NZ786010A - Malassezin and analogs thereof as skin brightening agents - Google Patents

Abstract

The present invention relates to compounds, compositions, and methods for brightening skin. The compounds, compositions, and methods of the present invention generally involve compounds produced by a Malassezia yeast, and chemical analogs thereof. In addition to skin brightening applications, the compounds, compositions, and methods of the present invention may be used to modulate melanocyte activity, induce melanocyte apoptosis, agonize an arylhydrocarbon receptor (AhR), improve hyperpigmentation caused by a hyperpigmentation disorder, and modulate melanin production, melanosome biogenesis, and melanosome transfer.

Description

MALASSEZIN AND ANALOGS F AS SKIN BRIGHTENING AGENTS CROSS-REFERENCE TO RELATED ATIONS The present invention claims benefit to U.S. provisional application serial number ,468, filed March 10, 2016. The entire contents of that application are incorporated by reference.

FIELD OF INVENTION The present invention relates to chemical analogs of compounds produced by a Ma/assezia yeast. The invention includes compositions comprising compounds produced by a Ma/assezia yeast as well as chemical analogs of compounds produced by a Ma/assezia yeast. Methods of using the compounds (including analogs thereof) and compositions of the present invention are also contemplated.

BACKGROUND OF THE INVENTION Individuals around the world use skin brightening agents to achieve a number of cosmetic goals, including producing an anti—aging , correcting sun damage, and meeting certain cultural standards of . Many commercially ble skin brightening products, while effective to varying degrees, contain l ingredients, some of which have been linked to cancer.

Thus, there exists a need for novel skin brightening agents and formulations that exhibit higher levels of safety and/or efficacy than agents currently on the market.

Ma/assezia is a genus of lipophilic yeast commonly found in the normal flora of human skin. Ma/assezia is responsible for a number of skin diseases, including tinea versicolor (pityriasis versicolor), seborrheic dermatitis, and atopic dermatitis.

Tinea versicolor is a ntagious skin disease caused by Ma/assezia overgrowth that locally alters pigmentation levels. Ma/assezia yeasts have two metabolic pathways for synthesizing melanin and tryptophan—derived indole pigments. The indole pigments e malassezin, a tryptophan metabolite of Ma/assezia that may elicit cyte apoptosis and contribute to the entation characteristic of Ma/assezia overgrowth.

The ion disclosed herein utilizes compounds produced by Ma/assezia yeast, including malassezin, and chemical analogs thereof, as the basis for safe and efficacious skin brightening compositions.

SUMMARY OF THE INVENTION One embodiment of the present invention is a compound for ening skin. The compound is a al analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or ceutically acceptable salt thereof.

Another embodiment of the present invention is a compound for inducing melanocyte apoptosis. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a nd for modulating cyte activity. The compound is a al analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

An additional ment of the present invention is a compound for agonizing the arylhydrocarbon receptor (AhR). The nd is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Another ment of the present invention is a compound for improving hyperpigmentation caused by a hyperpigmentation disorder. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the t invention is a compound for modulating melanin production. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a lline form, hydrate, or cosmetically or pharmaceutically able salt thereof.

WO 56424 2017/021843 An additional embodiment of the present invention is a compound for modulating melanosome biogenesis. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a nd for modulating melanosome transfer. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a lline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a composition.

The composition comprises a Ma/assezia yeast and a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier.

An additional embodiment of the present invention is a composition.

The composition comprises a compound isolated or isolatable from a Ma/assezia yeast and a cosmetically or ceutically able vehicle, diluent or carrier.

Another embodiment of the present invention is a composition. The composition comprises any of the compounds, including analogs, disclosed herein and a ically or ceutically acceptable vehicle, diluent or carrier.

A further embodiment of the present invention is a method of brightening skin in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

An additional embodiment of the present invention is a method for inducing me|anocyte sis in a subject. The method comprises contacting the subject with any of the compounds or compositions sed herein.

Another embodiment of the t invention is a method for modulating me|anocyte activity in a subject. The method comprises ting the subject with any of the compounds or compositions disclosed herein.

A further ment of the present invention is a method for agonizing an arylhydrocarbon receptor (AhR) in a subject. The method comprises ting the subject with any of the compounds or itions disclosed herein.

An additional embodiment of the present invention is a method for improving hyperpigmentation caused by a hyperpigmentation disorder in a subject in need thereof. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

Another embodiment of the present invention is a method for ting melanin production in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for modulating some biogenesis in a subject. The method comprises contacting the subject with any of the nds or compositions disclosed herein.

An additional embodiment of the present invention is a method for modulating melanosome transfer in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

Another embodiment of the present invention is a nd. The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of en and methyl, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 is methyl; or a crystalline form, e, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present ion is a compound. The compound has the structure of a (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

An additional embodiment of the present invention is a compound for ening skin. The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or ically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound for brightening skin. The compound has the structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and ; or a crystalline form, e, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a compound for inducing melanocyte apoptosis. The compound has the ure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt f.

An additional embodiment of the present invention is a compound for inducing melanocyte sis. The compound has the structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently ed from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a compound for agonizing the arylhydrocarbon receptor (AhR). The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently ed from the group consisting of en and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further ment of the present invention is a compound for agonizing the arylhydrocarbon receptor (AhR). The compound has the structure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a lline form, hydrate, or ically or pharmaceutically able salt thereof.

An additional embodiment of the present invention is a composition. The ition comprises a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof, and a ically or pharmaceutically acceptable vehicle, diluent or carrier.

Another embodiment of the present invention is a composition. The composition comprises a compound having the structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group ting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt f, and a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier.

A further embodiment of the present invention is a method for brightening skin in a t. The method comprises: contacting the subject with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or ceutically acceptable salt f.

An additional embodiment of the present invention is a method for brightening skin in a subject. The method comprises: contacting the subject with a compound having the ure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a lline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is a method for inducing melanocyte apoptosis in a subject. The method comprises: contacting the subject with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are ndently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present ion is a method for inducing melanocyte apoptosis in a subject. The method comprises: contacting the subject with a compound having the structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or ceutically acceptable salt thereof.

Another embodiment of the present invention is a method for agonizing an arylhydrocarbon or (AhR) in a t. The method comprises: contacting the t with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

A further embodiment of the present invention is a method for agonizing an arylhydrocarbon receptor (AhR) in a subject. The method comprises: contacting the subject with a compound having the ure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group ting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt f.

BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon t and payment of the necessary fee.

Fig. 1A is a schematic diagram of the skin’s component layers. The inset diagram shows the cellular makeup of the epidermis and dermis. Fig. 18 is a schematic diagram showing potential mechanisms of action of hypopigmentation—causing agents.

Fig. 2 is a set of synthetic schemes for malassezin and malassezin derivatives: Fig. 2A: malassezin and indolo[3,2—b] ole; Fig. 28: compounds land IV; Fig. 20: compound II.

Fig. 3A is a summary chart showing E050 values of annexin V ion for certain compounds of the present invention in MeWo and WM115 cells. Figs. 38—3M are line graphs showing the percentage of MeWo (Figs. 38— 3G) or WM115 (Figs. 3H—3M) cells d with annexin V after exposure to various concentrations of the listed compounds.

Figs. 4A—4D are charts showing relative annexin V levels (%) in MeWo and WM115 cells after exposure to s concentrations of the listed compounds for 6, 24, 48, and 72 hours. Figs. 4E—4J are histograms showing results from Figs. 4A—4D. Figs. 4K and 4L are histograms showing the percentage of MeWo (Fig. 4K) and WM115 (Fig. 4L) cells labeled with annexin V after 6—hour exposure to the listed compounds at the concentrations shown.

Figs. 5A—5K are micrographs showing MeWo cell logy after 6 hours of treatment with various concentrations of CV—8684, CV—8685, CV—8688, DMSO, and staurosporine.

Figs. 6A—6K are micrographs showing MeWo cell morphology after 24 hours of treatment with s trations of CV—8684, CV—8685, CV— 8688, DMSO, and staurosporine.

Figs. 7A—7K are micrographs showing MeWo cell morphology after 48 hours of treatment with various concentrations of CV—8684, CV—8685, CV— 8688, DMSO, and staurosporine.

Figs. 8A—8K are micrographs g MeWo cell morphology after 72 hours of treatment with s concentrations of CV—8684, CV—8685, CV— 8688, DMSO, and staurosporine.

Figs. 9A—9K are micrographs showing WM115 cell morphology after 6 hours of treatment with s concentrations of 4, CV—8685, CV—8688, DMSO, and staurosporine.

Figs. 10A—10K are micrographs showing WM115 cell morphology after 24 hours of treatment with various concentrations of CV—8684, CV—8685, CV—8688, DMSO, and staurosporine.

Figs. 11A—11K are micrographs showing WM115 cell morphology after 48 hours of treatment with various concentrations of CV—8684, CV—8685, 8, DMSO, and sporine.

Figs. 12A—12K are micrographs showing WM115 cell morphology after 72 hours of treatment with various concentrations of CV—8684, CV—8685, CV—8688, DMSO, and staurosporine.

Figs. 13A—13D are charts showing the percentage of viable MeWo and WM115 cells remaining after treatment with various concentrations of CV— 8684 (Fig. 13A), CV—8685 (Fig. 138), CV—8688 (Fig. 130), or staurosporine (Fig. 13D) for 6, 24, 48, and 72 hours. Cell viability was assayed using CellTiter—Glo®.

Figs. 13E—13J are histograms showing results from Figs. 13A—13D. Fig. 13K is a summary chart comparing percentages of viable MeWo and WM115 cells after WO 56424 re to the listed trations of malassezin, indolocarbazole, compound II, and staurosporine for 24, 48, and 72 hours.

Figs. 14A—14D are charts showing levels of lactate dehydrogenase ("LDH") release from MeWo and WM115 cells after treatment with various concentrations of CV—8684 (Fig. 14A), CV—8685 (Fig. 148), CV—8688 (Fig. 140), or staurosporine (Fig. 14D) for 6, 24, 48, and 72 hours. Figs. 14E—14J are histograms showing results from Figs. 14A—14D. Figs. 14K and 14L are histograms showing lactate dehydrogenase levels after exposing MeWo (Fig. 14K) and WM115 (Fig. 14L) cells to the listed concentrations of malassezin, carbazole, compound II, and staurosporine for 24 hours.

Figs. 15A—15E show raw data and line graphs of arylhydrocarbon receptor ("AhR") activation in HepG2 cells stably transfected with an AhR— responsive luciferase reporter gene plasmid upon exposure to various trations of omeprazole (Fig. 15A), CV—8684 (Fig. 158), CV—8685 (Fig. 150), CV—8686 (Fig. 15D), and CV—8688 (Fig. 15E). Fig. 15F shows E05O values for each compound tested.

Figs. 16A—16K are photographs of MelanoDermTM matrices at either day 0 or day 7 after exposure to no treatment (Fig. 16A), sterile zed water (Fig. 168), 1% kojic acid (Fig. 160), 0.2% DMSO (Fig. 16D), 0.05% DMSO (Fig. 16E), 200 pM CV—8684 (Fig. 16F), 50 pM CV—8684 (Fig. 16G), 200 pM CV—8686 (Fig. 16H), 50 [1M CV—8686 (Fig. 16l), 200 pM CV—8688 (Fig. 16J), and 50 pM CV—8688 (Fig. 16K).

Figs. 17A—17K are 15X magnification icrographs of DermTM matrices at either day 0 or day 7 after exposure to no treatment (Fig. 17A), sterile deionized water (Fig. 178), 1% kojic acid (Fig. 170), 0.2% DMSO (Fig. 17D), 0.05% DMSO (Fig. 17E), 200 (M CV—8684 (Fig. 17F), 50 (M CV—8684 (Fig. 17G), 200 pM CV—8686 (Fig. 17H), 50 pM CV—8686 (Fig. 17l), 200 pM 8 (Fig. 17J), and 50 pM CV—8688 (Fig. 17K).

Figs. 18A—18F are photographs of zebrafish exposed to no treatment (Fig. 18A), DMSO (Fig. 188), phenylthiourea ("PTU") (Fig. 180), and compound II at 2.5 [M (Fig. 18D), 5 [M (Fig. 18E), and 10 (M (Fig. 18F). Red arrows indicate normal melanocytes.

Figs. 19A—19F are photographs of zebrafish exposed to no treatment (Fig. 19A), DMSO (Fig. 198), phenylthiourea ("PTU") (Fig. 190), and compound II at 0.3 (M (Fig. 19D), 1 (M (Fig. 19E), and 3 (M (Fig. 19F). Red arrows indicate normal melanocytes. Yellow arrows indicate abnormally small cytes.

Fig. 20 is a summary chart showing the number and percent of zebrafish with decreased skin pigmentation after exposure to the listed ions. The final six rows show the effects of various concentrations of compound ll.

Figs. 21A—21E are photographs of zebrafish treated with no treatment (Fig. 21A), DMSO (Fig. 218), PTU (Fig. 210), 0.5 (M (Fig. 21D), and 1.5 (M (Fig. 21 E). Bottom panels include regions of color scheme inversion.

Figs. 22A and 22B are histograms showing pigmentation density as measured by pigmented pixels / mm3 (Fig. 22A) and total pixels (Fig. 228) from photographs of zebrafish embryos, ified in Figs. 21A—21 E.

