQ ID NO: 2); SOXlO enhancer Forward: ggg gta cca gag aat ggt tct ctt gtt acc-3' (sense) (SEQ ID NO: 3) and 5'-ggg age teg aac aaa agg tec ctt tgt gg-3' (antisense) (SEQ ID NO: 4); mutated SOXlO enhancer Forward: 5'-ggg gta cca gag aat ggt tct ctC gtt ace aac a-3' (sense) (SEQ ID NO: 5) and 5'-ggg age teg aac Gaa agg tec ctt Cgt ggc get gc-3' (antisense) (SEQ ID NO: 6). The restriction enzyme sequences was changed for the reverse direction. Reporter plasmids containing the SOXlO promoter, pGL4.12-hSOX10p, were constructed by inserting 1.2-kb of the human SOXlO promoter region into pGL4.12. To construct the reporter plasmids containing the SOXlO enhancer or mutated SOXlO enhancer, the PCR products were inserted into the Kpnl/Sacl site of pGL3-TK or pGL4.12-hSOX10p.

Growth assays. Cell metabolic activity was assessed by Cell Proliferation Reagent WST-I (Roche Diagnostics) according to the manufacturer's instruction. Briefly, the UACC62, UACC257, normal human melanocytes, UACC62/GFP, UACC62/SOX10, or UACC62/SOX10-mut (1 x 10³ cells/well) were cultured for 24 h after plating in 96-well dishes and then the HDAC inhibitors, NaB (2.5 mM) or TSA (1 μM), were added to each well. After 24 or 48 h, the WST-I solution was added. The cells were incubated for an additional 1 h with the WST-I solution. Finally, the absorbance of 460 nm was measured using a microplate reader. Absorbance of cells treated with HDAC inhibitors was normalized to cells treated with the appropriate control, either PBS or ethanol vehicle.

Western blotting analysis. Whole cell extracts (10 μg / lane) were prepared using the method of Schreiber, E. et al. (44) and then subjected to western blotting analysis using anti-SOX10 polyclonal antibody (Santa Cruz Biotechnology), anti-MITF monoclonal antibody (45), anti-hemagglutinin (HA) monoclonal antibody (Roche Diagnostics), anti-acetyled histone 3 (AcH3) polyclonal antibody (Upstate Biotechnology) or anti-α-tubulin monoclonal antibody (Sigma).

Real-time RT-PCR analysis. The cDNAs were synthesized from the total RNAs of TSA-treated or untreated UACC62 or 501mel human melanoma cells. Realtime RT-PCR was performed by using iQ SYBR Green supermix (Bio-Rad) from cDNA or Quantitect probe RT-PCR Kit (Qiagen) from total RNA. The primers used for human SOXlO were 5 '-get get gaa cga aag tga ca-3' (sense) (SEQ ID NO: 7) and 5 '-gee tgg get ggt act tgt ag-3' (antisense) (SEQ ID NO: 8). In the case of M-MITF, we used the Taqman probe system. The primers for human M-MITF were 5 '-cat tgt tat get gga aat get aga a-3' (sense) (SEQ ID NO: 9) and 5'-ggc ttg ctg tat gtg gta ctt gg-3' (antisense) (SEQ ID NO: 10). The probe was 5'-6-FAM (6-carboxyfluorescein)-tca eta tea ggt gca gac cc acct cg-3' (SEQ ID NO: 11). All reactions were run in triplicate on an iCycler instrument (Bio-Rad), and SOXlO and M-MITFmRNA levels were normalized to glyceraldehyde-3- phosphate dehydrogenase expression.

RNA stability experiments. UACC62 melanoma cells were cultured for 24 h in fresh medium and exposed to actinomycin D (1 μg/ml) (Calbiochem). After incubation for 15 min, TSA (1 μM) was added and total RNA was collected at the indicated times and subjected to qPCR analyses.

