WO2011056542A1 - Thérapie cancéreuse avec combinaisons de fts et d'inhibiteurs hdac - Google Patents

Thérapie cancéreuse avec combinaisons de fts et d'inhibiteurs hdac Download PDF

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WO2011056542A1
WO2011056542A1 PCT/US2010/054042 US2010054042W WO2011056542A1 WO 2011056542 A1 WO2011056542 A1 WO 2011056542A1 US 2010054042 W US2010054042 W US 2010054042W WO 2011056542 A1 WO2011056542 A1 WO 2011056542A1
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cancer
fts
ras
hdac inhibitor
cells
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Yoel Kloog
Michael Brownstein
Roni Haklai
Anat Biran
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Ramot At Tel-Aviv University Ltd.
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    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Definitions

  • Ras proteins are central to the control of growth in both normal cells and cancerous or malignant cells alike. They are known to be mutated in approximately 30% of all human cancers . In order to exert their activity, normal Ras proteins must undergo at least three changes, the first of which is a chemical modification that entails attachment of a prenyl group. When that prenyl group is the triterpenoid farnesyl group, the attachment step is known as farnesylation . This reaction is catalyzed by an enzyme known as farnesyl transferase. Normal farnesylated Ras then undergoes additional enzyme-catalyzed cleavage of several amino acid residues, a step that is promoted by its interaction with growth factor receptors. Then, the Ras proteins must become activated, which entails docking or anchoring to galectin which is a protein located on the inner surface of the cell membrane.
  • cell division is controlled in part by the amount of activated farnesylated Ras attached to the cell membrane, which is in balance with inactive farnesylated Ras in the cytosol.
  • that balance is abnormal and is shifted toward greater amounts of membrane-bound Ras, especially mutated Ras, which remains anchored to the cell membrane. This imbalance results in uncontrolled cell division that is characteristic of this disease .
  • Ras proteins Activation of Ras proteins and the ensuing downstream events have been extensively researched, particularly in the context of cancer.
  • inhibitors of Ras proteins have been developed with the goal of suppressing Ras activity.
  • farnesyl transferase inhibitors growth factor receptor inhibitors which hinder the maturation of farnesylated Ras
  • Ras antagonists that target the anchoring of farnesylated Ras to galectin have all been the subject of human clinical trials.
  • the Ras antagonist S-trans trans- farnesylthiosalicylic acid (FTS) has proven effective in clinical trials to date in connection with several cancers.
  • FTS trans- farnesylthiosalicylic acid
  • a first aspect of the present invention is directed to a composition that includes S-trans , trans- farnesylthiosalicylic acid (also referred to herein as FTS or Salirasib) or an FTS analog, which together are defined by the formula described herein, an inhibitor of a histone deacetylase enzyme (referred to herein as an HDAC inhibitor) , and a pharmaceutically acceptable carrier.
  • FTS and the HDAC inhibitor are present in the composition in effective amounts.
  • a second aspect of the present invention is directed to a method of treating a cancer patient that includes co ⁇ administration of FTS or an FTS analog, and the HDAC inhibitor.
  • the treatment is carried out by administration of these anti-cancer agents in a single composition .
  • Fig. 1 is a bar graph that shows growth inhibition of three different cancer cell lines, namely human A549 cells (non-small cell lung cancer cells), DLD1 cells (human colon adenocarcinoma cells) and ARO cells (thyroid carcinoma cells), in the presence of 0.1% Me 2 S0 4 (control) or the indicated concentrations of FTS, and the HDAC inhibitor valproic acid (VPA) or a combination of the two inhibitors, wherein the number of viable cells in the treated cultures is expressed as a percentage of the total viable number of cells counted in the control .
  • human A549 cells non-small cell lung cancer cells
  • DLD1 cells human colon adenocarcinoma cells
  • ARO cells thyroid carcinoma cells
  • Figs. 2A and B are graphs that show synergistic growth inhibitory effect of VPA plus FTS on A549 and DLDl cells which had been incubated with FTS and VPA for 16 days, and wherein cells were counted at days 7, 12 and 16 , and wherein the number of cells that are in S-Phase of mitosis in the treated cultures was determined by FACS analysis and is expressed as a percentage of the control number of S-phase cells .
  • Fig. 2C is a bar graph showing, via FACS analysis, that VPA plus FTS achieved a synergistic growth inhibition of A549, DLDl and ARO cells in the S-phase (after 72 hrs) .
  • Figs. 3A and B show that combined treatment of FTS and VPA inhibits signaling in A549 and DLDl cells following treatment with the indicated concentrations of FTS and VPA for 72h, wherein Fig. 3A shows photos of electrophoretic gels showing total levels of Cyclin Dl, Ras, Survivin and ⁇ -Tubulin after lysing the cells and then immunoblotting, and wherein Fig. 3B is a bar graph showing real-time PCR analysis of survivin transcripts in control A549 cells and in FTS, VPA and VPA plus FTS A549-treated cells, wherein data are expressed as normalized survivin transcripts in drug-treated cells relative to the normalized control levels in percentage.
