WO2008127659A2 - Combination therapy for cancer - Google Patents

Combination therapy for cancer Download PDF

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Publication number
WO2008127659A2
WO2008127659A2 PCT/US2008/004719 US2008004719W WO2008127659A2 WO 2008127659 A2 WO2008127659 A2 WO 2008127659A2 US 2008004719 W US2008004719 W US 2008004719W WO 2008127659 A2 WO2008127659 A2 WO 2008127659A2
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WIPO (PCT)
Prior art keywords
romidepsin
erlotinib
cell
cancer
effective amount
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PCT/US2008/004719
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French (fr)
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WO2008127659A3 (en
Inventor
Eugene P. Frenkel
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University Of Texas Southwestern Medical Center
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Publication of WO2008127659A2 publication Critical patent/WO2008127659A2/en
Publication of WO2008127659A3 publication Critical patent/WO2008127659A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Romidepsin is a natural product which was isolated from Chromobacterium violaceum by Fujisawa Pharmaceuticals. See Published Japanese Patent Application Hei 7 (1995)-64872; U.S. Patent 4,977,138, issued December 1 1 , 1990, which is incorporated herein by reference. It is a bicyclic peptide consisting of four amino acid residues (D-valine, D-cysteine, dehydrobutyrine, and L-valine) and a novel acid (3-hydroxy-7-mercapto-4- heptenoic acid). Romidepsin is a depsipeptide which contains both amide and ester bonds. In addition to fermentation from C.
  • romidepsin can also be prepared by synthetic or semi-synthetic means.
  • the total synthesis of romidepsin reported by Kahn et al. involves 14 steps and yields romidepsin in 18% overall yield. J. Am. Chem. Soc. 1 18:7237-7238, 1996.
  • the structure of romidepsin is shown below:
  • Romidepsin has been shown to have anti-microbial, immunosuppressive, and anti-tumor activities. It is thought to act by selectively inhibiting deacetylases (e.g., histone deacetylase (HDAC), tubulin deacetylase (TDAC)), promising new targets for the development of anticancer therapies. Nakaj ima et al. , Experimental Cell Res. 241 : 126- 133, 1998. One mode of action is thought to involve the inhibition of one or more classes of histone deacetylases (HDAC). [0003] Histone deacetylase is a metallodeacetylation enzyme having zinc in its active site.
  • HDAC histone deacetylase
  • Histone deacetylase is a metallodeacetylation enzyme having zinc in its active site.
  • HDAC histone deacetylation-dependent chromatin relaxation
  • chromatin relaxation generally, but not universally, transcriptional activation.
  • HDAC inhibitors have been found to trigger apoptosis in tumor cells through diverse mechanisms, including the up-regulation of death receptors, Bid cleavage, ROS generation, Hsp90 dysregulation, and ceramide generation, among others.
  • HDAC inhibitors have entered the clinical arena and are demonstrating activity in both hematologic and non-hematologic malignancies.
  • Romidepsin has shown impressive activity in certain hematologic malignancies, particularly T-cell lymphoma (Piekarz et al. "A review of depsipeptide and other histone deacetylase inhibitors in clinical trials" Curr. Pharm. Des. 10:2289-98, 2004; incorporated herein be reference).
  • Erlotinib (TARCEV A ® ) is a tyrosine kinase inhibitor. It is thought to act by inhibiting the intracellular phosphorylation of the tyrosine kinase associated with epidermal growth factor receptor. Erlotinib is indicated for use in the treatment of patients with non- small cell lung cancer and pancreatic cancer.
  • Lung cancer is one of the leading causes of cancer-related deaths worldwide. There are 1.6 million cases of lung cancers every year in the world, and 1.1 million people will die from their disease. Non-small cell lung cancer accounts for approximately 85% of lung cancer cases. The EGFR signaling pathway is deregualted in over 50% of non-small cell lung cancers. There is a desperate need for the development of effective therapies for the treatment of patients with lung cancer, particularly non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • the present invention encompasses the finding that combinations of deacetylase (DAC) inhibitors with tyrosine kinase inhibitors have particular utility in the treatment of proliferative diseases.
  • the invention establishes the particular utility of DAC inhibitor/tyrosine kinase inhibitor combination therapy in treatment of lung cancer, and particularly in the treatment of non-small cell lung cancer (NSCLC), particularly wild type EGFR and KRAS NSCLCs.
  • the DAC inhibitor is romidepsin.
  • the tyrosine kinase inhibitor is erlotinib (TARCEVA ;.
  • Combination therapy with romidepsin and erlotinib is provided, for example for use in the treatment of proliferative disorders (e.g., cancer and other neoplasms) generally.
  • the combination of romidepsin and erlotinib is used in the treatment of lung cancer, particularly non-small cell lung cancer.
  • the present invention provides methods of treating a proliferative disorder by administering a combination of one or more DAC inhibitors and one or more tyrosine kinase inhibitors.
  • the invention provides a method of treating cancer in a subject (e.g., human) by administering therapeutically effective amounts of romidpesin and a tyrosine kinase inhibitor to the subject.
  • the combination includes romidepsin and erlotinib. Both of these agents have been used in the clinic to treat human subjects with cancer.
  • the romidpesin and erlotinib may be used in combination at dosages lower than when each is used individually.
  • the additive nature of the combination is particularly useful in treating cancer or other neoplasms.
  • the romidpesin is administered at a dosage of 0.5 mg/m 2 to 15 mg/m 2
  • erlotinib is administered at a dosage of approximately 25 mg/day to approximately 200 mg/day.
  • the two drugs may be administered together, or one after another.
  • the method is particular useful in treating lung cancer (e.g., non-small cell lung cancer).
  • the romidpesin and a tyrosine kinase inhibitor are administered in conjunction with another anti-neoplastic agent.
  • the romidepsin is administered intravenously, and the erlotinib is administered orally.
  • each of the romidepsin and the tyrosine kinase inhibitor is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.
  • the romidepsin is administered weekly, and the tyrosine kinase inhibitor is administered daily.
  • the present invention further provides methods of treating lung cancer (e.g., non- small cell lung cancer) by administering a DAC inhibitor together with a tyrosine kinase inhibitor.
  • the present invention provides a method of treating non- small cell lung cancer in a subject (e.g., human) by administering a therapeutically effective amount of romidepsin and erlotinib to a subject with non-small cell lung cancer.
  • the therapeutically effective amount of romidepsin ranges from 4 mg/m 2 to 15 mg/m 2 or from 8 mg/m 2 to 10 mg/m 2 .
  • the therapeutically effective amount of erlotinib (TARCEVA ® ) ranges from approximately 25 mg to 200 mg.
  • therapeutically effective amount of erlotinib is approximately 100 mg.
  • therapeutically effective amount of erlotinib is approximately 150 mg.
  • the therapeutically effective amount of romidepsin ranges from 8 mg/m 2 to 10 mg/m 2 , and the therapeutically effective amount of erlotinib (TARCEVA ® ) is approximately 150 mg per day.
  • the romidepsin is administered weekly, and the erlotinib is administered daily.
  • the invention provides methods of treating cells in vitro by contacting cells with a combination of romidepsin and a tyrosine kinase inhibitor such as erlotinib.
  • the cells may be treated with a sufficient concentration of the combination to kill the treated cells.
  • a sufficient concentration of the combination is used to induce apoptosis as evidenced by changes in levels of cellular markers of apoptosis.
  • the cells are neoplastic cells.
  • the cells may be from human cancers or derived from cancer cell lines (e.g., lung cancer, non-small cell lung cancer).
  • the cells are lung cancer cells, in particular non-small cell lung cancer cells.
  • the cells may be at any stage of differentiation or development.
  • the methods are particularly useful for assessing the cytotoxicity of a given combination under certain conditions (e.g., concentration of each agent, combination with other pharmaceutical agents).
  • the inventive methods may be used to ascertain the susceptibility of a subject's cancer or neoplasm to the combination therapy.
  • the inventive methods using the inventive combinations may be for clinical or research purposes.
  • the present invention provides combination regimens, and unit dosages of pharmaceutical compositions useful in such regimens.
  • pharmaceutical compositions or preparations comprising romidepsin and a tyrosine kinase inhibitor are provided.
  • the composition or preparation comprises romidepsin and erlotinib.
  • the pharmaceutical composition includes a therapeutically effective amount of each pharmaceutical agent for the treatment of cancer (e.g.. lung cancer, non-small cell lung cancer).
  • the pharmaceutical composition may include other cytotoxic agents or other anti-neoplastic agents.
  • the pharmaceutical composition may also include other agents to alleviate pain, nausea, hair loss, weight loss, weight gain, neuropathy, cardiac arrhythmias, electrolyte deficiencies or imbalances, anemia, thrombocytopenia, immunosuppression, skin conditions, or other conditions associated with cancer or the treatment of cancer.
  • the present invention further provides kits for combination therapy of DAC inhibitors and tyrosine kinase inhibitors.
  • the invention provides kits including the inventive pharmaceutical compositions in a convenient dosage form.
  • the agents may be packaged together or separately in the kit.
  • the kit may include multiple doses of each agent.
  • the kits include a sufficient amount of each agent for a full course of chemotherapy in the treatment of a subject's cancer.
  • the kit may also include excipients or devices for use in administering the inventive combination.
  • the kit may also include instructions for administering the inventive combination.
  • Figures 1-3 depict structures of certain DAC inhibitors that, like other DAC inhibitors available in the art and/or described herein, may be utilized in some embodiments of the present invention.
  • Figure 4 shows materials and methods utilized in a study that demonstrates the effectiveness of combination therapy with a DAC inhibitor (romidepsin) and a tyrosine kinase inhibitor (erlotinib) on non-small cell lung cancer cells.
  • DAC inhibitor romidepsin
  • erlotinib tyrosine kinase inhibitor
  • Figure 5 illustrates the IC 50 for romidepsin in non-small cell lung cancer cell lines.
  • Figure 6 shows enhanced sensitivity of non-small cell lung cancer cell lines (HCC 193 (EGFR, KRAS wt); NCl-H 1299 (EGFR, KRAS wt); NCI-H 157 (KRAS mutant); NCI-H 1975 (EGFR mutant) to a combination of romidepsin and erlotinib.
  • NSCLC cells lines were treated with various concentrations of erlotinib either in the absence or presence of romidepsin (1 ng/mL) for 72 hours. Cell viability was measured by MTS assay, and four representative pairs of cell viability curves are shown.
  • Figure 7 demonstrates that the combination of romidepsin and erlotinib induced apoptosis in a HCC 15 NSCLC cell line.
  • Figure 7 A Nuclei of HCC 15 cells treated with 5 ⁇ M erlotinib and/or 2 ng/mL romidepsin were stained with DNA dye Hoechst 33258 and examined by microscopy.
  • Figure 7 B HCC 15 cells treated with 5 ⁇ M erlotinib and/or 2 ng/mL romidepsin were analyzed for apoptosis using cell death detection kit (Roche). *, P ⁇ 0.05 vs. control; **, P ⁇ 0.01 vs. control (Welch nest).
  • FIG. 8 shows that co-treatment with romidepsin and erlotinib gives greater growth inhibition of NCI-H 1299 cell xenografts than does treatment with either agent alone.
  • 5 x 10 6 NCI-H 1299 cells were injected sub-cutaneously into each of twenty BALB/c athymic nude mice. These mice were divided into four groups at day 7 after tumor development. They were injected with either Ix PBS, romidepsin alone, erlotinib alone, or the combination of romidepsin and erlotinib.
  • Romidepsin was administered 3 times at 4-day intervals (1.2 mg/kg body weight).
  • Erlotinib was administered five days a week (50 mg/kg body weight).
  • Tumor sizes were measured at the indicated days. Results are shown as mean + SEM of groups of five mice. Statistical significance was determined by Welch t test (*, P ⁇ 0.05 vs. control).
  • Figure 9 demonstrates that romidepsin's ability to inhibit MAPK correlates with its ability to increase efficacy of erlotinib.
  • FIG. 10 Romidepsin down-regulated MAPK (ERK 1/2) and AKT pathways, increased p21 and Bim expression in NSCLC cell lines. HCC15, HC193, and NCI-H1299 cell lines were treated with erlotinib (5 ⁇ M) or romidepsin (1 ng/mL) or in combination for
  • Protein lysates were prepared and subjected to western blot analysis. Antibodies against phosphorylated or total ERK1/ERK2, phosphorylated or total AKT, p21, and Bim were used as indicated. Beta-actin was used as a loading control.
  • Figure 11 shows the cytotoxic effect of romidepsin (1 ng/mL) on NSCLC cells lines. Sixteen NSCLC cell lines were either in the presence or absence of romidepsin (1 ng/mL) for 72 hours. MTS assays were performed to determine cell viability.
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to a human, at any stage of development.
  • animal refers to a non-human animal, at any stage of development.
  • animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms.
  • the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig).
  • an animal may be a transgenic animal, genetically-engineered animal, and/or clone.
  • Alicyclic denotes a monovalent group derived from a monocyclic or bicyclic saturated carbocyclic ring compound by the removal of a single hydrogen atom. Examples include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl. Such alicyclic groups may be further substituted.
  • Aliphatic An "aliphatic group" is non-aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds.
  • An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms.
  • aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted.
  • Aryl refers to a mono- or polycyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted.
  • Cell Proliferative Disorder, Disease, or Condition The term "cell proliferative disease or condition" is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation.
  • a DAC inhibitor may desirably be administered in combination with one or more other therapeutic agents, such as, for example, a tyrosine kinase inhibitor.
  • Such therapy will commonly involve administration of multiple individual doses of a DAC inhibitor and/or of other agent (e.g., a tyrosine kinase inhibitor), spaced out over time.
  • Doses of a DAC inhibitor and other agent may be administered in the same amounts and/or according to the same schedule or alternatively may be administered in different amounts and/or according to different schedules.
  • DAC Inhibitor In general, any agent that specifically inhibits a deacetylase is considered to be a DAC inhibitor.
  • DAC inhibitors may be administered in any form such as, for example, salts, esters, prodrugs, metabolites, etc.
  • DAC inhibitors that contain chiral centers may be administered as single stereoisomers or as mixtures, including racemic mixtures, so long as the single stereoisomer or mixture has DAC inhibitor activity.
  • DAC inhibitor therapy refers to the regimen by which a DAC inhibitor is administered to an individual.
  • DAC inhibitor therapy will involve administration of multiple individual doses of a DAC inhibitor, spaced out over time. Such individual doses may be of different amounts or of the same amount.
  • dosing regimens e.g., number of doses, amount(s) of doses, spacing of doses
  • different dosing regimens are typically employed with different DAC inhibitors.
  • Depsipeptide The term "depsipeptide”, as used herein, refers to polypeptides that contain both ester and amide bonds. Naturally occurring depsipeptides are usually cyclic. Some depsipeptides have been shown to have potent antibiotic activity. Examples of depsipeptides include actinomycin, enniatins, valinomycin, and romidepsin.
  • Effective amount In general, the "effective amount" of an active agent or combination of agents refers to an amount sufficient to elicit the desired biological response.
  • the effective amount of an inventive combination may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the agents being delivered, the disease being treated, the mode of administration, and the patient.
  • the effective amount of an inventive combination e.g., romidepsin and erlotinib
  • the effective amount of an inventive combination is the amount that results in reducing the tumor burden, causing a remission, or curing the patient.
  • Electrolyte refers to physiologically relevant free ions.
  • Representative such free ions include, but are not limited to sodium(Na + ), potassium (K + ), calcium (Ca 2+ ), magnesium (Mg 2+ ), chloride (Cl-), phosphate (PO4 3 ), and bicarbonate (HCO 3 ' ).
  • Electrolyte Supplementation refers to administration to a subject of a composition comprising one or more electrolytes in order to increase serum electrolyte levels in the subject.
  • electrolyte supplementation when electrolyte supplementation is administered "prior to, during, or after” other therapy (e.g., DAC inhibitor therapy and/or combination therapy), it may be administered prior to initiation of that therapy (i.e., prior to administration of any dose), or prior to, concurrently with, or after any particular dose or doses.
  • Halogen refers to an atom selected from fluorine, chlorine, bromine, and iodine.
  • Heteroaryl refers to a mono- or polycyclic (e.g. bi-, or tri-cyclic or more) aromatic radical or ring having from five to ten ring atoms of which one or more ring atom is selected from, for example, S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized.
  • a mono- or polycyclic e.g. bi-, or tri-cyclic or more
  • aromatic radical or ring having from five to ten ring atoms of which one or more ring atom is selected from, for example, S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized.
  • Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
  • Heterocyclic refers to a non-aromatic 5- , 6- or 7-membered ring or a bi- or tri-cyclic group fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (iv) any of the above rings may be fused to a benzene ring, and (v) the remaining ring atoms are carbon atoms which may be optionally oxo-substituted.
  • heterocycloalkyl groups include, but are not limited to, [l ,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and tetrahydrofuryl. Such heterocyclic groups may be further substituted.
  • initiation when applied to therapy can refer to a first administration of an active agent (e.g., a DAC inhibitor, tyrosine kinase inhibitor, or combinations thereof) to a patient who has not previously received the agent.
  • an active agent e.g., a DAC inhibitor, tyrosine kinase inhibitor, or combinations thereof
  • initiation can refer to administration of a particular dose of an agent (e.g., a DAC inhibitor, tyrosine kinase inhibitor, or combinations thereof) during therapy of a patient.
  • Peptide or protein comprises a string of at least three amino acids linked together by peptide bonds.
  • the terms “protein” and “peptide” may be used interchangeably.
  • Peptides preferably contain only natural amino acids, although non-natural amino acids ⁇ i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed.
  • one or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc.
  • the modifications of the peptide lead to a more stable peptide ⁇ e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide.
  • peptide refers to depsipeptide.
  • compositions comprising: a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • pharmaceutically acceptable ester As used herein, the term “pharmaceutically acceptable ester” refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
  • Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
  • prodrug refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention.
  • Prodrug as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of the invention.
  • prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrugs, Textbook of Drug Design and Development". Chapter 5, 1 13-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8: 1 -38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq.
  • compositions which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1 -19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid.
  • nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pam
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate.
  • Romidepsin The term "romidepsin”, refers to a natural product of the chemical structure:
  • Romidepsin is a deacetylase inhibitor and is also known in the art by the names FK228, FR901228, NSC630176, or depsipeptide.
  • the identification and preparation of romidepsin is described in U.S. Patent 4,977,138, issued December 1 1 , 1990, which is incorporated herein by reference.
  • the molecular formula is C 24 H 36 N 4 O 6 S 2 ; and the molecular weight is 540.71 g/mol.
  • Romidepsin has the chemical name, (l S,4S,10S,16E,21 R)-7-[(2Z)-ethylidene]-4,21 - diisopropyl-2-oxa- 12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9, 19,22- pentanone.
  • Romidepsin has been assigned the CAS number 128517-07-7.
  • romidepsin In crystalline form, romidepsin is typically a white to pale yellowish white crystal or crystalline powder.
  • the term "romidepsin" encompasses this compound and any pharmaceutically forms thereof.
  • romidepsin may also include salts, pro-drugs, esters, protected forms, reduced forms, oxidized forms, isomers, stereoisomers ⁇ e.g., enantiomers, diastereomers), tautomers, and derivatives thereof.
  • Stable refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject). In general, combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.
  • Substituted refers to aryl, heteroaryl. aliphatic groups as previously defined, substituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to, -F, -Cl, -Br, -I, -OH, protected hydroxyl.
  • Susceptible to refers to an individual having higher risk (typically based on genetic predisposition, environmental factors, personal history, or combinations thereof) of developing a particular disease or disorder, or symptoms thereof, than is observed in the general population.
  • Therapeutically effective amount of an active agent or combination of agents is intended to refer to an amount of agent(s) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect).
  • An effective amount of a particular agent may range from about 0.1 mg/Kg to about 500 mg/Kg, preferably from about 1 to about 50 mg/Kg. Effective doses may also vary depending on route of administration, as well as the possibility of co-usage with other agents.
  • any particular active agent utilized in accordance with the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.
  • Therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • treatment refers to any administration of a biologically active agent that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • the present invention demonstrates, among other things, that combinations of DAC inhibitors (e.g., romidepsin) and tyrosine kinase inhibitors (e.g., erlotinib) are particularly useful in the treatment of proliferative disorders (e.g., cancers such as lung cancer, including non-small cell lung cancer).
  • proliferative disorders e.g., cancers such as lung cancer, including non-small cell lung cancer.
  • the present invention provides a novel system for treating proliferative diseases by administering a combination of romidepsin and a tyrosine kinase inhibitor.
  • the combination of these agents may lead to an additive or synergistic effect.
  • the combination has been found to be particularly effective in treating cancers that have wild type EGFR and wild type KRAS.
  • a synergistic interaction between romidepsin and tyrosine kinase inhibitors in the treatment of cancer or other neoplasms has been demonstrated as described herein. See Figures 6-11. This synergistic effect is particularly pronounced in the case of lung cancer cells, particularly non-small cell lung cancer cells. Without wishing to be bound by any particular theory, the effect may be due to the induction of apoptosis by the combination of agents. [0054]
  • the combination of romidepsin and erlotinib has been found to be useful in treating lung cancer.
  • the inventive combination is particularly useful in treating non-small cell lung caner.
  • the inventive combination is useful in treating non-small cell lung caners that are wild type EGFR and wild type KRAS.
  • the inventive combination may also be effective in treating KRAS mutant cell lines.
  • the invention provides methods of treating cells with the inventive combinations both in vitro and in vivo.
  • the invention also provides pharmaceutical compositions and kits comprising the inventive combinations.
  • the inventive combination comprises romidepsin and erlotinib.
  • Deacetylase inhibitors are compounds which are capable of inhibiting the deacetylation of proteins in vivo, in vitro, or both.
  • the invention relates to HDAC inhibitors, which inhibit the deacetylation of histones.
  • the invention relates to TDAC inhibitors, which inhibit the deacetylation of tubulin.
  • DAC inhibitors often have a variety of biological activities, at least some of which may well be independent of histone deacetylase inhibition.
  • DAC inhibitors inhibit the activity of at least one deacetylase.
  • the DAC inhibitor is an HDAC inhibitor
  • an increase in acetylated histones occurs and accumulation of acetylated histones is a suitable biological marker for assessing the activity of HDAC inhibitors. Therefore, procedures which can assay for the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of agents of interest. Analogous assays can determine DAC inhibitory activity
  • Suitable DAC inhibitors include, for example, 1 ) hydroxamic acid derivatives; 2) short-chain fatty acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting deacetylase activity.
  • Examples of such DAC inhibitors include, but are not limited to:
  • HYDROXAMIC ACID DERIVATIVES such as Suberoylanilide Hydroxamic Acid (SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95:3003, 1998); M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Richon et al., supra); pyroxamide; CBHA; Trichostatin analogues such as Trichostatin A (TSA) and Trichostatin C (Koghe et al. Biochem. Pharmacol. 56: 1359, 1998); Salicylihydroxamic Acid (SBHA) (Andrews et al., International J.
  • CYCLIC TETRAPEPTIDES such as Trapoxin A (TPX)-Cyclic Tetrapeptide (cyclo-(L- phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amin-o-8-oxo-9,10-epoxy decanoyl)) (Kijima et al., J Biol. Chem. 268:22429, 1993); FR901228 (FK 228, FR901228, Depsipeptide, Romidepsin) (Nakajima et al., Ex. Cell Res.
