US 2015 0023915A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0023915 A1 MORRISON et al. (43) Pub. Date: Jan. 22, 2015

(54) TREATMENT FOR Publication Classification (71) Applicant: The Board of Regents of the University (51) Int. Cl. of Texas System, Austin, TX (US) A613 L/7048 (2006.01) A6II 45/06 (2006.01) (72) Inventors: Sean MORRISON, Dallas, TX (US); A638/20 (2006.01) Ugur ESKIOCAK, Dallas, TX (US) A638/2I (2006.01) A 6LX3/59 (2006.01) A 6LX39/395 (2006.01) (73) Assignee: The Board of Regents of the University (52) U.S. Cl. of Texas System, Austin, TX (US) CPC ...... A6 IK3I/7048 (2013.01); A61 K3I/519 (2013.01); A61 K39/3955 (2013.01); A61 K (21) Appl. No.: 14/280,376 38/2013 (2013.01); A61 K38/21 (2013.01); A61K 45/06 (2013.01) USPC ...... 424/85.4; 514/26: 424/130.1; 424/85.2 (22) Filed: May 16, 2014 (57) ABSTRACT Related U.S. Application Data The present invention relates to combination for melanoma, and in particular, metastatic melanoma. Drugs for (60) Provisional application No. 61/847,797, filed on Jul. use in such therapies in include MEK inhibitors combination 18, 2013. with cardiac glycosides. Patent Application Publication Jan. 22, 2015 Sheet 1 of 8 US 2015/0023915 A1

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TREATMENT FOR MELANOMA intraarterial, Subcutaneous, oral or intra-tumoral administra 0001. The present application claims benefit of priority to tion, or may comprise local, regional or systemic administra U.S. Provisional Application Ser. No. 61/847,797, filed Jul. tion, or may comprise continuous infusion over a period of 18, 2013, the entire contents of which are hereby incorporated time. The subject may have failed one or more standard by reference. melanoma therapies. 0008. The MEK inhibitor may be administered before the BACKGROUND OF THE INVENTION cardiac glycoside , at the same time as the cardiac glycoside therapy or after the cardiac glycoside therapy. The 0002 I. Technical Field MEK inhibitor may be administered more than once, and/or 0003. The present invention relates generally to the fields the cardiac glycoside may be administered more than once. of and genetics. More particularly, it The cardiac glycoside may be selected from digoxin, digi relates to the combined use of MEK inhibition and cardiac toxin, gitoxin, oleandrin, neriifolin, bufalin, marinobufage glycosides to treat melanoma, particularly metastatic mela nin, cinobufagenin, UNBS1450 and lanatoside C. The MEK Oa. inhibitor may be selected from trametinib, AS703026 (Pima 0004 II. Related Art sertib, MSC1936369B), AZD8330 (ARRY-424704), Selu 0005 Melanoma is a malignant tumor of melanocytes. metinib (AZD6244), PD-325901, GDC-0973 (XL518) and Melanocytes are cells that produce the dark pigment, mela CI-1040 (PD184352). The amount sufficient to inhibit said nin, which is responsible for the color of skin. They predomi melanoma may be less than the amount required for inhibi nantly occur in skin, but are also found in other parts of the tion by any single agent alone. The amount Sufficient to body, including the bowel, oral cavity and the eye. Melanin inhibit said melanoma may be less toxic than the amount also protects the deeper layers of the skin from the sun’s required for inhibition by any single agent alone. harmful ultraviolet (UV) rays. When people spend time in the 0009. The method may further comprising treating said Sunlight, the melanocytes make more melanin and cause the Subject with an additional agent, such as an immunomodula skin to tan. This also happens when skin is exposed to other tor (e.g., imiquimod, IL-2 or interferon), or a chemotherapeu forms of ultraviolet light (such as in a tanning booth). If the tic agent, Such as dacarbazine. The method may also further skin receives too much ultraviolet light, the melanocytes Suf comprise excising a melanoma lesion, where excising occurs fer genetic damage and can be transformed into cancerous prior to, after or concurrent with MEK inhibition/cardiac melanoma cells. glycoside treatment. The meloma may be BRAF wild-type or 0006 Because of the link to cumulative sun exposure, the BRAF mutated, such as where the BRAF mutation is V600E chance of getting melanoma increases with age, but many or V600K. The cardiac glycoside may be digitoxin or is young people also get melanoma. In fact, melanoma is one of digoxin. The immunomodulator may be an antibody against the most common cancers in young adults. Each year in the PD1 or CTLA4, such as Ipilimumab. U.S., more than 50,000 people young and old learn that 0010. It is contemplated that any method or composition they have melanoma. And, according to a WHO report, about described herein can be implemented with respect to any 48,000 melanoma related deaths occur worldwide each year. other method or composition described herein. The treatment includes surgical removal of the tumor, and if melanoma is found early while relatively small and thin, 0011. The use of the word “a” or “an when used in con complete removal gives a high cure rate. The chance of the junction with the term “comprising in the claims and/or the melanoma coming back or spreading depends on how deeply specification may mean “one but it is also consistent with it has invaded into the layers of the skin. Thus, the more the meaning of"one or more.” “at least one.” and “one or more progressed the lesion, the great the chancer for recurrence than one.” and/or metastasis. For that recur or spread, treat 0012. Other objects, features and advantages of the ments include chemo- plus immunotherapy or radiation present invention will become apparent from the following therapy, but the prognosis for Such patients, including those detailed description. It should be understood, however, that exhibiting metastatic disease (AJCC Stage III and IV) is poor, the detailed description and the specific examples, while indi with 5-year survival rates being less than 10%. As such, cating specific embodiments of the invention, are given by improved treatments for melanoma, particularly for advanced way of illustration only, since various changes and modifica metastatic disease, are urgently needed. tions within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed descrip SUMMARY OF THE INVENTION tion. 0007 Thus, in accordance with the present invention, BRIEF DESCRIPTION OF THE DRAWINGS there is provided a method of inhibiting melanoma in a Sub ject comprising administering to said subject (a) a MEK 0013 The following drawings form part of the present inhibitor, and (b) a cardiac glycoside, in an amount Sufficient specification and are included to further demonstrate certain to inhibit said melanoma. The Subject may be a human or a aspects of the present invention. The invention may be better non-human mammal. Inhibiting may comprise inhibiting the understood by reference to one or more of these drawings in growth of primary melanomas, inhibiting metastasis, inhib combination with the detailed description of specific embodi iting the growth of metastases, killing circulating melanoma ments presented herein. cells, inducing remission, extending remission, or inhibiting 0014 FIGS. 1A-C. Therapeutically achievable and safe recurrence. The melanoma may be AJCC stage III disease or levels of digitoxin are sufficient for anti-melanoma activity. AJCC stage IV disease. The subject may have previously FIG. 1A, ICso values of digitoxin for various melanomas and received a radiotherapy, a chemotherapy, an immunotherapy, human umbilical cord blood cells (hUCB). Melanomas that a molecularly targeted therapy or had surgical resection of a are sensitive to digitoxin at therapeutically achievable doses tumor. Administering may comprise topical, intravenous, are indicated in orange. FIG. 1B, Representative dose US 2015/0023915 A1 Jan. 22, 2015 response curves. FIG. 1C, Digitoxin concentrations mea vehicle only (200 ul 0.5% promethylcellulose, 0.2% Sured in the plasma of the mice 4 hours after 0.5 mg/kg Tween80 by gavage and 50% DMSO with osmotic pump). injection. Tumors were measured every 3-4 days with calipers. FIG. 6B, 0015 FIG. 2. Melanoma regression after treatment with a Average tumor weight (N=5) and standard deviation for each combination of MEK inhibitor (Trametinib, GSK1 120212) treatment. The statistical significance of differences between and digitoxin. The effect of digitoxin alone, or in combination mean values was assessed by the Student's t-test. FIG. 6C, with the MEK inhibitor, was tested in xenograft assays using Melanomas excised and photographed at the end of treat primary melanomas from patients. Melanoma cells were ment. FIG. 6D, Digoxin levels achieved in plasma of mice at injected Subcutaneously in the right flanks of immunocom the conclusion of therapy (21-days). promised NSG mice. When tumors became palpable, mice (0020 FIGS. 7A-D. Digoxin and MEK inhibitor combina were randomized to four different treatments. Mice were tion is effective against M481 (BRAF V600E). FIG. 7A, treated either with digitoxin (0.5 mg/kg/day in 200 ul of 0.5% M481 melanoma cells were injected subcutaneously in the promethylcellulose, 0.2% Tween80 and 5% DMSO by oral right flanks of immunocompromised NSG mice. When gavage), or GSK1 120212 (0.5 mg/kg/day in 200 ul of 0.5% tumors became palpable, mice were randomized to four dif promethylcellulose, 0.2% Tween80 and 5% DMSO by oral ferent treatments. Mice were treated either with MEKi gavage) alone, or in combination. Control mice received (trametinib, 0.5 mg/kg/day in 200 ul of 0.5% promethylcel vehicle only (200 ul 0.5% promethylcellulose, 0.2% lulose, 0.2% Tween80 by oral gavage) and/or digoxin (10 Tween80 and 5% DMSO per day). Tumors were measured mg/kg/day with osmotic pump). Control mice received every 2-3 days with calipers. vehicle only (200 ul 0.5% promethylcellulose, 0.2% 0016 FIGS. 3A-C. Digitoxin and MEK inhibitor combi Tween80 by gavage and 50% DMSO with osmotic pump). nation is effective against melanoma from patient M481 Tumors were measured every 3-4 days with calipers. FIG.7B, (BRAF V600E). FIG. 3A, Representative bioluminescence Average tumor weight (N-3-5) and standard deviation for images of M481 xenografts treated for 36 days with digitoxin each treatment. The statistical significance of differences and/or GSK1 120212. FIG. 3B, the average tumor mass and between mean values was assessed by the Student's t-test. standard deviation for each treatment. The statistical signifi FIG.7C, Melanomas excised and photographed at the end of cance of differences between mean values was assessed by treatments. FIG. 7D, Digoxin levels achieved in plasma of the Student's t-test. FIG. 3C, Melanomas excised and photo mice at the conclusion of therapy (15-days). graphed at the end of treatments. (0021 FIG.8. Combination of