  • Fig. 4 is a bar graph that shows that the combination of VPA and FTS synergistically inhibits aurora kinase A transcription and interferes with mitosis in A549 cells, via real-time PCR analysis of aurora A (Aurk-A) transcripts in control, FTS, VPA and VPA plus FTS A549 treated cells (72h) , wherein data are expressed as percentage of normalized aurora A transcripts in drug-treated cells relative to the normalized control levels .
  • Fig. 4 is a bar graph that shows that the combination of VPA and FTS synergistically inhibits aurora kinase A transcription and interferes with mitosis in A549 cells, via real-time PCR analysis of aurora A (Aurk-A) transcripts in control, FTS, VPA and VPA plus FTS A549 treated cells (72h) , wherein data are expressed as percentage of normalized aurora A transcripts in drug-treated cells relative to the normalized control levels .
  • FIG. 5 is a bar graph that shows real-time PCR analysis of NuSAP transcripts in control FTS, VPA and VPA-plus- FTS A549-treated cells (72h) , wherein the level of NuSAP transcripts was normalized to the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and wherein data are expressed as normalized AurK-A transcripts in drug-treated cells relative to the normalized control levels in percentage .
  • GPDH glyceraldehyde-3-phosphate dehydrogenase
  • Figs. 6A and B are graphs showing the synergistic growth inhibitory effect of SAHA plus FTS on A549 and SW-480 cells respectively, at the indicated concentrations, for 18 days, wherein all cells were counted at days 7, 14 and 18.
  • Ras proteins e.g., H-, N- and K- ras, act as on-off switches that regulate signal-transduction pathways controlling cell growth, differentiation, and survival.
  • They are anchored to the inner leaflet of the plasma membrane, where activation of cell-surface receptors, such as receptor tyrosine kinase, induces the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on Ras and the conversion of inactive Ras-GDP to active Ras-GTP.
  • GDP guanosine diphosphate
  • GTP guanosine triphosphate
  • the active Ras protein promotes oncogenesis through activation of multiple Ras effectors that contribute to deregulated cell growth, differentiation, and increased survival, migration and invasion.
  • Ras effectors that contribute to deregulated cell growth, differentiation, and increased survival, migration and invasion.
  • FTS is known as a Ras inhibitor that acts in a rather specific manner on the active, GTP-bound forms of H-, N-, and K- Ras proteins.
  • Ras inhibitor that acts in a rather specific manner on the active, GTP-bound forms of H-, N-, and K- Ras proteins.
  • FTS competes with Ras-GTP for binding to specific saturable binding sites in the plasma membrane, resulting in mislocalization of active Ras and facilitating Ras degradation.
  • Ras antaqonists useful in the present invention include FTS and its structural analoqs, are described below.
  • R represents farnesyl, or qeranyl- qeranyl
  • R 2 is COOR 7 , CONR 7 R 8 , or COOCHR 9 OR 10
  • R 7 and R 8 are each independently hydroqen, alkyl, or alkenyl, includinq linear and branched alkyl or alkenyl, which in some embodiments includes C1-C4 alkyl or alkenyl
  • R 9 represents H or alkyl
  • R 10 represents alkyl, includinq linear and branched alkyl and which in some embodiments represents C1-C4 alkyl
  • R 3 , R 4 , R 5 and R 6 are each independently hydroqen, alkyl, alkenyl, alkoxy (includinq linear and branched alkyl, alkenyl or alkoxy and which in some embodiments are Cl- C4 alkyl, alkenyl or alkoxy) , halo, trifluoromethyl, trifluorome
  • any of R 7 , R 8 , R 9 and/or R 10 represents alkyl, it is preferably a methyl or ethyl qroup .
  • the Ras antaqonists may be present in the form of a pharmaceutically acceptable salt, or any other form in which it is therapeutically effective.
  • the Ras antaqonist is S- trans , trans-farnesylthiosalicylic acid or FTS (wherein R 1 is farnesyl, R 2 is COOR 7 , and R 7 is hydroqen) .
  • the FTS analog is halogenated, e.g., 5-chloro-FTS (wherein R 1 is farnesyl, R 2 is COOR 7 , R 4 is chloro, and R 7 is hydrogen) , and 5-fluoro-FTS (wherein R 1 is farnesyl, R 2 is COOR 7 , R 4 is fluoro, and R 7 is hydrogen) .
  • the FTS analog is FTS-methyl ester (wherein R 1 represents farnesyl, R 2 represents COOR 7 , and R 7 represents methyl) , FTS-amide (wherein R 1 represents farnesyl, R 2 represents CONR 7 R 8 , and R 7 and R 8 both represent hydrogen) ; FTS-methylamide (wherein R 1 represents farnesyl, R 2 represents CONR 7 R 8 , R 7 represents hydrogen and R 8 represents methyl); and FTS-dimethylamide (wherein R 1 represents farnesyl, R 2 represents CONR 7 R 8 , and R 7 and R 8 each represents methyl) .