  • TPX Trapoxin A
  • FR901228 FK 228, FR901228, Depsipeptide, Romidepsin
  • Valerate (McBain et al., supra); 4 Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15:879, 1995); Phenylbutyrate (PB) (Wang et al., Cancer Research, 59:2766, 1999); Propionate (McBain et al., supra); Butyramide (Lea and Tulsyan, supra); Isobutyramide (Lea and Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra); 3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et al., Cancer Research, 60:749, 2000); Valproic acid and Valproate.
  • 4-PBA Phenylbutyrate
  • PB Phenylbutyrate
  • Propionate (McBain et al., supra); Butyramide (Lea and T
  • BENZAMlDE DERIVATIVES such as CI-994; MS-275 [N-(2-aminophenyl)-4-[N- (pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA 96:4592, 1999; 3'-amino derivative of MS-27-275 (Saito et al., supra); MGCDO 103 (MethylGene; see Figure 1), or related compounds (for example, see Figure
  • E) ELECTROPHILIC KETONE DERIVATIVES such as trifluoromethyl ketones (Frey et al, Bioorganic & Med. Chem. Lett., 12: 3443, 2002; U.S. 6,51 1,990) and ⁇ -keto amides such as N-methyl- ⁇ -ketoamides.
  • Suitable DAC inhibitors for use in accordance with the present invention particularly include, for example, CRA-094781 CCelera Genomics), PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCDO 103 (M ethyl Gene), MS-275
  • the DAC or HDAC inhibitor used in the method of the invention is represented by formula (V):
  • B is a substituted or unsubstituted, saturated or unsaturated aliphatic group, a substituted or unsubstituted, saturated or unsaturated alicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted heterocyclic group;
  • R 2 o is hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino, or alkyloxy group;
  • R 2 i and R 22 are independently selected from hydrogen, hydroxyl, a substituted or unsubstituted, saturated or unsaturated aliphatic group, a substituted or unsubstituted, saturated or unsaturated alicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted heterocyclic group.
  • R 20 is a hydroxylamino, hydroxyl, amino, methylamino, dimethylamino or methyloxy group and B is a C 6 -alkyl.
  • R 2 ⁇ is a hydrogen atom
  • R 22 is a substituted or unsubstituted phenyl and B is a C 6 -alkyl.
  • R 2 i is hydrogen and R 22 is an ⁇ -, ⁇ -, or ⁇ -pyridine.
  • DAC or HDAC inhibitors can be found in, for example, U.S. Pat. Nos. 5,369,108, issued on Nov. 29, 1994, 5,700,81 1 , issued on Dec. 23, 1997, 5,773,474, issued on Jun. 30, 1998, 5,932,616 issued on Aug. 3, 1999 and 6,51 1 ,990, issued Jan. 28, 2003 all to Breslow et al.; U.S. Pat. Nos. 5,055,608, issued on Oct. 8, 1991, 5,175,191, issued on Dec. 29, 1992 and 5,608,108, issued on Mar. 4, 1997 all to Marks et al.; U.S. Provisional Application No. 60/459,826, filed Apr.
  • DAC or HDAC inhibitors are provided in the table below. It should be noted that the present invention encompasses any compounds which both are structurally similar to the compounds represented below and are capable of inhibiting histone deacetylases.
  • DAC or HDAC inhibitors for use in accordance with the present invention may be prepared by any available means including, for example, synthesis, semi-synthesis, or isolation from a natural source.
  • DAC or HDAC inhibitors for use in accordance with the present invention may be isolated or purified.
  • synthesized compounds can be separated from a reaction mixture, and natural products can be separated from their natural source, by methods such as column chromatography, high pressure liquid chromatography, and/or recrystallization.
  • a variety of synthetic methodologies for preparing DAC or HDAC inihibitors are known in the art. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds.
  • Synthetic chemistry transformations and protecting group methodologies useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.
  • DAC or HDAC inhibitors for use in accordance with the present invention may be modified as compared with presently known DAC or HDAC inhibitors, for example, by appending appropriate functionalities to enhance selective biological properties.
  • modifications are known in the art and may include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
  • a DAC (e.g., HDAC) inhibitor for use in accordance with the present invention may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers. and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- , or as (D)- or (L)- for amino acids.
  • the present invention encompasses all such possible isomers, as well as their racemic and optically pure forms to the extent that they have DAC inhibitory activity.
  • optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures.
  • the resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981).
  • a DAC e.g., HDAC
  • a DAC for use in accordance with the present invention may contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry.
  • the present invention encompasses both E and Z geometric isomers or cis- and trans- isomers to the extent that they have DAC inhibitory activity.
  • the present invention likewise encompasses all tautomeric forms that have DAC inhibitory activity.
  • a carbon-carbon double bond or carbon-heteroatom double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.
  • Romidepsin is a cyclic depsipeptide of formula:
  • Romidepsin may be provided in any form. Pharmaceutically acceptable forms are particular preferred. Exemplary forms of romidepsin include, but are not limited to, salts, esters, prodrugs, isomers, stereoisomers ⁇ e.g., enantiomers, diastereomers), tautomers, protected forms, reduced forms, oxidized forms, derivatives, and combinations thereof, with the desired activity ⁇ e.g., deacetylase inhibitory activity, aggresome inhibition, cytotoxicity). In certain embodiments, the romidepsin used in the combination therapy is pharmaceutical grade material and meets the standards of the U.S. Pharmacopoeia, Japanese Pharmacopoeia, or European Pharmacopoeia.
  • the romidepsin is at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.95% pure. In certain embodiments, the romidepsin is at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.95% monomeric. In certain embodiments, no impurities are detectable in the romidepsin materials ⁇ e.g., oxidized material, reduced material, dimerized or oligomerized material, side products, etc.). The romidepsin typically includes less than 1.0%, less than 0.5%, less than 0.2%, or less than 0.1 % of total other unknowns.
  • the purity of romidepsin may be assessed by appearance, HPLC, specific rotation, NMR spectroscopy, IR spectroscopy, UVNisible spectroscopy, powder x-ray diffraction (XRPD) analysis, elemental analysis, LC-mass spectroscopy, and mass spectroscopy.
  • the inventive combination therapy may also include a derivative of romidepsin.
  • the derivative of romidepsin is of the formula (I):
  • n is 0, 1 , 2 or 3; p and q are independently 1 or 2;
  • X is O, NH, or NR 8 ;
  • Ri, R 2 , and R 3 are independently hydrogen; unsubstituted or substituted, branched or unbranched, cyclic or acyclic aliphatic; unsubstituted or substituted, branched or unbranched, cyclic or acyclic heteroaliphatic; unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl; and
  • R 4 , R 5 , R 6 , R 7 and R 8 are independently hydrogen; or substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; and pharmaceutically acceptable forms thereof.
  • m is 1.
  • n is 1.
  • p is 1.
  • q is 1.
  • X is O.
  • Ri, R 2 , and R 3 are unsubstituted, or substituted, branched or unbranched, acyclic aliphatic.
  • R 4 , R 5 , R 6 , and R 7 are all hydrogen.
  • the derivative of romidepsin is of the formula (II):
  • X is O, NH, Or NR 8 ;
  • Y is OR 8 , or SR 8 ;
  • R 2 and R 3 are independently hydrogen; unsubstituted or substituted, branched or unbranched, cyclic or acyclic aliphatic; unsubstituted or substituted, branched or unbranched, cyclic or acylic heteroaliphatic; unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl;
  • R 4 , R 5 , R 6 , R 7 and R 8 are independently selected from hydrogen; or substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; and pharmaceutically acceptable forms thereof.
  • m is 1.
  • n is 1.
  • q is 2.
  • X is O.
  • X is NH.
  • R 2 and R 3 are unsubstituted or substituted, branched or unbranched, acyclic aliphatic.
  • R 4 , R 5 , R 6 , and R 7 are all hydrogen.
  • the derivative of romidepsin is of the formula (III):
  • A is a moiety that is cleaved under physiological conditions to yield a thiol group and includes, for example, an aliphatic or aromatic acyl moiety (to form a thioester bond); an aliphatic or aromatic thioxy (to form a disulfide bond); or the like; and pharmaceutically acceptable forms thereof.
  • Such aliphatic or aromatic groups can include a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group.
  • Ri is independently hydrogen; substituted or unsubstituted amino; substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; substituted or unsubstituted aromatic group; substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group.
  • Ri is hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl, benzyl, or bromobenzyl.
  • R 2 is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group.
  • R 2 is methyl, ethyl, 2-hydroxyethyl, isobutyl, fatty acids, a substituted or unsubstituted benzyl, a substituted or unsubstituted aryl, cysteine, homocysteine, or glutathione.
  • the derivative of romidepsin is of formula (IV) or (IV):
  • Ri, Ri, R 3 . and R 4 are the same or different and represent an amino acid side chain moiety
  • each R 6 is the same or different and represents hydrogen or C1-C4 alkyl
  • Pr and Pr 2 are the same or different and represent hydrogen or thiol-protecting group.
  • the amino acid side chain moieties are those derived from natural amino acids. In other embodiments, the amino acid side chain moieties are those derived from unnatural amino acids.
  • each amino acid side chain is a moiety selected from - H, -C 1 -C 6 alkyl, -C 2 -C 6 alkenyl, -L-O-C(O)-R', -L-C(O)-O-R", -L-A, -L-NR 11 R", -L-Het- C(O)-Het-R", and -L-Het-R", wherein L is a C 1 -C 6 alkylene group, A is phenyl or a 5- or 6- membered heteroaryl group, each R' is the same or different and represents C 1 -C 4 alkyl, each R" is the same or different and represent H or C 1 -C 6 alkyl, each -Het- is the same or different and is a heteroatom spacer selected from -0-, -N(R"')-, and -S-, and each R'" is the same of different and represents H or C 1 -C 4 al
  • R 6 is -H.
  • Pr 1 and Pr 2 are the same or different and are selected from hydrogen and a protecting group selected from a benzyl group which is optionally substituted by C 1 -C 6 alkoxy, C 1 -C 6 acyloxy, hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, C 1 -C 6 acyloxymethyl, C 1 -C 6 alkoxymethyl, tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl, and C 1 -C 6 alkyl
  • the romidepsin or a derivate thereof is purified from a fermentation, for example, of Chromobacterium violaceum. See, e.g., Ueda et al, J. Antibiot. (Tokyo) 47:301-310, 1994; Nakajima et al, Exp. Cell Res. 241 : 126-133, 1998; WO 02/20817; U.S. Patent 4,977,138; each of which is incorporated herein by reference.
  • romidepsin or a derivative thereof is prepared by synthetic or semi-synthetic means. J. Am. Chem. Soc. 1 18:7237-7238, 1996; incorporated herein by reference.
  • the therapeutically effective amount of romidepsin included in the combination therapy will vary depending on the patient, the cancer or neoplasm being treated, stage of the cancer, pathology of the cancer or neoplasm, genotype of the cancer or neoplasm, phenotype of the cancer or neoplasm, the route of administration, etc.
  • the romidepsin is dosed in the range of 0.5 mg/ m 2 to 28 mg/m 2 .
  • the romidepsin is dosed in the range of 1 mg/ m 2 to 25 mg/m 2 .
  • the romidepsin is dosed in the range of 0.5 mg/ m 2 to 15 mg/m 2 .
  • the romidepsin is dosed in the range of 1 mg/ m 2 to 15 mg/m 2 . In certain embodiments, the romidepsin is dosed in the range of 1 mg/ m 2 to 8 mg/m 2 . In certain embodiments, the romidepsin is dosed in the range of 0.5 mg/ m 2 to 5 mg/m 2 . In certain embodiments, the romidepsin is dosed in the range of 2 mg/ m 2 to 10 mg/m 2 . In certain embodiments, the romidepsin is dosed in the range of 4 mg/ m 2 to 15 mg/m 2 .
  • the romidepsin is dosed in the range of 8 mg/ m 2 to 10 mg/m 2 . In other embodiments, the dosage ranges from 10 mg/m 2 to 20 mg/m 2 . In certain embodiments, the dosage ranges from 5 mg/m 2 to 10 mg/m 2 . In other embodiments, the dosage ranges from 10 mg/m 2 to 15 mg/m 2 . In still other embodiments, the dosage is approximately 8 mg/m 2 . In still other embodiments, the dosage is approximately 9 mg/m . In still other embodiments, the dosage is approximately 10 mg/m " . In still other embodiments, the dosage is approximately 1 1 mg/m 2 . In still other embodiments, the dosage is approximately 12 mg/m 2 .
  • the dosage is approximately 13 mg/m . In still other embodiments, the dosage is approximately 14 mg/m 2 . In still other embodiments, the dosage is approximately 15 mg/m 2 . In certain embodiments, increasing doses of romidepsin are administered over the course of a cycle. For example, in certain embodiments, a dose of approximately 8 mg/m 2 , followed by a dose of approximately 10 mg/m 2 , followed by a dose of approximately 12 mg/m 2 may be administered over a cycle. As will be appreciated by one of skill in the art, depending on the form of romidepsin being administered the dosing may vary. The dosages given herein are dose equivalents with respect to the active ingredient, romidepsin.
  • romidepsin is administered intravenously.
  • the romidepsin is administered intravenously over a 1 -6 hour time frame.
  • the romidepsin is administered intravenously over 1-2 hours.
  • the romidepsin is administered intravenously over 3-4 hours.
  • the romidepsin is administered intravenously over 5-6 hours.
  • the romidepsin is administered one day followed by several days in which the romidepsin is not administered. In certain embodiments, the romidepsin and the tyrosine kinase inhibitor are administered together. In other embodiments, the romidpesin and the tyrosine kinase inhibitor are administered separately. For example, the administration of romidepsin and a tyrosine kinase inhibitor may be separated by one or more days. In certain embodiments, romidepsin is administered twice a week. In certain embodiments, romidepsin is administered once a week. In other embodiments, romidepsin is administered every other week.
  • romidepsin is administered on days 1 , 8, and 15 of a 28 day cycle. In certain particular embodiments, an 8 mg/m 2 dose of romidepsin is administered on day 1, a 10 mg/m 2 dose of romidepsin is administered on day 8, and a 12 mg/m 2 dose of romidepsin is administered on day 15. In certain embodiments, romidepsin is administered on days 1 and 15 of a 28 day cycle.
  • the 28 day cycle may be repeated. In certain embodiments, the 28 day cycle is repeated 3-10 times.
  • the treatment includes 5 cycles. In certain embodiments, the treatment includes 6 cycles. In certain embodiments, the treatment includes 7 cycles. In certain embodiments, the treatment includes 8 cycles. In certain embodiments, greater than 10 cycles are administered. In certain embodiments, the cycles are continued as long as the patient is responding. The therapy may be terminated once there is disease progression, a cure or remission is achieved, or side effects become intolerable.
  • romidepsin may be administered orally.
  • romidepsin is dosed orally in the range of 10 mg/ m 2 to 300 mg/m 2 .
  • romidepsin is dosed orally in the range of 25 mg/ m to 100 mg/m .
  • romidepsin is dosed orally in the range of 100 mg/ m 2 to 200 mg/m 2 .
  • romidepsin is dosed orally in the range of 200 mg/ m 2 to 300 mg/m 2 .
  • romidepsin is dosed orally at greater than 300 mg/m .
  • romidepsin is dosed orally in the range of 50 mg/ m 2 to 150 mg/m 2 . In other embodiments, the oral dosage ranges from 25 mg/m 2 to 75 mg/m 2 . As will be appreciated by one of skill in the art, depending on the form of romidepsin being administered the dosing may vary. The dosages given herein are dose equivalents with respect to the active ingredient, romidepsin. In certain embodiments, romidepsin is administered orally on a daily basis. In other embodiments, romidepsin is administered orally every other day. In still other embodiments, romidepsin is administered orally every third, fourth, fifth, or sixth day.
  • romidepsin is administered orally every week. In certain embodiments, romidepsin is administered orally every other week. In certain embodiments, the romidepsin and the tyrosine kinase inhibitor are administered together. In other embodiments, the romidepsin and the tyrosine kinase inhibitor are administered separately. For example, the administration of romidepsin and a tyrosine kinase inhibitor may be separated by one or more days. In certain embodiments, both romidepsin and the tyrosine kinase inhibitor are administered orally. In certain embodiments, only romidepsin is administered orally. The administration of romidepsin alone or the combination of romidepsin and the tyrosine kinase inhibitor may be terminated once there is disease progression, a cure or remission is achieved, or side effects become intolerable.
  • Tyrosine kinases add phosphate groups to tyrosine residues in proteins or peptides. Such phosphorylation often affects the activity, folding, and/or subcellular localization of the target protein, and is commonly involved in signaling cascades that communicate information and allow cells to respond to molecular cues. For example, tyrosine kinase signaling cascades are involved in activating cellular proliferation. Tyrosine. kinase activity has be found associated with the epidermal growth factor receptor as well as other receptors.
  • Mutations in proteins that participate in tyrosine kinase signaling cascades sometimes cause continual activation of cellular proliferation pathways, so that unregulated cell growth occurs and a proliferative disorder, disease, or condition ⁇ e.g., cancer or benign neoplasm) results.
  • tyrosine kinases There are two basic categories of tyrosine kinases: receptor tyrosine kinases and cellular tyrosine kinases.
  • Receptor tyrosine kinases are transmembrane proteins that have an extracellular ligand-binding domain and an intracellular catalytic domain.
  • the extracellular ligand binding domain typically includes or more conserved structural motifs such as, for example, cysteine- rich regions, fibronectin Ill-like domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains, Factor Vlll-like domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains.
  • the intracellular domain includes the catalytic sequences, which may be continuous or separated.
  • receptor tyrosine kinases are activated by ligand binding, which triggers dimerization. Dimerization. in turn, results in autophosphorylation which in turn creates binding sites for other cellular components involved in signal transduction.
  • cellular components include, for example, RasGAP, P13-kinase, phospholipase C Y, phosphotyrosine phosphatase SHP and adaptor proteins such as She, Grb2 and Crk.
  • Cellular tyrosine kinases are located in the cytoplasm, nucleus, or inner leaflet of the plasma membrane (i.e., are not transmembrane).
  • SRC cellular tyrosine kinase
  • JAK JAK
  • ABL ABL
  • FAK homologous kinase domain
  • FPS FPS
  • CSK CSK
  • SYK SYK
  • BTK BTK
  • All cellular tyrosine kinase domains share homologous kinase domains (Src Homology 1 , or SHl domains); some also share protein-protein interaction domains (e.g., SH2 and SH3 domains).
  • SH2 and SH3 domains protein-protein interaction domains.
  • Some members of the cytokine receptor pathway interact with JAKs, which phosphorylate the transcription factors, STATs.
  • the SRC cellular tyrosine kinase inhibitors are involved in cell growth.
  • Tyrosine kinase inhibitors are agents that reduce the activity and/or amount of a tyrosine kinase in a cell. Such agents can be useful in the treatment of proliferative disorders, diseases, or conditions. Certain leukemias, as well as cancers of the breast, prostate, ovary, bladder, liver, pancreas, and lung, are among the cancers that are most responsive to therapy with tyrosine kinase inhibitors.
  • tyrosine kinase inhibitors include, for example, axitinib, cediranib (RECENTIN), dasatinib (SPRYLCEL), erlotinib (TARCEV A ® ), gefitinib (IRESSA), imatinib (GLEEVEC), lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, and vandetanib.
  • romidepsin is used in combination with axitinib.
  • romidepsin is used in combination with cediranib.
  • romidepsin is used in combination with dasatinib. In certain embodiments, romidepsin is used in combination with erlotinib. In certain embodiments, romidepsin is used in combination with gefitinib. In certain embodiments, romidepsin is used in combination with imatinib. In certain embodiments, romidepsin is used in combination with lapatinib. In certain embodiments, romidepsin is used in combination with lestaurtinib. In certain embodiments, romidepsin is used in combination with nilotinib. In certain embodiments, romidepsin is used in combination with semaxanib. In certain embodiments, romidepsin is used in combination with sunitinib. In certain embodiments, romidepsin is used in combination with vandetanib.
  • the tyrosine kinase inhibitor is erlotinib.
  • Erlotinib specifically targets the epidermal growth factor receptor tyrosine kinase, which is highly expressed and occasionally mutated in various forms of cancer. Erlotinib has been shown to improve survival in lung cancer patients, and has been approved for use in treating lung and pancreatic cancer.
  • Anti-neoplastic agents suitable for use in the present invention includes any agents that inhibit or prevent the growth of neoplasms, checking the maturation and proliferation of malignant cells. Growth inhibition can occur through the induction of stasis or cell death in the tumor cell(s).
  • anti-neoplastic agents include cytotoxic agents in general.
  • Exemplary anti-neoplastic agents include, but are not limited to, cytokines, ligands, antibodies, radionuclides, and chemotherapeutic agents.
  • such agents include interleukin 2 (1L-2), interferon (IFN) TNF; photosensitizers, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 ( 131 I), yttrium-90 ( 90 Y), bismuth-212 ( 212 Bi), bismuth-213 ( 213 Bi), technetium-99m (.”' 11 Tc), rhenium- 186 ( 186 Re), and rhenium- 188 ( 188 Re); chemotherapeutics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterot
  • the combination of romidepsin and a tyrosine kinase inhibitor may be used in vitro or in vivo.
  • the inventive combination is particularly useful in the treatment of neoplasms in vivo.
  • the combination may also be used in vitro for research or clinical purposes (e.g., determining the susceptibility of a patient's disease to the inventive combination, researching the mechanism of action, elucidating a cellular pathway or process).
  • the neoplasm is a benign neoplasm.
  • the neoplasm is a malignant neoplasm. Any cancer may be treated using the inventive combination.
  • the invention provides methods for treating cell proliferative disorders, diseases or conditions.
  • cell proliferative disorders, diseases or conditions encompass a variety of conditions characterized by aberrant cell growth, preferably abnormally increased cellular proliferation.
  • cell proliferative disorders, diseases, or conditions include, but are not limited to, cancer, immune-mediated responses and diseases (e.g., transplant rejection, graft vs host disease, immune reaction to gene therapy, autoimmune diseases, pathogen-induced immune dysregulation, etc.), certain circulatory diseases, and certain neurodegenerative diseases.
  • the invention relates to methods of treating cancer.
  • cancer is a group of diseases which are characterized by uncontrolled growth and spread of abnormal cells. Examples of such diseases are carcinomas, sarcomas, leukemias, lymphomas, and the like.
  • cancers include, but are not limited to leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndrome, mesothelioma, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular, rectal and
  • HTLV human
  • the invention relates to treatment of solid tumors.
  • the invention relates to treatment of solid tumors such as lung, breast, colon, liver, pancreas, renal, prostate, ovarian, or brain cancer.
  • the invention relates to treatment of pancreatic cancer.
  • the invention relates to treatment of renal cancer.
  • the invention relates to treatment of lung cancer.
  • the invention relates to treatment of non- small cell lung cancer.
  • the invention relates to treatment of prostate cancer.
  • the invention relates to treatment of sarcomas.
  • the invention relates to treatment of soft tissue sarcomas.