digoxin and MEKi is differ 0017 FIGS. 4A-C. Digitoxin and MEK inhibitor combi entially toxic to melanoma cells compared to normal cells. nation is effective against melanoma from patient M214 Melanomas (M498 and M214) and normal human cells (fi (BRAF wild-type). FIG. 4A, Representative biolumines broblasts, melanocytes, cord blood cells, colonic epithelial cence images of M214 xenografts treated for 9 days with cells and bronchial epithelial cells) were treated with 50 digitoxin and/or GSK1 120212. FIG. 4B, The average tumor ng/mL digoxin and 25 nM trametinib for 72 h in quadripli weight and standard deviation for each treatment. The statis cates. Viability was measured with CellTiter-Glo (Promega). tical significance of differences between mean values was Error bars denote standard deviation. assessed by the Student's t-test. FIG. 4C, Melanomas excised and photographed at the end of treatments. DETAILED DESCRIPTION OF THE INVENTION 0018 FIGS.5A-D. Digoxin and MEK inhibitor combina tion is effective against M214 (BRAF wild-type). FIG. 5A, 0022. In previous studies, the inventors have identified M214 melanoma cells were injected subcutaneously in the cardiac glycosides as highly potent anti-melanoma agents. right flanks of immunocompromised NSG mice. When Here, they extend this work in pre-clinical studies combining tumors became palpable, mice were randomized to four dif cardiac glycosides (e.g., digitoxin) with a MEK inhibitor ferent treatments. Mice were treated either with MEKi (Trametinib) to inhibit human melanomas Xenografted in (trametinib, 0.5 mg/kg/day in 200 ul of 0.5% promethylcel mice. The data show that this combination of agents almost lulose, 0.2% Tween80 by oral gavage) and/or digoxin (10 completely eradicated the tumors, whereas either agent alone mg/kg/day with osmotic pump). Control mice received slowed tumor growth, but did not cause net regression. The vehicle only (200 ul 0.5% promethylcellulose, 0.2% effect was observed regardless of the BRAF status of the Tween80 by gavage and 50% DMSO with osmotic pump). tumors. Thus, the inventors propose this unique combination Tumors were measured every 3-4 days with calipers. FIG.5B, of agents as a new melanomatherapy. These and other aspects Average tumor weight (N=5) and standard deviation for each of the disclosure are described in greater detail below. treatment. The statistical significance of differences between mean values was assessed by the Student's t-test. FIG. 5C, I. CANCERTHERAPEUTICAGENTS Melanomas excised and photographed at the end of treat ment. FIG. 5D, Digoxin levels achieved in plasma of mice at 0023 A. Cardiac Glycosides the conclusion of therapy (28-days). 0024 Cardiac glycosides are drugs used in the treatment 0019 FIGS. 6A-D. Digoxin and MEK inhibitor combina of congestive heart failure and cardiac arrhythmia. These tion is effective against M491 (BRAF V600E). FIG. 6A, glycosides are found as secondary metabolites in several M491 melanoma cells were injected subcutaneously in the plants, but also in some animals, such as the milkweed but right flanks of immunocompromised NSG mice. When terflies. Therapeutic uses of cardiac glycosides primarily tumors became palpable, mice were randomized to four dif involve the treatment of cardiac failure. Their utility results ferent treatments. Mice were treated either with MEKi from an increased cardiac output by increasing the force of (trametinib, 0.5 mg/kg/day in 200 ul of 0.5% promethylcel contraction. By increasing intracellular calcium as described lulose, 0.2% Tween80 by oral gavage) and/or digoxin (10 below, cardiac glycosides increase calcium-induced calcium mg/kg/day with osmotic pump). Control mice received release and thus contraction. US 2015/0023915 A1 Jan. 22, 2015

0.025 Drugs such as ouabain and digoxin are cardiac gly failure, maximizing other therapies (e.g., beta blockers) first cosides. Digoxin from the foxglove plant is used clinically, is recommended before using digoxin. whereas ouabain is used only experimentally due to its 0035 Digoxin is usually given by mouth, but can also be extremely high potency. given by IV injection in urgent situations (the IV injection 0026 Normally, sodium-potassium pumps in the mem should be slow, and heart rhythm should be monitored). brane of cells (in this case, cardiac myocytes) pump potas While IV therapy may be better tolerated (less nausea), sium ions in and sodium ions out. Cardiac glycosides inhibit digoxin has a very long distribution half-life into the cardiac this pump by stabilizing it in the E2-P transition state, so that tissue, which will delay its onset of action by a number of Sodium cannot be extruded: intracellular Sodium concentra hours. The half-life is about 36 hours, and digoxin is given tion therefore increases. A second membrane ion exchanger, once daily, usually in 125-lug or 250-lug doses. NCX, is responsible for pumping calcium ions out of the cell 0036. In patients with decreased kidney function, the half and sodium ions in (3Na/Ca); raised intracellular sodium life is considerably longer, calling for a reduction in dose or a levels inhibit this pump, so calcium ions are not extruded and Switch to a different glycoside, such as digitoxin (not avail will also begin to build up inside the cell. able in the United States), which has a much longer elimina 0027 Increased cytoplasmic calcium concentrations tion half-life of around seven days, but is mainly eliminated cause increased calcium uptake into the sarcoplasmic reticu from the body via the liver, and thus not affected by changes lum via the SERCA2 transporter. Raised calcium stores in the in kidney function. SR allow for greater calcium release on stimulation, so the 0037 Effective plasma levels vary depending on the medi myocyte can achieve faster and more powerful contraction by cal indication. For congestive heart failure, levels between 0.5 cross-bridge cycling. The refractory period of the AV node is and 1.0 ng/ml are recommended. This recommendation is increased, so cardiac glycosides also function to regulate based on post hoc analysis of prospective trials, suggesting heart rate. higher levels may be associated with increased mortality 0028 Binding of cardiac glycoside to Na KATPase is rates. For heart rate control (atrial fibrillation), plasma levels slow, and also, after binding, intracellular calcium increases are less defined and are generally titrated to a goal heart rate. gradually. Thus, the action of digitalis (even on IV injection) Typically, digoxin levels are considered therapeutic for heart is delayed. rate control between 1.0 and 2.0 ng/ml. In suspected toxicity 0029 Raised extracellular potassium decreases binding of or ineffectiveness, digoxin levels should be monitored. cardiac glycoside to Na—KATPase. Consequently, increased Plasma potassium levels also need to be closely controlled toxicity of these drugs is observed in the presence of (see side effects below). Hypokalemia. 0038 Quinidine, Verapamil, and amiodarone increases 0030) 1. Digoxin plasma levels of digoxin (by displacing tissue binding sites 0031 Digoxin is a purified cardiac glycoside extracted and depressing renal digoxin clearance), so plasma digoxin from the foxglove plant, Digitalis lanata. Its corresponding must be monitored carefully. aglycone is digoxigenin, and its acetyl derivative is acetyl 0039. The occurrence of adverse drug reactions is com digoxin. Digoxin is widely used in the treatment of various mon, owing to its narrow therapeutic index (the margin heart conditions, namely atrial fibrillation, atrial flutter and between effectiveness and toxicity). Adverse effects are con sometimes heart failure that cannot be controlled by other centration-dependent, and are rare when plasma digoxin con medication. Digoxin preparations are commonly marketed centration is <0.8 ug/l. They are also more common in under the trade names Lanoxin, Digitek, and Lanoxicaps. It is patients with low potassium levels (hypokalemia), since also available as a 0.05 mg/ml oral solution and 0.25 mg/ml or digoxin normally competes with Kions for the same binding 0.5 mg/ml injectable solution. It is marketed by GlaxoSmith site on the Na"/K ATPase pump. Kline and many other pharmaceutical manufacturers. 0040 Common adverse effects (>1% of patients) include: 0032 Today, the most common indications for digoxin are loss of appetite, nausea, vomiting and diarrhea as gastrointes atrial fibrillation and atrial flutter with rapid ventricular tinal motility increases. Other common effects are blurred response. Beta blockers and/or calcium channel blockers vision, visual disturbances (yellow-green halos and problems should be the first choice. High ventricular rate leads to insuf with color perception), confusion, drowsiness, dizziness, ficient diastolic filling time. By slowing down the conduction insomnia, nightmares, agitation, and depression, as well as a in the AV node and increasing its refractory period, digoxin higher acute sense of sensual activities. Less frequent adverse can reduce the ventricular rate. The arrhythmia itself is not effects (0.1%-1%) include: acute psychosis, delirium, amne affected, but the pumping function of the heart improves sia, convulsions, shortened QRS complex, atrial or Ventricu owing to improved filling. lar extrasystoles, paroxysmal atrial tachycardia with AV 0033. The use of digoxin in heart problems during sinus block, ventricular tachycardia or fibrillation, and heart block. rhythm was once standard, but is now controversial. In theory, Rarely, digoxin has been shown to cause thrombocytopenia. the increased force of contraction should lead to improved Gynaecomastia (enlargement of breast tissue) is mentioned in pumping function of the heart, but its effect on prognosis is many textbooks as a side effect, thought to be due to the disputable, and other effective treatments are now available. estrogen-like Steroid moiety of the digoxin molecule, but Digoxin is no longer the first choice for congestive heart when systematically sought, the evidence for this is equivo failure, but can still be useful in patients who remain Symp cal. The pharmacological actions of digoxin usually result in tomatic despite proper diuretic and ACE inhibitor treatment. non-toxic electrocardiogram changes, including ST depres 0034 Digitalis/digoxin has recently fallen out of favor sion or T wave inversion. PR interval prolongation, however, because it did not demonstrate a mortality benefit in patients may be a sign of digoxin toxicity. Additionally, increased with congestive heart failure; however, it did demonstrate a intracellular Ca" may cause a type of arrhythmia called reduction in hospitalizations for this condition. Because other bigeminy (coupled beats), eventually ventricular tachycardia therapies have shown a mortality benefit in congestive heart or fibrillation. The combination of increased (atrial) arrhyth US 2015/0023915 A1 Jan. 22, 2015