Chromatin immunoprecipitation (ChIP) assay. ChIP was performed in UACC62 human melanoma cells treated with PBS, NaB, Ethanol, or TSA for 24 h as described previously (46). Chromatin was immunoprecipitated using antibodies against RNA polymerase II antibody (Covance) and human placental protein 14 antibody (Assay Designs Inc.) as a control. Quantitative PCR was performed on samples using primers for SOXlO (5'-TGG TTG GTG GTA AGG ATT CAG GCT-3' (SEQ ID NO: 12) and 5'- GGG CTC GTC CTT AGG AAG TGG AAA-3' (SEQ ID NO: 13)) andβ-actin (5'-CAT CCT CAC CCT GAA GTA CCC-3' (SEQ ID NO: 14) and 5'-TAG AAG TGT GGT GCC AGA TT-3'(SEQ ID NO: 15)).

Luciferase assays. Transciptional reporter activity of the human SOXlO enhancer was assessed by transient expression of firefly luciferase genes in UACC62, 501mel, and HeLa cells as described previously (30). Cells used were cultured for 24 h after plating in 24-well dishes and then transfected with each reporter plasmid (50 ng) and pRL-CMV (Promega) (1 ng) by the Lipofectamine2000 protocol (Invitrogen). pRL- CMV contains Renilla luciferase. After 24 h of transfection, cells were harvested in 100 μl of lysis buffer was and assayed by Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized by corresponding Renilla luciferase activity.

Colony forming assays. UACC62, UACC62/GFP, UACC62/SOX10, or UACC62/SOX10-mut (2 x 10² cells/well) were cultured for 24 h after plating in 6-well dishes and exposed to TSA (10 nM). After 24 h, the culture medium was changed into fresh medium without TSA and cultured for 7 days. Finally, the cells were fixed, stained by crystal violet, and counted. Example 1: HDACi drugs repress cell growth as well as M-MITF and SOXlO levels in melanoma cell lines

In order to examine the effects of HDAC inhibitors (HDACi) on human melanoma cell growth, WST-I assays were performed using primary human melanocytes (l^omel) or human melanoma cells treated with the HDACi drugs sodium butyrate (NaB) or Trichostatin A (TSA) (Fig. IA). Both NaB (2.5 mM) and TSA (1 μM) repressed cell growth at 48 h in UACC62 melanoma cells, UACC257 melanoma cells, and 1 "mel. The same growth inhibition was observed in other tested melanoma cell lines (data not shown). Since MITF is central to growth and proliferation of melanocytes and melanoma cells, we examined if the HDACi had any effects expression levels of MITF or its upstream regulator SOXlO. The melanocyte-specific isoform of MITF (M-MITF) migrates as a doublet (Fig IB, arrows) due to serine 73 phosphorylation by MAPK (47), whereas higher migrating MITF isoforms (asterisk) reflect non-melanocyte-specific forms of MITF derived from distinct promoters (15, 16). HDACi treatments were seen to reproducibly suppress expression of M-MITF doublet bands, but not the non- melanocytic band (in some cases even being associated with some degree of upregulation). M-MITF suppression was observed in all melanoma cell lines examined (Fig IB and data not shown). Other HDACi drugs, suberoylanilide hydroxamic acid (SAHA) and LBH589, also repressed M-MITF expression in melanoma cells (Fig IB). This M-MITF specific suppression pattern suggested the possibility that HDACi drugs were influencing M-MITF promoter activity. The acetylation status of histone H3 by HDACi in human melanoma cells was examined to confirm the activity of the HDACi drugs. The effects of HDACi in human clear cell sarcoma cells were also examined, which have also been shown to express M-MITF (30) (Fig. 1C). M-MITF suppression was observed in all three tested clear cell sarcoma cell lines. Since SOXlO is a key transcriptional regulator of the M-MITF promoter, we examined SOXlO expression after the same treatments. As shown in Fig. IB and 1C, HDACi treatments were seen to potently suppress SOXlO protein levels, following a parallel pattern seen for M-MITF.