Figs. 23A—230 are mass spectra of CV—8684 in DMSO (Fig. 23A), RPMI media (Fig. 238), and DMEM (Fig. 230). Figs. 23D—23F are mass spectra of CV-8686 in DMSO (Fig. 230), RPMI media (Fig. 23E), and DMEM (Fig. 23F).

Figs. 23G—23l are mass spectra of CV—8688 in DMSO (Fig. 23G), RPMI media (Fig. 23H), and DMEM (Fig. 23l). Fig. 23J is a summary chart showing percent of test compound remaining in the listed solvent after 2—hour incubation.

DETAILED DESCRIPTION OF THE INVENTION One embodiment of the present invention is a compound for brightening skin. The compound is a al analog of a nd produced by a Ma/assezia yeast, or a crystalline form, e, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the term "compound" refers to two or more atoms that are ted by one or more chemical bonds. In the present invention, chemical bonds include, but are not limited to, nt bonds, ionic bonds, hydrogen bonds, and van der Waals interactions. Covalent bonds of the present invention e single, double, and triple bonds. Compounds of the present invention include, but are not limited to, organic molecules.

Organic compounds/molecules of the present ion include linear, branched, and cyclic hydrocarbons with or without functional groups. The term "Cx.y’ when used in conjunction with a al moiety, such as, alkyl, l, alkynyl or alkoxy is meant to include groups that contain from x to y carbons in the chain. For example, the term "CX.y alkyl" means substituted or unsubstituted ted hydrocarbon groups, including straight—chain alkyl and branched—chain alkyl groups that contain from x to y carbons in the chain, including haloalkyl groups such as oromethyl and 2,2,2—trifluoroethyl, etc.

The terms "CX.y alkenyl" and "CX.y alkynyl" refer to tuted or unsubstituted unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but containing at least one double or triple bond respectively.

The term "aliphatic", as used , means a group composed of carbon and hydrogen atoms that does not contain aromatic rings. Accordingly, aliphatic groups include alkyl, alkenyl, alkynyl, and carbocyclyl groups.

The term "alkyl" means the radical of saturated aliphatic groups that does not have a ring structure, including straight chain alkyl groups, and branched chain alkyl groups.

The term "alkenyl", as used , means an aliphatic group containing at least one double bond.

The term yl", as used herein, means an aliphatic group containing at least one triple bond.

As used herein, an "aromatic compound , aromatic", or compound containing an "aromatic ring" is an aryl or a heteroaryl compound. The term "aryl" as used herein es substituted or unsubstituted single—ring aromatic groups in which each atom of the ring is carbon. Preferably the ring is a 3— to 8—membered ring, more preferably a 6—membered ring. The term "aryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groups include benzene, alene, phenanthrene, phenol, aniline, and the like. The term "heteroaryl" includes substituted or tituted aromatic single ring structures, preferably 3— to 8—membered rings, more preferably 5— to 7—membered rings, even more preferably 5— to 6—membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. The term oaryl" also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, lkynyls, aryls, heteroaryls, and/or heterocyclyls. Heteroaryl groups e, for example, e, furan, thiophene, indole, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Preferably, certain compounds of the present invention include at least one, preferably two, indole groups as well as at least one aldehyde group.

The term "substituted" means moieties having at least one tuent that replaces a hydrogen atom on one or more carbons of the backbone. It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously o transformation such as by rearrangement, cyclization, elimination, etc. The permissible substituents can be one or more and the same or different for appropriate organic compounds.

As used herein, "skin brightening" and grammatical variations thereof refers generally to any actual or ved reduction in skin pigmentation.

Skin brightening methods have been used to reduce pigmentation of hyperpigmented areas of skin resulting from age, sun re, or a hyperpigmentation disorder. Application of the compounds and compositions of the present invention to, for e, a subject’s skin, can reduce pigmentation so that the skin appears lighter or whiter than before said application. Skin pigmentation can be assessed in a number of ways, including, but not limited to, visual assessments using, for example, the von Luschan chromatic scale, the trick skin typing test (Fitzpatrick et a/., 1988) and the Taylor Hyperpigmentation Scale (Taylor et a/., 2005) and reflectance spectrophotometry methods (Zonios, et a/., 2001). For example, the Fitzpatrick skin typing test includes six types of skin (l—Vl), and Type Vl skin that becomes Type V or less has been tened" as the term is used . As discussed further below, skin ening can result due to a number of phenomena, including, but not limited to, modulation of melanocyte activity, induction of melanocyte apoptosis, agonism of an arylhydrocarbon or (AhR), or modulation of melanin production, melanosome biogenesis, or melanosome transfer.

Certain compounds of the present invention are produced by, isolated from, or isolatable from a ezia yeast. Ma/assezia yeasts are yeasts of the genus Ma/assezia and include, but are not limited to, Ma/assezia globosa, Ma/assezia restricta, Ma/assezia furfur, Ma/assezia sympodia/is, Ma/assezia s/ooffiae, Ma/assezia obtusa, Ma/assezia ermatis, Ma/assezia dermatis, Ma/assezia japonica, Ma/assezia nana, Ma/assezia yamatoensis, Ma/assezia equine, Ma/assezia , and Ma/assezia /i. (Guého, et a/., 1996; Gaitanis, et a/., 2013). Ma/assezia yeast are part of the normal human cutaneous flora and typically produce no pathogenic effects. However, Ma/assezia yeast can cause a number of diseases, including, but not limited to pityriasis versicolor (both the hyperpigmented and hypopigmented varieties), seborrheic itis, dandruff, atopic dermatitis, Ma/assezia folliculitis, psoriasis, and confluent and reticulated papillomatosis. (Gaitanis, eta/., 2013).

As used herein, the term "chemical analog" refers to a compound that is structurally related to a parent compound and contains different functional groups or substituents. For e, a parent compound of the present ion is malassezin, and chemical analogs of malassezin contain certain functional groups and substituents that are ct from malassezin. Chemical analogs of the present invention may have icant advantages over a given parent compound, including a pharmacokinetic profile suitable for cosmetic use.

In some embodiments, a chemical analog is generated from a parent molecule by one or more chemical reactions. In other embodiments, ative synthesis s that do not ate with a parent compound can be used to generate al analogs of the present invention.

A compound of the present invention is "produced by a Ma/assezia yeast" if, over the course of its lifecycle, a Ma/assezia yeast would synthesize, secrete, accumulate, or otherwise generate the compound under appropriate growth ions. Ma/assezia yeast secrete different compounds depending on what their growth media is supplemented with. (Nazzaro—Porro, et a/., 1978).

The present invention includes any compound produced by a Ma/assezia yeast WO 56424 under any growth condition, but preferred compounds include, for e, malassezin and chemical analogs thereof.

In one aspect of this embodiment, the compound produced by a Malassezia yeast has the structure of formula (I): In another aspect of this embodiment, the nd is a chemical analog of malassezin.

Malassezin is one example of a compound produced by a Malassezia yeast of the present invention. Malassezin, also known as 2—(1H— indol—3—ylmethyl)—1H—indole—3—carbaldehyde, is a tryptophan lite originally isolated from Malassezia furfur. Malassezin is a known agonist of the arylhydrocarbon receptor (AhR), a receptor implicated in cell growth, differentiation, and gene expression. (Wille et al., 2001). Malassezin also induces apoptosis in y human melanocytes. (Kramer, et al., 2005).

Recently, certain chemical analogs of malassezin were synthesized by Winston— McPherson and colleagues, who examined the analogs’ AhR t activity.

(Winston—McPherson, eta/., 2014).

Another embodiment of the t invention is a compound for inducing melanocyte apoptosis. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the term "melanocyte" refers to a dendritic cell of the epidermis that normally synthesizes tyrosinase and, within melanosomes, the pigment melanin. Melanocytes of the present ion exhibit upregulation of certain genes, including, but not limited to, one or more of the following: tyrosinase (oculocutaneous albinism IA), microphthalmia—associated transcription factor, alpha—2—macroglobulin, tyrosinase—related protein 1, solute carrier family 16, G83955 protein, v—kit Hardy—Zuckerman 4 feline sarcoma, ocular albinism 1, Rag D protein, glycogenin 2, G—protein—coupled or, family C, oculocutaneous albinism ll, deleted in geal cancer 1, A, SRY—box , ATPase, Class V, type 100, matrix metalloproteinase 1, latent transforming growth factor beta b, ATP—binding te, sub—family C, yprostaglandin dehydrogenase 15, transmembrane 7 superfamily member 1, glutaminyl—peptide cyclotransferase, and other genes identified by Lee and colleagues. (Lee, et a/., 2013).

Melanocytes, like many other cell types, undergo programmed cell death or, sis. Melanocyte apoptosis pathways are known to those of skill in the art (Wang, et a/., 2014), and apoptosis pathways generally have been reviewed by Elmore (Elmore, 2007). A compound or composition of the present invention "induces" cyte sis by, for example, causing the activation of certain optotic signal transduction ys or causing the repression of certain anti—apoptotic pathways in a melanocyte. It is envisioned that the compound or composition of the present invention can directly activate/repress an apoptosis—related pathway by directly interacting with a signaling molecule of the pathway or by indirectly interacting with a molecule of the pathway via direct interaction with one or more intermediary molecules that do not typically function within the pathway.

In one aspect of this embodiment, the compound produced by a Ma/assezia yeast has the structure of formula (I): In another aspect of this embodiment, the compound is a chemical analog of malassezin.

A further ment of the present invention is a compound for ting melanocyte activity. The compound is a chemical analog of a compound produced by a ezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

Melanocyte activity can be modulated in a number of ways contemplated in the present invention, including, but not limited to, inducing melanocyte sis or altering melanocyte gene expression, cell ty, cell growth, n production, melanosome biogenesis, or melanosome transfer.

In one aspect of this embodiment, the compound produced by a Malassezia yeast has the structure of formula (I): In another aspect of this embodiment, the compound is a chemical analog of malassezin.

An additional embodiment of the present ion is a compound for agonizing the arylhydrocarbon receptor (AhR). The nd is a chemical analog of a compound produced by a Malassezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

As used herein, the terms "agonist", "agonizing", and tical variations thereof refer to a molecule that triggers (e.g., initiates or promotes), partially or fully enhances, stimulates or tes one or more biological ties. Agonists of the present invention include naturally occurring substances as well as synthetic nces.

An arylhydrocarbon receptor (AhR) of the present invention is any arylhydrocarbon receptor that naturally exists in a subject as described herein.

Arylhydrocarbon receptors are known to those of skill in the art. (Noakes, 2015).

Agonists of arylhydrocarbon receptors include, but are not d to, tryptophan— related compounds such as kynurenine, kynurenic acid, cinnabarinic acid, and 6— formylindolo [3,2—b] carbazole (FICZ). Malassezin is also known as an aryl hydrocarbon receptor agonist. (Wille, eta/., 2001).

In one aspect of this embodiment, the compound ed by a Malassezia yeast has the structure of formula (I): In another aspect of this embodiment, the nd is a chemical analog of malassezin.

Another embodiment of the present invention is a compound for improving hyperpigmentation caused by a hyperpigmentation disorder. The compound is a chemical analog of a compound ed by a Ma/assezia yeast, or a crystalline form, e, or cosmetically or pharmaceutically able salt thereof.

As used herein, the compounds, compositions, and methods of the present invention can be used to improve igmentation caused by a hyperpigmentation disorder by, for example, ng the level of hyperpigmentation in areas ed by a hyperpigmentation disorder, slowing further hyperpigmentation, or preventing further hyperpigmentation from occurring. However, because every subject may not respond to a particular dosing protocol, regimen, or process, improving hyperpigmentation caused by a hyperpigmentation disorder does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population. Accordingly, a given subject or subject population may fail to respond or respond inadequately to dosing, but other ts or subject populations may respond and, therefore, experience improvement in their hyperpigmentation disorder.

As used herein, the term "hyperpigmentation" is an actual or a perceived skin disorder of excessive dark color. The skin impairment can be actual, for example, uted to age, excessive sun exposure, or a disease or condition leading to dark skin areas. The dark skin areas can be in the form of spots, es, or relatively large areas of dark color. The skin impairment also can be perceived, for example, a perception by an individual that r skin shade is too dark. The individual may have a cosmetic desire to lighten the skin shade.

Hyperpigmentation disorders are ers in which hyperpigmentation is the primary symptom as well as disorders in which hyperpigmentation occurs as a secondary symptom. Hyperpigmentation disorders of the present invention include, but are not limited to, congenital hyperpigmentation disorders and acquired hyperpigmentation disorders.