  • the invention relates to methods of treating one or more immune-mediated responses and diseases.
  • the invention relates to treatment of disorders, diseases or conditions associated with chromatin remodeling.
  • the invention relates to treatment of lung cancer. In some embodiments, the invention relates to treatment of non small cell lung cancer. In some embodiments, the invention relates to treatment of wild type EGFR non-small cell lung cancer. In some embodiments, the invention relates to treatment of wild type KRAS non- small cell lung cancer. In some embodiments, the invention relates to treatment of wild type EGFR and wild type ICRAS non-small cell lung cancer. In some embodiments, the invention relates to treatment of mutant KRAS non-small cell lung cancer.
  • the inventive combinations of romidepsin plus a tyrosine kinase inhibitor may also be used to treat and/or kill cells in vitro.
  • a cytotoxic concentration of the combination of agents is contacted with the cells in order to kill them.
  • a sublethal concentration of the combination of agents is used to treat the cells.
  • the combination of agents acts additively to kill the cells.
  • the combination of agents acts synergistically to kill the cells. Therefore, a lower concentration of one or both agents is needed to kills the cells than would be needed if either agent were used alone.
  • the concentration of each agent ranges from 0.01 nM to 100 nM.
  • the concentration of each agent ranges from 0.1 nM to 50 nM. In certain embodiments, the concentration of each agent ranges from 1 nM to 10 nM. In certain embodiments, the concentration of romidepsin ranges from 1 nM to 10 nM, more particularly 1 nM to 5 nM. In certain embodiments, the concentration of the tyrosine kinase inhibitor ranges from 1 nM to 10 nM, more particularly 1 nM to 5 nM
  • the cells may be derived from any animal, plant, bacterial, or fungal source.
  • the cells may be at any stage of differentiation or development.
  • the cells are animal cells.
  • the cells are vertebrate cells.
  • the cells are mammalian cells.
  • the cells are human cells.
  • the cells may be derived from a male or female human in any stage of development.
  • the cells are primate cells.
  • the cells are derived from a rodent (e.g., mouse, rat, guinea pig, hamster, gerbil).
  • the cells are derived from a domesticated animal such as a dog, cat, cow, goat, pig, etc.
  • the cells may also be derived from a genetically engineered animal or plant, such as a transgenic mouse.
  • the cells used may be wild type or mutant cells.
  • the cells may be genetically engineered.
  • the cells are normal cells.
  • the cells are hematological cells.
  • the cells are white blood cells.
  • the cells are precursors of white blood cells (e.g., stem cells, progenitor cells, blast cells).
  • the cells are neoplastic cells.
  • the cells are cancer cells.
  • the cells are derived from a hematological malignancy.
  • the cells are derived from a solid tumor.
  • the cells are derived from a lung cancer.
  • the cells are derived from a non-small cell lung cancer.
  • the cells may be derived from a patient's tumor (e.g., from a biopsy or surgical excision).
  • cytotoxicity may be useful in determining whether a patient will respond to a particular combination therapy.
  • Such testing may also be useful in determining the dosage needed to treat the malignancy.
  • This testing of the susceptibility of a patient's cancer to the combination therapy would prevent the unnecessary administration of drugs with no effect to the patient.
  • the testing may also allow the use of lower doses of one or both of the drugs if the patient's cancer is particularly susceptible to the combination.
  • the cells are derived from cancer cells lines.
  • the cells are from lung cancers such as those discussed herein.
  • Lung cancer cell lines include ABC-I, A549, PC3, PC7, RERF-LCMS, RERF-LCKJ, LCD, LCOK, PC9, PC14, QG56, EBC-I , LK-2, LC-l/sq, PCl , RERF-LCAI, PCl O, SQ5, NCI-H69, SBC3, NCI- N23 I , Lul35, and MS-I .
  • the present invention demonstrates the particular utility of administering a combination of a DAC inhibitor and a tyrosine kinase inhibitor.
  • the DAC inhibitor is romidepsin (aka, depsipeptide, FK228, FR901228).
  • the DAC inhibitor is selected from the group consisting of CRA-024781 (Celera Genomics), phenylbutarate, PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCDO 103 (MethylGene), MS-275 (Schering AG), romidepsin (Gloucester Pharmceuticals), SAHA (Alton Pharma/Merck), and combinations thereof.
  • the DAC inhibitor is romidepsin (aka depsipeptide, FK228, FR901228).
  • the DAC inhibitor is SAHA.
  • the DAC inhibitor is phenylbutyrate.
  • the DAC inhibitor comprises a combination of DAC inhibitors.
  • the tyrosine kinase inhibitor is erlotinib (TARCEV A ® ).
  • the tyrosine kinase inhibitor is selected from the group consisting of axitinib, cediranib (RECENTIN), dasatinib (SPRYLCEL), erlotinib (TARCEVA), gefitinib (IRESSA), imatinib (GLEEVEC), lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, vandetanib, and combinations thereof [00100] As will be appreciated by those of skill in the art, and as is otherwise addressed herein, either or both of the DAC inhibitor and tyrosine kinase inhibitor may be provided in any useful form including, for example, as a salt, ester, active metabolite, prodrug, etc.
  • either or both agents may be provided as a pure isomer stereoisomer or as a combination of stereoisomers, including a racemic combination, so long as relevant activity is present.
  • either or both agents may be provided in crystalline form, whether a pure polymorph or a combination of polymorphs, or in amorphous form, so long as relevant activity is present.
  • combination therapy of DAC inhibitors and tyrosine kinase inhibitor will typically involve administration of multiple individual doses spaced out in time.
  • individual DAC inhibitor doses and tyrosine kinase inihbitor doses will be administered together, according to the same schedule.
  • DAC inhibitor doses and tyrosine kinase inhibitor doses will be administered according to different schedules.
  • the total daily dose of any particular active agent administered to a human or other animal in single or in divided doses in accordance with the present invention can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight.
  • Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. In certain embodiments, about 10-100 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 100-500 mg of the compound is administered per day in single or multiple doses.
  • a DAC inhibitor is typically dosed at 1 -30 mg/m 2 . In certain embodiments, a DAC inhibitor is dosed at 1-15 mg/m 2 . In certain embodiments, a DAC inhibitor is dosed at 5-15 mg/m 2 . In certain particular embodiments, a DAC inhibitor is dosed at 4, 6, 8, 10, 12, 14, 16, 18, or 20 mg/m 2 . A DAC inhibitor is typically administered in a 28 day cycle with the agent being administered on days 1, 8 and 15.
  • the DAC is administered on days 1 and 15 with day 8 being skipped.
  • the dosage and timing of administration of the dosage of the DAC inhibitor may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of DAC inhibitor administered.
  • Typical dosing schedules have been established for certain exemplary DAC inhibitors (e g., HDAC inhibitors).
  • SAHA is commonly administered within a range of about 300-400 mg daily orally
  • PXDlOl is commonly administered within a range of about up to 2000 mg/m 2 /day intravenously (e.g., on days 1 to 5 of a 21 day cycle), and may possibly be administered orally
  • MGCDO 103 is commonly administered at doses up to about 27 mg/m 2 given orally (e.g., daily for about 14 days)
  • LBH589 is commonly administered at doses up to about 14 mg/m 2 as an intravenous infusion (e.g., on days 1-7 of a 21 day cycle)
  • MS-275 is commonly administered within a dose range of about 2-12 mg/m 2 intravenously (e.g., every 14 days).
  • romidepsin is typically dosed at 1 -28 mg/m 2 . In certain embodiments, romidepsin is dosed at 1 -15 mg/m 2 . In certain embodiments, romidepsin is dosed at 5-14 mg/m 2 . In certain particular embodiments, romdiepsin is dosed at 8, 10, 12, or 14 mg/m 2 . Romidepsin is typically administered in a 28 day cycle with romidepsin being administered on days 1 , 8 and 15. In certain embodiments, romidepsin is administered on days 1 and 15 with day 8 being skipped.
  • erlotinib is typically administered orally at a dose of 150 mg/day.
  • erlotinib in combination with a DAC inhibitor is administered orally at a dose of approximately 100 mg/day. It is generally recommended that erlotinib be taken one hour before or two hours after the ingestion of food. Erlotinib is currently available in 150 mg, 100 mg, and 25 mg doses.
  • the dosage and timing of administration of any particular DAC inhibitor or tyrosine kinase inhbitor dose, or the dosage amount and schedule generally may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of one or the other agent, or of both agents, being administered.
  • the dosage schedule i.e., amount and timing of individual doses
  • the dosage schedule for the tyrosine kinase inhibitor may be different according to inventive combination therapy regimens than would be utilized in monotherapy (even for the same disorder, disease or condition).
  • a DAC inhibitor e.g., romidepsin
  • a tyrosine kinase inhibitor e.g., erlotinib
  • a DAC inhibitor e.g., romidepsin
  • a tyrosine kinase inhibitor e.g., erlotinib
  • dosing is adjusted based on a patient's response to therapy, and particularly to development of side effects.
  • inventive combination therapy with one or more DAC inhibitors and one or more tyrosine kinase inhibitors is further combined with administration of one or more other agents.
  • subjects receiving inventive combination therapy with one or more DAC inhibitors and one or more tyrosine kinase inhibitors further receive electrolyte supplementation for example as is described in co-pending United States Provisional Patent application, U. S. S.N. 60/909,780, , filed April 3, 2007; and U.S. non-provisional patent application.
  • U. S. S.N. 1 1/759,471 filed June 7, 2007; each of which is incorporated herein by reference.
  • Serum concentrations of potassium are generally considered to be "normal” when they are within the range of about 3.5 - 5.5 mEq/L or about 3.5 - 5.0 mEq/L. According to the present invention, it is often desirable to ensure that an individuals' serum potassium concentration is within this range prior to (and/or during) administration of DAC inhibitor and/or combination therapy.
  • Serum concentrations of magnesium are generally considered to be "normal” when they are within the range of about 1.5 - 2.5 mEq/L or about 1.5 - 2.2 mEq/L or about 1.25 - 2.5 mEq/L or about 1.25 - 2.2 mEq/L. According to the present invention, it is often desirable to ensure that an individual's serum magnesium concentration is within this range prior to (and/or during) administration of DAC inhibitor and/or combination therapy. [00115] In some embodiments of the invention, an individual's serum potassium and/or magnesium concentration(s) is/are at the high end of the normal range prior to (and/or during) administration of DAC inhibitor and/or combination therapy.
  • an individual's serum potassium concentration is at least about 3.8, 3.9, 4.0 mEq/L, or more prior to and/or during administration of DAC inhibitor and/or combination therapy. In some embodiments, care is taken not to increase serum potassium concentration above about 5.0, 5.2, or 5.5 mEq/L. In some embodiments, an individual's serum magnesium concentration is at least about 1.9 mEq/L or more prior to and/or during administration of DAC inhibitor and/or combination therapy. In some embodiments, care is taken not to increase magnesium concentration above about 2.5 mEq/L.
  • an individual's serum potassium concentration is at least about 3.5 mEq (in some embodiments at least about 3.8, 3.9, 4.0 mEq/L or above) and the individual's serum magnesium concentration is at least about 1.85 mEq/L (in some embodiments at least about 1.25, 1.35, 1.45, 1.55, 1.65, 1.75, 1.85, 1.95, etc) prior to and/or during administration of DAC inhibitor and/or combination therapy.
  • electrolyte levels ⁇ e.g., potassium and/or magnesium levels, optionally calcium levels
  • electrolyte levels are assessed more than once during the course of DAC inhibitor and/or combination therapy; in some embodiments, different assessments are separated by a regular interval (e.g., 0.5 days or less, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc.).
  • electrolyte levels are assessed prior to each administration of DAC inhibitor or tyrosine kinase inhibitor.
  • DAC inhibitors and/or tyrosine kinase inhibitors for use in accordance with the present invention are often administered as pharmaceutical compositions comprising therapeutically effective amounts of DAC inhibitor and tyrosine kinase inhibitor, respectively, that are useful in the inventive combination therapy (which amounts may be different from, including less than, amounts required for either agent to be effective alone).
  • a DAC inhibitor and tyrosine kinase inhibitor are present together in a single pharmaceutical composition; in some embodiments these agents are provided in separate pharmaceutical compositions.
  • inventive pharmaceutical compositions are prepared in unit dosage forms.
  • a pharmaceutical composition of the present invention includes one or more active agents (i.e., one or more DAC inhibitors and/or one or more tyrosine kinase inhibitors) formulated with one or more pharmaceutically acceptable carriers or excipients.
  • This invention also provides pharmaceutical compositions, preparations, or kits comprising romidepsin and/or a tyrosine kinase inhibitor as described herein, which combination shows cytostatic or cytotoxic activity against neoplastic cells such as lung cancer.
  • the compositions, preparations, or kits typically include amounts appropriate for the administration of romidepsin and/or the tyrosine kinase inhibitor.
  • the romidepsin and the tyrosine kinase inhibitor are not mixed together in the same composition.
  • the two agents are not part of the same solution or powder.
  • the two agents are kept separate in two different compositions and are delivered separately.
  • a kit may contain a pharmaceutical composition of romidepsin and a separate pharmaceutical composition of a tyrosine kinase inhibitor.
  • the pharmaceutical compositions, preparations, or kits comprise romidepsin and erlotinib.
  • the amount of one or both agents is lower than the amount that is typically administered when the agent is administered alone. In certain embodiments, the amount of both agents is lower.
  • the amount administered is sufficient to achieve nanomolar levels in the bloodstream of the subject. In certain embodiments, the amount administered is sufficient to achieve nanomolar concentrations at the site of the cancer or other neoplasm in the subject.
  • the dosing of each of romidepsin and erlotinib is described in more detail herein.
  • the agents act synergistically to kill cancer cells. In other embodiments, the agents act additively to kill cancer cells.
  • the inventive pharmaceutical compositions, preparations, or kits may include other therapeutic agents.
  • the other pharmaceutical agent may be any other therapeutic agent that would be useful to administer to the subject.
  • the other therapeutic agent preferably does not interact adversely with romidepsin or the tyrosine kinase inhibitor being administered
  • the invention provides for the administration of romidepsin and a tyrosine kinase inhibitor in combination with one or more other therapeutic agents, e.g., another cytotoxic agent, analgesic, etc.
  • the other therapeutic agent is another chemotherapeutic agent.
  • the other therapeutic agent may include an agent for alleviating or reducing the side effects of romidepsin and/or the tyrosine kinase inhibitor.
  • the other therapeutic agent is an anti-inflammatory agent such as aspirin, ibuprofen, acetaminophen, etc., pain reliever, anti-nausea medication, or anti-pyretic.
  • the other therapeutic agent is an agent to treat gastrointestinal disturbances such as nausea, vomiting, stomach upset, and diarrhea.
  • additional agents may include anti-emetics, anti-diarrheals, fluid replacement, electrolyte replacement, etc.
  • the other therapeutic agent is an electrolyte replacement or supplementation such as potassium, magnesium, and calcium, in particular, potassium and magnesium.
  • the other therapeutic agent is an anti-arrhythmic agent.
  • the other therapeutic agent is a platelet booster, for example, an agent that increases the production and/or release of platelets.
  • the other therapeutic agent is an agent to boost the production of blood cells such as erythropoietin.
  • the other therapeutic agent is an agent to prevent hyperglycemia.
  • the other therapeutic agent is an immune system stimulator.
  • the invention does not include the administration of another HDAC inhibitor besides romidepsin.
  • a pharmaceutically acceptable form includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, protected forms, stereoisomers, isomers, reduced forms, oxidized forms, tautomers, or any other adduct or derivative which upon administration to a patient in need is capable of providing, directly or indirectly, an agent as otherwise described herein, or a metabolite or residue thereof, e.g., a prodrug.
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19, 1977; incorporated herein by reference.
  • the salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base functionality with a suitable organic or inorganic acid.
  • Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid
  • organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate
  • alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate.
  • ester refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof.
  • Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms.
  • esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates.
  • the esters are cleaved by enzymes such as esterases.
  • pharmaceutically acceptable prodrugs refers to those prodrugs of the compounds utilized in accordance with the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use.
  • 'prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
  • the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier or excipient, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, permeation enhancers, solubilizing agents, and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable carrier or excipient includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, permeation enhancers, solubilizing agents, and the like, as suited to the particular dosage form desired.
  • Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975
  • any conventional carrier medium is incompatible with the anti-cancer compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.
  • materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; Cremophor (polyethoxylated caster oil); Solutol (poly-oxyethylene esters of 12-hydroxystearic acid); excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; algin
  • the pharmaceutically acceptable carrier is selected from the group consisting of sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminun hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions; non-toxic compatible lubricants such as
  • compositions of this invention may be administered can be administered by any appropriate means including, for example, orally, parenterally, by inhalation spray, topically, rectally. nasally, buccally, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • pharmaceutical compositions are administered orally or by injection in accordance with the present invention.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • liquid dosage forms of pharmaceutical compositions may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can also include adj
  • Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • a sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U. S. P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid are used in the preparation of injectables.
  • Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • the rate of drug release can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
  • compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the active agents with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules.
  • the active agent(s) is/are typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting
  • the dosage form may also comprise buffering agents, permeation enhancers, and/or other agents to enhance absorption of the active agent(s).
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. [00131] In certain embodiments, oral dosage forms are prepared with coatings or by other means to control release of active agent (e.g., DAC inhibitor and/or tyrosine kinase inhibitor) over time and/or location within the gastrointestinal tract. A variety of strategies to achieve such controlled (or extended) release are well known in the art, and are within the scope of the present invention.
  • active agent e.g., DAC inhibitor and/or tyrosine kinase inhibitor
  • Dosage forms for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • preparations are prepared by admixing active agent(s) under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulation ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
  • Ointments, pastes, creams and gels may contain, in addition to active agent(s), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to active agent(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • Transdermal patches have often can provide controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • active agent(s) is/are formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system.
  • Solid or liquid particulate forms of the active agent(s) prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, particularly aerosolized antibiotics, is known in the art (see. for example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat.
  • compositions for use in accordance with the present invention can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneal ⁇ , intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, for example with a dosage ranging from about 0.1 to about 500 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug.
  • compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
  • any particular active agent that may be combined with pharmaceutically acceptable excipients or carriers to produce a single dosage form may vary depending upon the host treated and the particular mode of administration.
  • a typical preparation will contain from about 5% to about 95% active compound (w/w).
  • such preparations may contain from about 20% to about 80% active compound.
  • preparations may commonly contain about 20-50%, 25-45%, 30-40%, or approximately 32%, 33%, 34%, or 35% active compound.
  • the dosage or frequency of administration, or both may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.
  • each agent is present at dosage levels of between about 1 to 100%, for example about 5 to 95%, of the level normally administered in a monotherapy regimen.
  • NSCLCs non-small cell lung cancers
  • TKIs EGFR tyrosine kinase inhibitors
  • HDAC histone deacetylase
  • NSCLC cell lines were treated with erlotinib alone or in combination with romidepsin. MTS assays were performed to determine the ICs 0 of erlotinib in these cell lines 72 hours after drug addition.
  • NCI-H 1299 xenografts were inoculated subcutaneously into nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured.
  • romidepsin increased the sensitivity of erlotinib in 13 of the 16 NSCLC cell lines, especially in EGFR and KRAS wild type NSCLC cells.
  • the epidermal growth factor receptor belongs to the EGFR family of tyrosine kinase receptors including EGFR, HER2/neu (ERRB2), HER3 (ERRB3) and HER4 (ERRB4) (1 ).
  • EGFR can promote cell proliferation and survival through Ras/MEK/MAPK and/or PI3K/AKT signaling pathways (2).
  • NSCLCs non-small cell lung cancers
  • TKIs EGFR tyrosine kinase inhibitors
  • TARCEVA erlotinib
  • IRESSA gefitinib
  • HATs histone acetyltransferases
  • HDACs histone deacetylases
  • Many nonhistone proteins can be HDAC substrates such as p53, cMyc, Stat3, and Hsp90 (9).
  • HDAC inhibitors can induce growth arrest, differentiation, and apoptosis in tumor cells (10).
  • Romidepsin (previously termed FK228, depsipeptide) is a cyclic peptide HDAC inhibitor that has shown important inhibition of in vitro growth of several tumor cell types including lung and prostate cancers, lymphomas and leukemias (12-14). Romidepsin is now in a phase II clinical trial for treatment to cutaneous T cell lymphoma (15).
  • NSCLC cell lines 16 NSCLC cell lines (see table below) were cultured in RPMl 1640 (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum and incubated in humidified air and 5% CO 2 at 37 °C. The cell lines were established in our lab. The NSCLC lines were all DNA fingerprinted and free of mycoplasma by molecular tests.
  • Romidepsin was provided by Fujisawa Pharmaceutical Co. (Japan).
  • Erlotinib (TARCEVA) was purchased from OSl Pharmaceuticals, Inc. (NY).
  • OSl Pharmaceuticals, Inc. NY
  • romidepsin was dissolved in ethanol and erlotinib was dissolved in DSMO.
  • the mutation status of NSCLC cell lines were determined as described in Materials and Methods.
  • the IC50 of erlotinib was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves.
  • Bold values indicate sensitivity to erlotinib (see text).
  • SQ squamous carcinoma
  • AD adenocarcinoma
  • LC large cell
  • wt wild type
  • mut mutant.
  • DNA isolation and PCR DNA was isolated from above NSCLC cell lines and polymerase chain reaction (PCR) was performed to determine the KRAS and EGFR mutation status of each cell line according to previously reported methods (16).
  • MTS assays Drug sensitivity was measured by a 3-(4, 5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, Wl). 50 ⁇ l of an exponentially growing cell suspension (cell numbers vary from 500 to 3000 /ml) was seeded into a 96-well microtiter plate, and 50 ⁇ l of various concentrations of each drug was added. After incubation for 72 hr at 37 °C, 20 ⁇ l of MTS solution was added to each well, and the plates were incubated for one hour at 37 °C.
  • Optical density was measured at 562 and 630 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnywale, CA). Each experiment was carried out in 8 replicate wells for each drug concentration. The IC 5 Q-value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves.
  • Apoptosis assays was preformed using cell death detection ELISA (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. For Hoechst staining, cells were fixed with cold methanol for 2 minutes, stained with DNA dye Hoechst 33258 for 10 minutes, washed with Ix PBS for 10 minutes, and observed by microscopy.
  • p21 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • Bim, phosphorylated or total p42/p44 antibody, and phosphorylated or total AKT antibody were purchased from Cell signaling Technology (Danvers, MA).
  • Actin antibody was purchased from Sigma (St. Louis, MO).
  • Romidepsin decreases the IC 5 0 of erlotinib in NSCLC cell lines.
  • NSCLC cell lines Sixteen NSCLC cell lines were used including fourteen EGFR wild type cell lines, and two EGFR mutant cell lines known to be resistant to erlotinib. Among EGFR wild type cell lines, seven cell lines contained KRAS mutations.
  • the IC 50 values of romidepsin in the NSCLC cell lines examined ranged from 1.2 to 3.5 ng/ml. We used romidepsin at a concentration of 1 ng/ml for the combined treatment. At this concentration, romidepsin alone only showed low cytotoxic effect.