  • the Ras antagonist is an alkoxyalkyl S-prenylthiosalicylate or an FTS-alkoxyalkyl ester (wherein R 2 represents COOCHR 9 OR 10 ) .
  • Representative examples include methoxymethyl S-farnesylthiosalicylate (wherein R 1 is farnesyl, R 9 is H, and R 10 is methyl); methoxymethyl S- geranylgeranylthiosalicylate (wherein R 1 is geranylgeranyl, R 9 is H, and R 10 is methyl) ; methoxymethyl 5-fluoro-S- farnesylthiosalicylate (wherein R 1 is farnesyl, R 5 is fluoro, R 9 is H, and R 10 is methyl) ; and ethoxymethyl S- farnesylthiosalicyate (wherein R 1 is farnesyl, R 9 is methyl and R 10 is ethyl) .
  • each of R 3 , R 4 , R 5 and R 6 represents hydrogen.
  • Histones are small proteins that complex with DNA.
  • Two of each of the histones known as H2A, H2B, H3 and H4 are tightly complexed with DNA, typically in amounts of about 150 base pairs, to form a nucleosome. This structure is further connected by linker DNA to form a solenoid.
  • Histone acetyltransferases (HATs) and histone deacetylases (HDACs) represent two enzyme families that control the level of histone tail acetylation by the addition and removal respectively, of an acetyl group from the lysine residues of core nucleosomal histories.
  • Histones extending from the nucleosomal core are thus enzymatically modified, affecting chromatin structure and gene expression.
  • HATs via acetylation of histones, allow transcription and gene expression since acetylated histones can recruit transcription factors and other co-activator proteins.
  • HDACs are usually associated with DNA hyper-methylation and gene silencing.
  • HDACs play an important role in cell proliferation and differentiation. Acetylated histones are relatively less effective in facilitating DNA transcription, whereas deacetylated histones are relatively more effective in facilitating this process. Acetylation and deacetylation are in balance in normal cells. However, in malignant cells, there is an excess of HDAC activity compared to HAT activity, resulting in an imbalance of deacetylated histones to acetylated histones. This imbalance results in excessive DNA transcription and uncontrolled cell proliferation. The involvement of HDACs in the control of cell proliferation and differentiation suggests that aberrant HDAC activity may play a role in cancer .
  • the HDAC enzyme family includes at least 18 enzymes, grouped in four (4) classes (Classes I, Ila, lib, III and IV) .
  • Class I HDACs include HDACs 1, 2, 3, and 8.
  • Class I HDACs can be found in the nucleus and are believed to be involved with transcriptional control repressors and co-factors.
  • Class Ila HDACs include HDACS 4, 5, 7 and 9, and
  • Class lib HDACs include HDACs 6 and 10. These enzymes can be found in both the cytoplasm as well as the nucleus.
  • Certain class I and class II HDACs are overexpressed in tumors relative to normal tissues. See, Johnstone, Nature Reviews Drug Disovery 1:287-99 (2002) .
  • Class III HDACs are believed to be NAD-dependent proteins and include members of the sirtuin family of proteins .
  • Non-limiting examples of sirtuin proteins include SIRT1-7.
  • Class IV HDACs include HDAC 11.
  • HDAC inhibitor refers to a compound that has the ability to inhibit histone deacetylase activity. This therapeutic class is able to block angiogenesis and cell cycling, and promote apoptosis and differentiation. HDAC inhibitors exhibit targeted anticancer activity and as disclosed herein, act synergistically with FTS and its analogs in the treatment of cancer.
  • Selective and non-selective HDAC inhibitors alike may be useful in the present invention.
  • the term "selective HDAC inhibitor” refers to an HDAC inhibitor that does not significantly interact with all three HDAC classes.
  • a "Class I selective HDAC” refers to an HDAC inhibitor that interacts with one or more of HDACs 1, 2, 3 or 8, but does not significantly interact with the Class II HDACs (i.e., HDACs 4-7, 9 and 10) .
  • the HDAC inhibitor is selective HDAC inhibitor, such as a Class I selective HDAC inhibitor.
  • a Class I selective HDAC inhibitor Representative examples of such inhibitors that may be useful in the present invention include benzamides such as MGCD-0103 (N- (2-amino-phenyl) -4- [ (4-pyridin- 3-yl-pyrimidin-2-ylamino) -methyl] -benzamide) , also known as Mocetinostat , and related compounds as disclosed in U.S.
