WO2005065666A1

WO2005065666A1 - Us of a pkc-delta inhibitor for the inhibition of metastatic melanoma

Info

Publication number: WO2005065666A1
Application number: PCT/US2004/041610
Authority: WO
Inventors: Denise Perry Simmons
Original assignee: Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services
Priority date: 2003-12-22
Filing date: 2004-12-13
Publication date: 2005-07-21

Abstract

A method of inhibiting metastatic melanoma cells comprising administering to the cells an effective amount of a PKC-delta inhibitor, preferably rottlerin, whereupon the metastatic melanoma cells are inhibited, and a pharmaceutical composition comprising a PKC-delta inhibitor in an amount effective to inhibit metastatic melanoma in a host and a pharmaceutically acceptable carrier.

Description

OF A PKC-DELTA INHIBITOR FOR THE INHIBITION OF METASTATIC MELANOMA FIELD OF THE INVENTION [0001] The present invention pertains to a method and a composition for inhibiting metastatic melanoma. More particularly, the present invention pertains to the use of a protein kinase C delta inhibitor (PKC-δ), such as rottlerin, alone or in further combination with one or more other active agents, to inhibit metastatic melanoma.

BACKGROUND OF THE INVENTION [0002] Melanoma is the fastest rising cancer in developing countries, and the death rate from melanoma has continued to increase approximately two percent annually since 1960 (Susarla et al., J. Neurochem. 86:635-45 (2003)). Importantly, although cutaneous melanoma represents only 4% of all cancers diagnosed in the United States in 2000 (Greenlee RT et al., CA Cancer J. Clin. 50: 7-33 (2000)), it accounts for nearly 75% of all deaths from skin cancer (Anonymous, Deaths from melanoma-United States 1973-1992, MMWR 44: 343- 47 (1995)).

[0003] Standard treatment for primary melanoma is surgical excision with clear margins, which is curative in the absence of micrometastatic disease. However, early detection and definitive therapy are directly related to survival rates of patients with malignant melanoma (Dancey et al., Nature Reviews Drug Discovery 2:296-313) and, to date, suppressive, rather than curative, treatments are available to patients with cutaneous metastatic melanoma. Moreover, it is well established that metastatic melanoma is a chemotherapeutic refractory and chemically-induced, apoptosis-resistant tumor (Tamura et al., Pigment Cell Res. 16: 470-76 (2003)). Indeed, toxicity to conventional radiation and chemotherapies, along with resistance to various immunotherapies, clearly indicate a need for novel therapies.

[0004] hi view of the foregoing, there is a need in the art for novel methods and compositions for treating metastatic melanoma. The present invention provides such a method of treatment and such a composition. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION [0005] The present invention provides a method of inhibiting metastatic melanoma cells. The method comprises administering to the cells an effective amount of a PKC-δ inhibitor, whereupon the metastatic melanoma cells are inhibited.

[0006] Also provided by the present invention is a pharmaceutical composition comprising a PKC-δ inhibitor in an amount effective to inhibit metastatic melanoma in a host and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS [0007] Figure 1 sets forth the amino acid sequences of SEQ ID NOS: 1-5, which are presented from N-terminus to C-terminus from left to right in accordance with convention.

DETAILED DESCRIPTION OF THE INVENTION [0008] The present invention is predicated on the surprising aiid unexpected discovery that PKC-δ inhibitors, such as rottlerin, can inhibit melanoma. Accordingly, the present invention provides a method of inhibiting metastatic melanoma cells. The method comprises administering to the cells an effective amount of a PKC-δ inhibitor, whereupon the metastatic melanoma cells are inhibited. The method can be used in combination with other known treatment methods, such as radiation, surgery, or the administration of other active agents, such as adjuvants or other cytotoxic agents and their prodrugs. [0009] The term "inhibiting" means that the proliferation of cells is diminished, such as by about 1 %, 5 %, 10 %, 20 %, 30%, 40 %, 50%, 75 % or more. Preferably, the proliferation is reduced by about 90 % or more. The reduction in proliferation is witnessed by an increase in the number of dead melanoma cells, which is evidenced by rounding, detachment, and floating of cells without re-attachment to the substrate. The term "inhibiting" additionally or alternatively means the induction of differentiation of the cells. The metastatic melanoma cell can have epithelioid or fibroblastoid morphology. Differentiation refers to changes in cellular morphology, including cellular processes. Differentiation of the cells is evidenced by an increase in branching and in the number of processes of the melanoma cells.