Congenital hyperpigmentation disorders of the present invention include, but are not d to, those involving epidermal hyperpigmentation (nevus cell nevus, Spitz nevus, and nevus spilus), dermal hyperpigmentation (blue nevus, nevus Ohta, dermal melanosis, nevus Ito, and ian spot), ephelides, acropigmentation reticularis, Spitzenpigment/acropigmentation, and lentiginosis (generalized inosis, LEOPARD syndrome, inherited patterned lentiginosis, Carney complex, Peutz—Jeghers me, Laugier—Hunziker—Baran syndrome, and Cronkhite—Canada syndrome). uchi, et a/., 2014). Acquired hyperpigmentation disorders of the present invention include, but are not limited to, senile lentigines/lentigo, melasma/chloasma, s melanosis, labial melanotic macule, penile/vulvovaginal melanosis, erythromelanosis follicularis faciei Kitamura, UV—induced pigmentation (tanning and pigmentation petaloides actinica), flammatory pigmentation (friction melanosis and ashy dermatosis), chemical/drug—induced pigmentation (polychlorinated biphenyl, arsenic, 5—FU, bleomycin, cyclophosphamide, methotrexate, chlorpromazine, oin, tetracycline, and chloroquine), pigmentary demarcation lines, and foreign material deposition (such as carotene, silver, gold, mercury, h, and tattoos). Hyperpigmentation related with systemic disorders es metabolism/enzyme disorders (hemochromatosis, Wilson’s e, Gaucher’s disease, Niemann—Pick’s disease, amyloidosis, ochronosis, acanthosis nigricans, and porphyria cutanea tarda), endocrine ers (Addison’s disease, Cushing syndrome, and hyperthyroidism), nutritional disorders gra, vitamin Bf2 deficiency, folic acid ency, vagabond’s disease, and prurigo pigmentosa), mastocytosis, collagen diseases, liver dysfunction, and kidney dysfunction.

Hyperpigmentation can also be related with infectious diseases (measles, syphilis, and Ma/assezia ) and syndromes (von Recklinghausen’s disease, Sotos syndrome, POEMS me, Naegeli syndrome, Cantu syndrome, McCune—Albright syndrome, Watson syndrome, and Bloom syndrome).

(Yamaguchi, eta/., 2014).

In one aspect of this embodiment, the compound produced by a Ma/assezia yeast has the structure of formula (I): o H In another aspect of this embodiment, the compound is a chemical analog of malassezin.

A r embodiment of the present invention is a compound for modulating melanin production. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or ceutically acceptable salt thereof.

Melanin is a naturally produced pigment that gives color to skin and hair. A schematic diagram of the skin is shown in Fig. 1A. Melanin is produced by melanocytes in organelles known as melanosomes. A nd or composition of the present invention modulates melanin production in a subject by, for example, modulating melanosome biogenesis and directly or indirectly inhibiting melanin synthesis at the tic level.

Melanosome biogenesis occurs via four stages: Stage I is characterized by lanosomes, which are essentially gmented es. In stage II, pre—melanosomes develop striations on which melanin is deposited in stage III. Stage IV results in mature melanosomes that are rich in melanin content. Compounds and compositions of the present invention modulate melanosome biogenesis by ting or attenuating the biological processes that normally promote any or all of these stages. (Wasmeier, et a/., 2008) Melanin synthesis primarily involves three s: tyrosinase, nase related protein—1, and dopachrome tautomerase. Additional factors that affect intracellular trafficking of these enzymes include, but are not limited to, BLOC—1, 0A1, and SLC45A2. The compounds and compositions of the present invention can modulate melanin production by, for e, inhibiting or attenuating the activity of any of these s or factors. (Yamaguchi, et a/., 2014).

Once melanosomes have formed and melanin has been synthesized, melanosomes need to be transferred from epidermal melanocytes to skin and hair keratinocytes. Melanosomes originate near the nucleus of melanocytes and are transported to the periphery of melanocytes along microtubules and actin filaments. nds and compositions of the present invention modulate melanosome transfer by interfering with any of the ical processes that result in the transport of melanosomes from the perinuclear region, to the melanocyte ery, and into adjacent nocytes. A schematic diagram of melanin synthesis, melanin transport, and melanocyte sis is shown in Fig. 18.

In one aspect of this embodiment, the compound produced by a Ma/assezia yeast has the structure of formula (I): In another aspect of this embodiment, the compound is a chemical analog of malassezin.

An onal embodiment of the present invention is a compound for modulating melanosome biogenesis. The compound is a chemical analog of a compound produced by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt f.

In one aspect of this embodiment, the compound produced by a ezia yeast has the structure of formula (I): In r aspect of this embodiment, the compound is a chemical analog of malassezin.

Another embodiment of the present invention is a compound for modulating melanosome transfer. The compound is a chemical analog of a compound ed by a Ma/assezia yeast, or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this ment, the compound produced by a Ma/assezia yeast has the structure of formula (I): In another aspect of this embodiment, the nd is a chemical analog of ezin.

A further embodiment of the present invention is a composition.

The composition comprises a Ma/assezia yeast and a cosmetically or pharmaceutically acceptable vehicle, t or carrier.

An additional embodiment of the present invention is a composition.

The composition comprises a nd ed or isolatable from a Ma/assezia yeast and a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier.

A compound isolated from a Ma/assezia yeast of the present invention arily exists, before isolation, in a Ma/assezia yeast or is produced by a Ma/assezia yeast. Therefore, a compound isolated from a Ma/assezia yeast is d from actual yeast cells. Standard protocols for extracting compounds from cellular material are known to those of skill in the art.

A compound isolatable from a Ma/assezia yeast need not be derived from actual yeast cells. lnstead, synthetic reactions can be used to generate compounds produced in yeast without the involvement of actual yeast WO 56424 cells. Organic synthesis reactions are well known to those of skill in the art and can be used in this regard.

Another embodiment of the present invention is a composition. The composition comprises any of the compounds disclosed herein, including analogs, and a cosmetically or pharmaceutically acceptable vehicle, diluent or A further embodiment of the present invention is a method of brightening skin in a subject. The method comprises contacting the t with any of the compounds or compositions disclosed herein.

As used herein, the term "contacting" and grammatical variations thereof refer to bringing two or more materials into close enough proximity that they can interact. Thus, for illustrative purposes only, a compound of the present invention can contact a melanocyte by, for example, interacting with a receptor on the surface of the melanocyte. Similarly, a composition of the present invention can contact a human subject by, for example, being applied ly to the t’s skin.

As used herein, a "subject" means a mammalian cell, , organism, or populations thereof. Subjects of the present invention are preferably human, including human cells, s, and beings, but otherwise include, primates, farm animals, ic animals, laboratory animals, etc. Some examples of agricultural animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc.

An additional embodiment of the present ion is a method for ng melanocyte apoptosis in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

Another embodiment of the present ion is a method for modulating melanocyte activity in a subject. The method comprises ting the subject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for agonizing an arylhydrocarbon or (AhR). The method comprises contacting the subject with any of the compounds or itions disclosed herein.

An additional embodiment of the present invention is a method for ing hyperpigmentation caused by a hyperpigmentation disorder in a subject in need f. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

As used herein, a subject "in need" of improvement in igmentation caused by a hyperpigmentation disorder includes subjects with a real or perceived need of improvement.

Another embodiment of the present invention is a method for modulating melanin production in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

A further embodiment of the present invention is a method for modulating melanosome biogenesis in a subject. The method comprises contacting the subject with any of the compounds or itions disclosed herein.

An additional embodiment of the present invention is a method for modulating melanosome transfer in a subject. The method comprises contacting the subject with any of the compounds or compositions disclosed herein.

Another embodiment of the t invention is a compound. The compound has the ure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, R10 and R11 is methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically able salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: CHO \ I2 and CHO \ A further embodiment of the present ion is a compound. The compound has a structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl, and at least one of R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 is ; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is: N C An additional embodiment of the present ion is a compound for brightening skin. The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently ed from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ Another embodiment of the present invention is a compound for brightening skin. The compound has the ure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or ically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is ed from the group consisting of: H and A further embodiment of the present invention is a compound for inducing melanocyte apoptosis. The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a lline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ An additional embodiment of the present invention is a compound for ng melanocyte apoptosis. The compound has the structure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, e, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: H and Another embodiment of the present invention is a nd for agonizing the arylhydrocarbon receptor (AhR). The compound has the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of en and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ A further embodiment of the present invention is a nd for agonizing the arylhydrocarbon receptor (AhR). The compound has the structure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of en and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: H and An onal embodiment of the present invention is a composition.

The composition comprises a compound having the structure of formula (II) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a lline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof, and a cosmetically or pharmaceutically acceptable vehicle, diluent or r.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ Another embodiment of the present invention is a composition. The ition comprises a compound having the structure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or ically or pharmaceutically acceptable salt thereof, and a ically or pharmaceutically acceptable vehicle, diluent or carrier.

In one aspect of this embodiment, the compound is selected from the group consisting of: H and A further embodiment of the present invention is a method for brightening skin in a subject. The method comprises: ting the subject with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or ically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ An additional embodiment of the present invention is a method for brightening skin in a t. The method comprises: contacting the subject with a compound having the structure of formula (III): n: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, e, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the nd is selected from the group consisting of: H and Another embodiment of the present invention is a method for inducing melanocyte apoptosis in a subject. The method comprises: contacting the subject with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are ndently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: M and CHO \ A further embodiment of the present invention is a method for inducing melanocyte apoptosis in a subject. The method comprises: contacting the subject with a compound having the structure of formula (III): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, e, or cosmetically or ceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: H and An additional embodiment of the present invention is a method for agonizing an arylhydrocarbon receptor (AhR) in a subject. The method comprises: contacting the subject with a compound having the structure of formula (II): wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically able salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: CHO \ CHO \ M and CHO \ Another embodiment of the present invention is a method for ing an arylhydrocarbon receptor (AhR) in a subject. The method comprises: contacting the subject with a compound having the structure of formula (III): (111) wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 are independently ed from the group consisting of hydrogen and methyl; or a crystalline form, hydrate, or cosmetically or pharmaceutically acceptable salt thereof.

In one aspect of this embodiment, the compound is selected from the group consisting of: I2 and WO 56424 As used herein, the term "composition" means an entity comprising a compound of the present invention, as well as any entity which results, directly or indirectly, from combinations of a nd of the present invention with other ingredients. Compositions of the present invention can be used as, for example, in vitro or in vivo research reagents. Compositions of the present invention can also be applied directly to the skin of a human or man subject for a cosmetic effect.

A composition of the present invention may be administered in any desired and effective manner: for oral ingestion or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, ulmonary, rectal, vaginal, gual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a composition of the t invention may be stered in conjunction with other compositions. A composition of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired.

The compositions of the invention comprise one or more active ingredients in admixture with one or more cosmetically or ceutically acceptable carriers and, ally, one or more other compounds, ingredients and/or materials. Regardless of the route of administration selected, the compounds and compositions of the present invention are formulated into cosmetically or pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

Cosmetically or pharmaceutically acceptable vehicles, diluents and carriers are well known in the art and include materials suitable for contact with the tissues of humans and non—humans t undue toxicity, incompatibility, instability, irritation, allergic response and the like. Cosmetically or pharmaceutically able vehicles, diluents and carriers include any substantially non—toxic substance conventionally usable, for example, for topical, oral, peritoneal, or subcutaneous administration of cosmetics or pharmaceuticals in which the nds and compositions of the present invention will remain stable and bioavailable when applied, injested, injected, or otherwise administered to a human or non—human subject. ically or pharmaceutically acceptable carriers suitable for l application are known to those of skill in the art and include cosmetically or pharmaceutically acceptable liquids, creams, oils, lotions, ointments, gels, or solids, such as tional cosmetic night creams, foundation creams, suntan s, sunscreens, hand lotions, p and make—up bases, masks and the like. Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

The compositions of the present invention can n other ingredients conventional in cosmetics including perfumes, estrogen, Vitamins A, C and E, alpha—hydroxy or keto acids such as pyruvic, lactic or glycolic acids, lanolin, vaseline, aloe vera, methyl or propyl paraben, pigments and the like. Non—limiting cosmetically or pharmaceutically acceptable vehicles, diluents and carriers of the present invention include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, ose preparations, calcium phosphates (e.g., dicalcium ate, tricalcium ate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's ion, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl l, propyl alcohol, and benzyl alcohol), polyols (e.g., ol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and triglycerides), biodegradable polymers (e.g., polylactide—polyglycolide, poly(orthoesters), and nhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc.

The itions of the invention may, optionally, contain additional ingredients and/or materials commonly used in cosmetic compositions.

These ingredients and materials are well known in the art and include, for example, (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and c acid; (2) s, such as carboxymethylcellulose, alginates, gelatin, polyvinyl idone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar—agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross—linked sodium carboxymethyl cellulose and sodium WO 56424 carbonate; (5) solution ing agents, such as paraffin; (6) tion accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, m stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar—agar and tragacanth; (11) ing agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, hylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface—active agents; (16) dispersing agents; (17) control—release or absorption—delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21) emulsifying and ding agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl l, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl te, ene glycol, 1,3—butylene glycol, oils (in ular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, ydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening ; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, ing and preservative agents.

Each such ingredient or material must be "acceptable" in the sense of being ible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and ed route of administration are well known in the art, and able ients and materials for a chosen dosage form and method of administration may be determined using ry skill in the art.

Compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, es, a solution or a suspension in an aqueous or non—aqueous liquid, an oil— in—water or water—in—oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan—coating, mixing, granulation or lyophilization processes.

Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be ed, e.g., by mixing the active ingredient(s) with one or more cosmetically or pharmaceutically acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type may be employed as fillers in soft and hard—filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable , lubricant, inert t, vative, disintegrant, surface—active or dispersing agent. Molded s may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as capsules, pills and granules, may optionally be scored or ed with coatings and shells, such as enteric coatings and other coatings well known in the cosmetic formulating art. They may also be ated so as to e slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria—retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

Liquid dosage forms for oral stration include cosmetically or pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may n suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, ng, ing and preservative agents. Suspensions may contain suspending agents.