  • Patent 6, 897, 220 benzamides such as MS-275 ( (N- ( 2-aminophenyl ) -4- (N- (pyridin-3-ylmethoxycarbonyl) aminomethyl) benzamide), also known as entinostat or "SNDX-275”), and related compounds as disclosed in e.g., U.S. Patent 6,174,905), spiruchostatin A, SK7041 and SK7068 (Class I HDAC inhibitors; Park, et al . , Clin. Cancer Res. 10:5271 (2004) ) and 6-amino nicotinamides.
  • the HDAC is a non-selective HDAC inhibitor.
  • HDAC inhibitors include hydroxamic acids including trichostatin analogs such as trichostatin A (TSA) and trichostatin C (TSC) (Koghe, et al., Biochem. Pharmacol. 55:1359-64 (1998), salicylihydroxamic acid (SBHA) (U.S.
  • Patent 5,608,108 azelaic bishydroxamic acid (ABHA) , azelaic-1- hydroxamate-9-analide (AAHA) , 6- (3-chlorophenylureido) carpoic hydroxamic acid (3C1-UCHA) , and ' -hydroxy-N-phenyl- octanediamide (suberoylanilide hydroxamic acid, also known as "SAHA” or vorinostat, and related hydroxamic acid compounds as disclosed in U.S.
  • TPX trapoxin
  • CBHA m- carboxycinnamic acid bishydroxamide
  • CBHA Richon, et al . , PNAS 95:3003-7 (1998)
  • oxamflatin A-161906, GCK1026
  • the non-selective HDAC inhibitor is a small-molecular weight carboxylate such as valproic acid (2-n-propylpentanoic acid, VPA) or a derivative thereof.
  • VPA has been reported to inhibit HDACs 1-3 (Class I) and HDACs -8 (Class II) .
  • Valproic acid is represented by the
  • VPA derivatives suitable for use in the present invention are represented by the formula
  • R 1 and R 2 each independently represents a linear or branched, saturated or unsaturated aliphatic 2 -25r preferably C3-25 hydrocarbon chain which optionally comprises one or several heteroatoms and which may be substituted, R 3 is hydroxyl, alkoxy or an optionally alkylated amino group.
  • the hydrocarbon chains R 1 and R 2 may contain one or several heteroatoms (e.g., 0, N, S) replacing carbon atoms in the hydrocarbon chain.
  • R 1 and R 2 may be substituted, e.g., with hydroxyl, amino, carboxylic and alkoxy groups as well as aryl and heterocyclic groups .
  • R 1 and R 2 independently contain 2 to 10, more preferably 3 to 10 or 5 to 10 carbon atoms. It is also preferred that R 1 and R 2 independently are saturated or contain one double bond or one triple bond.
  • VPA derivatives that may be particularly suitable include S-4-yn VPA and 2-EHXA (2- Ethyl-hexanoic acid) .
  • the HDAC inhibitor may be present in the form of a pharmaceutically acceptable salt, or any other form in which it is therapeutically effective.
  • HDAC inhibitors are now in use in clinical cancer trials. Aside from various HDAC inhibitors mentioned above, they include Panobinostat , Belinostat, ITF-2357, PC-24781, phenylbutyrate, SB-939 and JHJ-26481585.
  • the Ras antagonist and the HDAC inhibitor are co ⁇ administered, which as used herein, encompasses treatment regimens in which these anti-cancer agents are administered to the cancer patient at the same or different times (i.e., substantially simultaneously or sequentially) , and by the same or different route of administration, such that both agents and/or their metabolites are present in the patient at the same time in order to achieve the benefits of their combined therapeutic effect.
  • Co-administration thus includes simultaneous administration in separate compositions, administration at different times in separate compositions, and/or administration in a composition that contains both agents.
  • the Ras antagonist and the HDAC inhibitor are administered in a single composition.
  • the term "therapeutically effective amounts”, as used herein, refers to a sufficient amount of each of the Ras antagonist and the HDAC inhibitor that will ameliorate at least one symptom of the cancer and its associated manifestations, diminish extent or severity of the disease, delay or retard disease progression, achieve partial or complete remission, prolong survival and combinations thereof.
  • combinations of the Ras antagonist and the HDAC inhibitor achieve synergy, i.e., a greater than additive effect, at least with respect to inhibition of the growth of the cancer cells in vitro. Applicants believe that these results reflect tumor/cancer cell growth inhibitory activity in vivo, and ultimately result in more effective cancer therapy and a commensurate improvement in one or more of these clinical manifestations of the disease.
  • Appropriate "effective" amounts for any cancer patient can be determined using techniques, such as a dose escalation study. Specific dose levels for any particular patient will depend on several factors such as the potency of the Ras antagonist and the HDAC inhibitor, the age, weight, and general health of the patient, and the severity of the cancer.
  • the average daily dose of the Ras antagonists of the present invention generally ranges from about 200 mg to about 2000 mg (e.g., 200 mg, 400 mg, 600 mg, 800 mg, 1000 mg, 1200 mg, 1400 mg, 1600 mg, 1800 mg and 2000 mg) , in some embodiments from about 400 to about 1600 mg, and in some other embodiments from about 600 to about 1200 mg, and in yet other embodiments, from about 800 mg to about 1200 mg .