[0010] Metastasis is the dissemination of cancer cells to other parts of the host's body from the primary tumor site, where the tumor originated. Metastasized cells are cells from a umor that are disseminated to other parts of the host's body. "Melanoma," as used herein, refers to transformed melanocytes. Melanocytes, used in this context, are found in the basal layer of the epidermis. Melanoma is further characterized based on the tissue in which the positioned melanocytes are transformed, such as skin, epidermal, or cutaneous melanomas. [0011] PKC is a family of enzymes comprising eleven isoforms with different modalities of activation, tissue and intracellular localization, and phosphorylation of specific substrates. PKC-δ is one of the PKC isoforms. Different kinds of PKC-δ inhibitors are known to those of ordinary skill in the art. Preferably, the PKC-δ inhibitor is rottlerin (available from Calbiochem, San Diego, CA) and derivatives/analogues thereof. Other PKC-δ inhibitors include Go 6976 and Go 6983 (both available from Calbiochem, San Diego, CA), which are PKC inhibitors that are isozyme-selective dependent upon concentrations used. [0012] Derivatives and analogues of PKC-δ inhibitors also can be used in the present invention. One of ordinary skill in the art will recognize that derivatives and analogues will have the same inhibitory activity of the unaltered agent, optionally to a greater of lesser extent, but not negated. Such chemical modifications will be based on structure activity relationships (SAR) or molecular modeling. For example, functional groups can be substituted or eliminated. [0013] The PKC-δ inhibitor can be administered to a cell in vitro. As used herein, the term "in vitro" means that the cell to which the compound is being administered is not in a living organism. The PKC-δ inhibitor can be administered to the cell in vivo. As used herein, the term "in vivo" means that the cell is a part of a living organism or is the living organism. [0014] The PKC-δ inhibitor can be administered alone or co-administered with one or more other active agents. The term "co-administered" means administered concurrently with, or sequentially to, in either order, with an active agent. By "active agent" is meant an agent that inhibits metastatic melanoma cells or promotes the inhibition of metastatic melanoma cells by the PKC-δ inhibitor. In a preferred embodiment, the active agent can be a PKC inhibitor, such as a general PKC inhibitor, a PKC-c inhibitor, a PKC-/3 inhibitor, a PKC-γ inhibitor, PKC-δ inhibitor, PKC-e inhibitor, PKC-τj inhibitor, PKC- f inhibitor, and the like. General PKC inhibitors include, but are not limited to, safmgol (L-threo- dihydorsphingosine), Ro-1 (Bisindolylmaleimide), Ro32-0432 (Bisindolylmaleimide tertiary amine), and flavopiridol (L86-8275). The general PKC inliibitor is preferably staurosporine, a derivative of staurosporine, or a salt of either staurosporine or the derivative of staurosporine. Examples of staurosporine derivatives are UNC-01 (7-OH-staurosporine), CGP41251 (PKC412; 4'-N- benzoyl staurosporine), and the like. One of ordinary skill in the art will appreciate that a variety of derivatives and analogues as well as salts of a general PKC inhibitor can be synthesized or obtained commercially. [0015] Alternatively, but still preferably, the active agent can be a cytotoxic agent, such as a chemotherapeutic agent. Examples of cyotoxic agents and their prodrugs include genistein, okadaic acid, 1-β-D-arabinofuranosyl-cytosine, arabinofuranosyl-5-aza-cytosine, cisplatin, carboplatin, actinomycin D, asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin, chlorambucil, cyclohexyl- chloro-ethyl-nitroso-urea, cytosine arabinoside, daunomycin, etoposide, hydroxyurea, melphalan, mercaptopurine, mitomycin C, nitrogen mustard, procarbazine, teniposide, thioguanine, thiotepa, vincristine, 5-fluorouracil, 5- fluorocytosine, adriamycin, cyclophosphamide, methotrexate, vinblastine, doxorubicin, leucovorin, taxol, anti-estrogen agents, such as tamoxifen, intracellular antibodies against oncogenes, the flavonol quercetin, Guan-mu-tong extract, retinoids, such as fenretinide, nόntoxid retinoid analogues, such as N-(4- hydroxyphenyl)-retinamide (HPR), and monoterpenes, such as limonene, perillyl alcohol and sobrerol. [0016] The cytotoxic agent can be a chemotherapeutic agent, e.g., a polyamine or an analogue thereof. Examples of therapeutic polyamines include those set forth in U.S. Patent Nos. 5,880,161, 5,541,230 and 5,962,533; Saab et al., J. Med. Chem. 36: 2998-3004 (1993); Bergeron et al., J. Med. Chem. 37(21): 3464-76 (1994); Casero et al., Cancer Chemother. Pharmacol. 36: 69-74 (1995); Bernacki et al., Clin. Cancer Res. 1: 847-57 (1995); Bergeron et al., J. Med. Chem. 40: 1475-94 (1997); Gabrielson et al., Clinical Cancer Res. 5: 1638-41 (1999); and Bergeron et al., J. Med. Chem. 43: 224-35 (2000), which can be administered alone or in combination with other active agents, such as anti-cancer agents, e.g., cis-diaminedichloroplatinum (II) and l,3-bis(2-chloroethyl)-l-nitrosourea. In an embodiment a chemotherapeutic agent is a cytoxic agent, such as mitomycin C, carboplatin, taxol, or doxorubicin.