  • the NSCLC cell lines were treated with varied concentrations of erlotinib either in the absence or presence of 1 ng/ml romidepsin. MTS assays were performed to determine the cell viability and four representative pairs of cell viability curves are presented in Figure 6.
  • the IC 50 value of erlotinib for each cell line was calculated based on the curves and is showed in the table above.
  • the fold decreases in sensitivity ranged from 1.9 to 164, with a median of 13.
  • NSCLC cell lines had different histologies including adenocarcinoma, squamous carcinoma, and large cell types. Furthermore, in five of the seven EGFR and KRAS wild type NSCLC cell lines, the IC50 values of erlotinib were decreased to less than 2.5 ⁇ M, which is the threshold to distinguish sensitive from resistant cell lines (17). It is known that KRAS mutant NSCLC cell lines are resistant to TKl inhibitors (18). Our data indicated that romidepsin could also sensitize KRAS mutant cell lines in response to erlotinib. However, only one of the seven KRAS mutant cell lines reached the sensitive threshold of less than 2.5 ⁇ M in the presence of 1 ng/ml romidepsin.
  • phosphorylated levels of ERK 1/2 and AKT protein decreased upon romidepsin treatment alone or with erlotinib, indicating that MAPK and AKT pathways were down-regulated by romidepsin.
  • the expression of the cyclin kinase inhibitor p2l dramatically increased after romidepsin alone or in combination while only modest changes Bim expression were seen.
  • EGFR TKIs such as erlotinib and gefitinib have been found to be efficient in the treatment of NSCLCs that express mutant EGFR (3, 4). While ethnic and geographic differences exist, about 20% NSCLCs contain mutant EGFR (19). The majority of NSCLCs containing wild type EGFR are resistant to EGFR TKIs (17). Several new TKIs have been developed to treat NSCLCs that have a broader spectrum kinase activity.
  • EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, has shown efficiency in therapy of both wild-type and mutant EGFR in vitro and in vivo (20).
  • combinational therapies have been used to overcome NSCLC resistance to EGFR TKIs.
  • erlotinib and the humanized vascular endothelial growth factor receptor monoclonal antibody bevacizumab in advanced, chemotherapy- refractory NSCLCs has shown promising results (21).
  • HDAC inhibitors have pleiotropic effects on gene expression, growth arrest and apoptosis, and therefore are excellent candidates for combination therapies (15). Synergistic or additive activity has been reported between HDAC inhibitors and TKIs. HDAC inhibitor MS 125 was shown to increase the sensitivity of gefitinib in NSCLCs (22).
  • IC 50 values less than 2.5 ⁇ M are considered to be sensitive to erlotinib in vitro (17), which is approximately corresponding to the plasma steady-state concentration of erlotinib in patients treated with a dose of 150 mg daily (24).
  • the IC 50 values of five NSCLC cell lines containing wild type EGFR decreased to around 1 ⁇ M and therefore are regarded as sensitive to erlotinib.
  • EMT epithelial to mesenchymal transition
  • mesenchymal markers such as vimentin or fibronectin
  • romidepsin increased the expression of the cyclin dependent kinase inhibitor p21.
  • Bim belongs to the BH3-only group of protein, can bind Bcl2 and inhibit its function (28). Induction of important players in the apoptosis pathway or cell cycle control by romidepsin may also be mechanisms accounting for the synergy.
  • DAC inhbitors e.g., HDAC inihibtors
  • tyrosine kinase inhibitors may have clinical use in the treatment of non-small cell lung cancers, especially in treatment of EGFR wild type tumors.
  • FR901228 a novel antitumor bicyclic depsipeptide produced by Chromobacterium violaceum no. 968, on Ha-ras transformed NIH3T3 cells. Biosci Biotechnol Biochem, 58:
  • Giaccone, G Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells.
  • Claims or descriptions that include "or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
  • compositions made according to any of the methods for preparing compositions disclosed herein.
  • any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims.
  • Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention can be excluded from any one or more claims.
  • the biologically active agent is not an anti-proliferative agent.
  • all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Abstract

The present invention provides compositions and methods for the treatment of cell proliferative disorders using at least one DAC inhibitor and at least one tyrosine kinase inhibitor. In particular, the invention provides a combination therapy for treating a proliferative disorder (e.g., cancer) including romidepsin and a tyrosine kinase inhibitor. When administered together, romidepsin and a tyrosine kinase inhibitor (e.g., erlotinib) interact to induce apoptosis. The effect is particularly pronounced in wild type non-small cell lung cancers, particularly wild type EGFR and wild type KRAS non-small cell lung cancer cells. The invention provides methods of killing malignant cells in vitro and in vivo. Pharmaceutical compositions, preparations, and kits including romidepsin and a tyrosine kinase inhibitor are also provided.

Description

COMBINATION THERAPY FOR CANCER
Related Applications
[0001] The present application claims priority under 35 U. S. C. § 1 19(e) to U.S. provisional application, USSN 60/923,249, filed April 13, 2007, which is incorporated herein by reference.
Background of the Invention
[0002] Romidepsin is a natural product which was isolated from Chromobacterium violaceum by Fujisawa Pharmaceuticals. See Published Japanese Patent Application Hei 7 (1995)-64872; U.S. Patent 4,977,138, issued December 1 1 , 1990, which is incorporated herein by reference. It is a bicyclic peptide consisting of four amino acid residues (D-valine, D-cysteine, dehydrobutyrine, and L-valine) and a novel acid (3-hydroxy-7-mercapto-4- heptenoic acid). Romidepsin is a depsipeptide which contains both amide and ester bonds. In addition to fermentation from C. violaceum, romidepsin can also be prepared by synthetic or semi-synthetic means. The total synthesis of romidepsin reported by Kahn et al. involves 14 steps and yields romidepsin in 18% overall yield. J. Am. Chem. Soc. 1 18:7237-7238, 1996. The structure of romidepsin is shown below:
Figure imgf000002_0001
Romidepsin has been shown to have anti-microbial, immunosuppressive, and anti-tumor activities. It is thought to act by selectively inhibiting deacetylases (e.g., histone deacetylase (HDAC), tubulin deacetylase (TDAC)), promising new targets for the development of anticancer therapies. Nakaj ima et al. , Experimental Cell Res. 241 : 126- 133, 1998. One mode of action is thought to involve the inhibition of one or more classes of histone deacetylases (HDAC). [0003] Histone deacetylase is a metallodeacetylation enzyme having zinc in its active site. Finnin et ai, Nature, 401 : 188-193, 1999. This enzyme is thought to regulate gene expression by enhancing the acetylation of histones, thereby inducing chromatin relaxation and generally, but not universally, transcriptional activation. Although these enzymes are known as HDACs, they have also been implicated in various other cellular processes. For example, HDAC inhibitors have been found to trigger apoptosis in tumor cells through diverse mechanisms, including the up-regulation of death receptors, Bid cleavage, ROS generation, Hsp90 dysregulation, and ceramide generation, among others. Several HDAC inhibitors have entered the clinical arena and are demonstrating activity in both hematologic and non-hematologic malignancies. Romidepsin has shown impressive activity in certain hematologic malignancies, particularly T-cell lymphoma (Piekarz et al. "A review of depsipeptide and other histone deacetylase inhibitors in clinical trials" Curr. Pharm. Des. 10:2289-98, 2004; incorporated herein be reference).
[0004] In addition to romidepsin, various derivatives have been prepared and studied. The following patents and patent applications describe various derivatives of romidepsin: U.S. Patent 6,548,479; WO 05/0209134; WO 05/058298; and WO 06/129105; each of which is incorporated herein by reference.
[0005] Erlotinib (TARCEV A®) is a tyrosine kinase inhibitor. It is thought to act by inhibiting the intracellular phosphorylation of the tyrosine kinase associated with epidermal growth factor receptor. Erlotinib is indicated for use in the treatment of patients with non- small cell lung cancer and pancreatic cancer.
[0006] Lung cancer is one of the leading causes of cancer-related deaths worldwide. There are 1.6 million cases of lung cancers every year in the world, and 1.1 million people will die from their disease. Non-small cell lung cancer accounts for approximately 85% of lung cancer cases. The EGFR signaling pathway is deregualted in over 50% of non-small cell lung cancers. There is a desperate need for the development of effective therapies for the treatment of patients with lung cancer, particularly non-small cell lung cancer (NSCLC).
Summary of the Invention
[0007] The present invention encompasses the finding that combinations of deacetylase (DAC) inhibitors with tyrosine kinase inhibitors have particular utility in the treatment of proliferative diseases. Among other things, the invention establishes the particular utility of DAC inhibitor/tyrosine kinase inhibitor combination therapy in treatment of lung cancer, and particularly in the treatment of non-small cell lung cancer (NSCLC), particularly wild type EGFR and KRAS NSCLCs. In certain particular embodiments, the DAC inhibitor is romidepsin. In certain particular embodiments, the tyrosine kinase inhibitor is erlotinib (TARCEVA ;. Combination therapy with romidepsin and erlotinib is provided, for example for use in the treatment of proliferative disorders (e.g., cancer and other neoplasms) generally. In certain embodiments, the combination of romidepsin and erlotinib is used in the treatment of lung cancer, particularly non-small cell lung cancer.
[0008] The present invention provides methods of treating a proliferative disorder by administering a combination of one or more DAC inhibitors and one or more tyrosine kinase inhibitors. In one aspect, the invention provides a method of treating cancer in a subject (e.g., human) by administering therapeutically effective amounts of romidpesin and a tyrosine kinase inhibitor to the subject. In certain embodiments, the combination includes romidepsin and erlotinib. Both of these agents have been used in the clinic to treat human subjects with cancer. In certain embodiments, the romidpesin and erlotinib may be used in combination at dosages lower than when each is used individually. In other embodiments, the additive nature of the combination is particularly useful in treating cancer or other neoplasms. In certain embodiments, the romidpesin is administered at a dosage of 0.5 mg/m2 to 15 mg/m2, and erlotinib is administered at a dosage of approximately 25 mg/day to approximately 200 mg/day. The two drugs may be administered together, or one after another. The method is particular useful in treating lung cancer (e.g., non-small cell lung cancer). In certain embodiments, the romidpesin and a tyrosine kinase inhibitor are administered in conjunction with another anti-neoplastic agent. In certain embodiments, the romidepsin is administered intravenously, and the erlotinib is administered orally. In certain embodiments, each of the romidepsin and the tyrosine kinase inhibitor is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals. In certain embodiments, the romidepsin is administered weekly, and the tyrosine kinase inhibitor is administered daily. [0009] The present invention further provides methods of treating lung cancer (e.g., non- small cell lung cancer) by administering a DAC inhibitor together with a tyrosine kinase inhibitor. In certain embodiments, the present invention provides a method of treating non- small cell lung cancer in a subject (e.g., human) by administering a therapeutically effective amount of romidepsin and erlotinib to a subject with non-small cell lung cancer. In certain embodiments, the therapeutically effective amount of romidepsin ranges from 4 mg/m2 to 15 mg/m2 or from 8 mg/m2 to 10 mg/m2. In certain embodiments, the therapeutically effective amount of erlotinib (TARCEVA®) ranges from approximately 25 mg to 200 mg. In certain embodiments, therapeutically effective amount of erlotinib is approximately 100 mg. In certain embodiments, therapeutically effective amount of erlotinib is approximately 150 mg. In certain embodiments, the therapeutically effective amount of romidepsin ranges from 8 mg/m2 to 10 mg/m2, and the therapeutically effective amount of erlotinib (TARCEVA®) is approximately 150 mg per day. In certain embodiments, the romidepsin is administered weekly, and the erlotinib is administered daily.
[0010] In another aspect, the invention provides methods of treating cells in vitro by contacting cells with a combination of romidepsin and a tyrosine kinase inhibitor such as erlotinib. The cells may be treated with a sufficient concentration of the combination to kill the treated cells. In certain embodiments, a sufficient concentration of the combination is used to induce apoptosis as evidenced by changes in levels of cellular markers of apoptosis. [0011] In certain embodiments, the cells are neoplastic cells. The cells may be from human cancers or derived from cancer cell lines (e.g., lung cancer, non-small cell lung cancer). In certain embodiments, the cells are lung cancer cells, in particular non-small cell lung cancer cells. The cells may be at any stage of differentiation or development. The methods are particularly useful for assessing the cytotoxicity of a given combination under certain conditions (e.g., concentration of each agent, combination with other pharmaceutical agents). The inventive methods may be used to ascertain the susceptibility of a subject's cancer or neoplasm to the combination therapy. The inventive methods using the inventive combinations may be for clinical or research purposes.
[0012] The present invention provides combination regimens, and unit dosages of pharmaceutical compositions useful in such regimens. In certain embodiments, pharmaceutical compositions or preparations comprising romidepsin and a tyrosine kinase inhibitor are provided. In certain particular embodiments, the composition or preparation comprises romidepsin and erlotinib. The pharmaceutical composition includes a therapeutically effective amount of each pharmaceutical agent for the treatment of cancer (e.g.. lung cancer, non-small cell lung cancer). The pharmaceutical composition may include other cytotoxic agents or other anti-neoplastic agents. The pharmaceutical composition may also include other agents to alleviate pain, nausea, hair loss, weight loss, weight gain, neuropathy, cardiac arrhythmias, electrolyte deficiencies or imbalances, anemia, thrombocytopenia, immunosuppression, skin conditions, or other conditions associated with cancer or the treatment of cancer. [0013] The present invention further provides kits for combination therapy of DAC inhibitors and tyrosine kinase inhibitors. The invention provides kits including the inventive pharmaceutical compositions in a convenient dosage form. The agents may be packaged together or separately in the kit. The kit may include multiple doses of each agent. In certain embodiments, the kits include a sufficient amount of each agent for a full course of chemotherapy in the treatment of a subject's cancer. The kit may also include excipients or devices for use in administering the inventive combination. The kit may also include instructions for administering the inventive combination.
Brief Description of the Drawing
[0014] Figures 1-3 depict structures of certain DAC inhibitors that, like other DAC inhibitors available in the art and/or described herein, may be utilized in some embodiments of the present invention.
[0015] Figure 4 shows materials and methods utilized in a study that demonstrates the effectiveness of combination therapy with a DAC inhibitor (romidepsin) and a tyrosine kinase inhibitor (erlotinib) on non-small cell lung cancer cells.
[0016] Figure 5 illustrates the IC50 for romidepsin in non-small cell lung cancer cell lines. [0017] Figure 6 shows enhanced sensitivity of non-small cell lung cancer cell lines (HCC 193 (EGFR, KRAS wt); NCl-H 1299 (EGFR, KRAS wt); NCI-H 157 (KRAS mutant); NCI-H 1975 (EGFR mutant) to a combination of romidepsin and erlotinib. NSCLC cells lines were treated with various concentrations of erlotinib either in the absence or presence of romidepsin (1 ng/mL) for 72 hours. Cell viability was measured by MTS assay, and four representative pairs of cell viability curves are shown.
[0018] Figure 7 demonstrates that the combination of romidepsin and erlotinib induced apoptosis in a HCC 15 NSCLC cell line. Figure 7 A. Nuclei of HCC 15 cells treated with 5 μM erlotinib and/or 2 ng/mL romidepsin were stained with DNA dye Hoechst 33258 and examined by microscopy. Figure 7 B. HCC 15 cells treated with 5 μM erlotinib and/or 2 ng/mL romidepsin were analyzed for apoptosis using cell death detection kit (Roche). *, P < 0.05 vs. control; **, P < 0.01 vs. control (Welch nest).
[0019] Figure 8 shows that co-treatment with romidepsin and erlotinib gives greater growth inhibition of NCI-H 1299 cell xenografts than does treatment with either agent alone. 5 x 106 NCI-H 1299 cells were injected sub-cutaneously into each of twenty BALB/c athymic nude mice. These mice were divided into four groups at day 7 after tumor development. They were injected with either Ix PBS, romidepsin alone, erlotinib alone, or the combination of romidepsin and erlotinib. Romidepsin was administered 3 times at 4-day intervals (1.2 mg/kg body weight). Erlotinib was administered five days a week (50 mg/kg body weight).
Tumor sizes were measured at the indicated days. Results are shown as mean + SEM of groups of five mice. Statistical significance was determined by Welch t test (*, P < 0.05 vs. control).
[0020] Figure 9 demonstrates that romidepsin's ability to inhibit MAPK correlates with its ability to increase efficacy of erlotinib.
[0021] Figure 10. Romidepsin down-regulated MAPK (ERK 1/2) and AKT pathways, increased p21 and Bim expression in NSCLC cell lines. HCC15, HC193, and NCI-H1299 cell lines were treated with erlotinib (5 μM) or romidepsin (1 ng/mL) or in combination for
24 hours. Protein lysates were prepared and subjected to western blot analysis. Antibodies against phosphorylated or total ERK1/ERK2, phosphorylated or total AKT, p21, and Bim were used as indicated. Beta-actin was used as a loading control.
[0022] Figure 11 shows the cytotoxic effect of romidepsin (1 ng/mL) on NSCLC cells lines. Sixteen NSCLC cell lines were either in the presence or absence of romidepsin (1 ng/mL) for 72 hours. MTS assays were performed to determine cell viability.
Definitions
[0023] Definitions of other terms used throughout the specification include:
[0024] As used herein and in the appended claims, the singular forms "a", "an", and "the" include the plural reference unless the context clearly indicates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells.
[0025] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to a human, at any stage of development.
In some embodiments, "animal" refers to a non-human animal, at any stage of development.
In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and/or worms. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or clone.
[0026] Alicyclic: The term "alicyclic," as used herein, denotes a monovalent group derived from a monocyclic or bicyclic saturated carbocyclic ring compound by the removal of a single hydrogen atom. Examples include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo [2.2.1] heptyl, and bicyclo [2.2.2] octyl. Such alicyclic groups may be further substituted.
[0027] Aliphatic: An "aliphatic group" is non-aromatic moiety that may contain any combination of carbon atoms, hydrogen atoms, halogen atoms, oxygen, nitrogen or other atoms, and optionally contain one or more units of unsaturation, e.g., double and/or triple bonds. An aliphatic group may be straight chained, branched or cyclic and preferably contains between about 1 and about 24 carbon atoms, more typically between about 1 and about 12 carbon atoms. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, such as polyalkylene glycols, polyamines, and polyimines, for example. Such aliphatic groups may be further substituted.
[0028] Aryl: The term "aryl," as used herein, refers to a mono- or polycyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group. Aromatic groups can be substituted or unsubstituted. [0029] Cell Proliferative Disorder, Disease, or Condition: The term "cell proliferative disease or condition" is meant to refer to any condition characterized by aberrant cell growth, preferably abnormally increased cellular proliferation.
[0030] Combination Therapy: According to the present invention, a DAC inhibitor may desirably be administered in combination with one or more other therapeutic agents, such as, for example, a tyrosine kinase inhibitor. Such therapy will commonly involve administration of multiple individual doses of a DAC inhibitor and/or of other agent (e.g., a tyrosine kinase inhibitor), spaced out over time. Doses of a DAC inhibitor and other agent may be administered in the same amounts and/or according to the same schedule or alternatively may be administered in different amounts and/or according to different schedules. [0031] DAC Inhibitor: In general, any agent that specifically inhibits a deacetylase is considered to be a DAC inhibitor. Any agent that specifically inhibits a histone deacetylase is considered to be an HDAC inhibitor. Those of ordinary skill in the art will appreciate that, unless otherwise set forth herein or known in the art, DAC inhibitors may be administered in any form such as, for example, salts, esters, prodrugs, metabolites, etc. Furthermore, DAC inhibitors that contain chiral centers may be administered as single stereoisomers or as mixtures, including racemic mixtures, so long as the single stereoisomer or mixture has DAC inhibitor activity.
[0032] DAC Inhibitor Therapy: As used herein, the phrase "DAC inhibitor therapy" refers to the regimen by which a DAC inhibitor is administered to an individual. Commonly, DAC inhibitor therapy will involve administration of multiple individual doses of a DAC inhibitor, spaced out over time. Such individual doses may be of different amounts or of the same amount. Furthermore, those of ordinary skill in the art will readily appreciate that different dosing regimens (e.g., number of doses, amount(s) of doses, spacing of doses) are typically employed with different DAC inhibitors.
[0033] Depsipeptide: The term "depsipeptide", as used herein, refers to polypeptides that contain both ester and amide bonds. Naturally occurring depsipeptides are usually cyclic. Some depsipeptides have been shown to have potent antibiotic activity. Examples of depsipeptides include actinomycin, enniatins, valinomycin, and romidepsin. [0034] Effective amount: In general, the "effective amount" of an active agent or combination of agents refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of an inventive combination may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the agents being delivered, the disease being treated, the mode of administration, and the patient. For example, the effective amount of an inventive combination (e.g., romidepsin and erlotinib) is the amount that results in reducing the tumor burden, causing a remission, or curing the patient.
[0035] Electrolyte: In general, the term "electrolyte", as used herein, refers to physiologically relevant free ions. Representative such free ions include, but are not limited to sodium(Na+), potassium (K+), calcium (Ca2+), magnesium (Mg2+), chloride (Cl-), phosphate (PO43 ), and bicarbonate (HCO3 ').
[0036] Electrolyte Supplementation: The term "electrolyte supplementation", as used herein, refers to administration to a subject of a composition comprising one or more electrolytes in order to increase serum electrolyte levels in the subject. For purposes of the present invention, when electrolyte supplementation is administered "prior to, during, or after" other therapy (e.g., DAC inhibitor therapy and/or combination therapy), it may be administered prior to initiation of that therapy (i.e., prior to administration of any dose), or prior to, concurrently with, or after any particular dose or doses. [0037] Halogen: The term "halogen", as used herein, refers to an atom selected from fluorine, chlorine, bromine, and iodine.
[0038] Heteroaryl: The term "heteroaryl", as used herein, refers to a mono- or polycyclic (e.g. bi-, or tri-cyclic or more) aromatic radical or ring having from five to ten ring atoms of which one or more ring atom is selected from, for example, S, O and N; zero, one or two ring atoms are additional heteroatoms independently selected from, for example, S, O and N; and the remaining ring atoms are carbon, wherein any N or S contained within the ring may be optionally oxidized. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, and the like.
[0039] Heterocyclic: The term "heterocyclic", as used herein, refers to a non-aromatic 5- , 6- or 7-membered ring or a bi- or tri-cyclic group fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, (iv) any of the above rings may be fused to a benzene ring, and (v) the remaining ring atoms are carbon atoms which may be optionally oxo-substituted. Representative heterocycloalkyl groups include, but are not limited to, [l ,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, and tetrahydrofuryl. Such heterocyclic groups may be further substituted.
[0040] Initiation: As used herein, the term "initiation" when applied to therapy can refer to a first administration of an active agent (e.g., a DAC inhibitor, tyrosine kinase inhibitor, or combinations thereof) to a patient who has not previously received the agent. Alternatively or additionally, the term "initiation" can refer to administration of a particular dose of an agent (e.g., a DAC inhibitor, tyrosine kinase inhibitor, or combinations thereof) during therapy of a patient.