  • the average daily dose of the HDAC inhibitor will vary, depending on the specific agent.
  • the average daily dose for valproic acid (and its derivatives) for example, generally ranges from about 500 mg to about 3500 mg, in some embodiments from about 750 to 3000 mg, and in some other embodiments from about 750 mg to about 1500 mg .
  • the average daily dose for vorinostat (and its derivatives), for example, generally ranges from about 100 mg to about 600 mg, in some other embodiments from about 200 to about 500 mg, and in some other embodiments from about 300 mg to about 400 mg .
  • the average weekly dose for SNDX-275 (and its derivatives) for example, generally ranges from about 2.5 mg to about 10 mg and in some embodiments from about 5 mg to about 10 mg, once every two weeks .
  • administration, " and the like, as used herein, refer to the methods that may be used to enable delivery of compounds or compositions to the desired site of biological action.
  • Medically acceptable administration techniques suitable for use in the present invention are known in the art. See, e.g., Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed . ; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.
  • at least one or both active agents are administered orally.
  • at least one or both active agents are administered parenterally (which for purposes of the present invention, includes intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular and infusion) .
  • Other administration routes such as topical and rectal administration may also be suitable.
  • pharmaceutically acceptable refers to a material, such as a carrier and other non-active excipients, which does not abrogate the biological activity or properties of the active agent (s), and is relatively nontoxic.
  • composition refers to the Ras antagonist and/or the HDAC inhibitor, optionally combined (e.g., mixed) with a pharmaceutically acceptable carrier. These ingredients are non-toxic, physiologically inert and do not adversely interact with the active agent (s) present in the composition. Carriers facilitate formulation and/or administration of the active agents. Pharmaceutical compositions of the present invention may further contain one or more excipients.
  • compositions for the Ras antagonist and/or the HDAC inhibitor can be prepared by bringing the agent (s) into association with (e.g., mixing with) the carrier, the selection of which is based on the mode of administration.
  • Carriers are generally solid or liquid. In some cases, compositions may contain solid and liquid carriers.
  • Compositions suitable for oral administration that contain the active are preferably in solid dosage forms such as tablets (e.g., including film-coated, sugar-coated, controlled or sustained release) , capsules, e.g., hard gelatin capsules (including controlled or sustained release) and soft gelatin capsules, powders and granules.
  • compositions may be contained in other carriers that enable administration to a patient in other oral forms, e.g., a liquid or gel. Regardless of the form, the composition is divided into individual or combined doses containing predetermined quantities of the active ingredient or ingredient s .
  • Oral dosage forms may be prepared by mixing the active pharmaceutical ingredient or ingredients with one or more appropriate carriers (optionally with one or more other pharmaceutically acceptable excipients), and then formulating the composition into the desired dosage form e.g., compressing the composition into a tablet or filling the composition into a capsule or a pouch.
  • Typical carriers and excipients include bulking agents or diluents, binders, buffers or pH adjusting agents, disintegrants (including crosslinked and super disintegrants such as croscarmellose) , glidants, and/or lubricants, including lactose, starch, mannitol, microcrystalline cellulose, ethylcellulose , sodium carboxymethylcellulose , hydroxypropylmethylcellulose, dibasic calcium phosphate, acacia, gelatin, stearic acid, magnesium stearate, corn oil, vegetable oils, and polyethylene glycols.
  • Coating agents such as sugar, shellac, and synthetic polymers may be employed, as well as colorants and preservatives. See, Remington' s Pharmaceutical Sciences, The Science and Practice of Pharmacy, 20th Edition (2000) .
  • Liquid form compositions include, for example, solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
  • the active agent (s) for example, can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent (and mixtures thereof), and/or pharmaceutically acceptable oils or fats.
  • liquid carriers for oral administration include water (particularly containing additives as above, e.g., cellulose derivatives, preferably in suspension in sodium carboxymethyl cellulose solution) , alcohols (including monohydric alcohols (including monohydric alcohols and polyhydric alcohols, e.g., glycerin and non-toxic glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil) .
  • the liquid composition can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colorants, viscosity regulators, stabilizers or osmoregulators .
  • Carriers suitable for preparation of compositions for parenteral administration include Sterile Water for Injection, Bacteriostatic Water for Injection, Sodium Chloride Injection (0.45%, 0.9%), Dextrose Injection (2.5%, 5%, 10%), Lactated Ringer's Injection, and the like. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, and in oils.
  • Compositions may also contain tonicity agents (e.g., sodium chloride and mannitol) , antioxidants (e.g., sodium bisulfite, sodium metabisulfite and ascorbic acid) and preservatives (e.g., benzyl alcohol, methyl paraben, propyl paraben and combinations of methyl and propyl parabens) .