mogenesis and inhibited atrioventricular conduction (for 0046. There is also evidence that digoxin increases vagal example paroxysmal atrial tachycardia) is said to be pathog activity, thereby decreasing heart rate by slowing depolariza nomonic (i.e., diagnostic) of digoxin toxicity. An often tion of pacemaker cells in the AV node. This negative chro described, but rarely seen, adverse effect of digoxin is a notropic effect would therefore be synergistic with the direct disturbance of color vision (mostly yellow and green) called effect on cardiac pacemaker cells. Digoxin is used widely in Xanthopsia. Digoxin has potentially dangerous interactions the treatment of various arrhythmias. with Verapamil, amiodarone, erythromycin, and epinephrine 0047 2. Digitoxin (as would be injected with a local anesthetic). 0048 Digitoxin has similar structure and effects to 0041. The main pharmacological effects of digoxin are on digoxin (though the effects are longer-lasting). Unlike the heart. Extracardiac effects are responsible for some of the digoxin (which is eliminated from the body via the kidneys), therapeutic and many of the adverse effects. It has mechanical it is eliminated via the liver, so could be used in patients with effects as it increases myocardial contractility; however, the poor or erratic kidney function. However, it is now rarely used duration of the contractile response is just slightly increased. in current Western medical practice. While several controlled Overall, the heart rate is decreased, while blood pressure trials have shown digoxin to be effective in a proportion of increases as the stroke Volume is increased, leading to patients treated for heart failure, the evidence base for digi increased tissue perfusion. Myocardial efficiency is due to toxin is not as strong, although it is presumed to be similarly improved hemodynamics, and the ventricular function curve effective. Digitoxin exhibits similar toxic effects to the more is improved. commonly used digoxin, namely: anorexia, nausea, vomit ing, diarrhea, confusion, visual disturbances, and cardiac 0042. Other, electrical effects are an initial brief increase arrhythmias. Antidigoxin antibody fragments, the specific in action potential, followed by a decrease as the K conduc tance increases due to an increased intracellular amounts of treatment for digoxin poisoning, are also effective in serious Ca" ions. The refractory period of theatria and ventricles is digitoxin toxicity. The first structural analysis of digitoxin decreased, while it increases in the sinoatrial and AV nodes. A was performed in 1925, but the full structure with an exact less negative resting membrane potential is made, leading to determination of the Sugar groups was not accomplished until increased excitability. Other, more indirect effects are choli 1962. nomimetic because of Vagal stimulation, giving rise to AV 0049. 3. Other Cardiac Glycosides nodal delay. 0050 Gitoxin (CHO) is a secondary glycoside derived from Digitalis purpurea used in medicine for coro 0043. The conduction velocity increases in the atria, but nary disease. Other cardiac glycosides include oleandrin, decreases in the AV node. The effect upon Purkinje fibers and neriifolin, bufalin, marinobufagenin, cinobufagenin, Ventricles is negligible. Automaticity is also increased, in the UNBS1450 and lanatoside C. atria, AV node, Purkinje fibers and ventricles. ECG changes 0051 B. MEK Inhibitors are increased PR interval, due to decreased AV conduction, 0.052 MEK is a dual-specificity kinase that phosphory and a decreased QT interval because of the altered duration of lates the tyrosine and threonine residues on ERKs 1 and 2 decreased action potential. Also, the T wave is inverted, required for activation. Two related genes encode MEK1 and accompanied by ST depression. It may cause AV junctional MEK2 which differ in their binding to ERKs and, possibly, in rhythm and ectopic beats (bigeminy) resulting in Ventricular their activation profiles. MEKs do not phosphorylate either tachycardia and fibrillation. Slight vasodilation is seen in SAPKorp38 MAPK. MEKs are substrates for several protein heart failure. This effect is contrary to effects that should be kinases including the Rafs (c-. A- and B-). Mos, Tpl-2, and seen as a result of increased intracellular calcium levels, but MEKK1. this occurs since digoxin improves hemodynamics, which 0053 MEKs are phosphorylated by these kinases at two leads to restored angiotensin levels and decreased sympa serine residues (218 and 222 in rat MEK1). Introduction of thetic discharge, causing indirect vasodilation. Digoxin also acidic residues and truncation of an alpha-helical region in the affects the kidney by increased renal blood flow and increased N-terminal domain causes constitutive activation of MEK. glomerular filtration rate. A mild diuretic effect is seen only in Such proteins are transforming. Mutation of the same resi heart failure. dues to alanine generates dominant-negative proteins that 0044 Digoxin binds to a site on the extracellular aspect of have been used to determine the requirement for the ERK the a-subunit of the Na/K ATPase pump in the membranes pathway in various processes. For example, when expressed of heart cells (myocytes) and decreases its function. This in T cells of transgenic mice, T cell maturation and selection causes an increase in the level of sodium ions in the myocytes, are disrupted. which leads to a rise in the level of intracellular calcium ions. 0054 MEK inhibitors inhibit MEK1 and/or MEK2. They This occurs because the sodium/calcium exchanger on the can be used to inhibit the MAPK/ERK pathway which is often plasma membrane depends on a constant inward Sodium gra overactive in some cancers, such as melanoma. Hence MEK dient to pump out calcium. Digoxin decreases sodium con inhibitors have potential for treatment of some cancers, espe centration gradient and the Subsequent calcium outflow, thus cially BRAF-mutated melanoma, and KRAS/BRAF mutated raising the calcium concentration in myocardiocytes and colorectal cancer. One MEK inhibitor is Trametinib pacemaker cells. (GSK1 120212), which is FDA-approved to treat BRAF-mu 0045 Increased intracellular calcium lengthens phase 4 tated melanoma. It is also studied in combination with BRAF and phase 0 of the cardiac action potential, which leads to a inhibitor dabrafenib to treat BRAF-mutated melanoma. decrease in heart rate. Increased amounts of Ca" also leads to 0055. The Phase II Trametinib trial (NCT01245062) was increased storage of calcium in the sarcoplasmic reticulum, conducted in patients with BRAF too mutant advanced or causing a corresponding increase in the release of calcium malignant melanoma. Patients were randomized 2:1 to during each action potential. This leads to increased contrac Trametinib (2 mg QD) or chemotherapy (dacarbazine or tility, the force of contraction, of the heart. paclitaxel). Patients were stratified by baseline LDH leveland US 2015/0023915 A1 Jan. 22, 2015

prior chemotherapy; patients in the chemotherapy arm were Worldwide, doctors diagnose about 160,000 new cases of allowed to crossover to receive Trametinib after confirmation melanoma yearly. It is more common in women than in men. of progressive disease. Primary endpoint was progression In women, the most common site is the legs and melanomas free survival in patients with BRAFoo mutation-positive in men are most common on the back. It is particularly com malignant melanoma and no prior brain metastasis; second mon among Caucasians, especially northern Europeans liv ary endpoints were overall Survival, overall response rate and ing in Sunny climates. There are high rates of incidence in safety in primary and intention to treat groups. Progression Australia, New Zealand, North America (especially Texas free Survival and overall Survival were compared using a and Florida), Latin America, and Northern Europe, with a stratified log-rank test. The study was designed with a 99% paradoxical decrease in Southern Italy and Sicily. This geo power and one-sided C=0.025 to detect 57% reduction in the graphic pattern reflects the primary cause, ultraviolet light risk of progressive disease or death in pts treated with Trame (UV) exposure crossed with the amount of skin pigmentation tinib vs chemotherapy. in the population. 0056 Between December of 2010 and July of 2011, 322 0061 1. Early Signs patients were randomized to Trametinib (n=214) or chemo 0062 Early signs of melanoma are changes to the shape or therapy (n=108): 273 patients were BRAF too mutation color of existing moles or, in the case of nodular melanoma, positive with no prior brain metastasis. HR for primary popu the appearance of a new lump anywhere on the skin (Such lation for progression free Survival by investigator was 0.44 lesions should be referred without delay to a dermatologist). (95% CI 0.31-0.64; p<0.0001) in favor of Trametinib with a At later stages, the mole may itch, ulcerate or bleed. Early median progression free survival of 4.8 mo vs 1.4 mo with signs of melanoma are Summarized by the mnemonic chemotherapy. Progression free survival benefit in favor of “ABCDE: Trametinib was observed in the intention to treat group; this 0063 Asymmetry was confirmed by an independent review. The confirmed 0064. Borders (irregular) overall response rate was 24% with Trametinib and 7% with 0065 Color (variegated) chemotherapy. HR for interim overall survival was 0.53 (95% 0.066 Diameter (greater than 6 mm (0.24 in), about the CI 0.30-0.94: p=0.0181), in favor of Trametinib in primary size of a pencil eraser) population. Overall survival benefit was consistent in the 0067 Evolving over time intention to treat population despite 51 patients crossover These classifications do not, however, apply to the most dan from chemotherapy to Trametinib. The most frequent adverse gerous form of melanoma, nodular melanoma, which has its events with Trametinib were skin rash, diarrhea, edema, own classifications: hypertension, fatigue. Known MEKi class effects were 0068 Elevated above the skin surface observed, e.g., chorioretinopathy (<1%) and decreased ejec 0069 Firm to the touch tion fraction (7%). Grade 3 adverse effects in the Trametinib (0070 Growing arm were hypertenstion (12%) and rash (7%). The study Metastatic melanoma may cause nonspecific paraneoplastic concluded that Trametinib was the first in class MEKi asso symptoms, including loss of appetite, nausea, vomiting and ciated with a significant improvement of progression free fatigue. Metastasis of early melanoma is possible, but rela