[0041] Peptide or protein: According to the present invention, a "peptide" or "protein" comprises a string of at least three amino acids linked together by peptide bonds. The terms "protein" and "peptide" may be used interchangeably. Peptides preferably contain only natural amino acids, although non-natural amino acids {i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain) and/or amino acid analogs as are known in the art may alternatively be employed. Also, one or more of the amino acids in a peptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. In certain embodiments, the modifications of the peptide lead to a more stable peptide {e.g., greater half-life in vivo). These modifications may include cyclization of the peptide, the incorporation of D-amino acids, etc. None of the modifications should substantially interfere with the desired biological activity of the peptide. In certain embodiments, peptide refers to depsipeptide.
[0042] Pharmaceutically acceptable carrier or excipient: As used herein, the term "pharmaceutically acceptable carrier or excipient" means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. [0043] Pharmaceutically acceptable ester: As used herein, the term "pharmaceutically acceptable ester" refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include, but are not limited to, formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
[0044] Pharmaceutically acceptable prodrug: The term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention. "Prodrug", as used herein means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of the invention. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al., (ed). "Design and Application of Prodrugs, Textbook of Drug Design and Development". Chapter 5, 1 13-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8: 1 -38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, "Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology," John Wiley and Sons, Ltd. (2002).
[0045] Pharmaceutically acceptable salt; As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1 -19 (1977). The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable include, but are not limited to, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include, but are not limited to, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and aryl sulfonate. [0046] Romidepsin: The term "romidepsin", refers to a natural product of the chemical structure:
Figure imgf000013_0001
Romidepsin is a deacetylase inhibitor and is also known in the art by the names FK228, FR901228, NSC630176, or depsipeptide. The identification and preparation of romidepsin is described in U.S. Patent 4,977,138, issued December 1 1 , 1990, which is incorporated herein by reference. The molecular formula is C24H36N4O6S2; and the molecular weight is 540.71 g/mol. Romidepsin has the chemical name, (l S,4S,10S,16E,21 R)-7-[(2Z)-ethylidene]-4,21 - diisopropyl-2-oxa- 12,13-dithia-5,8,20,23-tetraazabicyclo[8.7.6]tricos-16-ene-3,6,9, 19,22- pentanone. Romidepsin has been assigned the CAS number 128517-07-7. In crystalline form, romidepsin is typically a white to pale yellowish white crystal or crystalline powder. The term "romidepsin" encompasses this compound and any pharmaceutically forms thereof. In certain embodiments, the term "romidepsin" may also include salts, pro-drugs, esters, protected forms, reduced forms, oxidized forms, isomers, stereoisomers {e.g., enantiomers, diastereomers), tautomers, and derivatives thereof.
[0047] Stable: The term "stable", as used herein, refers to compounds which possess stability sufficient to allow manufacture and which maintains the integrity of the compound for a sufficient period of time to be useful for the purposes detailed herein (e.g., therapeutic or prophylactic administration to a subject). In general, combinations of substituents and variables envisioned by this invention are only those that result in the formation of stable compounds.
[0048] Substituted: The terms "substituted aryl", "substituted heteroaryl", or "substituted aliphatic," as used herein, refer to aryl, heteroaryl. aliphatic groups as previously defined, substituted by independent replacement of one, two, or three or more of the hydrogen atoms thereon with substituents including, but not limited to, -F, -Cl, -Br, -I, -OH, protected hydroxyl. -NO2, -CN, -C1-C12-alkyl optionally substituted with, for example, halogen, C2- C12-alkenyl optionally substituted with, for example, halogen, -C2-C12-alkynyl optionally substituted with, for example, halogen, -NH2, protected amino, -NH -C1-C12-alkyl, -NH -C2- C,2-alkenyl, -NH -C2-C12-alkenyl, -NH -C3-C12-cycloalkyl, -NH -aryl, -NH -heteroaryl, -NH -heterocycloalkyl, -dialkylamino, -diarylamino, -diheteroarylamino, -O-C1- C12-alkyl, -0-C2- C,2-alkenyl, -O-C2-C12-alkenyl, -O-C3-C12-cycloalkyl, -O-aryl, -O-heteroaryl, -O- heterocycloalkyl, -C(O)- C1-C,2-alkyl, -C(O)- C2-C12-alkenyl, -C(O)- C2- C12-alkenyl, -C(O)- C3-C12-cycloalkyl, -C(O)-aryl, -C(O)-heteroaryl, -C(O)-heterocycloalkyl, -CONH2, -CONH- C,-C12-alkyl, -CONH- C2-C12-alkenyl, -CONH- C2-C,2-alkenyl, -CONH-C3-C12-cycloalkyl, - CONH-aryl, -CONH-heteroaryl, -CONH-heterocycloalkyl, -OCO2- C1-C12-alkyl, -OCO2- C2-C12-alkenyl, -OCO2- C2-C12-alkenyl, -OCO2-C3-C)2-cycloalkyl, -OCO2-aryl, -OCO2- heteroaryl, -OCO2-heterocycloalkyl, -OCONH2, -OCONH- C-C2-alkyl, -OCONH- C2-C2- alkenyl, -OCONH- C2-C12-alkenyl, -OCONH- C3-C12-cycloalkyl, -OCONH- aryl, -OCONH- heteroaryl, -OCONH- heterocycloalkyl, -NHC(O)- C1-C12-alkyl, -NHC(O)-C2- C12-alkenyl, - NHC(O)-C2-C, 2-alkenyl, -NHC(O)-C3-C, 2-cycloalkyl, -NHC(O)-aryl, -NHC(O)-heteroaryl, - NHC(O)-heterocycloalkyl, -NHCO2- C,-C!2-alkyl, -NHCO2- C2-C,2-alkenyl, -NHCO2- C2- C]2-alkenyl, -NHCO2- C3-C,2-cycloalkyl, -NHCO2- aryl, -NHCO2- heteroaryl, -NHCO2- heterocycloalkyl, -NHC(O)NH2, -NHC(O)NH- C,-C,2-alkyl, -NHC(O)NH-C2-C,2-alkenyl, - NHC(O)NH-C2-C12-alkenyl, -NHC(O)NH-C3-C,2-cycloalkyl, -NHC(O)NH-aryl, - NHC(O)NH-heteroaryl, -NHC(O)NH-heterocycloalkyl, NHC(S)NH2, -NHC(S)NH- C-C2- alkyl, -NHC(S)NH-C2-C, 2-alkenyl, -NHC(S)NH-C2-C, 2-alkenyl, -NHC(S)NH-C3-C12- cycloalkyl, -NHC(S)NH-aryl, -NHC(S)NH-heteroaryl, -NHC(S)NH-heterocycloalkyl, - NHC(NH)NH2, -NHC(NH)NH- C,-C,2-alkyl, -NHC(NH)NH-C2-C12-alkenyl, - NHC(NH)NH-C2-C, 2-alkenyl, -NHC(NH)NH-C3-C12-cycloalkyl, -NHC(NH)NH-aryl, - NHC(NH)NH-heteroaryl, -NHC(NH)NH-heterocycloalkyl, -NHC(NH)-C,-C12-alkyl, - NHC(NH)-C2-C,2-alkenyl, -NHC(NH)-C2-C, 2-alkenyl, -NHC(NH)-C3-C 12-cycloalkyl, - NHC(NH)-aryl, -NHC(NH)-heteroaryl, -NHC(NH)-heterocycloalkyl, -C(NH)NH-C1-C12- alkyl, -C(NH)NH-C2-C12-alkenyl, -C(NH)NH-C2-C,2-alkenyl, -C(NH)NH-C3-C12-cycloalkyl, -C(NH)NH-aryl, -C(NH)NH-heteroaryl, -C(NH)NH-heterocycloalkyl, -S(O)-C1 -C, 2-alkyl, - S(O)-C2-C,2-alkenyl, - S(O)-C2-C,2-alkenyl, - S(O)-C3-C,2-cycloalkyl, - S(O)-aryl, - S(O)- heteroaryl, - S(O)-heterocycloalkyl -SO2NH2, -SO2NH- C,-C12-alkyl, -SO2NH- C2-C2- alkenyl, -SO2NH- C2-C12-alkenyl, -SO2NH- C3-C,2-cycloalkyl, -SO2NH- aryl, -SO2NH- heteroaryl, -SO2NH- heterocycloalkyl, -NHSOrC-Cn-alkyl, -NHSO2-C2-C12-alkenyl, - NHSO2-C2-C, 2-alkenyl, -NHSO2-C3-C12-cycloalkyl, -NHSO2-aryl, -NHSO2-heteroaryl, - NHSCh-heterocycloalkyl, -CH2NH2, -CH2SO2CH3, -aryl, -arylalkyl, -heteroaryl, - heteroarylalkyl, -heterocycloalkyl, -C3-C12-cycloalkyl, polyalkoxyalkyl, polyalkoxy, - methoxymethoxy, -methoxyethoxy, -SH, -S-C1-C12-alkyl, -S-C2-C12-alkenyl, -S-C2-C12- alkenyl, -S-C3-C i2-cycloalkyl, -S-aryl, -S-heteroaryl, -S-heterocycloalkyl, or methylthiomethyl. It is understood that the aryls, heteroaryls, alkyls, and the like can be further substituted.
[0049] Susceptible to: The term "susceptible to", as used herein refers to an individual having higher risk (typically based on genetic predisposition, environmental factors, personal history, or combinations thereof) of developing a particular disease or disorder, or symptoms thereof, than is observed in the general population.
[0050] Therapeutically effective amount: The term "therapeutically effective amount" of an active agent or combination of agents is intended to refer to an amount of agent(s) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of a particular agent may range from about 0.1 mg/Kg to about 500 mg/Kg, preferably from about 1 to about 50 mg/Kg. Effective doses may also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of any particular active agent utilized in accordance with the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.
[0051] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
[0052] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a biologically active agent that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of and/or reduces incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
Detailed Description of Certain Embodiments of the Invention [0053] The present invention demonstrates, among other things, that combinations of DAC inhibitors (e.g., romidepsin) and tyrosine kinase inhibitors (e.g., erlotinib) are particularly useful in the treatment of proliferative disorders (e.g., cancers such as lung cancer, including non-small cell lung cancer). In particular, the present invention provides a novel system for treating proliferative diseases by administering a combination of romidepsin and a tyrosine kinase inhibitor. The combination of these agents may lead to an additive or synergistic effect. The combination has been found to be particularly effective in treating cancers that have wild type EGFR and wild type KRAS. In certain embodiments, a synergistic interaction between romidepsin and tyrosine kinase inhibitors in the treatment of cancer or other neoplasms has been demonstrated as described herein. See Figures 6-11. This synergistic effect is particularly pronounced in the case of lung cancer cells, particularly non-small cell lung cancer cells. Without wishing to be bound by any particular theory, the effect may be due to the induction of apoptosis by the combination of agents. [0054] In certain embodiments, the combination of romidepsin and erlotinib has been found to be useful in treating lung cancer. The inventive combination is particularly useful in treating non-small cell lung caner. The inventive combination is useful in treating non-small cell lung caners that are wild type EGFR and wild type KRAS. The inventive combination may also be effective in treating KRAS mutant cell lines.
[0055] Based on these discoveries, the invention provides methods of treating cells with the inventive combinations both in vitro and in vivo. The invention also provides pharmaceutical compositions and kits comprising the inventive combinations. In certain particular embodiments, the inventive combination comprises romidepsin and erlotinib. Deacetylase Inhibitors
[0056] Deacetylase inhibitors, as that term is used herein are compounds which are capable of inhibiting the deacetylation of proteins in vivo, in vitro, or both. In many embodiments, the invention relates to HDAC inhibitors, which inhibit the deacetylation of histones. In certain embodiments, the invention relates to TDAC inhibitors, which inhibit the deacetylation of tubulin. However, those of ordinary skill in the art will appreciate that DAC inhibitors often have a variety of biological activities, at least some of which may well be independent of histone deacetylase inhibition.
[0057] As indicated, DAC inhibitors inhibit the activity of at least one deacetylase. Where the DAC inhibitor is an HDAC inhibitor, an increase in acetylated histones occurs and accumulation of acetylated histones is a suitable biological marker for assessing the activity of HDAC inhibitors. Therefore, procedures which can assay for the accumulation of acetylated histones can be used to determine the HDAC inhibitory activity of agents of interest. Analogous assays can determine DAC inhibitory activity
[0058] It is understood that agents which can inhibit deacetylase activity (e.g., histone deacetylase activity) typically can also bind to other substrates and as often can inhibit or otherwise regulate other biologically active molecules such as enzymes. [0059] Suitable DAC inhibitors according to the present invention include, for example, 1 ) hydroxamic acid derivatives; 2) short-chain fatty acids (SCFAs); 3) cyclic tetrapeptides; 4) benzamides; 5) electrophilic ketones; and/or any other class of compounds capable of inhibiting deacetylase activity. Examples of such DAC inhibitors include, but are not limited to:
A) HYDROXAMIC ACID DERIVATIVES such as Suberoylanilide Hydroxamic Acid (SAHA) (Richon et al., Proc. Natl. Acad. Sci. USA 95:3003, 1998); M-Carboxycinnamic Acid Bishydroxamide (CBHA) (Richon et al., supra); pyroxamide; CBHA; Trichostatin analogues such as Trichostatin A (TSA) and Trichostatin C (Koghe et al. Biochem. Pharmacol. 56: 1359, 1998); Salicylihydroxamic Acid (SBHA) (Andrews et al., International J. Parasitology 30:761 , 2000); Azelaic Bishydroxamic Acid (ABHA) (Andrews et al., supra); Azelaic- l-Hydroxamate-9-Anilide (AAHA) (Qiu et al., MoI. Biol. Cell 1 1 :2069, 2000); 6-(3-Chlorophenylureido) carpoic Hydroxamic Acid (3Cl- UCHA), Oxamflatin [(2E)-5-[3-[(phenylsuibnyl-)amino phenyl]-pent-2-en-4- ynohydroxamic acid (Kim et al. Oncogene, 18: 2461 , 1999); A-161906, Scriptaid (Su et al. 2000 Cancer Research, 60:3137, 2000); PXD-101 (Prolifix); LAQ-824; CHAP; MW2796 (Andrews et al., supra); and MW2996 (Andrews et al., supra).
B) CYCLIC TETRAPEPTIDES such as Trapoxin A (TPX)-Cyclic Tetrapeptide (cyclo-(L- phenylalanyl-L-phenylalanyl-D-pipecolinyl-L-2-amin-o-8-oxo-9,10-epoxy decanoyl)) (Kijima et al., J Biol. Chem. 268:22429, 1993); FR901228 (FK 228, FR901228, Depsipeptide, Romidepsin) (Nakajima et al., Ex. Cell Res. 241 :12, 1998); FR225497 Cyclic Tetrapeptide (Mori et al., PCT Application WO 00/08048, Feb. 17, 2000); Apicidin Cyclic Tetrapeptide [cyclo (NO-methyl-L-tryptophanyl-L-isoleucinyl-D-pipe- colinyl-L-2-amino-8oxodecanoyl)] (Darkin-Rattray et al., Proc. Natl. Acad. Sci. USA 93:13143, 1996); Apicidin Ia, Apicidin Ib, Apicidin Ic, Apicidin Ha, and Apicidin Hb (P. Dulski et al., PCT Application WO 97/1 1366); CHAP, HC-Toxin Cyclic Tetrapeptide (Bosch et al., Plant Cell 7:1941 , 1995); WF27082 Cyclic Tetrapeptide (PCT Application WO 98/48825); and Chiamydocin (Bosch et al., supra).
C) SHORT CHAIN FATTY ACID (SCFA) DERIVATIVES such as: Sodium Butyrate (Cousens et al., J. Biol. Chem. 254: 1716, 1979); Isovalerate (McBain et al., Biochem. Pharm. 53:1357, 1997); Valerate (McBain et al., supra); 4 Phenylbutyrate (4-PBA) (Lea and Tulsyan, Anticancer Research, 15:879, 1995); Phenylbutyrate (PB) (Wang et al., Cancer Research, 59:2766, 1999); Propionate (McBain et al., supra); Butyramide (Lea and Tulsyan, supra); Isobutyramide (Lea and Tulsyan, supra); Phenylacetate (Lea and Tulsyan, supra); 3-Bromopropionate (Lea and Tulsyan, supra); Tributyrin (Guan et al., Cancer Research, 60:749, 2000); Valproic acid and Valproate.
D) BENZAMlDE DERIVATIVES such as CI-994; MS-275 [N-(2-aminophenyl)-4-[N- (pyridin-3-ylmethoxycarbonyl)aminomethyl]benzamide] (Saito et al., Proc. Natl. Acad. Sci. USA 96:4592, 1999; 3'-amino derivative of MS-27-275 (Saito et al., supra); MGCDO 103 (MethylGene; see Figure 1), or related compounds (for example, see Figure
2).
E) ELECTROPHILIC KETONE DERIVATIVES such as trifluoromethyl ketones (Frey et al, Bioorganic & Med. Chem. Lett., 12: 3443, 2002; U.S. 6,51 1,990) and α-keto amides such as N-methyl-α-ketoamides.
F) OTHER DAC Inhibitors such as Depudecin (Kwon et al., Proceedings of the National Academy of Sciences USA, 95:3356, 1998), and compounds depicted in Figure 3.
[0060] Suitable DAC inhibitors for use in accordance with the present invention particularly include, for example, CRA-094781 CCelera Genomics), PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCDO 103 (M ethyl Gene), MS-275
(Schering AG), romidepsin (Gloucester Pharmceuticals), and/or SAHA (Alton
Pharma/Merck).
[0061] In some embodiments, the DAC or HDAC inhibitor used in the method of the invention is represented by formula (V):
Figure imgf000019_0001
wherein
B is a substituted or unsubstituted, saturated or unsaturated aliphatic group, a substituted or unsubstituted, saturated or unsaturated alicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted heterocyclic group; R2o is hydroxylamino, hydroxyl, amino, alkylamino, dialkylamino, or alkyloxy group; R2i and R22 are independently selected from hydrogen, hydroxyl, a substituted or unsubstituted, saturated or unsaturated aliphatic group, a substituted or unsubstituted, saturated or unsaturated alicyclic group, a substituted or unsubstituted aromatic group, a substituted or unsubstituted heteroaromatic group, or a substituted or unsubstituted heterocyclic group. In a particular embodiment of Formula IV, R20 is a hydroxylamino, hydroxyl, amino, methylamino, dimethylamino or methyloxy group and B is a C6-alkyl. In yet another embodiment of Formula IV, R2ι is a hydrogen atom, R22 is a substituted or unsubstituted phenyl and B is a C6-alkyl. In further embodiments of Formula IV, R2i is hydrogen and R22 is an α-, β-, or γ-pyridine.
[0062] Other examples of DAC or HDAC inhibitors can be found in, for example, U.S. Pat. Nos. 5,369,108, issued on Nov. 29, 1994, 5,700,81 1 , issued on Dec. 23, 1997, 5,773,474, issued on Jun. 30, 1998, 5,932,616 issued on Aug. 3, 1999 and 6,51 1 ,990, issued Jan. 28, 2003 all to Breslow et al.; U.S. Pat. Nos. 5,055,608, issued on Oct. 8, 1991, 5,175,191, issued on Dec. 29, 1992 and 5,608,108, issued on Mar. 4, 1997 all to Marks et al.; U.S. Provisional Application No. 60/459,826, filed Apr. 1 , 2003 in the name of Breslow et al.; as well as, Yoshida, M., et al., Bioassays 17, 423-430 (1995): Saito, A., et al., PNAS USA 96, 4592- 4597, (1999); Furamai R. et al., PNAS USA 98 (1), 87-92 (2001); Komatsu, Y., et al., Cancer Res. 61(1 1), 4459-4466 (2001); Su, G. H., et al., Cancer Res. 60, 3137-3142 (2000); Lee, B. I. et al., Cancer Res. 61(3), 931 -934; Suzuki, T., et al., J. Med. Chem. 42(15), 3001-3003 (1999); published PCT Application WO 01/18171 published on Mar. 15, 2001 Sloan- Kettering Institute for Cancer Research and The Trustees of Columbia University; published PCT Application WO02/246144 to Hoffmann-La Roche; published PCT Application WO02/22577 to Novartis; published PCT Application WO02/30879 to Prolifix; published PCT Applications WO 01/38322 (published May 31, 2001), WO 01/70675 (published on Sep. 27, 2001) and WO 00/71703 (published on Nov. 30, 2000) all to Methylgene, Inc.; published PCT Application WO 00/21979 published on Oct. 8, 1999 to Fujisawa Pharmaceutical Co., Ltd.; published PCT Application WO 98/40080 published on Mar. 1 1, 1998 to Beacon Laboratories, L. L. C; and Curtin M. (Current patent status of histone deacetylase inhibitors Expert Opin. Ther. Patents (2002) 12(9): 1375-1384 and references cited therein).
[0063] Specific non-limiting examples of DAC or HDAC inhibitors are provided in the table below. It should be noted that the present invention encompasses any compounds which both are structurally similar to the compounds represented below and are capable of inhibiting histone deacetylases.
Figure imgf000021_0001
-continued
Figure imgf000022_0001
-continued
Figure imgf000023_0001
[0064] DAC or HDAC inhibitors for use in accordance with the present invention may be prepared by any available means including, for example, synthesis, semi-synthesis, or isolation from a natural source.
[0065] DAC or HDAC inhibitors for use in accordance with the present invention may be isolated or purified. For example, synthesized compounds can be separated from a reaction mixture, and natural products can be separated from their natural source, by methods such as column chromatography, high pressure liquid chromatography, and/or recrystallization. [0066] A variety of synthetic methodologies for preparing DAC or HDAC inihibitors are known in the art. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. [0067] DAC or HDAC inhibitors for use in accordance with the present invention may be modified as compared with presently known DAC or HDAC inhibitors, for example, by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and may include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
[0068] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in accordance with the present invention may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers. and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- , or as (D)- or (L)- for amino acids. The present invention encompasses all such possible isomers, as well as their racemic and optically pure forms to the extent that they have DAC inhibitory activity.
[0069] In general, optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). [0070] In some embodiments, a DAC (e.g., HDAC) inhibitor for use in accordance with the present invention may contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry. The present invention encompasses both E and Z geometric isomers or cis- and trans- isomers to the extent that they have DAC inhibitory activity. The present invention likewise encompasses all tautomeric forms that have DAC inhibitory activity. In general, where a chemical structure is presented, the configuration of any carbon- carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states or it is otherwise clear from context; thus a carbon-carbon double bond or carbon-heteroatom double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.