Compositions of the present ion for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. itions of the present invention which are suitable for l administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such cosmetically or pharmaceutically acceptable carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops, emulsions, suspensions, aerosols, and inhalants. Any desired conventional vehicles, assistants and optionally r active ingredients may be added to the formulation.

Preferred assistants originate from the group comprising preservatives, antioxidants, stabilisers, lisers, ns, colorants, odour improvers, film s, thickeners and humectants.

Solutions and emulsions can comprise the conventional vehicles, such as solvents, solubilisers and emulsifiers, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3—butyl , oils, in particular cottonseed oil, groundnut oil, maize oil, olive oil, castor oil and sesame oil, glycerol fatty acid esters, polyethylene glycols and fatty acid esters of sorbitan, or mixtures of these substances.

The emulsions may exist in various forms. Thus, they can be, for example, an emulsion or microemulsion of the water—in—oil (W/O) type or of the oil—in—water (O/W) type, or a multiple emulsion, for example of the water—in—oil—in— water (W/O/W) type.

The compositions ing to the invention may also be in the form of emulsifier—free, se ations. They can be, for example, hydrodispersions or Pickering emulsions.

Suspensions may comprise conventional vehicles, such as liquid diluents, for example water, ethanol or ene glycol, suspension media, for example ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline cellulose, aluminium droxide, bentonite, agar—agar and tragacanth, or mixtures of these nces.

Pastes, ointments, gels and creams may comprise conventional vehicles, for example animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene s, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures of these substances.

Face and body oils may comprise the conventional vehicles, such as synthetic oils, such as fatty acid esters, fatty alcohols, silicone oils, natural oils, such as vegetable oils and oily plant extracts, paraffin oils, lanolin oils, or mixtures of these substances.

Sprays may comprise the conventional propellants, for example chlorofluorocarbons, propane/butane or dimethyl ether.

Compositions of the present invention suitable for parenteral administrations comprise one or more compounds in combination with one or more cosmetically or pharmaceutically acceptable sterile isotonic aqueous or non—aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable ons or sions just prior to use, which may n suitable antioxidants, buffers, solutes which render the formulation ic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These itions may also contain suitable adjuvants, such as wetting , emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In on, prolonged absorption of the injectable cosmetic form may be brought about by the inclusion of agents which delay absorption.

In some cases, in order to g the effect, it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility.

The rate of tion of the active agent/drug then s upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally—administered composition may be accomplished by dissolving or suspending the active composition in an oil vehicle. lnjectable depot forms may be made by g microencapsule matrices of the active ient in biodegradable polymers.

Depending on the ratio of the active ingredient to r, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also ed by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial— retaining filter.

The compositions of the present invention may be ted in unit—dose or multi—dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.

Extemporaneous ion solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

In the present invention, the term "crystalline form" means the crystal structure of a compound. A compound may exist in one or more crystalline forms, which may have different structural, physical, pharmacological, or chemical characteristics. Different crystalline forms may be obtained using variations in nucleation, growth kinetics, agglomeration, and ge.

Nucleation results when the phase—transition energy barrier is overcome, thereby allowing a particle to form from a supersaturated solution. Crystal growth is the ement of crystal particles caused by deposition of the al compound on an existing e of the l. The relative rate of nucleation and growth ine the size distribution of the crystals that are formed. The thermodynamic driving force for both nucleation and growth is supersaturation, which is defined as the deviation from thermodynamic equilibrium. Agglomeration is the formation of larger particles through two or more particles (e.g., crystals) sticking together and forming a larger crystalline structure.

The term "hydrate", as used herein, means a solid or a semi—solid form of a chemical compound containing water in a molecular complex. The water is lly in a stoichiometric amount with respect to the chemical compound.

As used herein, "cosmetically or pharmaceutically acceptable salt" refers to a derivative of the compounds disclosed herein wherein the compounds are modified by making acid or base salts thereof. Examples of ically or pharmaceutically acceptable salts include, but are not d to, mineral or c acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. For example, such salts include salts from ammonia, L—arginine, betaine, benethamine, benzathine, calcium hydroxide, choline, deanol, diethanolamine (2,2'—iminobis(ethanol)), lamine, 2—(diethylamino)—ethanol, 2—aminoethanol, ethylenediamine, N— ethyl—glucamine, hydrabamine, 1H—imidazole, , magnesium hydroxide, 4—(2— hydroxyethyl)—morpholine, piperazine, potassium hydroxide, ydroxy—ethyl)— pyrrolidine, sodium hydroxide, triethanolamine (2,2‘,2"—nitrilotris(ethanol)), trometh—amine, zinc hydroxide, acetic acid, 2.2—dichloro—acetic acid, adipic acid, alginic acid, ascorbic acid, L—aspartic acid, benzenesulfonic acid, benzoic acid, 2,5—dihydroxybenzoic acid, 4—acetamido—benzoic acid, (+)—camphoric acid, (+)— camphor—10—sulfonic acid, carbonic acid, cinnamic acid, citric acid, ic acid, ic acid, dodecylsulfuric acid, ethane—1,2—disulfonic acid, ethanesulfonic acid, 2—hydroxy—ethanesulfonic acid, ethylenediamonotetraacetic acid, formic acid, fumaric acid, galacaric acid, gentisic acid, D—glucoheptonic acid, D—gluconic acid, D—glucuronic acid, glutamic acid, glutantic acid, glutaric acid, 2—oxo—glutaric acid, o—phosphoric acid, glycine, glycolic acid, hexanoic acid, hippuric acid, hydrobromic acid, hydrochloric acid isobutyric acid, tic acid, lactobionic acid, lauric acid, lysine, maleic acid, (—)—L—malic acid, malonic acid, delic acid, methanesulfonic acid, galactaric acid, naphthalene—1,5—disulfonic acid, naphthalene—2—sulfonic acid, 1—hydroxy—2—naphthoic acid, nicotinic acid, nitric acid, octanoic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid ic acid), phosphoric acid, propionic acid, (—)—L—pyroglutamic acid, salicylic acid, 4—amino—salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, WO 56424 tannic acid, (+)—L—tartaric acid, thiocyanic acid, p—toluenesulfonic acid and undecylenic acid. Further ically or pharmaceutically acceptable salts can be formed with cations from metals like aluminum, calcium, lithium, magnesium, potassium, sodium, zinc and the like.

The cosmetically or pharmaceutically acceptable salts of the present invention can be synthesized from a compound disclosed herein which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be ed by ng the free acid or base forms of these compounds with a sufficient amount of the appropriate base or acid in water or in an organic diluent like ether, ethyl acetate, ethanol, panol, or acetonitrile, or a mixture thereof.

It is envisioned that the compounds and compositions of the present invention may be included in cosmetic or pharmaceutical compositions.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in the specification and the appended claims, the singular forms "a, an," and "the" include plural referents unless the context clearly dictates otherwise.

For recitation of numeric ranges herein, each intervening number there n with the same degree of precision is explicitly contemplated. For e, for the range of 6—9, the numbers 7 and 8 are plated in addition to 6 and 9, and for the range 6.0—7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not ed to limit the scope of the invention in any way.

EXAMPLES Example 1 Materials and Methods Isolation of Compounds Produced by Malassezia Malassezin is isolated using, for example, the procedures outlined in Wille eta/., 2001. The protocol is briefly outlined below.

Medium A medium consisting of Tween 80 (30 mL), eximide (0.5 g), chloramphenicol (0.05 g), agar (20 g), and a volume of water sufficient for a 1000 mL mixture is ized and mixed with 0.3% sterile filtered L—tryptophan at a concentration of 0.3 g% at 50°C. 10 mL portions are poured into 10 cm Petri dishes and the pH is ed to 5.5 using 0.1 M HCI.

Cultivating Malassezia furfur and Isolating Compounds Produced By M. furfur Malassezia furfur is swabbed on the medium described above and incubated for 14 days at 30°C. The contents of the Petri dish are pureed and extracted with ethyl acetate for 12 hours. The extract is ed over glass wool, evaporated to dryness, and dissolved in methanol. The extract is then fractionated by chromatography on Sephadex LH—20 with methanol as the eluent.

WO 56424 Further separation is accomplished with preparative thin—layer chromatography with toluene:ethyl formate:formic acid (10:5:3). Main zones are partitioned between water and ethyl acetate. Fractions are ed for ty of interest.

Compounds from fractions of interest are isolated by HPLC.

Synthesis of Malassezin and Chemical Analogs of Malassezin Malassezin is synthesized according to the protocol set forth in Wille et al., 2001. Chemical analogs of malassezin are synthesized according to novel synthesis protocols, as well as those described in n—McPherson, et al., 2014.

Screening Protocols Effective skin brightening compounds are evaluated using both ing protocols known to those of skill in the art and novel screening methods. For example, malassezin and chemical analogs thereof are evaluated by a nase bioassay, as described above. Other screening ols involving both in vitro cell and in vivo tissue models are utilized, including aryl hydrocarbon receptor (AhR) binding assays.

Tyrosinase Bioassay nase bioassays are performed as described in Wille et al., 2001. Briefly, L—DOPA is mixed with tyrosinase enzyme. Extinction is measured over 1 minute, indicating the formation of dopaquinone. Using, for example, the fractions discussed above, these fractions are dissolved in DMSO and added ly to the tyrosinase reaction, with pure DMSO as a control. Tyrosinase inhibitory activity is measured as reduced increase in extinction compared to control.

Ary/ Hydrocarbon Receptor Binding Assay AhR binding assays are performed according to the protocol described in, for example, Song, et a/., 2002. Briefly, human and murine AhRs are expressed in vitro using, for example, a TnT Quick—coupled Reticulocyte Lysate Systems reaction (Promega, Madison, WI). or ligand binding studies utilize velocity sedimentation on sucrose nts as described in Karchner, eta/., 1999.

EFl’OD Assay Compounds, compositions, and formulations of the present invention are also evaluated using the ethoxyresorufin—O—deethylase (EROD) assay known to those of skill in the art. (Donato, eta/., 1993; Whyte, eta/., 2000; Wille eta/., 2001).

Melanocyte sis Assays ate compounds are evaluated for apoptosis—inducing ty in melanocytes. Human epidermal melanocytes are cultured in Medium 254 supplemented with Human Melanocyte Growth ment (HMGS) (Thermo— Fisher Scientific, Waltham, MA) or Dermal Cell Basal Medium (ATCC, as, VA). Additional components of human melanocyte growth media can include, but are not limited to, insulin (5 ug/ml), ascorbic acid (50 ug/ml), L—glutamine (6 mM), epinephrine (1.0 uM), and calcium chloride (0.2 mM). Human melanocyte cultures are maintained at 37°C in 5% 002.

Candidate compounds are diluted in DMSO and mixed directly into melanocyte cultures. Equivalent volumes of pure DMSO are used as controls. xicity assays known to those of skill in the art are performed according to manufacturer’s instructions. Cytotoxicity assays that are used in the present invention include, but are not limited to, CellToxTM Green Cytotoxicity Assay, Apo—ONE fluorescent caspase assays, ApoTox—GloTM assay, and Caspase—Glo® assays (Promega, Madison, WI). Fluorescence detection is accomplished using standard FACS or microscopy assays known to those in the art, including those described in Kramer, eta/., 2005. onal means of assessing apoptosis are used, including FACS es for annexin V and Western blots for caspase—9 expression. Western blotting is performed according to methods known to those of skill in the art.

Mouse Xenograft Assays Mouse xenograft models of human skin are generated according to protocols known in the art. (Black, et a/., 1985; Manning eta/., 1973; Reed, et a/., 1973; Plenat, eta/., 1992; Scott eta/., 1998; Otulakowski, eta/., 1994). Once ished, mouse xenograft models are exposed to compounds of the present invention and changes in pigmentation are ed as ed to controls.

Changes in skin pigmentation are ed using various pigmentation scales known to those of skill in the art, including, but not limited to, the Fitzpatrick skin typing test and the Taylor Hyperpigmentation Scale. (Taylor, et a/., 2005).

Human Assays Compounds, compositions, and formulations of the present invention are applied to humans, for example, on human skin, and compared to control substances. Changes in skin pigmentation are assessed using s pigmentation scales known to those of skill in the art, ing, but not limited to, the Fitzpatrick skin typing test and the Taylor Hyperpigmentation Scale.

Example 2 Biochemical Target of Malassezin and Its Analogs It is expected that the compounds and compositions of the present invention will t, for example, tyrosinase inhibition and AhR agonist ty comparable to malassezin. Compounds and compositions of the present invention are expected to exhibit, for example, more potent tyrosinase inhibition and stronger AhR agonism compared to malassezin. Likewise, n of the compounds and compositions of the present invention are expected to be less effective tyrosinase inhibitors and AhR agonists than malassezin. Such compounds, compositions, and formulations may have more favorable toxicity profiles compared to more potent nds.