[0017] The PKC-δ inhibitor can be conjugated to a delivery agent, such as a polypeptide. For example, the polypeptide can have the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5. (Fig. 1). While the polypeptides of SEQ ID NOS: 1- 5 are preferred, conservative and neutral amino acid substitutions can be introduced into the polypeptides, thereby generating variants. A polypeptide variant can comprise one, two, or three conservative or neutral amino acid substitutions, provided that amino acids 8 and 9 in SEQ ID NO: 1, amino acids 1, 8 and 9 in SEQ ID NO: 2, amino acids 7 and 8 in SEQ ID NO: 3, amino acids 1 and 8 in SEQ ID NO: 4, and amino acid 7 in SEQ ID NO: 5 remain unchanged. Additionally or alternatively, the variant can comprise one, two, or three amino acid additions at the N-terminus and/or C-terminus. Preferably, not more than a total of one, two, or three amino acids are added. Desirably, the variant has activity characteristic of the unaltered polypeptide, optionally to a greater or lesser extent, but not negated.

[0018] Techniques of conjugating compounds to delivery agents are known in the art, and conjugate kits are commercially available. (See, e.g., Wadwa et al., J. Drug Targeting 3: 111 (1995) and U.S. Patent No. 5,156,840). The term "delivery agent" as used herein, refers to any molecule or agent that can freely cross membranes as well as any molecule or agent that specifically recognizes and binds to an intracellular and/or a cell-surface receptor, such that the delivery agent directs the delivery of the compound to cells where the receptor is expressed. Delivery agents include, but are not limited to, peptide nucleic acids (PNA's), antibodies, or antigenically-reactive fragments thereof, peptides, polypeptides, hormones, growth factors, cytokines, and any other naturally- or hon-naturally- existing ligands, which bind to cell surface receptors. A "linker" can be used to attach the compound to the delivery agent. One of ordinary skill in the art recognizes that sites on the compound, which are not necessary for the function of the compound, are ideal sites for attaching a linker and/or a delivery agent, provided that the linker and/or delivery agent, once attached to the compound, do(es) not interfere with the function of the compound, i.e., the ability to reduce proliferation of a cell, induce differentiation of a cell, or inhibit metastasis. [0019] The cell can be in a host. Preferably, the host is a mammal. For purposes of the present invention, mammals include, but are not limited to, mammals of the order Rodentia, such as mice, mammals of the order Logomorpha, such as rabbits, mammals of the order Carnivora, including Felines (cats) and Canines (dogs), mammals of the order Artiodactyla, including Bovines (cows) and Swines (pigs), and mammals of the order Perssodactyla, including Equines (horses). It is preferred that the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). An especially preferred mammal is the human.