Example 2: Overexpression of SOXlO can rescue the repression of M-MITF

To test whether the repression of M-MITF by HDACi is dependent on the suppression of SOXlO, stable melanoma cell lines were generated expressing CMV promoter-driven SOXlO, or SOX10-mut, (encoding a non-functional SOXlO mutant), and GFP (as a negative control) by retroviral transduction. Whereas endogenous SOXlO was suppressed by HDACi in all of these cell lines, M-MITF expression was suppressed by HDACi treatment of GFP (control) and SOXIO-mut (control) cells, but M-MITF levels were rescued from HDACi-induced suppression in SOXlO overexpressing cells (Fig 2). In addition, CMV-promoter-driven SOXlO levels (HA-tagged) were not altered by HDACi treatment, in the same cells exhibiting suppression of endogenous SOXlO. This observation suggests that HDACi affect SOXlO levels via effects on transcription or RNA processing, rather than post-translationally. Moreover the repression of M-MITF by HDACi is likely to be mediated by SOXlO in human melanoma cells since M-MITF levels are rescued by ectopic SOXlO.

Example 3: HDAC inhibitors repress transcription of the SOXlO gene

To analyze by which mechanism HDACi influences SOXlO and M-MITF mRNA levels, quantitative RT-PCR (Fig. 3A) were carried out. In UACC62 and 501mel cells, SOXlO and M-MITF mRNA were both potently suppressed by TSA. The effects of HDACi on SOXlO and M-MITF mRNA stability were also examined using Actinomycin D (measuring mRNA decay kinetics). Presence or absence of HDACi treatment did not measurably affect SOXlO or M-MITFmKNA stability (Fig. 3B). To study the effects of HDACi on SOXlO transcription, quantitative chromatin immunoprecipitation (q-ChIP) was performed of RNA polymerase II (PoI-II) to directly assess ongoing transcriptional activity at the SOXlO locus in UACC62 cells treated with vehicle (PBS or ethanol) or HDACi (NaB or TSA) (Fig. 3C). The PCR primer sets were designed to amplify a DNA segment within exon 1 of SOXlO and exon 3 of β-actin as control. The ChIP assay revealed significantly lower occupancy of PoI-II at the SOXlO gene in HDACi-treated UACC62 cells than in vehicle-treated UACC62 cells. The β-actin gene showed only small changes in the same assay from the same cells treated with the same drugs (Fig 3C). These data suggest that transcription of the SOXlO gene is significantly repressed by HDACi.

Example 4: SOXlO enhancer-element suppression by HDACi A SOXlO enhancer region has been described and shown to activate bi- directionally with the reverse direction showing higher enhancer activity (42, 43). We examined the effects of HDACi on the activity of the SOXlO enhancer using a luciferase reporter construct (Fig. 4A). UACC62 and 501mel cells (melanomas) showed activity of SOXlO enhancer, but HeLa cells (non-melanoma), which do not express SOXlO, did not. Reporter activity from the SOXlO enhancer was reduced by HDACi. These data are consistent with the possibility that HDACi may repress SOXlO expression via effects at the SOXlO enhancer region. In this particular enhancer region, there are three TCF/LEF/SOX family/SRY-binding sites. We examined which TCF/LEF/SOX family/SRY-binding sites are required for enhancer function by using several mutated enhancer constructs. The enhancer sequence mutated in each TCF/LEF/SOX family/SRY-binding site did not show any activity in reporter assay (data not shown), suggesting that all three TCF/LEF/SOX family/SRY-binding sites are required for the SOXlO enhancer function. Next we examined the effects of the bi-directional enhancer on SOXlO promoter activity (Fig. 4B). As previously reported, the SOXlO enhancer worked bi-directionally when spliced next to the SOXlO promoter. Moreover, the enhancer reporter which was mutated in TCF/LEF/SOX family/SRY-binding sites did not show enhancer activity. These results suggest that TCF/LEF/SOX family/SRY- binding sites are important for enhancer activity.