In Vitro Efficacy It is expected that the compounds and compositions of the present invention will induce melanocyte apoptosis and modulate melanocyte activity, melanin production, some biogenesis, and/or some transfer at least as potently as ma|assezin. It is also contemplated that certain of the compounds and compositions of the present invention will effect these biological processes less potently than ma|assezin. Such nds and compositions may have more favorable toxicity profiles compared to more potent species.

Example 4 In Vivo Efficacy It is expected that the nds and compositions of the present invention will be at least as effective as malassezin for brightening skin and improving hyperpigmentation caused by hyperpigmentation disorders. It is further expected that the compounds and compositions of the present invention will t favorable pharmacokinetic profiles in terms of, for example, half—life and absorption. Certain compounds will exhibit a longer half—life, whereas others will exhibit a shorter half—life. Similarly, certain compounds will exhibit ent absorption profiles, with some compounds taking longer to be fully absorbed and others taking less time to be fully absorbed.

Example 5 Synthesis of Malassezin and Malassezin Derivatives WO 56424 Malassezin ("CV—8684") and its cyclized derivative indolo[3,2—b] carbazole ("CV—8685") were synthesized according to the scheme shown in Fig.

Synthesis of uty/ (2-iodo-pheny/)carbamate, compound 1 To a solution of 2—iodo—aniline (25.0 g, 0.114 mol) in tetrahydrofuran (250 mL) at 0°C was added LiHMDS (251.0 mL, 1 M in THF, 0.251 moi) slowly while maintaining the internal temperature below 5°C over 40 min.. After 30 min stirring at 0°C, a solution of 800 anhydride (27.0 g, 0.125 mol) in THF (50 mL) was slowly added while maintaining the internal temperature below 5°C over 40 min. The reaction mixture was warmed to ambient temperature and stirred 1 hr.

Saturated NH4C| (250 mL) was added to quench the reaction. The organic layer was separated and washed with water (150 mL). The combined s layer was extracted with ethyl acetate (2 x 150 mL), the layers were separated. The ethyl acetate layer was combined with the original organic layer and concentrated in vacuo to give as brown oil. The crude compound purified by column chromatography (0—5% ethyl acetate/hexanes). Compound 1 was obtained as a light yellow liquid (29.0 g, 80%).

Synthesis of compound 2 Copper iodide (0.95 g, 10% mol) and PdCl2(PPh3)4 (1.75 g, 5% mol) was added to a degassed on of compound 1 (16.0 g, 0.05 mol), propargyl methyl ether (4.25 g, 0. 06 mol) in triethylamine (200 mL) at ambient temperature. After stirring at ambient temperature over 2 hr, the reaction was complete (monitored by TLC using 10% ethyl acetate/hexanes). The reaction mixture diluted with ethyl e (300mL), reaction e was washed with water, ted NaCl and dried over Na2804. The solvent was filtered and concentrated in vacuo to give as brown oil. The crude compound purified by column chromatography (10% ethyl acetate/hexane). Compound 2 was obtained as a light yellow liquid (13.0 g, 99%). sis of compound 3 To an oven—dried flask was added PtCl2 (0.26 g, 0.001 mol), Na2CO3 (1.6 g, 0.015 mol), indole (2.32 g, 0.02 mol) and compound 2 (2.6 g, 0.01 mol) in dioxane (120 mL). The flask was degassed with nitrogen, sealed and heated to 100°C overnight. After the reaction was complete (monitored by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under d pressure. The reaction mixture diluted with ethyl acetate (200mL), reaction mixture was washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and concentrated in vacuo to give as brown oil.

This on was repeated using compound 2 (2.6 g, 0.01 mol) in different batch. Both batches crude compounds were combined and purified by column chromatography (10% ethyl e/hexane). Compound 3 was obtained as a light brown solid (3.8 g, 55%).

Synthesis of compound 4 Potassium carbonate (4.6 g, 0.0329 mol) was added to a solution of compound 3 (3.8 g, 0.0109 mol) in methanol (150 mL) and water (50 mL) mixture at ambient temperature. The resulting sion was heated to reflux overnight.

After the reaction was complete (monitored by TLC using 20% ethyl acetate/hexanes). The on e was cooled to ambient temperature and solvent concentrated in vacuo. The residue taken in ethylacetate (200 mL) and washed with water and brine then dried (sodium sulfate), filtered, solvent concentrated in vacuo to give as a brown solid. Crude nd purified by column chromatography (20% ethyl acetate/hexane. Compound 4 was obtained as an orange color solid (2.2 g, 81%).

Synthesis of compound Malassezin (CV-8684) To a dried 100 mL two neck round-bottom flask under argon at 00C}, dimethylformamide (20 mL) was added. POClg (0.75 g, 0.0048 moi) slowly added while maintaining the internal temperature below 5°C over 10 min. After min stirring at 0°C, a solution of compound 4 (1.0 g, 0.004 mol) in dimethylformamide (5 mL) was slowly added while maintaining the al temperature below 5°C over 10 min. The resulting e was stirred at ambient temperature overnight. After the reaction was complete (monitored by TLC using % ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 mL) and stirred for 1 hr. Resulting mixture was extracted with ethyl acetate (2 x 100 mL). The c layers were combined and washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and concentrated in vacuo to give as brown solid. The crude compound purified by column chromatography (0—20% ethyl acetate/hexanes). Compound Malassezin (CV-8684) was obtained as a light pink solid (0.82 g, 74%).

HPLC purity: 97.8% (area%). 1H—NMR, 13C spectrum tent with the structure. ESl—MS: Calc. for C18H15N20 (M + H)+: 275, found: 275.2 Synthesis of compound |ndolo[3,2-b] carbazole (CV-8685).

Concentrated HCI (0.25 mL) was added to a on of malassezin (0.75 g) in tetrahydrofuran (120 mL) at ambient temperature. The resulting mixture was heated to reflux overnight. After the reaction was complete ored by TLC using 40% ethyl acetate/hexanes). The reaction e was cooled to ambient temperature and stirred for 1 hr. Filtered the solid, washed with tetrahydrofuran (20 mL) and dried to give Indolo[3,2-b] carbazole (CV-8685) light yellow solid (0.55 g, 78 %).

HPLC purity: 96.22% (area%). 1H—NMR, 13C spectrum consistent with the structure. ESl—MS: Calc. for C18H13N2 (M + H)+: 257, found: 257.5.

Compound I ("CV—8686") and compound lV ("CV—8687") were synthesized according to the scheme shown in Fig. 2B.

Synthesis of compound 5 To an oven—dried flask was added PtCl2 (1.0 g, 0.0038 mol), Na2C03 (6.1g, 0.057 mol), 6—methyl indole (10.0 g, 0.076 mol) and compound 2 (10.0 g, 0.038 mol) in dioxane (250 mL). The flask was degassed with en, sealed and heated to 100°C overnight. After the reaction was complete (monitored by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under reduced pressure. The reaction mixture diluted with ethyl acetate ), on mixture was washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and trated in vacuo to give as brown oil. Crude compound purified by column chromatography (10% ethyl acetate/hexane). Compound 5 was obtained as a light brown solid (6.5 g, 47%).

Synthesis of nd 6 Potassium carbonate (7.4 g, 0.054 mol) was added to a solution of compound 5 (6.5 g, 0.018 mol) in methanol (150 mL) and water (50 mL) mixture at ambient temperature. The resulting suspension was heated to reflux overnight.

After the on was complete (monitored by TLC using 20% ethyl acetate/hexanes). The reaction e was cooled to ambient temperature and solvent concentrated in vacuo. The residue taken in ethylacetate (200 mL) and washed with water and brine then dried (sodium sulfate), filtered, solvent concentrated in vacuo to give as brown solid. Crude compound purified by column chromatography (20% ethyl acetate/hexane). Compound 6 was obtained as an orange color solid (3.3 g, 72%).

Synthesis of compound compound I (CV-8686) To a dried 100 mi. two neck round—bottom flask under argon at 0°C, dimethylformamide (20 mL) was added. POCig (0.6 g, 0.0038 moi) slowly added while maintaining the internal temperature below 5°C over 10 min.. After min stirring at 0°C, a solution of compound 6 (1.0 g, 0.0038 mol) in dimethylformamide (5 mL) was slowly added while maintaining the internal temperature below 5°C over 10 min. The resulting mixture was stirred at ambient temperature overnight. After the reaction was complete (monitored by TLC using % ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 mL) and stirred for 1 hr. Resulting mixture was extracted with ethyl e (2 x 100 mL). The organic layers were combined and washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and trated in vacuo to give as brown solid. The crude compound purified by column tography (0—20% ethyl acetate/hexanes). Compound I (CV-8686) was obtained as a light pink solid (0.84 g, 75%).

HPLC purity: 97.01% (area%). 1H—NMR, 13C spectrum consistent with the structure. ESl—MS: Calc. for N20 (M + H)+: 289, found: 289.1 Synthesis of compound compound IV (CV-8687) Concentrated HCI (0.3 mL) was added to a solution of compound I (1.0 g) in tetrahydrofuran (125 mL) at ambient temperature. The resulting mixture was heated to reflux overnight. After the reaction was complete (monitored by TLC using 40% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and stirred for 1 hr. ed the solid, washed with ydrofuran (20 mL) and dried to give compound IV 87) light yellow solid (0.84 g, 89 %).

HPLC purity: 98.4% (area%). 1H—NMR, 13C spectrum consistent with the structure. : Calc. for C19H15N2 (M + H)+: 271, found: 271.3.

Compound ll ("CV—8688") was synthesized according to the scheme shown in Fig. 2C.

Synthesis of compound 7 Copper iodide (0.53 g, 10% mol) and PdCl2(PPh3)4 (1.0 g, 5% mol) was added to a degassed solution of compound 1 (9.0 g, 0.03 mol), 3—methoxy— 1—butyne (2.8 g, 0. 035 mol) in triethylamine (150 mL) at ambient temperature.

After stirring at t temperature over 2 hr. The reaction was te (monitored by TLC using 10% ethyl acetate/hexanes). The reaction mixture diluted with ethyl acetate (300mL), reaction mixture was washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and concentrated in vacuo to give as brown oil. The crude compound purified by column tography (10% ethyl e/hexane). Compound 7 was obtained as a light yellow liquid (7.0 g, 90%).

Synthesis of compound 8 To an oven—dried flask was added PtCl2 (0.68 g, 0.0025 mol), Na2CO3 (4.0 g, 0.038 mol), indole (6.0 g, 0.05 mol) and compound 7 (10.0 g, 0.025 mol) in dioxane (250 mL). The flask was degassed with nitrogen, sealed and heated to 100°C overnight. After the reaction was complete (monitored by TLC using 10% ethyl acetate/hexanes). The solvent was evaporated under reduced pressure. The reaction mixture diluted with ethyl acetate ), reaction mixture was washed with water, saturated NaCl and dried over Na2804.

The solvent was filtered and concentrated in vacuo to give as brown oil. Crude compound ed by column chromatography (10% ethyl acetate/hexane).

Compound 8 was obtained as a light brown solid (3.5 g, 77%).

Synthesis of compound 9 Potassium carbonate (3.8 g, 0.027 mol) was added to a on of compound 8 (3.3 g, 0.0091 mol) in methanol (75 mL) and water (25 mL) e at ambient temperature. The resulting suspension was heated to reflux overnight.

After the reaction was complete ored by TLC using 20% ethyl acetate/hexanes). The reaction mixture was cooled to ambient temperature and solvent concentrated in vacuo. The residue taken in ethylacetate (200 mL) and washed with water and brine then dried (sodium sulfate), filtered, solvent concentrated in vacuo to give as brown solid. Crude compound purified by column chromatography (20% ethyl acetate/hexane). Compound 9 was obtained as an orange color solid (2.1 g, 88%).

Synthesis of compound compound II (CV-8688) To a dried 100 mL two neck round—bottom flask under argon at 0 °C, ylformamide (20 mL): was added. P0013 (0.75 g, 0.005 moi) slowly added while maintaining the internal temperature below 5°C over 10 min. After min stirring at 0°C, a solution of compound 9 (1.3 g, 0.005 mol) in dimethylformamide (5 mL) was slowly added while maintaining the internal ature below 5°C over 10 min. The resulting mixture was stirred at ambient temperature overnight. After the reaction was complete (monitored by TLC using % ethyl acetate/hexanes). The reaction mixture was poured into saturated aqueous sodium bicarbonate (150 mL) and stirred for 1 hr. Resulting e was extracted with ethyl acetate (2 x 100 mL). The organic layers were combined and washed with water, saturated NaCl and dried over Na2804. The solvent was filtered and concentrated in vacuo to give as brown solid. The crude compound crystallized in chloroform (25 mL). Compound || (CV-8688) was obtained as a light pink solid (0.81 g, 53%).

HPLC purity: 98.94% (area%). 1H—NMR, 13C spectrum consistent with the structure. : Calc. for C19H17N20 (M + H)+: 289, found: 289.0.

Example 6 Cell Morphology Typical cell morphology after various ents is shown in Figs. 5A—5K, 6A—6K, 7A—7K, 8A—8K, 9A—9K, 10A—10K, 11A—11K, and 12A—12K. The morphology of both cell lines was significantly affected by 100 [M of 4 and CV—8688, as well as sporine treatment at 6 hours. CV—8685 appeared to only affect WM1 15 at 100 uM.