[0020] An "effective amount" of the PKC-δ inhibitor is an amount sufficient to, e.g., inhibit metastatic melanoma cells over a reasonable time frame. An "effective amount" can be determined by one skilled in the art. In particular, the effective amount will be determined by the strength of the particular agent(s) or composition(s) administered or co-administered. The skilled artisan will recognize that an effective amount in vitro depends on a variety of factors, such as the type of cells, the type and amount of PKC-δ inhibitors, the type and amount of active agents, the condition of the cells; etc.

[0021] To describe effective amounts of a PKC-δ inhibitor, it can be useful, in some contexts, to describe the inhibitor's IC₅₀, the concentration at which there is 50 % reduction of proliferation of cells. For Go 6976 (U.S. Patent No. 5,489,608) IC50 with respect to PKC-c is 2.3 nM, but the amounts needed to inhibit PKC-δ are in the micromolar range. For Go 6983 (Wang et al., J Biol. Chem. 273:33027 (1998)) IC₅₀ with respect to PKC-α and PKC-/3 is 7 nM and with respect to PKC-γ is 6 nM, but the amounts needed to achieve IC₅₀ for PKC-δ is 10 nM. These molar amounts are all cell type dependent, and, therefore, the IC₅₀ is tested empirically. [0022] hi so far as the PKC-δ inhibitor, such as rottlerin, is effective against metastatic melanoma, which includes epithelioid melanoma, one of ordinary skill in the art will recognize that the PKC-δ inhibitor alone or in combination with one or more other active agents can be used to inhibit epithelial-type cancers. Examples of epithelial-type cancers include, but are not limited to, prostate, intestinal (colon), and breast cancers.

[0023] In a pharmaceutical composition, the PKC-δ inhibitor can be rottlerin or a derivative thereof. The pharmaceutical composition can further comprise one or more other active agents wherein the one or more other active agents is a PKC inhibitor. The PKC inliibitor is preferably staurosporine, a derivative of staurosporine, or a salt of either staurosporine or the derivative of staurosporine. Further, the pharmaceutical composition can have a PKC-δ inhibitor, which is not a general PKC inhibitor. Additionally, the pharmaceutical composition can comprise a PKC-δ inhibitor conjugated to a^'delivery agent. The delivery agent can be a polypeptide, such as a polypeptide having amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

[0024] The "effective amount" for in vivo treatment depends on the condition of the mammal (e.g., human), as well as the body weight (e.g., human) to be treated. The size of the effective amount will also be determined by the existence, nature, and extent of any adverse side effects that might accompany administration of agents. Many assays for determining an administered amount are known in the art. Ultimately, the attending physician will decide the dosage of the PKC-δ inhibitor to be administered alone or to be co-administered with one or more other active agents with which to treat each individual patient, taking into consideration a variety of factors, such as age, body weight, general health, diet, sex, inhibitor to be administered, route of administration, and the severity of the condition being treated. [0025] The present invention further provides for a pharmaceutical composition comprising a PKC-δ inhibitor in an amount effective to inhibit metastatic melanoma and pharmaceutically acceptable carrier. The pharmaceutical composition can have the PKC-δ inhibitor, alone or in combination with one or more other agents, such as a general PKC-inhibitor and/or a chemotherapeutic agent.

[0026] With respect to pharmaceutical compositions, the carrier can be any of those conventionally used and is limited only by chemico-physical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. It will be appreciated by one of skill in the art that, in addition to the following described pharmaceutical composition, the inhibitors of the present inventive methods can be formulated as inclusion complexes, such as cyclodextrin inclusion complexes, or liposomes.

[0027] The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. It is preferred that the pharmaceutically acceptable carrier be one which is chemically inert to the active compound(s) and one which has no detrimental side effects or toxicity under the conditions of use. [0028] The choice of carrier will be determined in part by the particular PKC-δ inhibitor, as well as by the particular method used to administer the compound. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition. The following formulations for oral, aerosol, parenteral, subcutaneous, intravenous, intramuscular, interperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering a compound of the present invention or a pharmaceutical composition are known, and, although more than one route can be used to administer a particular compound, a particular route can provide a more immediate and more effective response than another route. [0029] The requirements for effective pharmaceutical carriers for injectable compositions are well-known to those of ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-50 (1982), and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-30 (1986)). [0030] Topical formulations are well-known to those of skill in the art. Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers; aqueous, powder, or oily bases; thickeners; and the like may be necessary or desirable.