Survival and overall Survival compared to chemotherapy in tively rare: less than a fifth of melanomas diagnosed early pts with BRAF took mutant malignant melanoma. become metastatic. Brain metastases are particularly com 0057. Others MEK inhibitors include selumetinib, which mon in patients with metastatic melanoma. It can also spread had a phase 2 clinical trial for non-Small cell lung cancer to the liver, bones, abdomen or distant lymph nodes. (NSCLC), MEK162, which had a phase 1 trial for biliary tract (0071) 2. Development cancer and melanoma, PD-325901, indicated for breast can 0072 The earliest stage of melanoma starts when the mel cer, colon cancer, and melanoma, as well as XL518, CI-1040, anocytes begin to grow out of control. Melanocytes are found AS703026 (Pimasertib, MSC1936369B), AZD8330(ARRY between the outer layer of the skin (the epidermis) and the 424704), Selumetinib (AZD6244), and PD035901. next layer (the dermis). This early stage of the disease is II. METHODS OF TREATMENT called the radial growth phase, and the tumor is less than 1 mm thick. Because the cancer cells have not yet reached the 0058. In a particular aspect, the present invention provides blood vessels lower down in the skin, it is very unlikely that methods for the treatment of melanoma. Treatment methods this early-stage cancer will spread to other parts of the body. will involve administering to an individual having Such a If the melanoma is detected at this stage, then it can usually be disease an effective amount of a composition containing the completely removed with . When the tumor cells start compounds of the present invention. An effective amount is to move in a different direction vertically up into the epi described, generally, as that amount Sufficient to detectably dermis and into the papillary dermis—the behavior of the and repeatedly to ameliorate, reduce, minimize or limit the cells changes dramatically. extent of the disease or its symptoms. More specifically, it is 0073. The next step in the evolution is the invasive radial envisioned that the treatment with compounds of the present growth phase, which is a confusing term; however, it explains invention kill cancer cells, inhibit their growth, reduce or the next step in the process of the radial growth, when indi inhibit metastasis, inhibit or reduce or delay recurrence, or vidual cells start to acquire invasive potential. This step is otherwise provide clinical benefit. Also, combinations my important—from this point on the melanoma is capable of reduce toxicity due to lower dosing and or reduced frequency spreading. The Breslow's depth of the lesion is usually less of administration. than 1 mm (0.04 in), the Clark level is usually 2. 0059 A. Melanoma 0074 The following step in the process is the invasive 0060 Melanoma is less common than other skin cancers. melanoma the vertical growth phase (VGP). The tumor However, it is much more dangerous if it is not found early. It attains invasive potential, meaning it can grow into the Sur causes the majority (75%) of deaths related to . rounding tissue and can spread around the body through US 2015/0023915 A1 Jan. 22, 2015

blood or lymph vessels. The tumor thickness is usually more around trial evidence for this procedure. Treatment of than 1 mm (0.04 in), and the tumor involves the deeper parts advanced malignant melanoma is performed from a multidis of the dermis. The host elicits an immunological reaction ciplinary approach. against the tumor (during the VGP), which is judged by the (0093 4. Staging presence and activity of the tumor infiltrating lymphocytes 0094 Melanoma stages are listed below with their 5 year (TILs). These cells sometimes completely destroy the pri survival rates: mary tumor; this is called regression, which is the latest stage (0095 Stage 0: Melanoma in situ (Clark Level I), 99.9% of the melanoma development. In certain cases, the primary survival tumor is completely destroyed and only the metastatic tumor (0096 Stage I/II: Invasive melanoma, 85-99% survival is discovered. 0097 T1a: Less than 1.00 mm primary tumor thickness, 0075 3. Detection without ulceration, and mitosis <1/mm 0076 Visual diagnosis of melanomas is still the most com 0.098 T1b: Less than 1.00 mm primary tumor thickness, mon method employed by health professionals. Moles that with ulceration or mitoses >1/mm are irregular in color or shape are often treated as candidates 0099 T2a: 1.00-2.00 mm primary tumor thickness, of melanoma. The diagnosis of melanoma requires experi without ulceration ence, as early stages may look identical to harmless moles or 0100 Stage II: High risk melanoma, 40-85% survival not have any color at all. People with a personal or family 0101 T2b: 1.00-2.00 mm primary tumor thickness, history of skin cancer or of dysplastic nevus syndrome (mul with ulceration tiple atypical moles) should see a dermatologist at least once 0102 T3a: 2.00-4.00 mm primary tumor thickness, a year to be sure they are not developing melanoma. There is without ulceration no blood test for detecting melanomas. Metastatic melano 0.103 T3b: 2.00-4.00 mm primary tumor thickness, mas can be detected by X-rays, CT scans, MRIs, PET and with ulceration PET/CTs, ultrasound, LDH testing and photoacoustic detec 0.104 T4a: 4.00 mm or greater primary tumor thickness tion. without ulceration 0077. Many melanomas present themselves as lesions 0105 T4b: 4.00 mm or greater primary tumor thickness Smaller than 6 mm in diameter, and all melanomas were with ulceration malignant on day 1 of growth, which is merely a dot. Anastute 0106 Stage III: Regional metastasis, 25-60% survival will examine all abnormal moles, including ones 0107 N1: Single positive lymph node less than 6 mm in diameter. Seborrheic keratosis may meet some or all of the ABCD criteria, and can lead to false alarms 0108) N2: Two to three positive lymph nodes or regional among laypeople and sometimes even . An expe skin/in-transit metastasis rienced doctor can generally distinguish Seborrheic keratosis 0.109 N3: Four positive lymph nodes or one lymph from melanoma upon examination, or with dermatoscopy. node and regional skin/in-transit metastases 0078 Total body photography, which involves photo 0110 Stage IV: Distant metastasis, 9-15% survival graphic documentation of as much body Surface as possible, 0111 M1a: Distant skin metastasis, normal LDH is often used during follow-up of high-risk patients. The 0112 M1b: Lung metastasis, normal LDH technique has been reported to enable early detection and 0113 M1c. Other distant metastasis or any distant provides a cost-effective approach (being possible with the metastasis with elevated LDH use of any digital camera), but its efficacy has been questioned 0114 5. Prognosis due to its inability to detect macroscopic changes. The diag 0115 Features that affect prognosis are tumor thickness in nosis method should be used in conjunction with (and not as millimeters (Breslow's depth), depth related to skin struc a replacement for) dermoscopic imaging, with a combination tures (Clark level), type of melanoma, presence of ulceration, of both methods appearing to give extremely high rates of presence of lymphatic/perineural invasion, presence of detection. tumor-infiltrating lymphocytes (if present, prognosis is bet 0079 Melanoma is divided into the following types: ter), location of lesion, presence of satellite lesions, and pres 0080 Lentigo maligna ence of regional or distant metastasis. Certain types of mela 0081 Lentigo maligna melanoma noma have worse prognoses but this is explained by their thickness. Interestingly, less invasive melanomas even with 0082 Superficial spreading melanoma lymph node metastases carry a better prognosis than deep 0083 Acral lentiginous melanoma melanomas without regional metastasis at time of staging. 0084. Local recurrences tend to behave similarly to a primary unless 0085 Nodular melanoma they are at the site of a wide local excision (as opposed to a I0086 Polypoid melanoma staged excision or punch/shave excision) since these recur 0087. Desmoplastic melanoma rences tend to indicate lymphatic invasion. 0088 0116. When melanomas have spread to the lymph nodes, one of the most important factors is the number of nodes with 0089 Soft-tissue melanoma malignancy. Extent of malignancy within a node is also 0090 Melanoma with small nevus-like cells important; micrometastases in which malignancy is only 0091 Melanoma with features of a microscopic have a more favorable prognosis than macrome 0092 Uveal melanoma tastases. In some cases micrometastases may only be detected Confirmation of the clinical diagnosis is achieved with a skin by special staining, and if malignancy is only detectable by a biopsy. This is usually followed up with a wider excision of rarely employed test known as the polymerase chain reaction the scar or tumor. Depending on the stage, a sentinel lymph (PCR), the prognosis is better. Macrometastases in which node biopsy is done, as well, although controversy exists malignancy is clinically apparent (in some cases cancer com US 2015/0023915 A1 Jan. 22, 2015 pletely replaces a node) have a far worse prognosis, and if that many patients with a negative sentinel lymph node actu nodes are matted or if there is extracapsular extension, the ally had a small number of positive cells in their nodes. prognosis is still worse. Alternatively, a fine-needle aspiration biopsy may be per 0117. When there is distant metastasis, the cancer is gen formed and is often used to test masses. erally considered incurable. The five year survival rate is less 0.124. If a lymph node is positive, depending on the extent than 10%. The median survival is 6 to 12 months. Treatment of lymph node spread, a radical lymph node dissection will is palliative, focusing on life-extension and quality of life. In often be performed. If the disease is completely resected, the Some cases, patients may live many months or even years with patient will be considered for adjuvant therapy. Excisional metastatic melanoma (depending on the aggressiveness of the skin biopsy is the management of choice. Here, the Suspect treatment). Metastases to skin and lungs have a better prog lesion is totally removed with an adequate (but minimal, nosis. Metastases to brain, bone and liver are associated with usually 1 or 2 mm) ellipse of Surrounding skin and tissue. To a worse prognosis. avoid disruption of the local lymphatic drainage, the pre 0118. There is not enough definitive evidence to ferred surgical margin for the initial biopsy should be narrow adequately stage, and thus give a prognosis for ocular mela (1 mm). The biopsy should include the epidermal, dermal, noma and melanoma of soft parts, or mucosal melanoma and subcutaneous layers of the skin. This