Example 7 Apoptosis-lnducing Activity of Malassezin and Malassezin Derivatives — inary Annexin V Assays Materials and Reagents n V—FITC assay kit was purchased from Beyotime Biotechnology, RPMI 1640 medium and Dulbecco’s modified Eagle medium ("DMEM") were purchased from Gibco, fetal bovine serum ("FBS") was purchased from lnvitrogen, stabilized antibiotic antimycotic solution (100x) was purchased from Sigma, and 0.25% trypsin—EDTA (1X), phenol red was purchased from lnvitrogen.

Cell Culture MeWo (ATCC® HTB—65TM) and WM115 (ATCC® CRL—1675) cells were purchased from ATCC sas, VA) and maintained in the following: for MeWo: DMEM supplemented with 10% FBS; for WM115: RPMI 1640 supplemented with 10% FBS (10% FBS, 1% stabilized antibiotic anti—mycotic solution).

Study Summary In the intermediate stages of apoptosis, phosphatidylserine ("P8") is translocated from the inner to the outer leaflet of the cell membrane, exposing PS to the extracellular environment, where it can be detected. Highly fluorescent annexin V conjugates e quick and reliable detection methods for ng the externalization of PS.

During the first set of studies, both MeWo and WM115 cells were treated with test compounds at 10 doses starting from 100 [M with 3—fold dilution.

Staurosporine was used as positive l. After 6—hour ent, cell apoptosis was assessed using an annexin V assay. The test compounds evaluated were CV—8684, CV—8685, 6, CV—8687, and CV—8688.

Assay Procedures For cell seeding, cells were harvested and the cell number was determined using Countess® cell counter. Cells were then diluted with culture medium to the desired density. 40 uL of cell suspension per well was added to the required number of wells in a 384—well plate (Corning 3712 — clear bottom plate). The final cell density was 6,000 cells / well. After plating, the plates were incubated at 37°C and 5% 002 overnight.

For preparation of compound source plate, each test compound was dissolved in DMSO to 10 mM stock. 3—fold serial dilution was performed using an EVO200T'V' liquid r (TECAN) to te ten concentrations of test compound. 0.1% DMSO was employed as vehicle (negative) control. The nd source plate was then spun at room temperature at 1,000 RPM for 1 minute and ed using a plate shaker for 2 minutes.

For compound treatment, 40 nL of compound were erred from the compound source plate to the 384—well e plate using liquid handler Ech0550 (LabCyte lnc.). After 6—hour incubation, the plates were removed from the incubator for detection.

For the preliminary annexin V assay, the plates were removed from the incubator and allowed to equilibrate at room temperature for 15 minutes.

Culture media was then removed. 20 uL of pre—mixed annexin V—FITC and Hoechst33342 dye working solution were added to each well. The cells were then incubated at room temperature for 20 minutes. The plates were sealed and centrifuged for 1 minute at 1,000 RPM to remove bubbles. Afterward, the plate was read using an Acumen eX3 plate reader. The relative activity was ated ing to the following formula: Activity (%) = 100% x (CountAmxin v / CountTotal cell), and E050 was calculated using GraphPad Prism (v. 5.01 ).

Results In the preliminary screen discussed above, CV—8688 markedly increased annexin V staining of MeWo cells, with an E050 of 908.57 nM.

Staurosporine, the positive control, greatly increased annexin V staining in both cell lines. (Figs. 3A—3M).

Example 8 Apoptosis-lnducing Activity of Malassezin and Malassezin Derivatives — Additional Evaluation Using Annexin V Assays Study Summary To further investigate the impact of test compounds on apoptosis, multiple readouts, covering different stages of sis, were d out on both MeWo and WM115 cells. Both cell types were treated with test compounds at 3 doses (100 uM, 10 uM, and 1 uM). Staurosporine was used as a positive control. After the desired ent period (6, 24, 48, or 72 hours), apoptosis was assessed by measuring percentages of cells trating annexin V binding after exposure to the test compounds. The test compounds evaluated were CV—8684, 5, and CV—8688.

Assay Procedures Cell seeding was med as discussed above with the following exceptions: the final cell density was 4,000 cells / well for 6—hour and 24—hour detections, whereas 2,000 cells / well were used for 48—hour and 72—hour detections. For each time point, 384—well clear bottom plates (Coming 3712) and solid white bottom plates (Coming 3570) were prepared. The plates were incubated as sed above.

For preparation of the compound source plate, each test nd was dissolved in DMSO to 10 mM stock. Two additional concentrations were generations by 10—fold dilution to 1 mM and 0.1 mM. sporine was used as ve control and 1% DMSO was employed as vehicle (negative) control. The compound source plate was spun at room temperature at 1,000 RPM for 1 minute and agitated using a plate shaker for 2 minutes. 400 nL of test compound was erred from the compound source plate to 384—well culture plates using Ech0550 liquid handler. After 6, 24, 48, and 72 hours, the plates were removed from the incubator for detections.

For the annexin V assay, plates were removed from the incubator and equilibrated at room temperature for 15 minutes. Culture media was d and cells were washed twice with PBS. 20 uL of pre—mixed annexin V— FITC working solution was added to each well. The cells were incubated at room temperature for 20 minutes. Plates were read using Acumen eX3 to count the number of FlTC—positive cells. The relative activity was calculated according to the following formula: Relative Activity (%) = 100% x (Countsample / Countvehicle).

Results CV—8684 d apoptosis at the highest concentration tested after 6 hours of treatment on both MeWo and WM115 cells. CV—8685 showed the induction effect with 24 hours of treatment on WM115, whereas 48 hours of treatment appeared to elicit sis in both cell types. Finally, CV—8688 showed the induction effect within 6 hours of treatment in a dose—dependent manner in both cell types. (Figs. 4A—4L).

Example 9 Cell Viability After Exposure to Malassezin and Malassezin tives — CellTiter-G|o® Assays Assay Procedures CellTiter—Glo® 2.0 assay was purchased from Promega. Cell seeding, preparation of the compound source plate, and exposure of cells to test compounds were performed as described in Example 8.

For the ter—Glo® assay, plates were removed from the incubator and equilibrated at room temperature for 15 minutes. ter—Glo® reagents were thawed and equilibrated to room temperature before the experiment. 40 uL of CellTiter—Glo® reagent was then added to each well for detection (at 1:1 ratio to culture medium). The plates were then ted at room temperature for 30 minutes and read using EnSpire (PerkinElmer) plate reader. The remaining activity was calculated according to the following formula: Remaining Activity (%) = 100% x (Lumsample — Lumbkg) / hicle — Lumbkg).

Results CV—8684 showed dose—dependent inhibition of cell viability in both cell lines tested, though the tory effect appeared to be more potent in MeWo cells. CV—8685 exhibited the inhibitory effect on WM115 cell ity in a dose— dependent manner only after 24—hour ent. CV—8688 inhibited viability of both cell types in a dose—dependent manner. Staurosporine, the positive control, exerted 100% inhibition of cell viability in both cell lines after 24—hour treatment.

(Figs. 13A—13K).

Example 10 Cytotoxicity of Malassezin and Malassezin Derivatives — Lactate Dehydrogenase e Assays Study Summary The LDH assay quantitatively measures lactate dehydrogenase ("LDH") released into the media from damaged cells as a biomarker for cytotoxicity and cytolysis.

Assay ures x—ONETM Homogenous Membrane Integrity Assay was purchased from Promega. Cell seeding, preparation of the compound source plate, and exposure of cells to test compounds were performed as bed in Example 8.

For the LDH release assay, plates were removed from the incubator and equilibrated at room temperature for 15 minutes. Plates were then centrifuged at 1,000 RPM for 1 minute. 20 uL of cell culture medium was transferred into a new 384—well black solid plate. Then, 20 uL of CytoTOX— ONETM was added into each well and incubated at room temperature for 10 minutes. Afterward, 10 uL of stop solution were added to each well, and the plates were agitated at 500 rpm for 1 minute. Plates were read using an excitation wavelength of 560 nm and an emission wavelength of 590 nm on EnSpire. The relative activity was calculated according to the following formula: Relative Activity (%) = 100% x (Lumsample — Lumbkg) / (Lumvehicle — ).

Results CV—8684 did not induce significant release in either cell line after 72—hour incubation. 5 showed a dose—dependent induction effect on LDH release from WM115, but not MeWo, cells after r treatment. CV—8688 induced LDH release at the highest concentration tested. (Figs. 14A—14L).

Example 11 Arylhydrocarbon Receptor Activation Potential of ezin and Malassezin Derivatives Assay Procedures HepG2—AhR—Luc cells were purchased from ron, One—Glo Luciferase assay system was purchased from Promega, DMEM was purchased from Hyclone, and penicillin / streptomycin was sed from o.

Culture media for stably ected HepG2 cells was prepared by supplementing DMEM with high glucose and L—glutamine, as well as 10% F88.

HepG2—AhR—Luc cells were cultured in T—75 flasks at 37°C, 5% 002, and 95% relative humidity. Cells were allowed to reach 80—90% confluence before detachment and splitting.

Cultivated cells were rinsed with 5 mL PBS. PBS was ted away, 1.5 mL n was added to the flask, and cells were incubated at 37°C for approximately 5 minutes or until the cells detached and floated. Trypsin was inactivated by adding excess serum—containing media.

The cell suspension was transferred to a conical tube and centrifuged at 120 g for 10 minutes to pellet the cells. Cells were ended in seeding media at a proper density. 40 uL of cells were transferred to a 384—well culture plate (5 x 103 cells / well). Plates were placed in the incubator at 37°C for 24 hours.

Afterward, stock solutions of test compounds and omeprazole positive control were prepared. 40 nL of compound solutions were transferred into the assay plate using Ech0550. The plate was then placed back into the incubator for compound treatment.

Later, after 24 hours of treatment, the plate was removed from the tor and allowed to cool at ambient ature. 30 uL One—Glo reagent equal to that of the culture medium was added in each well. Cells were allowed to lyse for at least 3 minutes, and then measured in a luminometer.

Dose responses were graphed using the non—linear regression is in XLfit, and EC50 values were also calculated.

Results AhR—Luciferase assay results are shown in Figs. 15A—15F.

Example 12 WO 56424 MelanoDermT'V' Assays Study Summary The purpose of this study is to evaluate the potential dermal irritation of the test article to the MelanoDermTM Skin Model after repeated exposures for dose selection for a subsequent study. Toxicity will be determined by measuring the relative conversion of MTT (3—[4,5 — dimethylthiazol—2—yl] — 2,5 — diphenyltetrazolium bromide) in the test article—treated tissues compared to the negative/solvent control—treated tissues.

The MelanoDermTM Skin Model provided by MatTek Corporation (Ashland, MA) will be used in this study. The MelanoDermTM tissue consists of normal, human—derived epidermal keratinocytes (NHEKs) and melanocytes (NHMs) which have been cultured to form a ayered, highly entiated model of the human epidermis. The NHMs within tures undergo neous melanogenesis leading to tissues of varying levels of pigmentation.

The cultures are grown on cell culture inserts at the quid interface, allowing for l application of skin modulators. The MelanoDermTM model exhibits in vivo—like morphological and ultrastructural characteristics. NHMs localized in the basal cell layer of MelanoDermTM tissues are dendritic and spontaneously produce melanin granules which progressively populate the layers of the tissue.

Thus the test system may be used to screen for als which may inhibit or stimulate the production of melanin relative to the negative controls.

The experimental design of this study consists of the determination of the pH of the neat test article if possible (and/or dosing solution as appropriate) and a definitive assay to determine the relative tissue viability after repeated exposures. The MelanoDermTM Skin Model will be exposed to the test article for a total of 7 days. The test article will be topically applied to the MelanoDermTM Skin Model every 48 hours (within a ame of 4812 hours from previous treatment). The toxicity of the test article will be determined by the NAD(P)H—dependent microsomal enzyme ion of MTT (and, to a lesser extent, by the succinate dehydrogenase ion of MTT) in control and test article—treated s. (Berridge eta/., 1996). Data will be presented in the form of relative survival (MTT conversion relative to the negative control).

Materials MelanoDermTM Maintenance Medium (EPl—100—LLMM) and MelanoDermTM Skin Model 00—A) were supplied by MatTek Corporation. 1% Kojic acid (prepared in sterile, deionized water) and MTT (3—[4,5 — dimethylthiazol—2—yl] — 2,5 — diphenyltetrazolium bromide) were supplied by Sigma. Dulbecco's ed Eagle's Medium (DMEM) containing 2mM L— glutamine (MTT Addition Medium) was supplied by Quality Biological. lsopropanol was ed by Aldrich. Sterile Ca++ and Mg++ Free Dulbecco’s Phosphate Buffered Saline (CMF—DPBS) was supplied by lnvitrogen or equivalent. Sterile Deionized Water was ed by Quality Biological or equivalent. DMSO was supplied by CiVenti Chem.

Assay ures Test es will generally be tested neat or as directed by the Sponsor (see Protocol Attachment 1). Ten microliters (10 uL) or 25 [L of each test article will be applied directly on the tissue so as to cover the upper e.

Depending on the nature of the test article (liquids, gels, creams, foams, etc.), the use of a dosing device, mesh or other aid to allow the uniform spreading of the test e over the e of the tissue may be necessary.