[0031] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the inhibitor dissolved in diluents, such as water, saline, or dextrose solutions; (b) capsules, sachets, tablets, lozenges, and troches, each containing a predetermined amount of the active ingredient, as solids or granules; (c) powders; (d) suspensions in an appropriate liquid; and (e) suitable emulsions. Liquid formulations may include diluents, such as water and alcohols, for example, ethanol, benzyl alcohol, and the polyethylene alcohols, either with or without the addition of a pharmaceutically acceptable surfactant. Capsule forms can be of the ordinary hard- or soft-shelled gelatin type containing, for example, surfactants, lubricants, and inert fillers, such as lactose, sucrose, calcium phosphate, and corn starch. Tablet forms can include one or more of lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, disintegrating agents, moistening agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia, emulsions, gels, and the like containing, in addition to the active ingredient, such excipients as are known in the art. [0032] The composition can be made into aerosol formulations to be administered via inhalation. These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer. Such spray formulations also may be used to spray mucosa.

[0033] Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The PKC-δ inhibitor can be administered in a physiologically acceptable diluent in a pharmaceutical carrier, such as a sterile liquid or. mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, such as 2,2-dimethyl-l,3-dioxolane-4-methanol, ethers, such as poly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as a soap or a detergent, suspending agent, such as pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifying agents and other pharmaceutical adjuvants.

[0034] Oils, which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral. Suitable fatty acids for use in parenteral formulations include oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.

[0035] Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include (a) cationic detergents, such as, for example, dimethyl dialkyl ammonium halides, and alkyl pyridinium halides, (b) anionic detergents, such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents, such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents, such as, for example, alkyl-b-aminopropionates, and 2- alkyl-imidazoline quaternary ammonium salts, and (e) mixtures thereof. [0036] The parenteral formulations will typically contain from about 0.5% to about 25% by weight of the active ingredient in solution. Preservatives and buffers may be used. In order to minimize or eliminate irritation at the site of injection, such compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17. The quantity of surfactant in such formulations will typically range from about 5% to about 15% by weight. Suitable surfactants include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol. The parenteral formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. [0037] Additionally, the composition can be made into suppositories by mixing with a variety of bases, such as emulsifying bases or water-soluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulas containing, in addition to the active ingredient, such carriers as are known in the art to be appropriate. [0038] Alternatively, the composition can be modified into a depot form, such that the manner in which the compound is released into the body to which it is administered is controlled with respect to time and location within the body (see, for example, U.S. Patent No. 4,450,150). Depot forms of compounds can be, for example, an implantable composition comprising the compound and a porous material, such as a polymer, wherein the compound is encapsulated by or diffused throughout the porous material. The depot is then implanted into the desired location within the body and the compound is released from the implant at a predetermined rate by diffusing through the porous material. EXAMPLE [0039] The following example serves to illustrate the present invention and should not be construed in any way as limiting its scope.

[0040] Example 1

[0041] This example describes the effects of rottlerin and staurosporine on metastatic melanoma cells.

[0042] Melanoma cells were purchased form the American Type Culture Collection (ATCC, Rockville, MD) and maintained as specified by the ATCC. Specifically, the melanoma cells were the matched tumor pairs [E:WM-115 and E:WM-266-4] and [F:Hs 688(A).T and F:Hs688(B).T]. Each pair was obtained from a single patient and consists of the primary site tumor cells and cells of a tumor that metastasized from the primary site. The "E" and "F" designations refer to epithelioid and fibroblastoid phenotypes as indicated by the ATCC specification sheets. Normal human primary epidermal melanocytes were obtained from the Yale Skin Diseases Research Center (New Haven, CT) and, upon arrival, were maintained in melanocyte growth medium from Clonetics (San Diego, CA). The purity of the normal human primary epidermal melanocyte cultures was achieved by growth media restriction of contaminating fibroblasts and keratinocytes. All cell types were identified as adult tissue in origin.