enables the histo (e.g., rectal melanoma), although these tend to metastasize pathologist to determine the thickness of the melanoma by more easily. Even though regression may increase Survival, microscopic examination. This is described by Breslow's when a melanoma has regressed, it is impossible to know its thickness (measured in millimeters). However, for large original size and thus the original tumor is often worse than a lesions, such as Suspected lentigo maligna, or for lesions in report might indicate. Surgically difficult areas (face, toes, fingers, eyelids), a small 0119 6. Standard of Care Treatments punch biopsy in representative areas will give adequate infor 0120 Excisional biopsies may remove the tumor, but fur mation and will not disrupt the final staging or depth deter ther Surgery is often necessary to reduce the risk of recur mination. In no circumstances should the initial biopsy rence. Complete Surgical excision with adequate Surgical include the final Surgical margin (0.5 cm, 1.0 cm, or 2 cm), as margins and assessment for the presence of detectable meta a misdiagnosis can result in excessive Scarring and morbidity static disease along with short- and long-term follow-up is from the procedure. A large initial excision will disrupt the standard. Often this is done by a wide local excision (WLE) local lymphatic drainage and can affect further lymphangio with 1 to 2 cm margins. Melanoma-in-situ and lentigo mali gram-directed lymph node dissection. A Small punch biopsy gnas are treated with narrower Surgical margins, usually 0.2 to can be used at any time where for logistical and personal 0.5 cm. Many surgeons consider 0.5 cm the standard of care reasons a patient refuses more invasive excisional biopsy. for standard excision of melanoma-in-situ, but 0.2 cm margin Small punch biopsies are minimally invasive and heal might be acceptable for margin controlled Surgery (Mohs quickly, usually without noticeable Scarring. Surgery, or the double-bladed technique with margin control). 0.125 High-risk melanomas may require adjuvant treat The wide excision aims to reduce the rate of tumor recurrence ment, although attitudes to this vary in different countries. In at the site of the original lesion. This is a common pattern of the United States, most patients in otherwise good health will treatment failure in melanoma. Considerable research has begin up to a year of high-dose interferon treatment, which aimed to elucidate appropriate margins for excision with a has severe side effects, but may improve the patient’s prog general trend toward less aggressive treatment during the last nosis slightly. However British Association of Dermatologist decades. guidelines on melanoma State that interferon is not recom 0121 Mohs surgery has been reported with cure rate as mended as a standard adjuvant treatment for melanoma. A low as 77% and as high as 98% for melanoma-in-situ. CCP 2011 meta-analysis showed that interferon could lengthen the DMA and the “double scalpel peripheral margin controlled time before a melanoma comes back but increased survival by Surgery is equivalent to Mohs Surgery in effectiveness on this only 3% at 5 years. The unpleasant side effects also greatly “intra-epithelial type of melanoma. decrease quality of life. In Europe, interferon is usually not 0122 Melanomas that spread usually do so to the lymph used outside the scope of clinical trials. nodes in the area of the tumor before spreading elsewhere. 0.126 Various chemotherapy agents also are used, includ Attempts to improve Survival by removing lymph nodes Sur ing dacarbazine (also termed DTIC), immunotherapy (with gically (lymphadenectomy) were associated with many com interleukin-2 (IL-2) or interferon (IFN)), as well as local plications, but no overall survival benefit. Recently, the tech perfusion, are used by different centers. The overall success in nique of sentinel lymph node biopsy has been developed to metastatic melanoma is quite limited. IL-2 (Proleukin) is the reduce the complications of lymph node Surgery while allow first new therapy approved for the treatment of metastatic ing assessment of the involvement of nodes with tumor. melanoma in 20 years. Studies have demonstrated that IL-2 0123. Although controversial and without prolonging sur offers the possibility of a complete and long-lasting remission vival, sentinel lymph node biopsy is often performed, espe in this disease, although only in a small percentage of cially for T1b/T2+ tumors, mucosal tumors, ocular mela patients. A number of new agents and novel approaches are noma and tumors of the limbs. A process called under evaluation and show promise. Clinical trial participa lymphoscintigraphy is performed in which a radioactive tion should be considered the standard of care for metastatic tracer is injected at the tumor site to localize the sentinel melanoma. node(s). Further precision is provided using a blue tracer dye, I0127. For lentigo maligna treatment, standard excision is and Surgery is performed to biopsy the node(s). Routine still being performed by most Surgeons. Unfortunately, the hematoxylin and eosin (H&E) and immunoperoxidase stain recurrence rate is exceedingly high (up to 50%). This is due to ing will be adequate to rule out node involvement. Poly the ill-defined visible Surgical margin, and the facial location merase chain reaction (PCR) tests on nodes, usually per of the lesions (often forcing the Surgeon to use a narrow formed to test for entry into clinical trials, now demonstrate Surgical margin). The narrow Surgical margin used, combined US 2015/0023915 A1 Jan. 22, 2015

with the limitation of the standard “bread-loafing technique 0.133 As is well known in the art, a specific dose level of offixed tissue histology, result in a high “false negative' error active compounds for any particular patient depends upon a rate, and frequent recurrences. Margin control (peripheral variety of factors including the activity of the specific com margins) is necessary to eliminate the false negative errors. If pound employed, the age, body weight, general health, sex, bread loafing is used, distances from sections should diet, time of administration, route of administration, rate of approach 0.1 mm to assure that the method approaches com excretion, drug combination, and the severity of the particular plete margin control. disease undergoing therapy. The person responsible for 0128. Some melanocytic nevi, and melanoma-in-situ (len administration will determine the appropriate dose for the tigo maligna), have resolved with an experimental treatment: individual subject. Moreover, for human administration, imiquimod (Aldara) topical cream, an immune enhancing preparations should meet Sterility, pyrogenicity, general agent. Some dermasurgeons are combining the 2 methods: safety and purity standards as required by FDA Office of Surgically excising the cancer and then treating the area with Biologics standards. Aldara cream postoperatively for three months. 0134) C. Formulations and Routes for Administration 0129 is often used after surgical resec 0.135 Pharmaceutical compositions of the present inven tion for patients with locally or regionally advanced mela tion comprise an effective amount of one or more candidate noma or for patients with unresectable distant metastases. It Substance or additional agent dissolved or dispersed in a may reduce the rate of local recurrence but does not prolong pharmaceutically acceptable carrier. The phrases “pharma Survival. Radioimmunotherapy of metastatic melanoma is ceutical orpharmacologically acceptable' refers to molecular currently under investigation. Radiotherapy has a role in the entities and compositions that do not produce an adverse, palliation of metastatic melanoma. allergic or other untoward reaction when administered to an 0130 B. Dosages animal. Such as, for example, a human, as appropriate. The 0131. In certain embodiments, the compound or com preparation of a pharmaceutical composition that contains at pounds of the present invention is/are administered to a Sub least one candidate Substance or additional active ingredient ject. In another embodiment of the invention, the dose range will be known to those of skill in the art in light of the present of the compound(s) will be measured by body weight, for disclosure, as exemplified by Remington’s Pharmaceutical example, about 0.5 mg/kg body weight to about 500 mg/kg Sciences, 18th Ed. Mack Printing Company, 1990, incorpo body weight. Those of skill will recognize the utility of a rated herein by reference. Moreover, for animal (e.g., human) Variety of dosage range, for example, 1 mg/kg body Weight to administration, it will be understood that preparations should 450 mg/kg body weight, 2 mg/kg body weight to 400 mg/kg meet sterility, pyrogenicity, general safety and purity stan body weighty, 3 mg/kg body weight to 350 mg/kg body dards as required by FDA Office of Biological Standards. weighty, 4 mg/kg body weight to 300 mg/kg body weight, 5 0.136. As used herein, “pharmaceutically acceptable car mg/kg body weight to 250 mg/kg body weighty, 6 mg/kg rier includes any and all solvents, dispersion media, coat body weight to 200 mg/kg body weight, 7 mg/kg body weight ings, Surfactants, antioxidants, preservatives (e.g., antibacte to 150 mg/kg body weighty, 8 mg/kg body weight to 100 rial agents, antifungal agents), isotonic agents, absorption mg/kg body weight, or 9 mg/kg body weight to 50 mg/kg delaying agents, salts, preservatives, drugs, drug stabilizers, body weight. Further, those of skill will recognize that a gels, binders, excipients, disintegration agents, lubricants, variety of different dosage levels will be of use, for example, Sweetening agents, flavoring agents, dyes, such like materials 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 and combinations thereof, as would be known to one of ordi mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 25 nary skill in the art (see, for example, Remington’s Pharma mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, ceutical Sciences, 18th Ed. Mack Printing Company, 1990, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 120 pp. 1289-1329, incorporated herein by reference). Except mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg, 200 insofar as any conventional carrier is incompatible with the mg/kg, 225 mg/kg, 250mg/kg, 275mg/kg, 300 mg/kg, 325 active ingredient, its use in the therapeutic or pharmaceutical mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 450 mg/kg, 500 compositions is contemplated. mg/kg, 550 mg/kg, 600 mg/kg, 700 mg/kg, 750 mg/kg, 800 0.137 The candidate substance may comprise different mg/kg, 900 mg/kg, 1000 mg/kg, 1250 mg/kg, 1500 mg/kg, types of carriers depending