In the days of dosing, each test article will be diluted at least 200— fold using the appropriate volume of EPl—100—LLMM (or ate solvent as determined during the solubility testing). A fresh dilution in EPl—100—LLMM will be prepared for each dosing. The final dilution to be performed for dosing solution preparation will be determined from the solubility assessment above and documented in the study workbook.

DMSO diluted as 0.5% (v/v) in EPILLMM will be used as vehicle control and dosed onto the tissues (10 [JL and 25 [JL doses) based on the same procedure used for the test articles and assay controls.

The test articles will be applied topically to the MelanoDermTM tissue every 48 hours (within a timeframe of 4812 hours from previous treatment) during a 7—day trial. Ten and 25 microliters, respectively, of each test article will be applied to each tissue. Twenty five iters of the positive and negative controls, respectively, will be applied to each tissue.

The pH of the neat liquid test article r dosing solution as appropriate) will be ined, if possible. The pH will be determined using pH paper (for example, with a pH range of 0 — 14 to estimate, and/or a pH range of 5 — 10 to ine a more precise value). The typical pH increments on the narrower range pH paper are approximately 0.3 to 0.5 pH units. The maximum increment on the pH paper is 1.0 pH units.

The definitive assay will include a negative control and a positive l. The MelanoDermTM tissues ated to the assay negative l will be treated with 25 pL of sterile, deionized water. Twenty five microliters of 1% Kojic acid (prepared in sterile, deionized water and filtered at the time of preparation) will be used to dose the tissues designated to the assay positive control. The 1% Kojic acid will be stored in a tube covered with aluminum foil until used within 2 hours of preparation. The negative and positive control exposure times will be identical to those used for the test articles.

It is necessary to assess the ability of each test article to directly reduce MTT. A 1.0 mg/mL MTT solution will be prepared in MTT Addition Medium as described below. Approximately 25 pL of the test article will be added to 1 mL of the MTT solution and the mixture incubated in the dark at 3790 J; 190 for one to three hours. A negative control, 25 pL of sterile, deionized water, will be tested concurrently. If the MTT solution color turns blue/purple, the test article is presumed to have reduced the MTT. Water insoluble test materials may show direct reduction (darkening) only at the interface between the test article and the medium.

The MTT direct reduction test for the test artic|e(s) may have been previously performed in an independent study. In such cases, the results of the MTT direct reduction test may be used for this specific study and the initial study will be referenced.

Tissue Exposure: At least 16 hours after ting the cultures, two MelanoDermTM tissues (considered untreated at Day 0) will be photographed using a digital camera to aid in the visual assessment of the degree of tation of the tissues at time zero of the assay. The exact procedures used to collect images of the tissues will be specified in the study workbook and report.

The MelanoDermTM tissues will be rinsed with CMF—DPBS, will be blotted dry on sterile absorbent paper and cleared of excess liquid. The MelanoDermTM tissues will be transferred to the appropriate MTT containing wells after rinsing and processed in the MTT assay as described in the MTT Assay section.

At least 16 hours after initiating the cultures, the s will be moved on a new 6—well plate ning 0.9 mL of fresh, pre—warmed 0— LLMM. The trial will be conducted over a 7—day timeframe. Two tissues will be treated topically on the first day, and every 48 hours n a timeframe of 48 +/— 2 hours from previous treatment) with 10 and 25 microliters, respectively, of each test e. The medium will be refreshed daily (within a timeframe of 24 +/— 2 hours from us refeeding); the tissues will be moved to a new 6—well plate containing 0.9 mL of fresh, pre—warmed EPl—100—LLMM.

Two tissues will be treated topically on the first day, and every 48 hours (within a timeframe of 48 +/— 2 hours from previous treatment) with 25 uL of positive and negative controls, respectively. The medium will be refreshed daily (within a timeframe of 24 +/— 2 hours from previous refeeding); the tissues will be moved to a new 6—well plate containing 0.9 mL of fresh, rmed EPl—100— LLMM. The tissues will be incubated at 371190 in a humidified atmosphere of 511% CO2 in air ard culture conditions) for the appropriate exposure times.

On the days of dosing, the MelanoDermTM tissue will be first gently rinsed three times using ~ 500 uL of BS to remove any residual test article. The tissues will then be moved to a new 6—well plate containing 0.9 mL of fresh, pre—warmed EPl—100—LLMM and dosed with the appropriate test e, negative or positive control. The tissues will be incubated at 371190 in a humidified atmosphere of 511% CO2 in air (standard culture conditions) for the appropriate exposure times. The exact rinsing procedure will be documented in the study workbook.

At the end of the 7—day trial, the MelanoDermTM tissues dosed with the negative or ve control, and with each test e will be photographed using a digital camera to aid in the visual assessment of the degree of pigmentation of the tissues at the end of the assay (Day 7). The exact procedures used to collect images of the tissues will be specified in the study workbook and report. Then, the viability of the tissues will be determined by MTT ion as indicated below.

MTT Assay: A 10X stock of MTT prepared in PBS (filtered at time of batch preparation) will be thawed and diluted in warm MTT Addition Medium to produce the 1.0 mg/mL on no more than two hours before use. Three hundred uL of the MTT solution will be added to each designated well of a pre— labelled 24—well plate.

After the exposure time, each MelanoDermTM tissue designated for the MTT assay will be rinsed with CMF—DPBS, blotted dry on sterile absorbent paper, and cleared of excess liquid. The MelanoDermTM tissues will be transferred to the appropriate MTT containing wells after rinsing. The 24—well plates will be incubated at standard conditions for 3 J; 0.1 hours.

After 3 J; 0.1 hours, the MelanoDermTM s will be blotted on sterile absorbent paper, cleared of excess liquid, and transferred to a pre—labelled 24—well plate containing 2.0 mL of isopropanol in each designated well. The plates will be covered with lm and stored in the erator ) until the last exposure time is harvested. If necessary, plates may be stored overnight (or up to 24 hours after the last exposure time is harvested) in the refrigerator prior to ting the MTT. Then the plates will be shaken for at least 2 hours at room temperature. At the end of the extraction period, the liquid within the cell culture inserts will be decanted into the well from which the cell culture insert was taken.

WO 56424 The extract solution will be mixed and 200 uL transferred to the appropriate wells of 96—well plate. Two hundred uL of panol will be added to the wells designated as . The absorbance at 550 nm (OD550) of each well will be measured with a Molecular Devices Vmax plate reader (with AUTOMIX function In cases where the test article is shown to reduce MTT, only test articles that remain bound to the tissue after rinsing, resulting in a false MTT reduction signal, present a problem. To demonstrate that possible residual test article is not acting to directly reduce the MTT, a functional check is performed in the definitive assay to show that the test material is not binding to the tissue and leading to a false MTT reduction signal.

To determine whether residual test article is acting to directly reduce the MTT, a freeze—killed control tissue is used. Freeze killed tissue is prepared at IIVS by placing ted MelanoDermTM/EpiDermTM (MelanodermTM without melanocytes) s in the —20‘-’C freezer at least overnight, thawing to room temperature, and then refreezing. Once killed, the tissue may be stored indefinitely in the freezer. Freeze killed tissues may be received already prepared from MatTek Corporation, and stored in the —20‘-’C freezer until use. To test for residual test e reduction, killed tissues are treated with the test article in the normal fashion. All assay procedures will be performed in the same manner as for the viable tissue. At least one killed control treated with e zed water (negative killed control) will be tested in parallel since a small amount of MTT reduction is expected from the residual NADH and associated enzymes within the killed tissue.

If little or no MTT reduction is observed in the test article—treated killed control, the MTT reduction observed in the test article—treated viable tissue may be ascribed to the viable cells. If there is appreciable MTT reduction in the treated killed control (relative to the amount in the treated viable tissue), additional steps must be taken to account for the chemical reduction or the test e may be considered untestable in this system. The OD550 values from the killed controls will be analyzed as described below The raw absorbance data will be ed and saved as a print—file and imported into an Excel spreadsheet. The mean OD550 value of the blank wells will be calculated. The corrected mean OD550 value of the negative control(s) will be determined by subtracting the mean OD550 value of the blank wells from their mean OD550 values. The corrected OD550 values of the individual test article exposures and the positive control res will be determined by subtracting from each the mean OD550 value for the blank wells.

All calculations will be performed using an Excel spreadsheet. gh the algorithms discussed are performed to ate the final endpoint analysis at the treatment group level, the same calculations can be applied to the dual replicates.

Corr. test e exposure OD550 = Test article exposure OD550 — Blank mean OD550 If killed controls (KC) are used, the following additional calculations will be performed to correct for the amount of MTT reduced directly by test article residues. The raw OD550 value for the negative control killed control will be subtracted from the raw OD550 values for each of the test article—treated killed ls, to determine the net OD550 values of the test article—treated killed controls.

Net OD550 for each test article KC = Raw OD550 test article KC — Raw OD550 negative control KC The net OD550 values represent the amount of d MTT due to direct reduction by test article residues at specific exposure times. In general, if the net OD550 value is greater than 0.150, the net amount of MTT reduction will be subtracted from the corrected OD550 values of the viable treated s to obtain a final corrected OD550 value. These final corrected OD550 values will then be used to determine the % of Control viabilities.

Final Corrected OD550 = Corrected test article OD550 (viable) — Net OD550 test e (KC) Finally, the following % of Control calculations will be made: % viability = [(Final ted OD550 of Test Article or Positive Control) / cted mean OD550 of Negative Control)] x 100 MelanoDermTM assay results are shown in Figs. 16A—16K.

Malassezin—, compound |—, and compound ll—treated tissues demonstrated reduced pigmentation on day 7 of the experiment. Figs. 17A—17K show 15X ication images of MelanoDermTM samples d to the listed treatment.

Example 13 Zebrafish Assays Assay Procedures Compounds: Compounds will be provided by Study Sponsor as Master Stock (MS) solution at the highest soluble concentration in water/PBS or DMSO.

Standard procedures for embryo collection: ix AB zebrafish will be generated by natural mating or using a Mass Embryo Production System (MEPS, Aquatic Habitats). Approximately 50 zebrafish will be generated per female zebrafish. Zebrafish will be maintained at 28°C in fish water. Zebrafish will be d (dead zebrafish removed) and sorted by developmental stage.

Because ish receive nourishment from an attached yolk sac, no feeding is required for 6 days post fertilization (dpf). nd Solubility: Master Stock (MS) (using the highest concentration) will be diluted in pure DMSO to sub—stock solutions (SS) ie: 10, 50, 100, 200, 300 mM, etc. Fish water [200 mg Instant Ocean Sea Salt (Aquarium Systems) per liter of deionized water; pH 6.6 — 7.0 maintained with 2.5 mg/liter Neutral Regulator (Seachem Laboratories Inc); conductivity 850 — 950 p8], supplied by Phylonix, will be dispensed into a testing vessel, 4 ml/vessel.

To generate test compound solution (T8), 4 pl of each 88 will be added directly to fish water. Example: 4 pl of 10 mM 88 added to fish water will generate 10 pM TS; final DMSO concentration will be 0.1%. Alternatively, to obtain the same final TS and DMSO trations, 10 pl 88 can be added to 10 ml/vessel of fish water. For assays that can tolerate DMSO up to 1%, 40 pl of 88 can be used to generate 100 pM TS. If 10 ml fish water is used, volume of 88 should be increased proportionally to obtain the same final TS and DMSO concentrations. The solution will be incubated at 28°C for the length of time specified for each assay and visually examined daily for presence of precipitation.

Maximum Tolerable Concentration (MTC): MTC (LC1o) will be used as the standard ion for compound lethality, determined using 10 compound trations. After determining the highest soluble compound concentration, Study Sponsor will select 10 concentrations.

Thirty ~2 dpf nated Phylonix wild—type AB zebrafish will be distributed into wells of 6—well microplates containing 4 ml/well fish water and DMSO at a concentration ranging from 0.1—1% depending on compound solubility. 10 concentrations (i.e.: 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, and 500 pM (or up to the concentration permitted by compound solubility), will be tested initially. If necessary, onal higher (up to 2000 pM) or lower (down to 0.001 pM) concentrations will be tested.

Zebrafish will be incubated with each concentration of test nd in the dark at 2890 for 3 days. Untreated and 0.1—1% DMSO treated zebrafish will be used as assay and vehicle controls. To calculate % lethality, after treatment, number of dead ish will be counted daily and removed. At dpf, dead animals will be counted to calculate % ity (= total number of dead zebrafish/30). Note, if dead zebrafish disintegrate, number of dead zebrafish will be deduced by counting number of live zebrafish.

To estimate MTC, lethality curves will be generated by plotting % lethality vs tration using EXCEL software. To obtain mean and SD of MTC, experiments will be performed 3 times.

Visually assess compound effect on zebrafish skin pigmentation: Zebrafish skin pigment cells including xanthophores, iridophores, and melanophores (melanocytes) originate from neural crest cells. In zebrafish, differentiated skin pigment precursor cells express pigment at ~ 24 hpf. The focus of this study is melanocytes which express melanin, the black t on the e of the skin. Melanocytes initially appear as small s of black color in the dorsal head region. As zebrafish develop, the number of patches increase and fuse to form bands which extend to the tail region. In contrast, mutant albino zebrafish exhibit sparse skin pigmentation. Compounds will be administered at 2 dpf, to assess if compounds arrest the continuous process of embryonic pigmentation, which is completed by 5 dpf. Three concentrations, MTC, 50% MTC, and 25% MTC, will be tested for each compound.