Romidepsin
[0071] Romidepsin is a cyclic depsipeptide of formula:
Figure imgf000025_0001
Romidepsin may be provided in any form. Pharmaceutically acceptable forms are particular preferred. Exemplary forms of romidepsin include, but are not limited to, salts, esters, prodrugs, isomers, stereoisomers {e.g., enantiomers, diastereomers), tautomers, protected forms, reduced forms, oxidized forms, derivatives, and combinations thereof, with the desired activity {e.g., deacetylase inhibitory activity, aggresome inhibition, cytotoxicity). In certain embodiments, the romidepsin used in the combination therapy is pharmaceutical grade material and meets the standards of the U.S. Pharmacopoeia, Japanese Pharmacopoeia, or European Pharmacopoeia. In certain embodiments, the romidepsin is at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.95% pure. In certain embodiments, the romidepsin is at least 95%, at least 98%, at least 99%, at least 99.9%, or at least 99.95% monomeric. In certain embodiments, no impurities are detectable in the romidepsin materials {e.g., oxidized material, reduced material, dimerized or oligomerized material, side products, etc.). The romidepsin typically includes less than 1.0%, less than 0.5%, less than 0.2%, or less than 0.1 % of total other unknowns. The purity of romidepsin may be assessed by appearance, HPLC, specific rotation, NMR spectroscopy, IR spectroscopy, UVNisible spectroscopy, powder x-ray diffraction (XRPD) analysis, elemental analysis, LC-mass spectroscopy, and mass spectroscopy. [0072] The inventive combination therapy may also include a derivative of romidepsin. In certain embodiments, the derivative of romidepsin is of the formula (I):
Figure imgf000026_0001
wherein m is 1 , 2, 3 or 4; n is 0, 1 , 2 or 3; p and q are independently 1 or 2;
X is O, NH, or NR8;
Ri, R2, and R3 are independently hydrogen; unsubstituted or substituted, branched or unbranched, cyclic or acyclic aliphatic; unsubstituted or substituted, branched or unbranched, cyclic or acyclic heteroaliphatic; unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl; and
R4, R5, R6 , R7 and R8 are independently hydrogen; or substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; and pharmaceutically acceptable forms thereof. In certain embodiments, m is 1. In certain embodiments, n is 1. In certain embodiments, p is 1. In certain embodiments, q is 1. In certain embodiments, X is O. In certain embodiments, Ri, R2, and R3 are unsubstituted, or substituted, branched or unbranched, acyclic aliphatic. In certain embodiments, R4, R5, R6, and R7 are all hydrogen. [0073] In certain embodiments, the derivative of romidepsin is of the formula (II):
Figure imgf000027_0001
wherein: m is 1 , 2, 3 or 4; n is 0, 1 , 2 or 3; q is 2 or 3;
X is O, NH, Or NR8;
Y is OR8, or SR8;
R2 and R3 are independently hydrogen; unsubstituted or substituted, branched or unbranched, cyclic or acyclic aliphatic; unsubstituted or substituted, branched or unbranched, cyclic or acylic heteroaliphatic; unsubstituted or substituted aryl; or unsubstituted or substituted heteroaryl;
R4, R5, R6 , R7 and R8 are independently selected from hydrogen; or substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; and pharmaceutically acceptable forms thereof. In certain embodiments, m is 1. In certain embodiments, n is 1. In certain embodiments, q is 2. In certain embodiments, X is O. In other embodiments, X is NH. In certain embodiments, R2 and R3 are unsubstituted or substituted, branched or unbranched, acyclic aliphatic. In certain embodiments, R4, R5, R6, and R7 are all hydrogen. [0074] In certain embodiments, the derivative of romidepsin is of the formula (III):
Figure imgf000027_0002
wherein A is a moiety that is cleaved under physiological conditions to yield a thiol group and includes, for example, an aliphatic or aromatic acyl moiety (to form a thioester bond); an aliphatic or aromatic thioxy (to form a disulfide bond); or the like; and pharmaceutically acceptable forms thereof. Such aliphatic or aromatic groups can include a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. A can be, for example, -CORi, -SC(=O)-O- Ri, or -SR2. Ri is independently hydrogen; substituted or unsubstituted amino; substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic; substituted or unsubstituted aromatic group; substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. In certain embodiment, Ri is hydrogen, methyl, ethyl, n-propyl, iso-propyl, n-butyl, isobutyl, benzyl, or bromobenzyl. R2 is a substituted or unsubstituted, branched or unbranched, cyclic or acyclic aliphatic group; a substituted or unsubstituted aromatic group; a substituted or unsubstituted heteroaromatic group; or a substituted or unsubstituted heterocyclic group. In certain embodiments, R2 is methyl, ethyl, 2-hydroxyethyl, isobutyl, fatty acids, a substituted or unsubstituted benzyl, a substituted or unsubstituted aryl, cysteine, homocysteine, or glutathione. [0075] In certain embodiments, the derivative of romidepsin is of formula (IV) or (IV):
Figure imgf000028_0001
wherein
Ri, Ri, R3. and R4 are the same or different and represent an amino acid side chain moiety, each R6 is the same or different and represents hydrogen or C1-C4 alkyl, and Pr and Pr2 are the same or different and represent hydrogen or thiol-protecting group. In certain embodiments, the amino acid side chain moieties are those derived from natural amino acids. In other embodiments, the amino acid side chain moieties are those derived from unnatural amino acids. In certain embodiments, each amino acid side chain is a moiety selected from - H, -C1-C6 alkyl, -C2-C6 alkenyl, -L-O-C(O)-R', -L-C(O)-O-R", -L-A, -L-NR11R", -L-Het- C(O)-Het-R", and -L-Het-R", wherein L is a C1-C6 alkylene group, A is phenyl or a 5- or 6- membered heteroaryl group, each R' is the same or different and represents C1-C4 alkyl, each R" is the same or different and represent H or C1-C6 alkyl, each -Het- is the same or different and is a heteroatom spacer selected from -0-, -N(R"')-, and -S-, and each R'" is the same of different and represents H or C1-C4 alkyl. In certain embodiments, R6 is -H. In certain embodiments, Pr1 and Pr2 are the same or different and are selected from hydrogen and a protecting group selected from a benzyl group which is optionally substituted by C1-C6 alkoxy, C1-C6 acyloxy, hydroxy, nitro, picolyl, picolyl-N-oxide, anthrylmethyl, diphenylmethyl, phenyl, t-butyl, adamanthyl, C1-C6 acyloxymethyl, C1-C6 alkoxymethyl, tetrahydropyranyl, benzylthiomethyl, phenylthiomethyl, thiazolidine, acetamidemethyl, benzamidomethyl, tertiary butoxycarbonyl (BOC), acetyl and its derivatives, benzoyl and its derivatives, carbamoyl, phenylcarbamoyl, and C1-C6 alkylcarbamoyl. In certain embodiments, Pr1 and Pr2 are hydrogen. Various romidepsin derivatives of formula (IV) and (IV) are disclosed in published PCT application WO 2006/129105, published December 7, 2006; which is incorporated herein by reference.
[0076] Processes for preparing romidepsin are known in the art. For example, exemplary processes of preparing romidepsin are described in U.S. Serial No. 1 1/966,258, filed on December 28, 2007; U.S. Serial No. 60/882,698, filed on December 29, 2006; U.S. Serial No. 60/882,704, filed on December 29, 2006; and U.S. Serial No. 60/882,712, filed on December 29, 2006, the teachings of all of which are incorporated by reference herein. Since romidepsin is a natural product, it is typically prepared by isolating it from a fermentation of a microorganism that produces it. In certain embodiments, the romidepsin or a derivate thereof is purified from a fermentation, for example, of Chromobacterium violaceum. See, e.g., Ueda et al, J. Antibiot. (Tokyo) 47:301-310, 1994; Nakajima et al, Exp. Cell Res. 241 : 126-133, 1998; WO 02/20817; U.S. Patent 4,977,138; each of which is incorporated herein by reference. In other embodiments, romidepsin or a derivative thereof is prepared by synthetic or semi-synthetic means. J. Am. Chem. Soc. 1 18:7237-7238, 1996; incorporated herein by reference.
[0001] The therapeutically effective amount of romidepsin included in the combination therapy will vary depending on the patient, the cancer or neoplasm being treated, stage of the cancer, pathology of the cancer or neoplasm, genotype of the cancer or neoplasm, phenotype of the cancer or neoplasm, the route of administration, etc. In certain embodiments, the romidepsin is dosed in the range of 0.5 mg/ m2 to 28 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 1 mg/ m2 to 25 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 0.5 mg/ m2 to 15 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 1 mg/ m2 to 15 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 1 mg/ m2 to 8 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 0.5 mg/ m2 to 5 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 2 mg/ m2 to 10 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 4 mg/ m2 to 15 mg/m2. In certain embodiments, the romidepsin is dosed in the range of 8 mg/ m2 to 10 mg/m2. In other embodiments, the dosage ranges from 10 mg/m2 to 20 mg/m2. In certain embodiments, the dosage ranges from 5 mg/m2 to 10 mg/m2. In other embodiments, the dosage ranges from 10 mg/m2 to 15 mg/m2. In still other embodiments, the dosage is approximately 8 mg/m2. In still other embodiments, the dosage is approximately 9 mg/m . In still other embodiments, the dosage is approximately 10 mg/m". In still other embodiments, the dosage is approximately 1 1 mg/m2. In still other embodiments, the dosage is approximately 12 mg/m2. In still other embodiments, the dosage is approximately 13 mg/m . In still other embodiments, the dosage is approximately 14 mg/m2. In still other embodiments, the dosage is approximately 15 mg/m2. In certain embodiments, increasing doses of romidepsin are administered over the course of a cycle. For example, in certain embodiments, a dose of approximately 8 mg/m2, followed by a dose of approximately 10 mg/m2, followed by a dose of approximately 12 mg/m2 may be administered over a cycle. As will be appreciated by one of skill in the art, depending on the form of romidepsin being administered the dosing may vary. The dosages given herein are dose equivalents with respect to the active ingredient, romidepsin. As will be appreciated by one of skill in the art, more of a salt, hydrate, co- crystal, pro-drug, ester, solute, etc. may need to be administered to deliver the equivalent number of molecules of romidepsin. In certain embodiments, romidepsin is administered intravenously. In certain embodiments, the romidepsin is administered intravenously over a 1 -6 hour time frame. In certain particular embodiments, the romidepsin is administered intravenously over 1-2 hours. In certain particular embodiments, the romidepsin is administered intravenously over 3-4 hours. In certain particular embodiments, the romidepsin is administered intravenously over 5-6 hours. In certain embodiments, the romidepsin is administered one day followed by several days in which the romidepsin is not administered. In certain embodiments, the romidepsin and the tyrosine kinase inhibitor are administered together. In other embodiments, the romidpesin and the tyrosine kinase inhibitor are administered separately. For example, the administration of romidepsin and a tyrosine kinase inhibitor may be separated by one or more days. In certain embodiments, romidepsin is administered twice a week. In certain embodiments, romidepsin is administered once a week. In other embodiments, romidepsin is administered every other week. In certain embodiments, romidepsin is administered on days 1 , 8, and 15 of a 28 day cycle. In certain particular embodiments, an 8 mg/m2 dose of romidepsin is administered on day 1, a 10 mg/m2 dose of romidepsin is administered on day 8, and a 12 mg/m2 dose of romidepsin is administered on day 15. In certain embodiments, romidepsin is administered on days 1 and 15 of a 28 day cycle. The 28 day cycle may be repeated. In certain embodiments, the 28 day cycle is repeated 3-10 times. In certain embodiments, the treatment includes 5 cycles. In certain embodiments, the treatment includes 6 cycles. In certain embodiments, the treatment includes 7 cycles. In certain embodiments, the treatment includes 8 cycles. In certain embodiments, greater than 10 cycles are administered. In certain embodiments, the cycles are continued as long as the patient is responding. The therapy may be terminated once there is disease progression, a cure or remission is achieved, or side effects become intolerable.
[0002] Alternatively, romidepsin may be administered orally. In certain embodiments, romidepsin is dosed orally in the range of 10 mg/ m2 to 300 mg/m2. In certain embodiments, romidepsin is dosed orally in the range of 25 mg/ m to 100 mg/m . In certain embodiments, romidepsin is dosed orally in the range of 100 mg/ m2 to 200 mg/m2. In certain embodiments, romidepsin is dosed orally in the range of 200 mg/ m2 to 300 mg/m2. In certain embodiments, romidepsin is dosed orally at greater than 300 mg/m . In certain embodiments, romidepsin is dosed orally in the range of 50 mg/ m2 to 150 mg/m2. In other embodiments, the oral dosage ranges from 25 mg/m2 to 75 mg/m2. As will be appreciated by one of skill in the art, depending on the form of romidepsin being administered the dosing may vary. The dosages given herein are dose equivalents with respect to the active ingredient, romidepsin. In certain embodiments, romidepsin is administered orally on a daily basis. In other embodiments, romidepsin is administered orally every other day. In still other embodiments, romidepsin is administered orally every third, fourth, fifth, or sixth day. In certain embodiments, romidepsin is administered orally every week. In certain embodiments, romidepsin is administered orally every other week. In certain embodiments, the romidepsin and the tyrosine kinase inhibitor are administered together. In other embodiments, the romidepsin and the tyrosine kinase inhibitor are administered separately. For example, the administration of romidepsin and a tyrosine kinase inhibitor may be separated by one or more days. In certain embodiments, both romidepsin and the tyrosine kinase inhibitor are administered orally. In certain embodiments, only romidepsin is administered orally. The administration of romidepsin alone or the combination of romidepsin and the tyrosine kinase inhibitor may be terminated once there is disease progression, a cure or remission is achieved, or side effects become intolerable.
Tyrosine Kinase Inhibitors
[0077] Tyrosine kinases add phosphate groups to tyrosine residues in proteins or peptides. Such phosphorylation often affects the activity, folding, and/or subcellular localization of the target protein, and is commonly involved in signaling cascades that communicate information and allow cells to respond to molecular cues. For example, tyrosine kinase signaling cascades are involved in activating cellular proliferation. Tyrosine. kinase activity has be found associated with the epidermal growth factor receptor as well as other receptors.
[0078] Mutations in proteins that participate in tyrosine kinase signaling cascades sometimes cause continual activation of cellular proliferation pathways, so that unregulated cell growth occurs and a proliferative disorder, disease, or condition {e.g., cancer or benign neoplasm) results.
[0079] There are two basic categories of tyrosine kinases: receptor tyrosine kinases and cellular tyrosine kinases.
[0080] Receptor tyrosine kinases are transmembrane proteins that have an extracellular ligand-binding domain and an intracellular catalytic domain. The extracellular ligand binding domain typically includes or more conserved structural motifs such as, for example, cysteine- rich regions, fibronectin Ill-like domains, immunoglobulin-like domains, EGF-like domains, cadherin-like domains, kringle-like domains, Factor Vlll-like domains, glycine-rich regions, leucine-rich regions, acidic regions and discoidin-like domains. The intracellular domain includes the catalytic sequences, which may be continuous or separated.
[0081] Typically, receptor tyrosine kinases are activated by ligand binding, which triggers dimerization. Dimerization. in turn, results in autophosphorylation which in turn creates binding sites for other cellular components involved in signal transduction. Such cellular components include, for example, RasGAP, P13-kinase, phospholipase C Y, phosphotyrosine phosphatase SHP and adaptor proteins such as She, Grb2 and Crk. [0082] Cellular tyrosine kinases are located in the cytoplasm, nucleus, or inner leaflet of the plasma membrane (i.e., are not transmembrane). There are at least eight known families (SRC, JAK, ABL, FAK, FPS, CSK, SYK, and BTK) of cellular tyrosine kinase, each of which has several members. All cellular tyrosine kinase domains share homologous kinase domains (Src Homology 1 , or SHl domains); some also share protein-protein interaction domains (e.g., SH2 and SH3 domains). Some members of the cytokine receptor pathway interact with JAKs, which phosphorylate the transcription factors, STATs. The SRC cellular tyrosine kinase inhibitors, among others, are involved in cell growth. [0083] Tyrosine kinase inhibitors are agents that reduce the activity and/or amount of a tyrosine kinase in a cell. Such agents can be useful in the treatment of proliferative disorders, diseases, or conditions. Certain leukemias, as well as cancers of the breast, prostate, ovary, bladder, liver, pancreas, and lung, are among the cancers that are most responsive to therapy with tyrosine kinase inhibitors.
[0084] Commercially available tyrosine kinase inhibitors include, for example, axitinib, cediranib (RECENTIN), dasatinib (SPRYLCEL), erlotinib (TARCEV A®), gefitinib (IRESSA), imatinib (GLEEVEC), lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, and vandetanib. In certain embodiments, romidepsin is used in combination with axitinib. In certain embodiments, romidepsin is used in combination with cediranib. In certain embodiments, romidepsin is used in combination with dasatinib. In certain embodiments, romidepsin is used in combination with erlotinib. In certain embodiments, romidepsin is used in combination with gefitinib. In certain embodiments, romidepsin is used in combination with imatinib. In certain embodiments, romidepsin is used in combination with lapatinib. In certain embodiments, romidepsin is used in combination with lestaurtinib. In certain embodiments, romidepsin is used in combination with nilotinib. In certain embodiments, romidepsin is used in combination with semaxanib. In certain embodiments, romidepsin is used in combination with sunitinib. In certain embodiments, romidepsin is used in combination with vandetanib.
[0085] In some embodiments of the present invention, the tyrosine kinase inhibitor is erlotinib. Erlotinib specifically targets the epidermal growth factor receptor tyrosine kinase, which is highly expressed and occasionally mutated in various forms of cancer. Erlotinib has been shown to improve survival in lung cancer patients, and has been approved for use in treating lung and pancreatic cancer.
Other Anti-neoplastic Agents
[0003] Anti-neoplastic agents suitable for use in the present invention includes any agents that inhibit or prevent the growth of neoplasms, checking the maturation and proliferation of malignant cells. Growth inhibition can occur through the induction of stasis or cell death in the tumor cell(s). Typically, anti-neoplastic agents include cytotoxic agents in general. Exemplary anti-neoplastic agents include, but are not limited to, cytokines, ligands, antibodies, radionuclides, and chemotherapeutic agents. In particular, such agents include interleukin 2 (1L-2), interferon (IFN) TNF; photosensitizers, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (."'11Tc), rhenium- 186 (186Re), and rhenium- 188 (188Re); chemotherapeutics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from Chinese cobra (naja naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis), and RNase; ly207702 (a difluorinated purine nucleoside); liposomes containing antitumor agents {e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc); and other antibodies or antibody fragments, such as F(ab). Any of the these anti-neoplastic agents may be used in combination with romidepsin and a tyrosine kinase inhibitor (e.g., erlotinib).
Uses in Cell Proliferative Disorders, Diseases, or Conditions
[0086] The combination of romidepsin and a tyrosine kinase inhibitor may be used in vitro or in vivo. The inventive combination is particularly useful in the treatment of neoplasms in vivo. However, the combination may also be used in vitro for research or clinical purposes (e.g., determining the susceptibility of a patient's disease to the inventive combination, researching the mechanism of action, elucidating a cellular pathway or process). In certain embodiments, the neoplasm is a benign neoplasm. In other embodiments, the neoplasm is a malignant neoplasm. Any cancer may be treated using the inventive combination.
[0087] In certain embodiments, the invention provides methods for treating cell proliferative disorders, diseases or conditions. In general, cell proliferative disorders, diseases or conditions encompass a variety of conditions characterized by aberrant cell growth, preferably abnormally increased cellular proliferation. For example, cell proliferative disorders, diseases, or conditions include, but are not limited to, cancer, immune-mediated responses and diseases (e.g., transplant rejection, graft vs host disease, immune reaction to gene therapy, autoimmune diseases, pathogen-induced immune dysregulation, etc.), certain circulatory diseases, and certain neurodegenerative diseases. [0088] In certain embodiments, the invention relates to methods of treating cancer. In general, cancer is a group of diseases which are characterized by uncontrolled growth and spread of abnormal cells. Examples of such diseases are carcinomas, sarcomas, leukemias, lymphomas, and the like.
[0089] For example, cancers include, but are not limited to leukemias and lymphomas such as cutaneous T-cell lymphomas (CTCL), peripheral T-cell lymphomas, lymphomas associated with human T-cell lymphotropic virus (HTLV) such as adult T-cell leukemia/lymphoma (ATLL), B-cell lymphoma, acute lymphocytic leukemia, acute nonlymphocytic leukemias, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphomas, multiple myeloma, myelodysplastic syndrome, mesothelioma, common solid tumors of adults such as head and neck cancers (e.g., oral, laryngeal and esophageal), genitourinary cancers (e.g., prostate, bladder, renal, uterine, ovarian, testicular, rectal and colon), lung cancer (e.g., non- small cell lung cancer), breast cancer, pancreatic cancer, melanoma and other skin cancers, stomach cancer, brain tumors, liver cancer and thyroid cancer, and/or childhood solid tumors such as brain tumors, neuroblastoma, retinoblastoma, Wilms' tumor, bone tumors, and soft- tissue sarcomas.
[0090] In some embodiments, the invention relates to treatment of solid tumors. In some such embodiments the invention relates to treatment of solid tumors such as lung, breast, colon, liver, pancreas, renal, prostate, ovarian, or brain cancer. In some embodiments, the invention relates to treatment of pancreatic cancer. In some embodiments, the invention relates to treatment of renal cancer. In some embodiments, the invention relates to treatment of lung cancer. For example, in some embodiments, the invention relates to treatment of non- small cell lung cancer. In some embodiments, the invention relates to treatment of prostate cancer. In some embodiments, the invention relates to treatment of sarcomas. In some embodiments, the invention relates to treatment of soft tissue sarcomas. In some embodiments, the invention relates to methods of treating one or more immune-mediated responses and diseases.
[0091] In some embodiments, the invention relates to treatment of disorders, diseases or conditions associated with chromatin remodeling.
[0092] In some embodiments, the invention relates to treatment of lung cancer. In some embodiments, the invention relates to treatment of non small cell lung cancer. In some embodiments, the invention relates to treatment of wild type EGFR non-small cell lung cancer. In some embodiments, the invention relates to treatment of wild type KRAS non- small cell lung cancer. In some embodiments, the invention relates to treatment of wild type EGFR and wild type ICRAS non-small cell lung cancer. In some embodiments, the invention relates to treatment of mutant KRAS non-small cell lung cancer.
[0093] The inventive combinations of romidepsin plus a tyrosine kinase inhibitor may also be used to treat and/or kill cells in vitro. In certain embodiments, a cytotoxic concentration of the combination of agents is contacted with the cells in order to kill them. In other embodiments, a sublethal concentration of the combination of agents is used to treat the cells. In certain embodiments, the combination of agents acts additively to kill the cells. In certain embodiments, the combination of agents acts synergistically to kill the cells. Therefore, a lower concentration of one or both agents is needed to kills the cells than would be needed if either agent were used alone. In certain embodiments, the concentration of each agent ranges from 0.01 nM to 100 nM. In certain embodiments, the concentration of each agent ranges from 0.1 nM to 50 nM. In certain embodiments, the concentration of each agent ranges from 1 nM to 10 nM. In certain embodiments, the concentration of romidepsin ranges from 1 nM to 10 nM, more particularly 1 nM to 5 nM. In certain embodiments, the concentration of the tyrosine kinase inhibitor ranges from 1 nM to 10 nM, more particularly 1 nM to 5 nM
[0094] Any type of cell may be tested or killed with the combination therapy {i.e., romidepsin and a tyrosine kinase inhibitor {e.g., erlotinib)). The cells may be derived from any animal, plant, bacterial, or fungal source. The cells may be at any stage of differentiation or development. In certain embodiments, the cells are animal cells. In certain embodiments, the cells are vertebrate cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the cells are human cells. The cells may be derived from a male or female human in any stage of development. In certain embodiments, the cells are primate cells. In other embodiments, the cells are derived from a rodent (e.g., mouse, rat, guinea pig, hamster, gerbil). In certain embodiments, the cells are derived from a domesticated animal such as a dog, cat, cow, goat, pig, etc. The cells may also be derived from a genetically engineered animal or plant, such as a transgenic mouse.