on whetherit is to be administered 1750 mg/kg, 2000 mg/kg, 2500 mg/kg, and/or 3000 mg/kg. in solid, liquid or aerosol form, and whether it need to be Of course, all of these dosages are exemplary, and any dosage sterile for Such routes of administration as injection. The in-between these points is also expected to be of use in the present invention can be administered intravenously, intrad invention. Any of the above dosage ranges or dosage levels ermally, intraarterially, intraperitoneally, intralesionally, may be employed for a compound or compounds of the intracranially, intraarticularly, intraprostatically, intrapleu present invention. rally, intratracheally, intranasally, intravitreally, intravagi 0132) The treatments may include various “unit doses.” nally, intrarectally, topically, intratumorally, intramuscularly, Unit dose is defined as containing a predetermined-quantity Subcutaneously, Subconjunctival, intravesicularly, mucos of the therapeutic composition calculated to produce the ally, intrapericardially, intraumbilically, intraocularally, desired responses in association with its administration, i.e., orally, locally, via inhalation (e.g., aerosol inhalation), via the appropriate route and treatment regimen. The quantity to injection, via infusion, via continuous infusion, via localized be administered, and the particular route and formulation, are perfusion bathing target cells directly, via a catheter, via a within the skill of those in the clinical arts. Also of import is lavage, in creams, in lipid compositions (e.g., liposomes), or the subject to be treated, in particular, the state of the subject by other method or any combination of the forgoing as would and the protection desired. A unit dose need not be adminis be known to one of ordinary skill in the art (see, for example, tered as a single injection but may comprise continuous infu Remington’s Pharmaceutical Sciences, 18th Ed. Mack Print sion over a set period of time. ing Company, 1990, incorporated herein by reference). US 2015/0023915 A1 Jan. 22, 2015

0.138. The actual dosage amount of a composition of the Suspensions, syrups, wafers, or combinations thereof. Oral present invention administered to an animal patient can be compositions may be incorporated directly with the food of determined by physical and physiological factors such as the diet. Preferred carriers for oral administration comprise body weight, severity of condition, the type of disease being inert diluents, assimilable edible carriers or combinations treated, previous or concurrent therapeutic interventions, idi thereof. In other aspects of the invention, the oral composition opathy of the patient and on the route of administration. The may be prepared as a syrup or elixir. A syrup or elixir, and may practitioner responsible for administration will, in any event, comprise, for example, at least one active agent, a Sweetening determine the concentration of active ingredient(s) in a com agent, a preservative, a flavoring agent, a dye, a preservative, position and appropriate dose(s) for the individual Subject. or combinations thereof. 0.139. In any case, the composition may comprise various 0144. In certain particular embodiments, an oral compo antioxidants to retard oxidation of one or more component. sition may comprise one or more binders, excipients, disin Additionally, the prevention of the action of microorganisms tegration agents, lubricants, flavoring agents, and combina can be brought about by preservatives Such as various anti tions thereof. In certain embodiments, a composition may bacterial and antifungal agents, including but not limited to comprise one or more of the following: a binder, such as, for parabens (e.g., methylparabens, propylparabens), chlorobu example, gum tragacanth, acacia, cornstarch, gelatin or com tanol, phenol, Sorbic acid, thimerosal or combinations binations thereof an excipient, Such as, for example, dical thereof. cium phosphate, mannitol, lactose, starch, magnesium Stear 0140. The candidate substance may be formulated into a ate, Sodium saccharine, cellulose, magnesium carbonate or composition in a free base, neutral or salt form. Pharmaceu combinations thereof a disintegrating agent, Such as, for tically acceptable salts, include the acid addition salts, e.g., example, corn starch, potato starch, alginic acid or combina those formed with the free amino groups of a proteinaceous tions thereof, a lubricant, Such as, for example, magnesium composition, or which are formed with inorganic acids Such Stearate; a Sweetening agent, such as, for example, Sucrose, as for example, hydrochloric or phosphoric acids, or Such lactose, Saccharin or combinations thereof a flavoring agent, organic acids as acetic, oxalic, tartaric or mandelic acid. Salts Such as, for example peppermint, oil of wintergreen, cherry formed with the free carboxyl groups can also be derived flavoring, orange flavoring, etc.; or combinations thereof the from inorganic bases such as for example, sodium, potas foregoing. When the dosage unit form is a capsule, it may sium, ammonium, calcium or ferric hydroxides; or Such contain, in addition to materials of the above type, carriers organic bases as isopropylamine, trimethylamine, histidine or Such as a liquid carrier. Various other materials may be procaine. present as coatings or to otherwise modify the physical form 0141. In embodiments where the composition is in a liquid of the dosage unit. For instance, tablets, pills, or capsules may form, a carrier can be a solvent or dispersion medium com be coated with shellac, sugar or both. prising but not limited to, water, ethanol, polyol (e.g., glyc (0145 Additional formulations which are suitable for other erol, propylene glycol, liquid polyethylene glycol, etc.), lip modes of administration include Suppositories. Suppositories ids (e.g., triglycerides, vegetable oils, liposomes) and are solid dosage forms of various weights and shapes, usually combinations thereof. The proper fluidity can be maintained, medicated, for insertion into the rectum, vagina or urethra. for example, by the use of a coating, such as lecithin; by the After insertion, Suppositories soften, melt or dissolve in the maintenance of the required particle size by dispersion in cavity fluids. In general, for Suppositories, traditional carriers carriers such as, for example liquid polyol or lipids; by the use may include, for example, polyalkylene glycols, triglycerides of Surfactants such as, for example hydroxypropylcellulose; or combinations thereof. In certain embodiments, supposito or combinations thereof such methods. In many cases, it will ries may be formed from mixtures containing, for example, be preferable to include isotonic agents, such as, for example, the active ingredient in the range of about 0.5% to about 10%, Sugars, sodium chloride or combinations thereof. and preferably about 1% to about 2%. 0142. In other embodiments, one may use eye drops, nasal 0146 Sterile injectable solutions are prepared by incorpo Solutions or sprays, aerosols or inhalants in the present inven rating the active compounds in the required amount in the tion. Such compositions are generally designed to be com appropriate solvent with various other ingredients enumer patible with the target tissue type. In a non-limiting example, ated above, as required, followed by filtered sterilization. nasal Solutions are usually aqueous Solutions designed to be Generally, dispersions are prepared by incorporating the vari administered to the nasal passages in drops or sprayS. Nasal ous sterilized active ingredients into a sterile vehicle which Solutions are prepared so that they are similar in many contains the basic dispersion medium and/or the other ingre respects to nasal Secretions, so that normal ciliary action is dients. In the case of sterile powders for the preparation of maintained. Thus, in preferred embodiments the aqueous sterile injectable solutions, Suspensions or emulsion, the pre nasal Solutions usually are isotonic or slightly buffered to ferred methods of preparation are vacuum-drying or freeze maintain a pH of about 5.5 to about 6.5. In addition, antimi drying techniques which yield a powder of the active ingre crobial preservatives, similar to those used in ophthalmic dient plus any additional desired ingredient from a previously preparations, drugs, or appropriate drug stabilizers, if sterile-filtered liquid medium thereof. The liquid medium required, may be included in the formulation. For example, should be suitably buffered if necessary and the liquid diluent various commercial nasal preparations are known and include first rendered isotonic prior to injection with sufficient saline drugs such as antibiotics or antihistamines. or glucose. The preparation of highly concentrated composi 0143. In certain embodiments the candidate substance is tions for direct injection is also contemplated, where the use prepared for administration by Such routes as oral ingestion. of DMSO as solvent is envisioned to result in extremely rapid In these embodiments, the Solid composition may comprise, penetration, delivering high concentrations of the active for example, Solutions, Suspensions, emulsions, tablets, pills, agents to a small area. capsules (e.g., hard or soft shelledgelatin capsules), Sustained 0147 The composition must be stable under the condi release formulations, buccal compositions, troches, elixirs, tions of manufacture and storage, and preserved against the US 2015/0023915 A1 Jan. 22, 2015 contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination ABAB, ABBBAAAABBAAAABBBBBABBAB should be kept minimally at a safe level, for example, less that AABBAB, ABABBABBAAB, ABABAABBBBA 0.5 ng/mg protein. AAABBAAAABAAAABAABBBBABBBBAB 0148. In particular embodiments, prolonged absorption of an injectable composition can be brought about by the use in 0155 E. Further Combinations the compositions of agents delaying absorption, such as, for 0156 Additional combinations with the agents/combina example, aluminum monostearate, gelatin or combinations tions set out above also are contemplated. Agents or factors thereof. Suitable for use in a further combined cancer therapy are any 014.9 The skilled artisan is directed to “Remington's chemical compound or treatment method that induces DNA Pharmaceutical Sciences' 15th Edition, chapter 33, in par damage when applied to a cell. Such agents and factors ticular pages 624-652. Some variation in dosage will neces include radiation and waves that induce DNA damage Such sarily occur depending on the condition of the Subject being as, Y-irradiation, X-rays, UV-irradiation, microwaves, elec treated. The person responsible for administration will, in any tronic emissions, and the like. A variety of chemical com event, determine the appropriate dose for the individual sub pounds, also described as “chemotherapeutic agents.” func ject. Moreover, for human administration, preparations tion to induce DNA damage, all of which are intended to be of should meet sterility, pyrogenicity, general safety and purity use in the combined treatment methods disclosed herein. standards as required by FDA Office of Biologics standards. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), 0150 D. Treatment camptothecin, actinomycin-D, mitomycin C, cisplatin 0151. In the context of the present invention, it is contem (CDDP) and even hydrogen peroxide. The invention also plated that the compounds may be used in combination with encompasses the use of a combination of one or more DNA each other to more effectively treat melanoma. When mul damaging agents, whether radiation-based or actual com tiple therapeutic agents are administered, as long as the dose pounds. Such as the use of X-rays with cisplatin or the use of of the additional therapeutic agent does not exceed previously cisplatin with etoposide. quoted toxicity levels, the effective amounts of the additional 0157. In treating cancer according to the invention, one therapeutic agent may simply be defined as that amount effec would contact a tumor or tumor cells with an agent according tive to exert a therapeutic effect when administered to an to the present invention along with the second agent or animal in combination with the primary agent. This may be therapy. This may be achieved by irradiating the localized easily determined by monitoring the animal or patient and tumor site with radiation Such as X-rays, UV-light, Y-rays or measuring those physical and biochemical parameters of even microwaves. Alternatively, the tumor or tumor cells may health and disease that are indicative of the Success of a given be contacted with the agent by administering to the Subject a treatment. Such methods are routine in animal testing and therapeutically effective amount of a pharmaceutical compo clinical practice. sition comprising a compound Such as, adriamycin, 5-fluo 0152 To kill or slow the growth of a cancer cell using the rouracil, etoposide, camptothecin, actinomycin-D, mitomy methods and compositions of the present invention, one can cin C, or more preferably, cisplatin. The agent may be provide to the subject the stated combination of agents. These prepared and used as a combined therapeutic composition, or compositions would be provided in a combined amount effec kit, by combining it with a compound according to the present tive to achieve a therapeutic benefit (inhibition of cancer cell invention. growth, reduction in tumor size, induction of apoptosis in a 0158 Agents that directly cross-link nucleic acids, spe cancer cell, etc.). This process may involve administering a cifically DNA, are envisaged to facilitate DNA damage lead combination at the same time. This may be achieved by ing to a synergistic, antineoplastic combination with com administering a single composition or pharmacological for pounds of the present invention. Agents such as cisplatin, and mulation that includes both agents, or by administering two other DNA alkylating agents may be used. Cisplatin has been distinct compositions or formulations, at the same time. widely used to treat cancer, with efficacious doses used in 0153. Alternatively, treatment with one agent may precede clinical applications of 20 mg/m for 5 days every three weeks or follow the additional agent treatment by intervals ranging for a total of three courses. Cisplatin is not absorbed orally from minutes to weeks. In embodiments where the additional and must therefore be delivered via injection intravenously, agent is administered separately to the patient, one would Subcutaneously, intratumorally or intraperitoneally. generally ensure that a significant period of time did not 0159 Agents that damage DNA also include compounds expire between the time of each delivery, such that the agents that interfere with DNA replication, mitosis and chromo would still be able to exert an advantageously combined Somal segregation. Such chemotherapeutic compounds effect on the cell. In Such instances, it is contemplated that one include adriamycin, also known as doxorubicin, etoposide, would contact the cell with both modalities within about Verapamil, podophyllotoxin, and the like. Widely used in a 12-24 hr of each other and, more preferably, within about clinical setting for the treatment of neoplasms, these com 6-12 hr of each other, with a delay time of only about 12 hr pounds are administered through bolus injections intrave being most preferred. In some situations, it may be desirable nously at doses ranging from 25-75 mg/m at 21 day intervals to extend the time period for treatment significantly, however, for adriamycin, to 35-50 mg/m for etoposide intravenously where several days (2,3,4, 5, 6 or 7) to several weeks (1,2,3, or double the intravenous dose orally. 4, 5, 6, 7 or 8) lapse between the respective administrations. 0160 Agents that disrupt the synthesis and fidelity of 0154 It also is conceivable that more than one adminis nucleic acid precursors and subunits also lead to DNA dam tration of either or both agents will be desired. Various com age. As such a number of nucleic acid precursors have been binations may be employed, where a MEK inhibitor is “A” developed. Particularly useful are agents that have undergone and the cardiac glycoside is “B” as exemplified below: extensive testing and are readily available. As such, agents US 2015/0023915 A1 Jan. 22, 2015

such as 5-fluorouracil (5-FU), are preferentially used by neo 0.165 For analysis of circulating melanoma cells, blood plastic tissue, making this agent particularly useful for target was collected from each mouse by cardiac puncture, using a ing to neoplastic cells. Although quite toxic, 5-FU, is appli Syringe pre-treated with citrate-dextrose solution (Sigma). cable in a wide range of carriers, including topical, however Red blood cells were precipitated by Ficoll sedimentation intravenous administration with doses ranging from 3 to 15 following the manufacturer's instructions (Ficoll Paque Plus, mg/kg/day being commonly used. GE Healthcare). Remaining cells were washed with HBSS 0161 Particular agents contemplated for use in melanoma (Invitrogen) prior to antibody staining and flow cytometric include interferon, IL-2, imiquimod, paclitaxel, Vemurafenib, analysis. ipilimumab and dacarbazine. 0166 All antibody staining was carried out for 20 min on ice, followed by washing and centrifugation. In order to select III. EXAMPLES live human melanoma cells and to exclude endothelial and hematopoietic cells, tumors obtained directly from patients 0162 The following examples are included to demon were stained with directly conjugated antibodies to human strate preferred embodiments of the invention. It should be CD45 (HI30-APC: BD Biosciences), human CD31 (WM59 appreciated by those of skill in the art that the techniques APC: eBiosciences), Glycophorin A (HIR2-APC: Bioleg disclosed in the examples which follow represent techniques end) and HLA-A.B.C (G46-2.6-PE, BD Biosciences). discovered by the inventor to function well in the practice of Tumors or blood obtained from mice were stained with anti the invention, and thus can be considered to constitute pre bodies against mouse CD45 (30-F11-APC: eBiosciences), ferred modes for its practice. However, those of skill in the art mouse CD31 (390-APC: Biolegend), mouse Ter119 (TER should, in light of the present disclosure, appreciate that many 119-APC: eBiosciences) and human HLA-A.B.C (G46-2.6- changes can be made in the specific embodiments which are PE, BD Biosciences). Cells were resuspended in 10 ug/ml disclosed and still obtain a like or similar result without DAPI (Sigma) and sorted on a FACSAria (Becton Dickin departing from the spirit and scope of the invention. son). 0.167 Transplanting melanoma cells. After sorting, cells Example 1 were counted and resuspended in staining medium (L15 medium containing 1 mg/ml BSA, 1% penicillin/streptomy Materials and Methods cin and 10 mM HEPES (pH7.4)) with 25% high protein Matrigel (product 354248; BD Biosciences). Subcutaneous 0163 Tissue banking. The tissue bank protocol used for injections of human melanoma cells were performed in each this study was developed and approved jointly by the clinical flank and the interscapular region of NOD.CB17 (NOD/ director of the University of Michigan (UM) melanoma pro SCIDIL2Ry". NSG) mice (Jackson Laboratories) in a final gram, the UM Cancer Center director of tissue procurement, volume of 50 ul. These experiments were performed accord the UM chief of anatomic pathology, and UM director of the ing to protocols approved by the animal use committee at the section of dermatopathology. The protocol was developed to University of Texas Southwestern Medical Center (protocol avoid any compromise in patient care, pathologic diagnosis, 2011-0118). tumor staging or treatment. Patient confidentiality was main (0168 Lentiviral transduction of human melanoma cells. tained by password and firewall protected access to all perti Replication incompetent lentiviruses were generated by the nent databases. Melanoma specimens were obtained with cotransfection of 293T cells with three plasmids (fuw, VSV informed consent from all patients according to protocols and delta), kindly provided by Dr. Alnawaz Rehemtulla (Uni approved by the Institutional Review Board of the UMMedi versity of Michigan). Forty-eight hours after transfection, cal School (IRBMED approvals HUMO0050754 and Supernatant with virus was collected and passed through a HUM00050085). All patients included in this study had clini 0.45 um low binding filter. Virus was concentrated by ultra cally apparent melanoma disease (biopsy-proven stage II, III, centrifugation and aliquots were frozen at -80° C. until use. or IV, or obvious clinical stage IV) from which a small (typi Around 150,000 sorted human melanoma cells (obtained cally 2-5mm) tissue sample not required for standard-of-care directly from the patients or from xenografts) were incubated pathology assessment was obtained. Most of the melanomas for 4 hat 37°C., 6.5% CO in a well of an ultra-low attach in this study were regional stage III lymph node or skin/soft ment 24-well plate (Corning) containing 300 ul of cell culture tissue disease with palpable, clinically enlarged node(s) or medium (50% DMEM-low glucose, 30% Neurobasal (Invit Soft tissues, undergoing definitive Surgical resection, with rogen), 15% chick embryo extract, 1% Penicillin/Streptomy biopsy-proven (most often needle core) diagnosis confirmed cin, 1% non-essential amino acids (Gibco), 117 nM retinoic before surgery. The tumors were then shared with the Morri acid, 50 uM2-mercaptoethanol (Sigma), 1% N2 supplement, son laboratory at the University of Texas Southwestern Medi 2% B27 supplement (Gibco), 20 ng/mL recombinant human cal Center under a material transfer agreement. bFGF, 20 ng/mL IGF-1 (R&D Systems)). Twenty ul lentivi 0164. Tumor cell preparation. Tumors were mechanically rus and 10 g/ml polybrene (Sigma) were added to the cul dissociated with a McIlwain tissue chopper (Mickle Labora tures. Cells were then washed twice with staining medium tory Engineering) before sequential enzymatic digestion in and approximately 25,000 cells (a mixture of infected and 200U/ml collagenase IV (Worthington) for 20 min followed non-infected cells) were injected into NSG mice. After grow by 0.05% trypsin-EGTA for 2 min, both at 37° C. DNase ing to 1-2 cm in diameter, tumors were excised, dissociated (50-100U/mL) was added to reduce clumping of cells during into a single cell suspension and luciferase-GFP+ cells were digestion. Cells were filtered (40 um cell strainer) to obtain a collected by flow cytometry for injections into secondary single cell suspension. Dead cells and debris were depleted by recipients. Metastasis was monitored by BLI in these second density centrifugation (1.1 g/ml Optiprep; Sigma) when nec ary recipients. essary. Cells were always passaged in vivo (in immunocom 0169 Bioluminescence imaging (BLI). Mice were promised mice as detailed below), not in vitro. injected with 100 luciferase-GFP+ cells on the right flank and US 2015/0023915 A1 Jan. 22, 2015