Example 5: HDACi affect cell growth/survival — rescue by ectopic SOXlO

The effects of HDACi on cell survival were examined using a colony-forming assay (Fig. 5). In this assay, melanoma cells were exposed to TSA for 24 hours, followed by drug wash-out and subsequent clonogenic growth assay. Under these conditions UACC62 cells displayed significant suppression of clonogenic growth, whereas SOXlO- overexpressing cells exhibited significant rescue of colony formation, relative to GFP- or SOXIO-mut overexpressing cells (Fig. 5, upper panel). The survival rate in SOXlO- overexpressing cells (0.84, normalized to ethanol-treated cells) after TSA treatment was significantly higher than that in UACC62, GFP-, or SOXIO-mut overexpressing cells (0.48, 0.51, or 0.51, respectively) (Fig. 5, lower panel). Collectively these data demonstrate suppression of SOXlO and M-MITF in melanoma cells, together with altered cell viability upon HDACi exposure, with the latter effect being largely rescued by the singular overexpression of SOXlO.

HDACi can suppress melanoma growth (Fig. IA) and repress M-MITF expression in human melanoma (Fig. IB) and clear cell sarcoma cells (Fig.1C). MITF is an oncogene in melanoma cells and is also critical for growth/survival of clear cell sarcoma cells (30). Clear cell sarcoma cells express EWS-ATFl (27-29), which induces "misexpression" of the melanocyte isoform of MITF in a fashion which retains a dependency on SOXlO (30). This melanocyte feature suggested the possibility that the growth/survival of clear cell sarcoma may, like melanoma cells, be susceptible to HDACi-mediated suppression.

Previously identified upstream SOXlO enhancer (42, 43) is specifically repressed by HDACi and that DNA consensuses sequence elements for TCF/LEF/SOX family/SRY-binding sites appear to be required for this enhancer activity. It remains uncertain how HDAC suppression modulates transcriptional activity of the SOXlO gene, including whether this is a direct or indirect effect. HDAC enzymes are involved in the acetylation of non- histone proteins (as well as histones). It is thus possible that the acetylation of transcriptional activators by HDACi inhibits such factors and their ability to stimulate the SOXlO enhancer region. HDACi drugs may also upregulate expression of a transcriptional repressor which acts on the SOXlO enhancer. Alternatively, HDACi might lead to an altered chromatin structure over the SOXlO regulatory region in which it becomes disabled for appropriate context-dependent transactivation through disrupted critical insulator or boundary effects.

SOXlO overexpression could partially rescue the cell growth inhibition induced by HDAC inhibition, a finding of some note, given the predicted ubiquitous consequences of suppressing histone deacetylation (Fig. 5). The rescue from the HDACi- induced repression of cell survival by SOXlO overexpression also suggests that SOXlO participates in melanoma survival. Mutation of SOXlO causes defects in vagal and trunk neural crest derivatives (enteric ganglia, melanocytes, and dorsal root ganglia) in humans, mice and zebrafϊsh (32, 48-50). However certain neural crest-derived embryonic structures are present in homozygous SOXlO⁰⁰"¹ mutant embryos (48, 49, 51, 52), suggesting that SOXlO is not necessarily required for the initial specification or migration of neural crest cells, but may participate in neural crest lineage survival. Example 6: Sirtinol induces p53

The effects of a class three HDAC inhibitor sirtinol were examined on p 53 induction in keratinocytes. Figure 6 clearly shows that sirtinol upregulated P53 in keratinocytes in a dose dependent manner.

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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.

Having thus described several aspects of at least one embodiment of this invention, it is to be appreciated various alterations, modifications, and improvements will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of this disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawings are by way of example only.

What is claimed is:

Claims

1. A method of inhibiting unwanted skin pigmentation, comprising locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase.

2. The method of claim 1, further comprising repeatedly locally administering to the area of skin of the subject an effective amount of the histone deacetylase inhibitor to maintain an inhibited amount of skin pigmentation, wherein the histone deacetylase inhibitor is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase.

3. A method of inhibiting unwanted skin pigmentation, comprising locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the histone deacetylase inhibitor is selected based on its inability to induce p53.

4. A method of inhibiting unwanted skin pigmentation, comprising locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor, wherein the effective amount of the histone deacetylase inhibitor is insufficent to induce p53.