Thirty 2 dpf self—hatched Phylonix ype AB zebrafish will be treated with each compound concentration for 3 days. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTU, 0.03%).

Zebrafish will be visually examined daily using a dissecting light microscope; compound and PTU treated zebrafish will be compared to untreated and vehicle treated control zebrafish. Number of zebrafish exhibiting decreased pigmentation will be counted daily and sed as % of test s; a representative image will be provided. To identify optimum compound tration and treatment time for decreased pigmentation, a kinetic curve will be generated by plotting % zebrafish exhibiting sed skin pigmentation vs. time (dpf). Fisher’s exact test will be used to determine if nd effect is icant (P < 0.05).

Additional visual assessment of compound effect on zebrafish skin pigmentation will be performed after treatment with: 0.1, 1, and 3 pM. Thirty 2 dpf self—hatched Phylonix wild—type AB zebrafish will be treated with each compound concentration for 3 days. Untreated and 0.1% DMSO d zebrafish will be used as controls. Positive control: phenylthiourea (PTU, 0.003%). Zebrafish will be visually examined daily using a dissecting light cope; compound and PTU treated zebrafish will be compared to untreated and vehicle treated control zebrafish.

At 5 dpf, number of zebrafish exhibiting decreased pigmentation will be counted and expressed as % of test animals; a representative image will be provided. To identify optimum compound concentration and ent time for decreased pigmentation, a kinetic curve will be generated by plotting % zebrafish exhibiting sed skin pigmentation vs concentration. Fisher’s exact test will be used to determine if compound effect is significant (P < 0.05).

Ouantitate compound effect on zebrafish skin pigmentation: Based on results from the visual assessment, we will use the optimum conditions (concentration, compound treatment time) to quantitate nd effect on zebrafish skin pigmentation.

Twenty Phylonix wild—type AB ish at the optimum stage ined by results from the visual assessment will be treated with optimum compound concentration. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTU, 0.03%).

Dorsal view image of whole zebrafish will be ed using a SPOT camera at 2X. Dorsal head and trunk region will be defined as region of interest (ROI) using Adobe Photoshop selection function. Black skin pigmentation in the ROI will be highlighted using Photoshop highlighting on.

Total pigment signal (P8) in pixels will be determined using the Photoshop histogram function.

If compound affects zebrafish growth, body length (L) and trunk width (W) will be smaller, which will affect ROI area and final PS. Therefore, we will normalize measurement of final signal (FS) using FS = P8/ LxW. ted and vehicle treated zebrafish are expected to t similar F8 to demonstrate that vehicle does not have an effect. PTU treated zebrafish are expected to exhibit low F8 to validate the assay. Compound treated zebrafish will be ed with vehicle treated control zebrafish.

To determine if compound effect is significant (P < 0.05), mean F8 for compound treated zebrafish will be compared to mean FS of vehicle treated ish using Student’s t test.

Additional tation of compound effect on zebrafish skin tation will be performed after treatment with: 0.5 and 1.5 pM compound concentration.

Twenty 2 dpf Phylonix wild—type AB zebrafish will be treated with 0.5 and 1.5 pM compound concentration. Untreated and 0.1% DMSO treated zebrafish will be used as controls. Positive control: phenylthiourea (PTU, 0.003%).

Dorsal view image of whole zebrafish will be ed using a SPOT camera at 2X. Dorsal head region will be defined as region of interest (ROI) using Adobe Photoshop selection function. Black skin pigmentation in the ROI will be ghted using Photoshop highlighting function. Total pigment signal (P8) in pixels will be determined using the Photoshop histogram function.

If compound affects zebrafish growth, body length (L) will be shorter and trunk width (W) will be smaller, which will affect ROI area and final PS. Therefore, we will normalize final signal (FS) measurement using FS = P8/ ted and vehicle treated zebrafish are expected to exhibit similar F8 to confirm no effect of vehicle. PTU treated zebrafish are expected to t low FS, validating the assay. Compound treated zebrafish will be compared with vehicle treated control zebrafish.

To determine if compound effect is significant (P < 0.05), mean F8 for compound d ish will be compared to mean FS of vehicle treated zebrafish using Student’s t test.

Results Visual assessment results for zebrafish exposed to compound II are shown in Figs. 18A—18F and Figs. 19A—19F. A chart summarizing results from the visual ment n of the study is shown in Fig. 20.

Quantitative assessment regions of interest and results for zebrafish exposed to compound II are shown in Figs. 21A—21E and Figs. 22A— Example 14 Stability of Malassezin and Malassezin Derivatives in DMSO and Cell Culture Media Tested compounds were prepared at 100 [M in DMSO and culture medium. The solutions were incubated at room temperature for 2 hours and analyzed using LC—MS. The peak area was used to evaluate the compound remaining in the solvent.

Results The LC—MS results are shown in Figs. 23A—23J. The s indicate that the compounds are stable in culture medium after 2—hour incubation.

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All documents cited in this application are hereby incorporated by reference as if recited in full herein.

Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art t departing from the scope or spirit of the invention.

Claims (18)

WHAT IS CLAIMED IS:

1. A method for inducing melanocyte apoptosis in a subject sing contacting the subject with a composition sing (a) a nd having the structure of formula (II): R1 R11 OHC R9 R6 R8 N R7 R3 R5 wherein: R1, R2, R3, R4, R5, R6, R7, R8, R9, R10, and R11 are independently selected from the group consisting of hydrogen and methyl; or a hydrate or cosmetically or pharmaceutically acceptable salt thereof; and (b) a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier.

2. The method of claim 1, wherein the compound is selected from the group consisting of: , and ; or a hydrate or cosmetically or pharmaceutically acceptable salt thereof.

3. The method of claim 2, n the compound is or a hydrate or cosmetically or pharmaceutically able salt f.

4. The method of claim 3, wherein the cosmetically or pharmaceutically able vehicle, diluent or carrier comprises one or more ingredients selected from the group consisting of lactose, sucrose, mannitol, sorbitol, a starch, a calcium phosphate, sodium citrate, ethyl alcohol, propyl alcohol, benzyl alcohol, a , a triglyceride, a biodegradable polymer, an elastomeric matrix, liposomes, microspheres, corn oil, germ oil, olive oil, castor oil, sesame oil, cottonseed oil, nut oil, cocoa butter, a wax, paraffin, silicone, talc, and silicylate.

5. The method of claim 3, wherein the composition further comprises one or more ingredients selected from the group consisting of perfumes, estrogen, vitamin A, vitamin C, vitamin E, pyruvic acid, lactic acid, glycolic acid, lanolin, vaseline, aloe vera, methyl paraben, propyl paraben, and pigments.

6. The method of claim 3, wherein the composition further comprises one or more cosmetically acceptable compounds, ingredients and/or materials selected from the group ting of: (i) starches, e, sucrose, glucose, mannitol, c acid; (ii) carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose, acacia; (iii) glycerol; (iv) cetyl alcohol, glycerol monostearate; (v) kaolin, bentonite clay; (vi) talc, m stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate; (vii) ethoxylated isostearyl alcohols, esters of polyoxyethylene sorbitol and sorbitan, microcrystalline ose, aluminum metahydroxide, bentonite, agar-agar, tragacanth; (viii) milk sugars, polyethylene glycols, animal fats, vegetable fats, oils, waxes, paraffins, cocoa , starches, cellulose derivatives, polyethylene , silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, polyamide powder; (ix) preservatives; (x) surface-active agents; (xi) ypropylmethyl cellulose, liposomes, microspheres, aluminum monostearate, gelatin, waxes; (xii) wetting agents; (xiii) ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl te, propylene glycol, 1,3-butylene glycol, cottonseed oil, groundnut oil, corn oil, germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofuryl alcohol, hylene glycols, fatty acid esters of sorbitan; and (xiv) lecithin.

7. The method of claim 3, n the composition comprises a cosmetically or pharmaceutically able carrier selected from the group consisting of a cosmetically or pharmaceutically acceptable liquid, cream, oil, lotion, ointment, gel, and solid.

8. The method of claim 3, wherein the composition is a topical or transdermal ition ed from the group consisting of a powder, spray, ointment, paste, cream, lotion, gel, patch, on, suspension, aerosol, and inhalant.

9. The method of claim 8, wherein the composition is an oil-in-water or water-in-oil emulsion.

10. The method of claim 3, wherein the composition is a dosage form for oral administration.

11. The method of claim 10, wherein the composition is a solid dosage form.

12. The method of claim 10, wherein the composition is a liquid dosage form.

13. A composition comprising: (a) a compound having the following structure: or a hydrate or cosmetically acceptable salt thereof, (b) a cosmetically acceptable vehicle, diluent or carrier, and (c) one or more cosmetically acceptable ingredients selected from the group consisting of perfumes, estrogen, vitamin A, vitamin C, vitamin E, c acid, lactic acid, glycolic acid, lanolin, vaseline, aloe vera, methyl paraben, propyl paraben, and pigments.

14. A composition comprising: (a) a compound having the following structure: or a hydrate or ically or pharmaceutically able salt thereof, and (b) a cosmetically or pharmaceutically acceptable vehicle, t or carrier, and (c) one or more cosmetically acceptable compounds, ingredients and/or materials selected from the group consisting of: (i) starches, lactose, sucrose, glucose, mannitol, silicic acid; (ii) ymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, e, acacia; (iii) glycerol; (iv) cetyl alcohol, glycerol monostearate; (v) kaolin, bentonite clay; (vi) talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate; (vii) ethoxylated isostearyl alcohols, esters of polyoxyethylene ol and sorbitan, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth; (viii) milk sugars, polyethylene s, animal fats, vegetable fats, oils, waxes, paraffins, cocoa , starches, cellulose derivatives, polyethylene , silicones, bentonites, c acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, polyamide powder; (ix) preservatives; (x) surface-active agents; (xi) hydroxypropylmethyl cellulose, liposomes, microspheres, aluminum monostearate, gelatin, waxes; (xii) wetting agents; (xiii) ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene , seed oil, groundnut oil, corn oil, germ oil, olive oil, castor oil, sesame oil, glycerol, tetrahydrofuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan; (xiv) lecithin, wherein the composition is a pharmaceutical or cosmetic ition.

15. A composition comprising: (a) a compound having the following structure: or a hydrate or cosmetically or pharmaceutically acceptable salt thereof, and (b) one or more ically or pharmaceutically acceptable carriers selected from the group consisting of a cosmetically or pharmaceutically acceptable cream, oil, lotion, ointment, gel, and solid, wherein the composition is a pharmaceutical or cosmetic composition.

16. A composition comprising: (a) a compound having the following structure: or a hydrate or cosmetically or pharmaceutically acceptable salt thereof, and (b) a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier, wherein the composition is a topical or transdermal composition selected from a , spray, ointment, paste, cream, lotion, gel, patch, emulsion, suspension, l, and inhalant wherein the composition is a pharmaceutical or cosmetic composition.

17. A composition comprising: (a) a nd having the following structure: or a e or cosmetically or pharmaceutically acceptable salt thereof, and (b) a cosmetically or pharmaceutically acceptable vehicle, diluent or carrier, wherein the cosmetically or pharmaceutically acceptable vehicle, t or carrier comprises one or more ients selected from the group consisting of lactose, sucrose, mannitol, sorbitol, a starch, a calcium phosphate, sodium citrate, ethyl alcohol, propyl alcohol, benzyl alcohol, a polyol, a ceride, a biodegradable polymer, an elastomeric matrix, liposomes, microspheres, corn oil, germ oil, olive oil, castor oil, sesame oil, cottonseed oil, groundnut oil, cocoa butter, a wax, paraffin, silicone, talc, and silicylate, wherein the composition is a pharmaceutical or cosmetic ition.

18. The composition of claim 16, wherein the composition is an oil-in-water or waterin-oil emulsion. ~~~~~~~~~~~~ Epéd‘ermés ~~~~~~~~~~v Dennis mt ”My~~~~~~~~~~~ Subcmanemss tissue Epidermis ““““muAuuAuuAuuAuuuuluuuuuu, wuuuuuluuuuu, , Dennis ~ SUBSTITUTE SHEET (RULE 26) WO 56424 mmggmw SUBSTITUTE SHEET (RULE 26) WIfig(300)20NaHMDS Q©\/\Pt0|z©¢PtI|(PPh3)ZCI2 Indole NH NH NH 1 o;\ij 2 OAOJV 3 |ndo|o[3,2-b] carbazole 4 SUBSTITUTE SHEET (RULE 26) é“ \O \ ZOIOJv 5 h 0 6 H l CHO \ N H nd IV Compound I SUBSTITUTE SHEET (RULE 26) G:I é \ O pth O \ NH d(PPh3)ZC| NH Indole N 1 ¢o Hoke8 Compound || SUBSTITUTE SHEET (RULE 26) Camgmumi m E656 {BM} an MeWU ECSS MM} cm wmms Staamsgmine 58328 881.5% 5933734 >1888fi game (CV-8585 >1SGQG >§GQQS CVISfiSsS £8883 )EGQSG S? >1£§$GQ >§8€§QG (IV-8688 988.57 >EG‘3SQ SUBSTITUTE SHEET (RULE 26)