monitored until tumor diameters approached 20mm, at which 0173 The mouse xenograft assays showed that digitoxin point they were imaged along with an uninjected control and GSK1 120212 individually slowed the growth of human mouse on an IVIS Imaging System 200 Series (Calliper Life melanoma xenografts. However, the combination of digitoxin Sciences) using Living Image software. Mice were injected and GSK1 120212 caused substantial regression in melano intraperitoneally with 100 ul of PBS containing 40 mg/ml mas obtained from multiple patients, some with BRAF muta D-luciferin monopotassium salt (Biosynth) ten minutes tions and some without (FIG. 2). The effects were signifi before imaging, followed by general anesthesia two minutes cantly better than digitoxin or GSK1 120212 alone. No before imaging. Following imaging of the whole mouse, the difference in efficacy for the combination was seen in BRAF mice were euthanized, and individual organs were quickly mutant versus BRAF wild-type tumors. Representative imaged. The exposure time of images ranged from 10 to 60 results from melanoma M481 carrying the BRAF-V600E seconds. The bioluminescence signal was quantified using mutation demonstrates that the effectiveness of the combina “region of interest’ measurement tools in Living Image soft tion therapy in decreasing the bioluminescence signal (FIG. ware. After imaging, tumors and organs were fixed in 10% 3A), final tumor weight (FIG.3B) and size (FIG.3C). Similar neutral buffered formalin for histopathology. observations were made with a BRAF wild-type melanoma, 0170 Histopathology and immunostaining. Melanomas M214 (FIGS. 4A-C). and mouse organs were fixed in 10% neutral buffered forma 0.174 Digoxin is another cardiac glycoside that is struc lin, paraffin embedded, sectioned, and stained with hema turally similar to digitoxin and has been used for the treatment toxylin and eosin for histopathology. Sections from paraffin of various heart conditions. The digoxin and trametinib com embedded tumors were stained for S100 and analysed by a bination was more effective against melanomas from patient dermatopathologist to confirm they were melanomas. Bind M214 (FIGS.5A-D), M491 (FIGS. 6A-D) and M481 (FIGS. ing of anti-S100 antibody (DAKO) was carried out for 30 min 7A-D) than trametinib alone. Importantly, the combination of at room temperature, detected by anti-rabbit secondary anti digoxin and trametinib was more toxic to two primary human body (30 min at room temperature) and revealed using DAB melanoma cells in culture than to a variety of normal human chromagen after quenching endogenous peroxidases. Slides cell lines including fibroblasts, melanocytes, cord blood cells, were counterstained with hematoxylin. colonic epithelial cells and bronchial epithelial cells (FIG. 8). 0.175. Although the present invention and its advantages Example 2 have been described in detail, it should be understood that various changes, Substitutions and alterations can be made Results herein without departing from the invention as defined by the 0171 High throughput screening of chemical libraries on appended claims. Moreover, the scope of the present appli primary human melanoma cells obtained from Surgical speci cation is not intended to be limited to the particular embodi mens revealed cardiac glycosides including digitoxin to ments of the process, machine, manufacture, composition of inhibit growth of melanoma cells in vitro. Digitoxin, has a matter, means, methods and steps described in the specifica long history of use in the treatment of various heart condi tion. As one will readily appreciate from the disclosure, pro tions. Plasma concentrations of digitoxin between 13 nMand cesses, machines, manufacture, compositions of matter, 45 nM need to be achieved for therapeutic benefit. The inven means, methods, or steps, presently existing or later to be tors used in vitro viability assays to determine whether doses developed that perform substantially the same function or that show activity against melanoma fall within this therapeu achieve Substantially the same result as the corresponding tic window. In these assays, cells isolated from primary mela embodiments described herein may be utilized. Accordingly, noma samples were treated with various doses of digitoxin or the appended claims are intended to include within their DMSO for 72 hours in triplicate. Viability was measured with Scope such processes, machines, manufacture, compositions CellTiter-Glo. These experiments revealed that therapeuti of matter, means, methods, or steps. cally achievable doses of digitoxin have activity against 9 out 1. A method of inhibiting melanoma in a subject compris of 16 melanomas (FIGS. 1A-B). In order to start testing the ing administering to said Subject: effect of digitoxin in vivo, the inventors determined condi (a) a MEK inhibitor; and tions that would yield doses in the plasma of mice that fell (b) a cardiac glycoside, within the therapeutic range in humans (FIG. 1C). in an amount Sufficient to inhibit said melanoma. 0172. The effect of digitoxin alone, or in combination with 2. The method of claim 1, wherein said subject is a human. the MEK inhibitor Trametinib (GSK1 120212), on in vivo 3. The method of claim 1, wherein said subject is a non melanoma growth was tested in Xenograft assays using pri human mammal. mary melanomas from patients. In these experiments, meta 4. The method of claim 1, wherein inhibiting comprises static melanoma cells were injected Subcutaneously in the inhibiting the growth of primary melanomas, inhibiting right flanks of immunocompromised NSG mice. When metastasis, inhibiting the growth of metastases, killing circu tumors became palpable, mice were randomized to four dif lating melanoma cells, inducing remission, extending remis ferent treatments. Mice were treated either with digitoxin (0.5 Sion, or inhibiting recurrence. mg/kg/day in 200 ul of 0.5% promethylcellulose, 0.2% 5. The method of claim 1, wherein said melanoma is AJCC Tween80 and 5% DMSO by oral gavage), or GSK1 120212 stage III or stage IV disease. (0.5 mg/kg/day in 200 ul of 0.5% promethylcellulose, 0.2% 6. (canceled) Tween80 and 5% DMSO by oral gavage) alone, or in combi 7. The method of claim 1, wherein said subject has previ nation. Control mice received vehicle only (200 ul 0.5% ously received a radiotherapy, a chemotherapy, an immuno promethylcellulose, 0.2% Tween80 and 5% DMSO per day). therapy, a molecularly targeted therapy or had Surgical resec Tumors were measured every 2-3 days with calipers and tion of a tumor. bioluminescence imaging was performed when the diameter 8. The method of claim 1, wherein said MEK inhibitor is of the tumors in vehicle treated mice reached 2 cm. administered before or after the cardiac glycoside therapy. US 2015/0023915 A1 Jan. 22, 2015

9. The method of claim 1, wherein said MEK inhibitor is for inhibition by either single agent alone, or (b) less toxic administered at the same time as the cardiac glycoside than the amount required for inhibition by either single agent therapy. alone. 10. (canceled) 20. (canceled) 21. The method of claim 1, further comprising treating said 11. The method of claim 1, wherein said MEK inhibitor Subject with an additional agent. and/or cardiac glycoside is administered more than once. 22. The method of claim 21, wherein said additional agent 12. (canceled) is an immunomodulator. 13. The method of claim 1, wherein said cardiac glycoside 23. The method of claim 22, wherein said immunomodu is selected from digoxin, digitoxin, gitoxin, oleandrin, neri lator is an antibody against PD1 or CTLA4, imiquimod, IL-2 folin, bufalin, marinobufagenin, cinobufagenin, UNBS1450 or interferon. and lanatoside C. 24. The method of claim 21, wherein said additional agent 14. The method of claim 1, wherein administering com is a chemotherapeutic agent. prises topical, intravenous, intraarterial. Subcutaneous, oral 25. (canceled) or intra-tumoral administration. 26. The method of claim 1, further comprising excising a melanoma lesion. 15. The method of claim 1, wherein administering com 27. The method of claim 26, wherein excising occurs prior prises local, regional or systemic administration. to or after MEK inhibition/cardiac glycoside treatment. 16. The method of claim 1, wherein administering com 28. (canceled) prises continuous infusion over a period of time. 29. The method of claim 26, wherein excising occurs con 17. The method of claim 1, wherein said subject has failed current with MEK inhibition/cardiac glycoside treatment. one or more standard melanoma therapies. 30. The method of claim 1, wherein said melanoma is 18. The method of claim 1, wherein said MEK inhibitor is BRAF wild-type. selected from trametinib, AS703026 (Pimasertib, 31. The method of claim 1, wherein said melanoma is MSC1936369B), AZD8330 (ARRY-424704), Selumetinib BRAF mutated. (AZD6244), PD-325901, GDC-0973 (XL518) and CI-1040 32. The method of claim31, wherein the BRAF mutation is (PD184352). V6OOE or V6OOK 19. The method of claim 1, wherein said amount sufficient 33-37. (canceled) to inhibit said melanoma is (a) less than the amount required