[0095] The cells used may be wild type or mutant cells. The cells may be genetically engineered. In certain embodiments, the cells are normal cells. In certain embodiments, the cells are hematological cells. In certain embodiments, the cells are white blood cells. In certain particular embodiments, the cells are precursors of white blood cells (e.g., stem cells, progenitor cells, blast cells). In certain embodiments, the cells are neoplastic cells. In certain embodiments, the cells are cancer cells. In certain embodiments, the cells are derived from a hematological malignancy. In other embodiments, the cells are derived from a solid tumor. In certain embodiments, the cells are derived from a lung cancer. In certain embodiments, the cells are derived from a non-small cell lung cancer. For example, the cells may be derived from a patient's tumor (e.g., from a biopsy or surgical excision). Such testing for cytotoxicity may be useful in determining whether a patient will respond to a particular combination therapy. Such testing may also be useful in determining the dosage needed to treat the malignancy. This testing of the susceptibility of a patient's cancer to the combination therapy would prevent the unnecessary administration of drugs with no effect to the patient. The testing may also allow the use of lower doses of one or both of the drugs if the patient's cancer is particularly susceptible to the combination.
[0096] In other embodiments, the cells are derived from cancer cells lines. In certain embodiments, the cells are from lung cancers such as those discussed herein. Lung cancer cell lines include ABC-I, A549, PC3, PC7, RERF-LCMS, RERF-LCKJ, LCD, LCOK, PC9, PC14, QG56, EBC-I , LK-2, LC-l/sq, PCl , RERF-LCAI, PCl O, SQ5, NCI-H69, SBC3, NCI- N23 I , Lul35, and MS-I .
Combination Therapy
[0097] The present invention demonstrates the particular utility of administering a combination of a DAC inhibitor and a tyrosine kinase inhibitor.
[0098] In some particular embodiments of the present invention, the DAC inhibitor is romidepsin (aka, depsipeptide, FK228, FR901228). In other particular embodiments, the DAC inhibitor is selected from the group consisting of CRA-024781 (Celera Genomics), phenylbutarate, PXD-101 (CuraGene), LAQ-824 (Novartis AG), LBH-589 (Novartis AG), MGCDO 103 (MethylGene), MS-275 (Schering AG), romidepsin (Gloucester Pharmceuticals), SAHA (Alton Pharma/Merck), and combinations thereof. In some particular embodiments of the present invention, the DAC inhibitor is romidepsin (aka depsipeptide, FK228, FR901228). In some particular embodiments, the DAC inhibitor is SAHA. In some particular embodiments, the DAC inhibitor is phenylbutyrate. In some particular embodiments, the DAC inhibitor comprises a combination of DAC inhibitors. [0099] In some particular embodiments of the present invention, the tyrosine kinase inhibitor is erlotinib (TARCEV A®). In other particular embodiments , the tyrosine kinase inhibitor is selected from the group consisting of axitinib, cediranib (RECENTIN), dasatinib (SPRYLCEL), erlotinib (TARCEVA), gefitinib (IRESSA), imatinib (GLEEVEC), lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, vandetanib, and combinations thereof [00100] As will be appreciated by those of skill in the art, and as is otherwise addressed herein, either or both of the DAC inhibitor and tyrosine kinase inhibitor may be provided in any useful form including, for example, as a salt, ester, active metabolite, prodrug, etc. Similarly, either or both agents (or salts, esters, or prodrugs thereof) may be provided as a pure isomer stereoisomer or as a combination of stereoisomers, including a racemic combination, so long as relevant activity is present. Comparably, either or both agents (or salts, esters, or prodrugs thereof) may be provided in crystalline form, whether a pure polymorph or a combination of polymorphs, or in amorphous form, so long as relevant activity is present.
[00101] As addressed above, combination therapy of DAC inhibitors and tyrosine kinase inhibitor will typically involve administration of multiple individual doses spaced out in time. In some embodiments, individual DAC inhibitor doses and tyrosine kinase inihbitor doses will be administered together, according to the same schedule. In other embodiments, DAC inhibitor doses and tyrosine kinase inhibitor doses will be administered according to different schedules.
[00102] The total daily dose of any particular active agent administered to a human or other animal in single or in divided doses in accordance with the present invention can be in amounts, for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1 to 25 mg/kg body weight. Single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. In certain embodiments, about 10-100 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 100-500 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 250-500 mg of the compound is administered per day in single or multiple doses. In certain embodiments, about 500-750 mg of the compound is administered per day in single or multiple doses. [00103] In the treatment of neoplasms such as cancer in a subject, a DAC inhibitor is typically dosed at 1 -30 mg/m2. In certain embodiments, a DAC inhibitor is dosed at 1-15 mg/m2. In certain embodiments, a DAC inhibitor is dosed at 5-15 mg/m2. In certain particular embodiments, a DAC inhibitor is dosed at 4, 6, 8, 10, 12, 14, 16, 18, or 20 mg/m2. A DAC inhibitor is typically administered in a 28 day cycle with the agent being administered on days 1, 8 and 15. In certain embodiments, the DAC is administered on days 1 and 15 with day 8 being skipped. As would be appreciated by one of skill in the art, the dosage and timing of administration of the dosage of the DAC inhibitor may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of DAC inhibitor administered.
[00104] Typical dosing schedules have been established for certain exemplary DAC inhibitors (e g., HDAC inhibitors). For example, SAHA is commonly administered within a range of about 300-400 mg daily orally; PXDlOl is commonly administered within a range of about up to 2000 mg/m2/day intravenously (e.g., on days 1 to 5 of a 21 day cycle), and may possibly be administered orally; MGCDO 103 is commonly administered at doses up to about 27 mg/m2 given orally (e.g., daily for about 14 days); LBH589 is commonly administered at doses up to about 14 mg/m2 as an intravenous infusion (e.g., on days 1-7 of a 21 day cycle); MS-275 is commonly administered within a dose range of about 2-12 mg/m2 intravenously (e.g., every 14 days).
[00105] In the treatment of neoplasms such as cancer in a subject, romidepsin is typically dosed at 1 -28 mg/m2. In certain embodiments, romidepsin is dosed at 1 -15 mg/m2. In certain embodiments, romidepsin is dosed at 5-14 mg/m2. In certain particular embodiments, romdiepsin is dosed at 8, 10, 12, or 14 mg/m2. Romidepsin is typically administered in a 28 day cycle with romidepsin being administered on days 1 , 8 and 15. In certain embodiments, romidepsin is administered on days 1 and 15 with day 8 being skipped. [00106] Acceptable dosing schedules have also been established for certain tyrosine kinase inhibitors. For example, in the treatment of non-small cell lung cancer, erlotinib is typically administered orally at a dose of 150 mg/day. In certain embodiments, erlotinib in combination with a DAC inhibitor is administered orally at a dose of approximately 100 mg/day. It is generally recommended that erlotinib be taken one hour before or two hours after the ingestion of food. Erlotinib is currently available in 150 mg, 100 mg, and 25 mg doses.
[00107] As would be appreciated by one of skill in the art, the dosage and timing of administration of any particular DAC inhibitor or tyrosine kinase inhbitor dose, or the dosage amount and schedule generally may vary depending on the patient and condition being treated. For example, adverse side effects may call for lowering the dosage of one or the other agent, or of both agents, being administered.
[00108] Moreover, those of ordinary skill in the art will readily appreciate that the dosage schedule (i.e., amount and timing of individual doses) by which any particular DAC inihbitor is administered may be different for inventive combination therapy with a tyrosine kinase inhibitor than it is alone. Comparably, the dosage schedule for the tyrosine kinase inhibitor may be different according to inventive combination therapy regimens than would be utilized in monotherapy (even for the same disorder, disease or condition). [00109] To give but one example, in some embodiments, a DAC inhibitor (e.g., romidepsin) and a tyrosine kinase inhibitor (e.g., erlotinib) are each dosed on days 1 and 15 of a 28 day cycle. Those of ordinary skill in the art will appreciate that any of a variety of other dosing regimens are within the scope of the invention. Commonly, dosing is adjusted based on a patient's response to therapy, and particularly to development of side effects. [00110] In some embodiments of the present invention, inventive combination therapy with one or more DAC inhibitors and one or more tyrosine kinase inhibitors is further combined with administration of one or more other agents.
[00111] In some embodiments, subjects receiving inventive combination therapy with one or more DAC inhibitors and one or more tyrosine kinase inhibitors further receive electrolyte supplementation for example as is described in co-pending United States Provisional Patent application, U. S. S.N. 60/909,780, , filed April 3, 2007; and U.S. non-provisional patent application. U. S. S.N. 1 1/759,471 , filed June 7, 2007; each of which is incorporated herein by reference. [00112] For example, as described in that application, an individual with a potassium serum concentration below about 3.5 mmol/L (3.5 mEq/L) and/or a serum magnesium concentration below about 0.8 mml/L (1.95 mEq/L) suffers an increased risk of developing cardiac repolarization effects and/or dysrhythmias.
[00113] Serum concentrations of potassium are generally considered to be "normal" when they are within the range of about 3.5 - 5.5 mEq/L or about 3.5 - 5.0 mEq/L. According to the present invention, it is often desirable to ensure that an individuals' serum potassium concentration is within this range prior to (and/or during) administration of DAC inhibitor and/or combination therapy.
[00114] Serum concentrations of magnesium are generally considered to be "normal" when they are within the range of about 1.5 - 2.5 mEq/L or about 1.5 - 2.2 mEq/L or about 1.25 - 2.5 mEq/L or about 1.25 - 2.2 mEq/L. According to the present invention, it is often desirable to ensure that an individual's serum magnesium concentration is within this range prior to (and/or during) administration of DAC inhibitor and/or combination therapy. [00115] In some embodiments of the invention, an individual's serum potassium and/or magnesium concentration(s) is/are at the high end of the normal range prior to (and/or during) administration of DAC inhibitor and/or combination therapy. For example, in some embodiments, an individual's serum potassium concentration is at least about 3.8, 3.9, 4.0 mEq/L, or more prior to and/or during administration of DAC inhibitor and/or combination therapy. In some embodiments, care is taken not to increase serum potassium concentration above about 5.0, 5.2, or 5.5 mEq/L. In some embodiments, an individual's serum magnesium concentration is at least about 1.9 mEq/L or more prior to and/or during administration of DAC inhibitor and/or combination therapy. In some embodiments, care is taken not to increase magnesium concentration above about 2.5 mEq/L.
[00116] In some embodiments of the present invention, an individual's serum potassium concentration is at least about 3.5 mEq (in some embodiments at least about 3.8, 3.9, 4.0 mEq/L or above) and the individual's serum magnesium concentration is at least about 1.85 mEq/L (in some embodiments at least about 1.25, 1.35, 1.45, 1.55, 1.65, 1.75, 1.85, 1.95, etc) prior to and/or during administration of DAC inhibitor and/or combination therapy. [00117] In some embodiments of the invention, electrolyte levels {e.g., potassium and/or magnesium levels, optionally calcium levels) are assessed more than once during the course of DAC inhibitor and/or combination therapy; in some embodiments, different assessments are separated by a regular interval (e.g., 0.5 days or less, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, etc.). In some embodiments, electrolyte levels are assessed prior to each administration of DAC inhibitor or tyrosine kinase inhibitor.
Pharmaceutical Compositions
[00118] DAC inhibitors and/or tyrosine kinase inhibitors for use in accordance with the present invention are often administered as pharmaceutical compositions comprising therapeutically effective amounts of DAC inhibitor and tyrosine kinase inhibitor, respectively, that are useful in the inventive combination therapy (which amounts may be different from, including less than, amounts required for either agent to be effective alone). In some embodiments, a DAC inhibitor and tyrosine kinase inhibitor are present together in a single pharmaceutical composition; in some embodiments these agents are provided in separate pharmaceutical compositions.
[0004] In some embodiments, inventive pharmaceutical compositions are prepared in unit dosage forms. In general, a pharmaceutical composition of the present invention includes one or more active agents (i.e., one or more DAC inhibitors and/or one or more tyrosine kinase inhibitors) formulated with one or more pharmaceutically acceptable carriers or excipients. This invention also provides pharmaceutical compositions, preparations, or kits comprising romidepsin and/or a tyrosine kinase inhibitor as described herein, which combination shows cytostatic or cytotoxic activity against neoplastic cells such as lung cancer. The compositions, preparations, or kits typically include amounts appropriate for the administration of romidepsin and/or the tyrosine kinase inhibitor. In certain embodiments, the romidepsin and the tyrosine kinase inhibitor are not mixed together in the same composition. For example, the two agents are not part of the same solution or powder. Typically, the two agents are kept separate in two different compositions and are delivered separately. A kit may contain a pharmaceutical composition of romidepsin and a separate pharmaceutical composition of a tyrosine kinase inhibitor. In certain particular embodiments, the pharmaceutical compositions, preparations, or kits comprise romidepsin and erlotinib. In certain embodiments, given the synergistic interactions between the two pharmaceutical agents, the amount of one or both agents is lower than the amount that is typically administered when the agent is administered alone. In certain embodiments, the amount of both agents is lower. In certain embodiments, the amount administered is sufficient to achieve nanomolar levels in the bloodstream of the subject. In certain embodiments, the amount administered is sufficient to achieve nanomolar concentrations at the site of the cancer or other neoplasm in the subject. The dosing of each of romidepsin and erlotinib is described in more detail herein. In certain embodiments, the agents act synergistically to kill cancer cells. In other embodiments, the agents act additively to kill cancer cells. [0005] The inventive pharmaceutical compositions, preparations, or kits may include other therapeutic agents. The other pharmaceutical agent may be any other therapeutic agent that would be useful to administer to the subject. The other therapeutic agent preferably does not interact adversely with romidepsin or the tyrosine kinase inhibitor being administered In certain embodiments, the invention provides for the administration of romidepsin and a tyrosine kinase inhibitor in combination with one or more other therapeutic agents, e.g., another cytotoxic agent, analgesic, etc. In certain embodiments, the other therapeutic agent is another chemotherapeutic agent. The other therapeutic agent may include an agent for alleviating or reducing the side effects of romidepsin and/or the tyrosine kinase inhibitor. In certain embodiments, the other therapeutic agent is an anti-inflammatory agent such as aspirin, ibuprofen, acetaminophen, etc., pain reliever, anti-nausea medication, or anti-pyretic. In certain embodiments, the other therapeutic agent is an agent to treat gastrointestinal disturbances such as nausea, vomiting, stomach upset, and diarrhea. These additional agents may include anti-emetics, anti-diarrheals, fluid replacement, electrolyte replacement, etc. In certain particular embodiments, the other therapeutic agent is an electrolyte replacement or supplementation such as potassium, magnesium, and calcium, in particular, potassium and magnesium. In certain embodiments, the other therapeutic agent is an anti-arrhythmic agent. In certain embodiments, the other therapeutic agent is a platelet booster, for example, an agent that increases the production and/or release of platelets. In certain embodiments, the other therapeutic agent is an agent to boost the production of blood cells such as erythropoietin. In certain embodiments, the other therapeutic agent is an agent to prevent hyperglycemia. In certain embodiments, the other therapeutic agent is an immune system stimulator. In certain embodiments, the invention does not include the administration of another HDAC inhibitor besides romidepsin.
[0006] It will also be appreciated that certain of the agents utilized in accordance with the present invention can exist in free form for treatment, or where appropriate, as a pharmaceutically acceptable form thereof. According to the present invention, a pharmaceutically acceptable form includes, but is not limited to, pharmaceutically acceptable salts, esters, salts of such esters, protected forms, stereoisomers, isomers, reduced forms, oxidized forms, tautomers, or any other adduct or derivative which upon administration to a patient in need is capable of providing, directly or indirectly, an agent as otherwise described herein, or a metabolite or residue thereof, e.g., a prodrug.
[0007] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19, 1977; incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or separately by reacting the free base functionality with a suitable organic or inorganic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate. undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate, and aryl sulfonate.
[0008] Additionally, as used herein, the term "pharmaceutically acceptable ester" refers to esters which hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids, particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl moiety advantageously has not more than 6 carbon atoms. Examples of particular esters include formates, acetates, propionates, butyrates, acrylates, and ethylsuccinates. In certain embodiments, the esters are cleaved by enzymes such as esterases. [0009] Furthermore, the term "pharmaceutically acceptable prodrugs" as used herein refers to those prodrugs of the compounds utilized in accordance with the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use. The term "'prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A. C. S. Symposium Series, and in Edward B. Roche, ed., Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
[00119] As described above, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier or excipient, which, as used herein, includes any and all solvents, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, permeation enhancers, solubilizing agents, and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical Sciences, Fifteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1975) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the anti-cancer compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention. Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; Cremophor (polyethoxylated caster oil); Solutol (poly-oxyethylene esters of 12-hydroxystearic acid); excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the composition, according to the judgment of the formulator.
[00120] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminun hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions; non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; releasing agents; coating agents; sweetening, flavoring and perfuming agents; preservatives and antioxidants; and combinations thereof. In some embodiments, the pH of the ultimate pharmaceutical formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. [00121] Pharmaceutical compositions of this invention may be administered can be administered by any appropriate means including, for example, orally, parenterally, by inhalation spray, topically, rectally. nasally, buccally, vaginally or via an implanted reservoir. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. In many embodiments, pharmaceutical compositions are administered orally or by injection in accordance with the present invention. [00122] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active agent(s), liquid dosage forms of pharmaceutical compositions may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
[00123] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. A sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U. S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables.
[00124] Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[00125] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from a site of subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. [00126] Alternatively, delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.
[00127] Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing the active agents with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
[00128] Solid dosage forms for oral administration include, for example, capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active agent(s) is/are typically mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or: a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents, permeation enhancers, and/or other agents to enhance absorption of the active agent(s). [00129] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[00130] Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. [00131] In certain embodiments, oral dosage forms are prepared with coatings or by other means to control release of active agent (e.g., DAC inhibitor and/or tyrosine kinase inhibitor) over time and/or location within the gastrointestinal tract. A variety of strategies to achieve such controlled (or extended) release are well known in the art, and are within the scope of the present invention.
[00132] Dosage forms for topical or transdermal administration include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. In general, such preparations are prepared by admixing active agent(s) under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
[00133] Ophthalmic formulation, ear drops, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.
[00134] Ointments, pastes, creams and gels may contain, in addition to active agent(s), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
[00135] Powders and sprays can contain, in addition to active agent(s), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
[00136] Transdermal patches have often can provide controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
For pulmonary delivery, active agent(s) is/are formulated and administered to the patient in solid or liquid particulate form by direct administration e.g., inhalation into the respiratory system. Solid or liquid particulate forms of the active agent(s) prepared for practicing the present invention include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. Delivery of aerosolized therapeutics, particularly aerosolized antibiotics, is known in the art (see. for example U.S. Pat. No. 5,767,068 to VanDevanter et al., U.S. Pat.
No. 5,508,269 to Smith et al., and WO 98/43,650 by Montgomery, all of which are incorporated herein by reference). A discussion of pulmonary delivery of antibiotics is also found in U.S. Pat. No. 6,014,969, incorporated herein by reference. [00137] Pharmaceutical compositions for use in accordance with the present invention can, for example, be administered by injection, intravenously, intraarterially, subdermally, intraperitoneal^, intramuscularly, or subcutaneously; or orally, buccally, nasally, transmucosally, topically, in an ophthalmic preparation, or by inhalation, for example with a dosage ranging from about 0.1 to about 500 mg/kg of body weight, alternatively dosages between 1 mg and 1000 mg/dose, every 4 to 120 hours, or according to the requirements of the particular drug.
[00138] The methods herein contemplate administration of an effective amount of active agent or pharmaceutical composition sufficient for a desired or stated effect. Typically, the pharmaceutical compositions of this invention will be administered from about 1 to about 6 times per day or alternatively, as a continuous infusion. Such administration can be used as a chronic or acute therapy.
[00139] The amount of any particular active agent that may be combined with pharmaceutically acceptable excipients or carriers to produce a single dosage form may vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w/w). Alternatively, such preparations may contain from about 20% to about 80% active compound. For romidepsin, preparations may commonly contain about 20-50%, 25-45%, 30-40%, or approximately 32%, 33%, 34%, or 35% active compound.
[00140] Lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician. [00141] Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms. [00142] When pharmaceutical compositions contain two or more active agents, it is generally the case that each agent is present at dosage levels of between about 1 to 100%, for example about 5 to 95%, of the level normally administered in a monotherapy regimen.
[00143] Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one of ordinary skill in the art. All publications, patents, published patent applications, and other references mentioned herein are hereby incorporated by reference in their entirety. The embodiments of the invention should not be deemed to be mutually exclusive and can be combined.
Examples
[00144] The present invention will be better understood in connection with the following Example, which is intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims.
Example 1: Histone Deacetylase Inhibitor Romidepsin Enhances Anti-Tumor Effect of
Erlotinib in Non-Small Cell Lung Cancer Cell Lines
[00145] Most EGFR mutant non-small cell lung cancers (NSCLCs) are sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib, but many EGFR wild type NSCLCs are resistant to TKIs. In this Example, we examined the effects of the histone deacetylase (HDAC) inhibitor, romidepsin, in combination with erlotinib, in the treatment of NSCLCs.
[00146] For in vitro studies, sixteen NSCLC cell lines were treated with erlotinib alone or in combination with romidepsin. MTS assays were performed to determine the ICs0 of erlotinib in these cell lines 72 hours after drug addition. For in vivo studies, NCI-H 1299 xenografts were inoculated subcutaneously into nude mice. Romidepsin and/or erlotinib were injected intraperitoneally after tumors developed and tumor sizes were measured. [00147] We found that romidepsin increased the sensitivity of erlotinib in 13 of the 16 NSCLC cell lines, especially in EGFR and KRAS wild type NSCLC cells. This effect was due to enhanced apoptosis. Furthermore, co-treatment of erlotinib and romidepsin inhibited tumor xenograft growth in nude mice. Finally, we showed that romidepsin down-regulated MAPK and AKT, while dramatically increasing the cyclin dependent kinase inhibitor p21 in NSCLC cell lines. These observations support a role for the combination of a HDAC inhibitor and a TKI in the treatment of NSCLCs, especially those cancers with wild type EGFR and KRAS. Introduction
[00148] The epidermal growth factor receptor (EGFR) belongs to the EGFR family of tyrosine kinase receptors including EGFR, HER2/neu (ERRB2), HER3 (ERRB3) and HER4 (ERRB4) (1 ). Upon activation, EGFR can promote cell proliferation and survival through Ras/MEK/MAPK and/or PI3K/AKT signaling pathways (2). In recent years, non-small cell lung cancers (NSCLCs) containing EGFR mutations have been shown to be sensitive to EGFR tyrosine kinase inhibitors (TKIs) such as erlotinib (TARCEVA) or gefitinib (IRESSA), and these drugs have been successfully utilized in the therapy of such patients (3, 4). Unfortunately, the patients with cancers containing EGFR mutations developed resistance to TKIs after a period of time. Although the mechanisms of resistance are not completely understood, the existence of the secondary T790M mutation activity appears to be at least one mechanism (5). Furthermore, most of the NSCLCs containing wild type EGFR receptor are resistant to therapy with EGFR TKIs (6). Thus, the role of erlotinib and gefitinib for the treatment of NSCLC is limited.