  • tonicity agents e.g., sodium chloride and mannitol
  • antioxidants e.g., sodium bisulfite, sodium metabisulfite and ascorbic acid
  • preservatives e.g., benzyl alcohol, methyl paraben, propyl paraben and combinations of methyl and propyl parabens
  • the pharmaceutical composition containing the Ras antagonist and the HDAC inhibitor, or first and second compositions containing the Ras antagonist and the HDAC inhibitor respectively, may be packaged and sold in the form of a kit.
  • the composition might be in the form of one or more oral dosage forms such as tablets or capsules (e.g., hard or soft gelatin capsules) containing one or both of the active agents.
  • the kit may also contain written instructions for carrying out the inventive methods as described herein.
  • the Ras antagonist is administered by dosing orally on a daily basis (in single or divided doses) for three weeks, followed by a one-week "off period", and repeating until remission is achieved.
  • the HDAC inhibitor may be present in the same composition.
  • VPA for example, is administered daily in single or divided dosages (e.g., 2 or 3 times daily) .
  • Vorinostat may be dosed once daily, e.g., at an initial dose of about 400 mg, which then is reduced to a daily dose of 300 mg and then may be continued at 300 mg every 5 consecutive days.
  • SNDX may be administered every two weeks in an amount of about 5 or 10 mg .
  • Cancer generally refers to a disease caused by the uncontrolled, abnormal growth of cells that can spread to adjoining tissues or other parts of the body.
  • Cancer cells can form a solid tumor, in which the cancer cells are massed together, or they can exist as dispersed cells, as in leukemia. Normal cells divide (reproduce) until maturation is attained and then only as necessary for replacement of damaged or dead cells. Cancer cells are often referred to as "malignant", because they divide endlessly, eventually crowding out nearby cells and spreading to other parts of the body. Malignant cancer cells eventually metastasize and spread to other parts of the body via the bloodstream or lymphatic system, where they can multiply and form new tumors. Malignant tumors are divided into carcinomas (which arise from epithelial precursor cells), sarcomas (which arise largely from mesenchymal tissues) and lymphomas (which arise from precursors of red and white blood cells) .
  • Cancers characterized by the presence of elevated wild-type Ras or the presence of mutated Ras proteins are amenable to treatment in accordance with the present invention.
  • These cancers include human lymphomas, sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, synovioma, mesothelioma, lymphangioendotheliosarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colorectal (e.g., colon) carcinoma, gastrointestinal (e.g., stomach) cancer, pancreatic cancer, thyroid cancer (e.g., follicular, papillary and anaplastic thyroid carcinomas), breast cancer, ovarian cancer, prostate cancer,
  • the invention is used to treat a patient afflicted with renal carcinoma, skin cancer, pancreatic cancer, colorectal (e.g., colon) cancer, NSCLC, ovarian cancer, hepatic (liver) cancer, thyroid cancer, seminoma, skin cancer, endometrial cancer, melanoma, leukemia, lymphoma, prostate cancer, bladder and urinary cancers, breast cancer, and brain metastases of these primary tumors, primary brain cancers (such as gliomas and neuroblastomas) and head and neck cancers. See, e.g., Bos, Cancer Res. 49:4682-89 (1989).
  • the combination of Ras antagonist and HDAC inhibitor may be used alone or in conjunction with other treatment agents such as biological anti-cancer agents (e.g., antibodies), chemotherapy and radiation.
  • the combination may be used as a front-line treatment strategy e.g., as a first course of treatment in a newly diagnosed cancer patient, and whether or not the cancer has metastasized.
  • the combination may also be used as a second-line treatment strategy e.g., in the treatment of a cancer patient who has been previously treated using at least one other agent but has not responded to the previous agent (s) or has developed a resistance thereto, which may have resulted in termination of the therapy even before it could achieve an appreciable therapeutic efficacy.
  • DLD1 and SW-480 which are human colon adenocarcinoma cells that also express oncogenic K-ras, were cultured in DMEM 10% FCS, 2mM L-Glutamine, 100 U/ml penicillin and 100 yg/ml streptomycin .
  • ARO, thyroid carcinoma cells wherein wild-type K-Ras is chronically active, and wherein B-Raf is constitutively active, were cultured in RPMI 1640 medium with 10% FCS, 2mM L- Glutamine, 100 U/ml penicillin and 100 yg/ml streptomycin.
  • FTS was prepared as previously described in Rotbalt, Meth. Enzymol. 439:467-89 (2008) .
  • VPA was stored at 4°C as a powder and before each experiment it was weighed and diluted with cell medium to the desired concentration.
  • SAHA was reconstituted in DMSO to a concentration of 5 mM, divided into 10 ⁇ stocks and stored at 4°C. For each experiment, one stock was diluted to the desired concentration using cell medium.