[0043] Cultures were seeded in 10% serum growth medium, in triplicate, in 6- well dishes to achieve a 50% confluence overnight. The next day, 50% cultures were rinsed with phosphate buffered saline (PBS) and incubated for one-hour in (i) serum- free medium or (ii) serum- free medium with 20 μM rottlerin or 40 nM staurosporine. Cultures were rinsed with PBS, then incubated with 2% serum medium supplemented with (i) solvent as vehicle control, or (ii) 16 nM 12-O- tetradecanolyphorbol- 13 -acetate (TPA) [phorbol myristate acetate (PMA)] as PKC receptor control. [0044] Cultures were observed by light microscopy for morphological changes. Cellular proliferation was assessed by hemacytometer counts and Trypan blue staining.

[0045] There were no apparent morphological changes when the medium was switched from growth medium containing 10% fetal bovine serum (FBS) to experimental medium containing 2% FBS.

[0046] The results indicate that 20 μM rottlerin affects morphological changes in the cells from the metastatic epithelioid melanoma [E:WM-266-4]. Rottlerin significantly reduced cell proliferation of E:WM-266-4. Furthermore, the observed rottlerin-induced cell death was reminiscent of frank apoptosis, while the 40 nM staurosporine induced morphological changes of elongated processes extending from the cell body of each cell, reflecting differentiation of E:WM-266- 4. Importantly, these rottlerin- and staurosporine-induced changes had no apparent similar effects on normal proliferating melanocytes. The above- described changes were not observed in the fibroblastoid matched tumor pair.

[0047] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0048] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. The phrase "consisting essentially of where used herein, is intended to only limit the scope of the invention to the specified materials or steps, and those that do not materially affect the basic and novel characteristics of the claimed invention as set forth above. As such, the scope of the invention where "consisting essentially of is recited is intended to be narrower than where "comprising" is used, however broader than where "consisting of is used. One of skill in the art, in reviewing the present specification, can readily identify those materials and steps that do not materially affect the basic and novel characteristics of the present invention. [0049] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

What is claimed is:

1. A method of inhibiting metastatic melanoma cells, which method comprises administering to the cells an effective amount of a protein kinase_. C delta (PKC-δ) inhibitor, whereupon the metastatic melanoma cells are inhibited.

2. The method of claim 1 , wherein the PKC-δ inhibitor is rottlerin or a derivative thereof.

3. The method of claim 1 or 2, wherein the PKC-δ inhibitor is co-administered with one or more other active agents.

4. The method of claim 3, wherein the one or more other active agents is a PKC inhibitor.

5. The method of claim 4, wherein the PKC inhibitor is staurosporine, a derivative of staurosporine, or a salt of either staurosporine or the derivative of staurosporine.

6. The method of claim 3, wherein the active agent is not a general PKC inhibitor.

7. The method of claim 1 or 2, wherein the PKC-δ inhibitor is conjugated to a delivery agent.

8. The method of claim 7, wherein the delivery agent is a polypeptide.

9. The method of claim 8, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

10. The method of claim 3, wherein the PKC-δ inhibitor is conjugated to a delivery agent.

11. The method of claim 10, wherein the delivery agent is a polypeptide.

12. The method of claim 11 , wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.

13. The method of claim 1, wherein the cells are in a host.

14. The method of claim 13 , wherein the host is a mammal.

15. The method of claim 14, wherein the mammal is a human.

16. A pharmaceutical composition comprising a PKC-δ inhibitor in an amount effective to inhibit metastatic melanoma in a host and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the PKC-δ inhibitor is rottlerin or a derivative thereof.

18. The pharmaceutical composition of claim 16 or 17, which further comprises one or more other active agents.

19. The pharmaceutical composition of claim 18, wherein the one or more other active agents is a PKC inhibitor.

20. The pharmaceutical composition of claim 19, wherein the PKC inhibitor is staurosporine, a derivative of staurosporine, or a salt of either staurosporine or the derivative of staurosporine.

21. The pharmaceutical composition of claim 18, wherein the PKC-δ inhibitor is not a general PKC inhibitor.

22. The pharmaceutical composition of claim 16 or 17, wherein the PKC-δ inhibitor is conjugated to a delivery agent.

23. The pharmaceutical composition of claim 22, wherein the delivery agent is a polypeptide.

24. The pharaiaceutical composition of claim 23, wherein the polypeptide has the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, or 5.