WO2018146316A1 - Combinaison d'un inhibiteur de voie mapk et d'un composé antisens ciblant kras - Google Patents

Combinaison d'un inhibiteur de voie mapk et d'un composé antisens ciblant kras Download PDF

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Publication number
WO2018146316A1
WO2018146316A1 PCT/EP2018/053463 EP2018053463W WO2018146316A1 WO 2018146316 A1 WO2018146316 A1 WO 2018146316A1 EP 2018053463 W EP2018053463 W EP 2018053463W WO 2018146316 A1 WO2018146316 A1 WO 2018146316A1
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kras
antisense compound
mapk pathway
compound targeted
cancer
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PCT/EP2018/053463
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English (en)
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Sarah Jane ROSS
Lyndsey HANSON
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Astrazeneca Ab
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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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic

Definitions

  • Sequence Listing is provided as a file entitled 200574_US_PSP_SequenceListing.txt created December 15, 2016, which is 76 kb in size.
  • the information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
  • methods, compounds, and compositions for treating cancer, for example KRAS mutant cancers, in an animal, by administering an agent capable of inhibiting expression of KRAS and another anti -tumour agent which is an inhibitor of the MAPK pathway, such as a MEK or ERK inhibitor, are provided herein.
  • the agent capable of inhibiting expression of KRAS is selective for inhibiting expression of KRAS.
  • the combination therapy involves administration to a patient in need thereof an antisense compound targeted to KRAS and an an inhibitor of the MAPK pathway.
  • Such methods, compounds, and compositions are useful to treat, prevent, or ameliorate KRAS mutant cancers, which may include, for example, certain lung, colon and pancreatic cancers.
  • MAP kinases play a key regulatory role in almost every aspect of cell biology.
  • the mammalian MAP kinases consist of cytoplasmic protein serine/threonine kinases that participate in the transduction of cellular signals from the plasma membrane to the nucleus.
  • MAPK signalling cascades each consisting of 3 components: a MAPK kinase (MAP3K), a MAPK kinase (MAP2K) and a MAPK.
  • MAP3K MAPK kinase
  • MAP2K MAPK kinase
  • the activated MAP kinases phosphorylate numerous substrates including other protein kinases, protein phosphatases, transcription factors and other functional proteins.
  • the RAS-RAF-MEK-ERK signalling cascade participates in the regulation of cell cycle progression, cell proliferation, survival, metabolism and transcription.
  • Kirsten Rat Sarcoma Viral Oncogene Homologue is one of three RAS protein family members (N, H and K-RAS) that are small membrane bound intracellular GTPase proteins. KRAS cycles between an inactive guanosine diphosphate (GDP)-bound state and an active guanosine triphosphate (GTP)-bound state. The process of exchanging the bound nucleotide is facilitated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). GEFs promote release of GDP from KRAS in exchange for GTP, resulting in active GTP -bound KRAS.
  • GDP inactive guanosine diphosphate
  • GTP active guanosine triphosphate
  • GEFs guanine nucleotide exchange factors
  • GAPs GTPase activating proteins
  • GAPs promote hydrolysis of GTP to GDP, resulting in inactive GDP-bound KRAS.
  • Active GTP -bound KRAS interacts with numerous effector proteins to stimulate signaling pathways regulating various cellular processes including proliferation and survival. Activating mutations render KRAS resistant to GAP-catalyzed hydrolysis of GTP and therefore lock the protein in an activated state.
  • KRAS is the most commonly mutated oncogene in human cancer. Approximately 20% of all human cancers have activating KRAS mutations with the highest incidence in colon, lung and pancreatic tumors, where KRAS mutation is also associated with poor prognosis.
  • the present disclosure features methods of treating KRAS mutant cancer (e.g., lung, colon and pancreatic) with an inhibitor of the MAPK pathway, such as a MEK or ERK inhibitor, and an antisense compound targeted to KRAS, in a subject in need thereof.
  • an inhibitor of the MAPK pathway such as a MEK or ERK inhibitor
  • an antisense compound targeted to KRAS in a subject in need thereof.
  • a particular MEK inhibitor is selumetinib.
  • the subject can be any mammal. In one embodiment the subject is a human.
  • combination products and kits each comprising one or more inhibitor of the MAPK pathway, such as MEK or ERK inhibitors and an antisense compound targeted to KRAS, for use in treating one or more types of cancer, particularly KRAS mutant cancers.
  • the antisense compound targeted to KRAS is a single-stranded antisense
  • ASO oligonucleotide
  • the MAPK pathway inhibitor is a MEK inhibitor, such as selumetinib.
  • the MAPK pathway inhibitor is an ERK inhibitor.
  • MEK inhibitors are known in the art, such as selumetinib (ARRY-142886).
  • ERK inhibitors are also known in the art and include those described in WO 2016/162325.