  • A549, DLD1, SW-480 and ARO Cells were plated at a density of 8xl0 3 cells per well in 24-well plates. After 24 hours cells were treated with VPA (Sigma), SAHA (Alexis) or FTS dissolved in 0.1% Me 2 S0 4 (Concordia Pharmaceuticals, Ft. Lauderdale, FL) or a combination of FTS with VPA, or a combination of FTS with SAHA, for the indicated periods of time. Control cells were treated with 0.1% Me 2 S0 4 . In the case of incubation times longer than 3 days the cells were detached, counted and re-plated at a density of 3xl0 5 cells per 10-cm plate. Below are the concentrations of VPA, SAHA and FTS that were used in the different cell lines:
  • A549, DLD1 and ARO cells (4xl0 5 cells per 10-cm plate) were plated for 24h and then incubated with VPA and FTS for additional 72h in 10% FCS-containing medium. The cells were then collected and resuspended in PBS containing propidium iodide (5C ⁇ g/mL; Sigma), 0.1% sodium citrate and 0.1% Triton x- 100 (BDH, Poole, United Kingdom) and incubated overnight in the dark at 4°C.
  • PBS propidium iodide
  • BDH Triton x- 100
  • A549, DLD1 and ARO cells were plated at density of 4xl0 5 cells per 10-cm plate and grown for 24h. The cells were then treated 0.1% Me 2 S0 4 (control), FTS, VPA, SAHA, a combined treatment of VPA plus FTS, or a combined treatment of FTS plus SAHA, for 72h.
  • the cells were lysed in 300 ⁇ 1 homogenization buffer (50 mmol/L Tris-HCl (pH 7.6), 20mM MgCl 2 , 200mM NaCl, 0.5% NP40, ImM DTT, and protease inhibitors) centrifuged for 10 min at 14,000 rpm at 4°C and the supernatant was collected. Equal amounts of proteins (50-100yg per lane) were subjected to SDS-PAGE, followed by immunoblotting with rabbit anti-cyclin Dl (1:1,000), mouse anti-pan-Ras antibody Ab (Calbiochem) , rabbit anti-Survivin Ab (Santa Cruz, CA) and rabbit anti- -tubulin Ab (Sigma) .
  • 300 ⁇ 1 homogenization buffer 50 mmol/L Tris-HCl (pH 7.6), 20mM MgCl 2 , 200mM NaCl, 0.5% NP40, ImM DTT, and protease inhibitors
  • A549 cells were plated on glass cover slips placed in 10-cm plates at a density of 4xl0 5 cells/plate for 24h before adding 0.1% Me 2 S0 4 , 75 ⁇ FTS or VPA 0.8mM or VPA+FTS for an additional 24 or 72 hours. Afterwards, the cells were fixed with formaldehyde at room temperature for 30 min and then treated with 0.2% Triton X-100.
  • phosphate-buffered saline PBS
  • slides were immersed in 1% bovine serum albumin (BSA) with 200yg/mL naive goat IgG (Jackson ImmunoResearch) for 30 min and then incubated with rabbit anti-Aurora A (1:50, Cell Signaling, Danvers, MA), anti- Aurora B (1:50, Bethyl Labs Montgomery, TX) or mouse anti- phosphor-H3 (1:50, Upstate, Charlottesville, VA) overnight at 4°C.
  • BSA bovine serum albumin
  • rabbit anti-Aurora A 1:50, Cell Signaling, Danvers, MA
  • anti- Aurora B 1:50, Bethyl Labs Montgomery, TX
  • mouse anti- phosphor-H3 1:50, Upstate, Charlottesville, VA
  • the cells were incubated with goat anti-mouse Cy3-conjugated Ab or with donkey anti-rabbit Cy2-conjugated Ab (1:200, Jackson ImmunoResearch) for 1 h in the dark. Then the cells were washed twice in PBS, and further incubated with anti-a-tubulin FITC Ab (1:50, sigma, F2168) for lh at room temperature. Finally, the cells were counterstained with Hoechst 33258 (Fluka AG, CH9470), and examined by fluorescent microscopy at 60x magnification with an LSM 510 META microscope.
  • Hoechst 33258 Fluka AG, CH9470
  • RNA samples were treated for 72 h with either 75 ⁇ FTS, 0.8mM VPA, 0.1% vehicle (control) or 75 ⁇ FTS plus 0.8mM VPA.
  • Total RNA was isolated from cultured cells using protocols and reagents contained in the RNeasy Plus Mini Kit (Qiagene) . The concentration of the RNA samples was determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer. Purified RNA was stored at -70°C in RNase- free water. The purified RNA was used for real-time PCR.
  • Extracts of total RNA (1 yg) were reverse-transcribed in a total volume of 20yL using the VersoTM RT-PCR Kits (ABgene) .
  • lpL cDNA samples were then used for real-time PCR (QPCR SYBR Green Mix Plus ROX Vial, ABgene) .