5. The method of any one of claims 1-4, wherein the unwanted skin pigmentation is hyperpigmentation associated with a condition selected from acanthosis nigricans, Addison's disease, biliary cirrhosis, cafe au lait spots, ectopic ACTH syndrome, eosinophilia-myalgia syndrome, ephelides (freckles), folate deficiency, hemochromatosis, junctional and compound nevi, lentigo, malabsorption, 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.

6. The method of any of claims 1-5, wherein the locally administering is topically administering.

7. The method of any of claims 1-6, wherein the area of skin is free of dermal malignancy.

8. A method of inhibiting unwanted skin pigmentation, comprising locally administering to an area of skin of a subject, wherein the area of skin has an unwanted amount of pigmentation, an effective amount of a histone deacetylase inhibitor in combination with an effective amount of skin lightening agent, wherein the skin lightening agent is not retinoid.

9. The method of claim 8 wherein the skin lightening agent is leptomycin B.

10. The method of claim 8 wherein the skin lightening agent is a tyrosinase inhibitor.

11. The method of claim 8 wherein the tyrosinase inhibitor is hydroquinone, kojic acid, kojic acid dipalmitate, arbutin, magnesium ascorbyl phosphate, or calcium D- pantetheine-S-sulfonate.

12. The method of claim 8 wherein the histone deacetylase inhibitor is a histone deacetylase inhibitor that is at least 10-fold more effective in inhibiting a class I or class II histone deacetylase in comparison to a class III histone deacetylase.

13. The method of claims 12 wherein the class III histone deacetylase inhibitor is sirtinol.

14. The method of claim 8, wherein the histone deacetylase inhibitor is selected based on its inability to induce p53.

15. The method of claim 8, further comprising repeatedly locally administering to the area of skin of the subject an effective amount of the histone deacetylase inhibitor in combination with an effective amount of skin lightening agent, wherein the skin lightening agent is not retinol, to maintain an inhibited amount of skin pigmentation.

16. The method of any of claims 8-15 wherein the locally administering is topically administering.

17. The method of any of claims 8- 16, wherein the area of skin is free of dermal malignancy.

18. The method of any one of claims 1-17, wherein the histone deacetylase inhibitor is selected from a short-chain fatty acid, a hydroxamic acid, an epoxyketone- containing cyclic tetrapeptide, a cyclic tetrapeptide, a benzamide, and Depudecin.

19. The method of claim 18, wherein the histone deacetylase inhibitor is a short-chain fatty acid.

20. The method of claim 19, wherein the short-chain fatty acid is selected from a butyrate, a phenylbutyrate, and a valproate.

21. The method of claim 19, wherein the short-chain fatty acid is sodium butyrate.

22. The method of claim 18, wherein the histone deacetylase inhibitor is a hydroxamic acid.

23. The method of claim 22, wherein the hydroxamic acid is selected from a trichostatin, SAHA, oxamflatin, ABHA, Scriptaid, Pyroxamide, LBH589, and Propenamide.

24. The method of claim 23, wherein the hydroxamic acid is Trichostatin A (TSA).

25. The method of claim 23, wherein the hydroxamic acid is SAHA.

26. The method of claim 23, wherein the hydroxamic acid is LBH589.

27. The method of claim 18, wherein the histone deacetylase inhibitor is an epoxyketone-containing cyclic tetrapeptide.

28. The method of claim 27, wherein the epoxyketone-containing cyclic tetrapeptide is selected from trapoxin, HC-toxin, Chlamydocin, Diheteropeptin, WF- 3161, CyI-I, and Cyl-2.

29. The method of claim 18, wherein the histone deacetylase inhibitor is a cyclic tetrapeptide.

30. The method of claim 29, wherein the cyclic tetrapeptide is selected from FR901228, Apicidin, and a cyclic-hydroxamic-acid-containing peptide (CHAP).

31. The method of claim 18, wherein the histone deacetylase inhibitor is a benzamide.

32. The method of claim 31 , wherein the benzamide is selected from MS-275 (MS-27-275) and CI-994.

33. The method of claim 18, wherein the histone deacetylase inhibitor is Depudecin.