[00149] The acetylation of histone plays an important role in chromatin organization and gene expression (7). Acetylation is determined by both histone acetyltransferases (HATs) and histone deacetylases (HDACs) (8). HDACs remove the acetyl groups from the lysine residue of the histone tail leading to compaction of chromatin and resultant repression of gene transcription (8). Many nonhistone proteins can be HDAC substrates such as p53, cMyc, Stat3, and Hsp90 (9). Such potential targets have provided a new strategy for the role of HDAC inhibitors in cancer therapy. HDAC inhibitors can induce growth arrest, differentiation, and apoptosis in tumor cells (10). Several HDAC inhibitors have been used in clinical trials (1 1). Romidepsin (previously termed FK228, depsipeptide) is a cyclic peptide HDAC inhibitor that has shown important inhibition of in vitro growth of several tumor cell types including lung and prostate cancers, lymphomas and leukemias (12-14). Romidepsin is now in a phase II clinical trial for treatment to cutaneous T cell lymphoma (15).
[00150] The present studies demonstrate that the HDAC inhibitor romidepsin enhanced the response of the TKl, erlotinib, in most of the EGFR wild type NSCLC cell lines examined, although in two EGFR mutant cell lines, the addition of romidepsin was not effective. The combination of romidepsin and erlotinib induced profound apoptosis in NSCLC cells. Furthermore, co-treatment with erlotinib and romidepsin inhibited tumor xenograft growth in nude mice. Finally, we showed that romidepsin down-regulated the MAPK and AKT pathways, increased the cyclin dependent kinase inhibitor p21, and the pro-apoptotic protein Bim expression in NSCLC cell lines. Materials and Methods
[00151] Cell lines and drugs. 16 NSCLC cell lines (see table below) were cultured in RPMl 1640 (Life Technologies, Rockville, MD) supplemented with 5% fetal bovine serum and incubated in humidified air and 5% CO2 at 37 °C. The cell lines were established in our lab. The NSCLC lines were all DNA fingerprinted and free of mycoplasma by molecular tests. Romidepsin was provided by Fujisawa Pharmaceutical Co. (Japan). Erlotinib (TARCEVA) was purchased from OSl Pharmaceuticals, Inc. (NY). For in vitro studies, romidepsin was dissolved in ethanol and erlotinib was dissolved in DSMO.
Effect of romidepsin on the IC50 values of erlotinib in NSCLC cell lines
Figure imgf000053_0001
H460
NCI- AD Wt mut 60.6 ± 4.3 6.8 ± 0.6 8.9
H2122
NCI- AD Wt mut 32.3 ± 0.4 2.9 ± 0.2 1 1
H2009
NCl- AD Wt mut 39.5 ± 4.4 12.9 ± 2.4 3.1
H1355
NCI- AD Wt mut 93.3 ± 7.8 49.2 ± 3.9 1.9
H I 792
NCI- AD mut Wt 24.1 ± 5.6 17.8 ± 2.7 1.4
H I 6450
NCI- AD mut Wt 10.2 ± 1.2 7.7 ± 0.7 1.3
HI 975
Note: * The mutation status of NSCLC cell lines were determined as described in Materials and Methods. ^ The IC50 of erlotinib was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves. Bold values indicate sensitivity to erlotinib (see text). SQ: squamous carcinoma; AD: adenocarcinoma; LC: large cell; wt: wild type; mut: mutant.
[00152] DNA isolation and PCR. DNA was isolated from above NSCLC cell lines and polymerase chain reaction (PCR) was performed to determine the KRAS and EGFR mutation status of each cell line according to previously reported methods (16).
[00153] MTS assays. Drug sensitivity was measured by a 3-(4, 5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (Promega, Madison, Wl). 50 μl of an exponentially growing cell suspension (cell numbers vary from 500 to 3000 /ml) was seeded into a 96-well microtiter plate, and 50 μl of various concentrations of each drug was added. After incubation for 72 hr at 37 °C, 20 μl of MTS solution was added to each well, and the plates were incubated for one hour at 37 °C. Optical density was measured at 562 and 630 nm using a SpectraMax 190 spectrophotometer (Molecular Devices, Sunnywale, CA). Each experiment was carried out in 8 replicate wells for each drug concentration. The IC5Q-value was defined as the concentration needed for a 50% reduction in the absorbance calculated based on the cell viability curves. [00154] Apoptosis assays. Apoptosis assay was preformed using cell death detection ELISA (Roche Applied Science, Indianapolis, IN) according to the manufacturer's protocol. For Hoechst staining, cells were fixed with cold methanol for 2 minutes, stained with DNA dye Hoechst 33258 for 10 minutes, washed with Ix PBS for 10 minutes, and observed by microscopy.
[00155] Xenograft animal models. 5 xl O6 H 1299 cells were re-suspended in 200 μl of RPMl and inoculated into the subcutaneous tissue of the flanks of twenty 5-week-old female nude athymic mice (Charles river Laboratories, Wilmington, MA). After tumors developed, romidepsin was injected intraperitoneally into the mice 3 times at 4 day intervals (1.2 mg/kg body weight). Erlotinib was injected 5 days a week (50 mg/kg body weight). The Ix PBS solution was used as a control solution. Tumor sizes were measured and calculated from the following formula: tumor size = L x W2/2, where L and W represent the length and the width of the tumor mass respectively.
[00156] Western blot analysis. Cells were lyzed in lysis buffer with protease inhibitor and phosphotase inhibitor cocktail added. Twenty micrograms of protein were subjected to a 12% SDS-PAGE (Bio-Rad, Hercules, CA). After transferring onto a nitrocellulose membrane (Osmonics, Minnetonka, MN), the membrane was blocked for 1 hr with Ix TBS containing 3% BSA and 0.1% Tween 20, then the primary antibody was incubated overnight at 4 °C followed by a corresponding secondary antibody. After three times of washing with IxTBS containing 0.1% Tween 20, the membranes were developed using ECL Plus (Amersham, Piscataway, NJ). p21 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Bim, phosphorylated or total p42/p44 antibody, and phosphorylated or total AKT antibody were purchased from Cell signaling Technology (Danvers, MA). Actin antibody was purchased from Sigma (St. Louis, MO).
[00157] Statistical analysis. The Welch t test was used to determine the potential differences between the control group and the drug treatment groups. PO.05 was considered statistically significant.
Results
[00158] Romidepsin decreases the IC50 of erlotinib in NSCLC cell lines. We first examined the cytotoxicity of romidepsin in NSCLC cell lines. Sixteen NSCLC cell lines were used including fourteen EGFR wild type cell lines, and two EGFR mutant cell lines known to be resistant to erlotinib. Among EGFR wild type cell lines, seven cell lines contained KRAS mutations. The IC50 values of romidepsin in the NSCLC cell lines examined ranged from 1.2 to 3.5 ng/ml. We used romidepsin at a concentration of 1 ng/ml for the combined treatment. At this concentration, romidepsin alone only showed low cytotoxic effect. The NSCLC cell lines were treated with varied concentrations of erlotinib either in the absence or presence of 1 ng/ml romidepsin. MTS assays were performed to determine the cell viability and four representative pairs of cell viability curves are presented in Figure 6. The IC50 value of erlotinib for each cell line was calculated based on the curves and is showed in the table above. We found that the IC5O values of erlotinib in thirteen out of the sixteen NSCLC cell lines decreased in the presence of 1 ng/ml romidepsin. The fold decreases in sensitivity ranged from 1.9 to 164, with a median of 13. These thirteen NSCLC cell lines had different histologies including adenocarcinoma, squamous carcinoma, and large cell types. Furthermore, in five of the seven EGFR and KRAS wild type NSCLC cell lines, the IC50 values of erlotinib were decreased to less than 2.5 μM, which is the threshold to distinguish sensitive from resistant cell lines (17). It is known that KRAS mutant NSCLC cell lines are resistant to TKl inhibitors (18). Our data indicated that romidepsin could also sensitize KRAS mutant cell lines in response to erlotinib. However, only one of the seven KRAS mutant cell lines reached the sensitive threshold of less than 2.5 μM in the presence of 1 ng/ml romidepsin. For the two erlotinib resistant EGFR mutant cell lines H 1975 and H1650, addition of romidepsin showed little effect on the sensitivity of the erlotinib. [00159] Combination of romidepsin and erlotinib induces profound apoptosis in HCC 15 NSCLC cell line. We next examined whether inducing apoptosis played a role in the ability of romidepsin to sensitize erlotinib. HCC 15 cells were treated with either 5 μM erlotinib or 2 ng/ml romodepsin, or the combination of these two drugs. After 72h, apoptosis was determined using cell death detection ELISA kit (Roche Applied Science). The mono- and oligonucleosomes indicating apoptosis were detected by antibodies against DNA and histones and quantitated by densitometry. As shown in Figure 7 B, while 5μM of erlotinib did not induce apoptosis and 2 ng/ml of romidepsin caused a 2.7 fold increase in apoptosis, co- treatment of erlotinib and romidepsin caused a 5.9 fold increase in apoptosis. We also stained the nuclei with a DNA dye Hoechst 33258. Apoptotic cells with shrink or fragmented nuclei were detected under microscope. As shown n Figure 7 A, 5 μM of erlotinib did not induce apoptosis; 2ng/ml of romidepsin induced apoptosis to a moderate degree; combined erlotinib and romidepsin caused profound cell death. These data indicated that erlotinib and romidepsin were synergistic in inducing apoptosis.
[00160] Co-administration of romidepsin and erlotinib shows inhibition on NCI-Hl 299 cell line xenografts. We further studied the combined effect of romidepsin and erlotinib on tumor growth in vivo. We injected 5x106 NCI-H 1299 cells subcutaneously into twenty BALE/c athymic nude mice. After visible tumors were seen at day seven, the mice were divided into four groups (five mice per group). One group was used as a PBS-treated control. The other three groups were intraperitoneal^ injected with either romidepsin alone, erlotinib alone or the combination of both agents. Tumor size was measured at the indicated days. After 20 days, the mice were sacrificed. As shown in Figure 8, erlotinib or romidepsin treatment alone failed to significantly suppress NCI-Hl 299 cell line xenograft growth (p > 0.05 vs. control group). Only combined treatment caused a significant decrease on tumor growth (p < 0.05 vs. control group).
[00161] Romidepsin down-regulates MAPK and AKT pathways, increases p21 and Bim expression. Finally, we studied the effects of erlotinib and/or romidepsin treatment on signaling pathways and gene expression patterns. NCl-H 1299, HCCl 93, and HCC 15 NSCLC cell lines were treated either with 5 μM erlotinib or 1 ng/ml romidepsin, or in combination. After 24 hours, cells were lysed and protein expression was studied by western blot analysis. As shown in Figure 10, phosphorylated levels of ERK 1/2 and AKT protein decreased upon romidepsin treatment alone or with erlotinib, indicating that MAPK and AKT pathways were down-regulated by romidepsin. The expression of the cyclin kinase inhibitor p2l dramatically increased after romidepsin alone or in combination while only modest changes Bim expression were seen.
Discussion
[00162] EGFR TKIs such as erlotinib and gefitinib have been found to be efficient in the treatment of NSCLCs that express mutant EGFR (3, 4). While ethnic and geographic differences exist, about 20% NSCLCs contain mutant EGFR (19). The majority of NSCLCs containing wild type EGFR are resistant to EGFR TKIs (17). Several new TKIs have been developed to treat NSCLCs that have a broader spectrum kinase activity. For example, EXEL-7647 (XL647), a novel spectrum-selective kinase inhibitor with potent activity against the EGF and vascular endothelial growth factor receptor tyrosine kinase families, has shown efficiency in therapy of both wild-type and mutant EGFR in vitro and in vivo (20). In addition, combinational therapies have been used to overcome NSCLC resistance to EGFR TKIs. For example, the combined use of erlotinib and the humanized vascular endothelial growth factor receptor monoclonal antibody bevacizumab in advanced, chemotherapy- refractory NSCLCs has shown promising results (21). HDAC inhibitors have pleiotropic effects on gene expression, growth arrest and apoptosis, and therefore are excellent candidates for combination therapies (15). Synergistic or additive activity has been reported between HDAC inhibitors and TKIs. HDAC inhibitor MS 125 was shown to increase the sensitivity of gefitinib in NSCLCs (22).
[00163] We have found that the HDAC inhibitor romidepsin sensitized the TKI erlotinib in thirteen of sixteen NSCLC cell lines examined. The concentration of romidepsin used for our in vitro combination treatment is far below 25 ng/ml that corresponds to 50% of the free drug concentration in plasma from lung cancer patients receiving this drug at the maximum tolerated dose (23). This suggested that the combined treatment of romidepsin and erlotinib was feasible in clinical trials. Our data indicated that romidepsin was effective in increasing the sensitivity of many NSCLC cell lines with wild type EGFR to erlotinib. IC50 values less than 2.5 μM are considered to be sensitive to erlotinib in vitro (17), which is approximately corresponding to the plasma steady-state concentration of erlotinib in patients treated with a dose of 150 mg daily (24). In our studies, in the presence of romidepsin, the IC50 values of five NSCLC cell lines containing wild type EGFR decreased to around 1 μM and therefore are regarded as sensitive to erlotinib. There are about 10% NSCLCs containing KRAS mutations(19). It is known that these tumors are resistant to EFGR TKIs (18). Our results showed that romidepsin could also increase the sensitivity of erlotinib in KRAS mutant NSCLC cell lines, but to a lesser degree compared with NSCLC cell lines containing wild type KRAS, indicating that there are resistant mechanisms existing in KRAS mutant cell lines which are not fully reversed by addition of romidepsin. Since most of EGFR mutant cell lines are very sensitive to erlotinib, we only examined two which exhibit resistance to erlotinib: H 1975 cell tine has a secondary T790M mutation, which is responsible for the resistance (5); H1650 cell line has a homozygous deletion of PTEN (25). We found that addition of romidepsin showed little effect on the sensitivity of the erlotinib in these NSCLCs, suggesting that romidepsin was unlikely to increase the sensitivity of erlotinib in TKI resistant EGFR mutant NSCLC cell lines.
[00164] Besides the secondary T790M mutation, several other mechanisms have been proposed to explain the resistance of NSCLCs to EGFR TKIs. It is reported that epithelial to mesenchymal transition (EMT) is a determinant of sensitivity of NSCLCs to EGFR inhibition (26). EMT is characterized by the combined loss of epithelial cell junction proteins such as E-cadherin and the gain of mesenchymal markers such as vimentin or fibronectin (27). Restoring E-cadherin expression has been shown to increase sensitivity to EGFR inhibitors in NSCLCs (22). Since we did not detect the induction of E-cadherin in romidepsin treated NSCLC cell lines, it is unlikely that our findings were due to E-cadherin induction by romidepsin. Persistent activity of MAPK or/and AKT pathway has also been related to EGFR TKl resistance. Treatment of specific inhibitors of MAPK or AKT pathway can increase the sensitivity of NSCLC cell lines to TKIs (6). Our data indicated that romidepsin down-regulated the MAPK and AKT pathways in NSCLC cell lines, suggesting that modification of EGFR downstream signaling pathway by romidepsin may be the basis for the synergistic effects seen with the combination of erlotinib and romidepsin. Furthermore, romidepsin increased the expression of the cyclin dependent kinase inhibitor p21. Bim belongs to the BH3-only group of protein, can bind Bcl2 and inhibit its function (28). Induction of important players in the apoptosis pathway or cell cycle control by romidepsin may also be mechanisms accounting for the synergy.
[00165] Our data have shown that romidepsin increased the sensitivity of erlotinib in wild type EGFR NSCLC cell lines, especially in the cell lines containing wild type KRAS, and including all of the major histological NSCLC types. EGFR and KRAS are mutually exclusive in NSCLCs (29). Although both mutations are subject to geographic variation, the overall occurrence of these two mutations in NSCLCs is about 30% (30). Therefore, about 70% of NSCLCs are wild type for both EGFR and KRAS. Our data indicate that erlotinib and romidepsin combination may benefit this large subgroup of NSCLC patients. [00166] Without wishing to be bound by any particular theory, we note that these data suggest that romidepsin enhances the anti-tumor effect of erlotinib through suppression of the MAPK pathway. According to the present invention, therefore, combination of DAC inhbitors (e.g., HDAC inihibtors) and tyrosine kinase inhibitors may have clinical use in the treatment of non-small cell lung cancers, especially in treatment of EGFR wild type tumors.
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Equivalents
[00197] The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims. [00198] To give but a few examples, in the claims articles such as "a", "an", and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.
[00199] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. In addition, the invention encompasses compositions made according to any of the methods for preparing compositions disclosed herein.
[00200] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It is also noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, steps, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, steps, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. Thus for each embodiment of the invention that comprises one or more elements, features, steps, etc., the invention also provides embodiments that consist or consist essentially of those elements, features, steps, etc.
[00201] Where ranges are given, endpoints are included unless otherwise indicated. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
[00202] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention can be excluded from any one or more claims. For example, in certain embodiments of the invention the biologically active agent is not an anti-proliferative agent. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

Claims

ClaimsWhat is claimed is:
1 . A method comprising steps of: administering to a subject suffering from or susceptible to a cell proliferative disorder, combination therapy of a DAC inhibitor and a tyrosine kinase inhibitor.
2. The method of claim 1 wherein the cell proliferative disorder involves a tumor.
3. The method of claim 2, wherein the tumor is a lung tumor.
4. The method of claim 2 or claim 3, wherein the tumor is a non-small cell lung cancer.
5. The method of any one of claims 1 -4 wherein the DAC inhibitor is romidepsin.
6. The method of any one of claims 1 -5 wherein the tyrosine kinase inhibitor is erlotinib.
7. The method of any one of claims 1-6 further comprising administering electrolyte supplementation.
8. A method of treating cancer or other neoplasm in a subject, the method comprising steps of: administering a therapeutically effective amount of romidepsin and a tyrosine kinase inhibitor to a subject with cancer or other neoplasm.
9. The method of claim 8, wherein romidepsin is of the formula:
Figure imgf000066_0001
10. The method of claim 1 , wherein the tyrosine kinase inhibitor is selected from the group consisting of axitinib, cediranib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, lestaurtinib, nilotinib, semaxanib, sunitinib, and vandetanib.
1 1. The method of claim 8, wherein the tyrosine kinase inhibitor is erlotinib.
12. The method of claim 8, wherein the cancer is a solid tumor.
13. The method of claim 8, wherein the cancer is a lung cancer.
14. The method of claim 8, wherein the cancer is a non-small cell lung cancer.
15. The method of claim 8, wherein the therapeutically effective amount of romidepsin ranges from approximately 0.5 mg/m to approximately 28 mg/m .
16. The method of claim 8. wherein the therapeutically effective amount of romidepsin ranges from approximately 1 mg/m2 to approximately 15 mg/m2.
17. The method of claim 8, wherein the therapeutically effective amount of romidepsin ranges from approximately 4 mg/m2 to approximately 15 mg/m2.
18. The method of claim 8, wherein the therapeutically effective amount of romidepsin ranges from approximately 8 mg/m" to approximately 14 mg/m".
19. The method of claim 8, wherein the therapeutically effective amount of romidepsin ranges from approximately 4 mg/m2 to approximately 10 mg/m2.
20. The method of claim 8, wherein the therapeutically effective amount of romidepsin is approximately 8 mg/m2.
21. The method of claim 8, wherein the therapeutically effective amount of romidepsin is approximately 10 mg/m2.
22. The method of claim 8, wherein the therapeutically effective amount of romidepsin is approximately 12 mg/m".
23. The method of claim 8, wherein the therapeutically effective amount of romidepsin is approximately 14 mg/m2.
24. The method of claim 8, wherein the therapeutically effective amount of erlotinib is approximately 150 mg/day.
25. The method of claim 8, wherein the therapeutically effective amount of erlotinib ranges from approximately 100 mg/day to approximately 150 mg/day.
26. The method of claim 8, wherein the therapeutically effective amount of romidepsin ranges from 4 mg/m2 to 15 mg/m2; and wherein the therapeutically effective amount of erlotinib is approximately 150 mg/day.
27. The method of claim 8, wherein either romidepsin or erlotinib is administered in combination at a dosage lower than when either is administered alone.
28. The method of claim 8, wherein both the dosages of romidepsin and bortezomib administered in combination are lower than the dosage of each administered alone.
29. The method of claim 8 further comprising administering another anti-neoplastic agent.
30. The method of claim 8 further comprising administering a cytotoxic agent.
31. The method of claim 8, wherein romidepsin is administered intravenously.
32. The method of claim 8, wherein romidepsin is administered bimonthly, monthly, triweekly, biweekly, weekly, twice a week, daily, or at variable intervals.
33. The method of claim 8, wherein romidepsin is administered weekly, and the tyrosine kinase inhibitor is administered daily.
34. A method of treating non-small cell lung cancer in a subject, the method comprising steps of: administering a therapeutically effective amount of romidepsin and erlotinib to a subject with multiple myeloma.
35. The method of claim 34, wherein the therapeutically effective amount of romidepsin ranges from 4 mg/m2 to 15 mg/m2.
36. The method of claim 34, wherein the therapeutically effective amount of romidepsin ranges from 8 mg/m2 to 10 mg/m2.
37. The method of claim 34, wherein the therapeutically effective amount of erlotinib is approximately 150 mg.
38. The method of claim 34, wherein the therapeutically effective amount of romidepsin ranges from 8 mg/m2 to 10 mg/m2; and wherein the therapeutically effective amount of erlotinib is approximately 150 mg.
39. The method of claim 34, wherein romidepsin is administered weekly, and erlotinib is administered daily.
40 The method of claim 34, wherein the non-small cell lung cancer is wild type EGFR and KRAS.
41. A method of treating cells, the method comprising steps of: administering a combination of romidepsin and erlotinib to a cell.
42. The method of claim 41 , wherein the step of administering comprises administering a combination of romidepsin and erlotinib to a cell at a concentration sufficient to kill the cell.
43. The method of claim 41 , wherein the cell is a cancer cell.
44. The method of claim 41 , wherein the cell is derived from a cancer cell line.
45. The method of claim 41 , wherein the cell is derived from a primary cancer.
46. The method of claim 41 , wherein the cell is a mammalian cell.
47. The method of claim 41 , wherein the cell is a rodent cell.
48. The method of claim 41 , wherein the cell is a human cell.
49. The method of claim 41 , wherein cell is a wild type EGFR and KRAS cell.
50. A method of inducing apoptosis in a cell, the method comprising: administering an amount of romidepsin and erlotinib effective to induce apoptosis in a cell.
51. A pharmaceutical composition for treating cancer comprising a therapeutically effect amount of romidepsin, and a therapeutically effective amount of erlotinib.
52. The pharmaceutical composition of claim 51 , wherein the romidepsin and erlotinib are packaged separately.
53. The pharmaceutical composition of claim 51 , wherein the amount of each of romidepsin and erlotinib is less than the amount of the agent alone used to treat a cancer.
54. The pharmaceutical composition of claim 51 , wherein the cancer is lung cancer.
55. The pharmaceutical composition of claim 51 , wherein the cancer is non-small cell lung cancer.
56. A kit comprising a pharmaceutical composition of claim 51.
57. The kit of claim 56, wherein the kit comprises multiple dosage units of the pharmaceutical composition of claim 48.
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