  • the primers used targeted Survivin, AurK-A and NuSap genes, and the housekeeping gene GAPDH. Primer sequences used for these experiments are set forth in the following table.
  • VPA and FTS synergistically in inhibit growth of cells with active Ras pathways.
  • VPA and FTS act synergistically to inhibit growth of NSCLC cells with active Ras pathways.
  • VPA and FTS synergistically down-regulate survivin in A549 and DLD1 cells
  • CPC chromosomal passenger complex
  • Typical results of these experiments indicated that FTS but not VPA caused a significant reduction in the levels of Ras and cyclin Dl in A549 and DLD1 cells. These results are in line with the known anti-Ras activity of FTS . Erlich, et al . , Biochem. Pharmacol. 72:427-36 (2006); Blum, et al . , Int. J. Cancer 119:527-38 (2006). VPA did not itself affect the levels of Ras and Cyclin Dl (Fig. 3A) . However, the effect of the combined treatment on Ras, Cyclin Dl, and survivin was clearly stronger than the effect of either drug alone (Fig. 3A) .
  • the positive control of Ras on survivin expression can be relieved by FTS (or by dominant-negative Ras) as manifested by a decrease in the level of survivin mRNA and protein in FTS- treated cells.
  • FTS or by dominant-negative Ras
  • the present results show a similar strong reduction in survivin protein level by FTS in A549 and DLD1 (Fig. 3A) . This result was unexpected at least from the standpoint that in addition to the cell growth cycle, the two drugs affected unrelated cellular pathways .
  • VPA and FTS synergistically inhibit Aurora kinase A transcription blocking mitosis in A549 cells
  • VPA and FTS synergistically block Aurora kinase B expression and histone-H3 phosphorylation
  • Aurora B is localized to the kinetechores during metaphase and aberrant aurora B expression coincides with reduced levels of phospho histone- H3 (serlO) which is critical for mitosis. Vader, Biochim. Biophys . Acta. 1786:60-12 (2008) .
  • Our previous gene profiling analysis in FTS-treated A549 cells (75 ⁇ FTS for 72 h) (Blum, et al . , Cancer Res. 57:3320-8 (2007)) showed a decrease in aurora B expression.
  • Other studies showed that HDAC inhibitors reduce the level of aurora B. Zhang, et al . , Cancer Biol. Ther. 7:1388-97 (2008) .
  • nucleolar and spindle associated protein is a novel microtubule- associated protein involved in mitotic spindle organization.
  • siRNA to NuSap disrupted formation of a normal spindle Raemaekers, et al., J. Cell. Biol. 152:1017-29 (2003).
  • FTS active pharmaceutical ingredient 2.0 kg
  • sodium valproate active pharmaceutical ingredient 2.3 kg
  • microcrystalline cellulose 2.0 kg
  • croscarmellose sodium 0.2 kg
  • magnesium stearate 0.1 kg
  • FTS active pharmaceutical ingredient (1.0 kg), sodium valproate (2.3 kg), microcrystalline cellulose (2.0 kg), croscarmellose sodium (0.2 kg), and magnesium stearate (0.1 kg) are blended to homogeneity and filled into hard gelatin capsules with an encapsulation machine. Assuming a 5% loss on material transfer and encapsulating machine start-up, adjustment, and shut-down, approximately 9,500 capsules are yielded .
  • FTS active pharmaceutical ingredient (2.0 kg), vorinostat active pharmaceutical ingredient (2.0 kg), microcrystalline cellulose (2.0 kg), croscarmellose sodium (0.2 kg), and magnesium stearate (0.1 kg) are blended to homogeneity and filled into hard gelatin capsules with an encapsulation machine. Assuming a 5% loss on material transfer and encapsulating machine start-up, adjustment, and shut-down, approximately 19,000 capsules are yielded.
  • FTS active pharmaceutical ingredient (2.0 kg) and VPA active pharmaceutical ingredient (2.0 kg) are dissolved in corn oil (4.0 kg) and stirred to form a homogeneous solution.
  • the solution is filled into soft gelatin capsules with a form- fill-seal encapsulation machine. Assuming a 5% loss on material transfers and soft-gelatin encapsulating machine start-up, adjustment, and shut-down, approximately 19,000 capsules containing FTS (100 mg) and VPA (100 mg) are yielded.

Abstract

La présente invention concerne des compositions pharmaceutiques qui contiennent des quantités efficaces de FTS (S-trans, acide trans-farnésylthiosalicylique ou Salirasib) ou un analogue de FTS, un inhibiteur de l'enzyme histone déacétylase (HDAC), et un vecteur pharmaceutiquement acceptable. L'invention porte en outre sur des méthodes de traitement du cancer par administration simultanée de quantités efficaces d'un antagoniste de Ras comprenant un FTS, ou un analogue de celui-ci, et d'un inhibiteur de HDAC à un patient atteint de cancer.
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