  • Ionis 651987 is an antisense oligonucleotide and an example of an antisense compound targeted to KRAS, and has the following chemical structure:
  • the combination involves the antisense oligonucleotide lonis 651987 or a salt thereof, and at least one kinase inhibitor selected from selumetinib and MAPK pathway inhibitors known in the art.
  • the combination involves selumetinib and the antisense oligonucleotide lonis 651987, or a salt thereof.
  • KRAS mutant cancer examples include: non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma and acute lymphoblastic leukemia. Definitions
  • NCBI National Center for Biotechnology Information
  • 2'-deoxynucleoside means a nucleoside comprising 2'-H furanosyl sugar moiety, as found naturally occurring in deoxyribonucleosides (DNA).
  • a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
  • 2 '-substituted nucleoside means a nucleoside comprising a substituent at the 2 '-position other than H or OH. Unless otherwise indicated, a 2 '-substituted nucleoside is not a bicyclic nucleoside.
  • 5-methylcytosine means a cytosine modified with a methyl group attached to the 5- position.
  • a 5-methylcytosine is a modified nucleobase.
  • the term “About” is understood as within a range of normal tolerance in the art, and generally means within ⁇ 10%, such as within 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%), 0.05%), or 0.01% of the stated value.
  • the compounds affected at least about 70% inhibition of KRAS it is implied that the KRAS levels are inhibited within a range of 63%> and 77%>. If it is stated that the compound is used at about 20mg/kg, it covers the range 18-22mg/kg inclusive. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • administerered concomitantly refers to the co-administration of two agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time.
  • Concomitant administration does not require that both agents be administered in a single
  • Agent refers to a substance, such as a compound, antisense oligonucleotide or antibody (and the like) that is capable of producting an effect.
  • Animal refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
  • Antisense compound means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
  • antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, miRNAs, and meroduplex RNAs (mdRNA).
  • An antisense compound targeted to KRAS means an oligomeric compound that is capable of undergoing hybridization to KRAS target nucleic acid through hydrogen bonding
  • Antisense oligonucleotide means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
  • Anti-tumor activity means any biological activity that reduces or stabilizes the
  • the anti-tumor activity is an anti-tumor immune response.
  • Bicyclic sugar means a furosyl ring modified by the bridging of two atoms.
  • a bicyclic sugar is a modified sugar.
  • cancer is meant a disease or disorder characterized by excess proliferation or reduced apoptosis.
  • Illustrative cancers for which the invention can be used include, but are not limited to leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease, DLBCL), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondros
  • craniopharyngioma ependymoma, pinealoma, hemangioblastoma, acoustic neuroma
  • oligodenroglioma oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.
  • Chimeric antisense compound means an antisense compound that has at least two chemically distinct regions.
  • Co-administration means administration of two or more pharmaceutical agents to an individual.
  • the two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions.
  • Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration.
  • Co-administration encompasses parallel or sequential administration. In one embodiment, the co-administration is carried out so as to result in exposure of the patient to both drugs at the same time based on the pharmacokinetics of the drugs.
  • Consstrained ethyl nucleoside (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH3)-0-2' bridge.
  • Contiguous nucleobases means nucleobases immediately adjacent to each other.
  • Disease is meant any condition or disorder that damages, interferes with or
  • a disease such as cancer (e.g., lung cancer) the normal function of a cell tissue or organ is subverted to enable immune evasion and/or escape.
  • “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable.
  • the diluent in an injected composition may be a liquid, e.g. saline solution.
  • Dose means a specified quantity of a pharmaceutical agent provided in a single
  • a dose may be administered in one, two, or more boluses, tablets, or injections.
  • the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose.
  • the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.
  • Effective amount means the amount of active pharmaceutical agent sufficient to effectuate a desired physiological outcome in an individual in need of the agent.
  • the effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the formulation of the composition, assessment of the individual's medical condition, and other relevant factors.
  • “Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions.
  • the internal region may be referred to as the "gap” and the external regions may be referred to as the "wings.”
  • “Hybridization” means the annealing of complementary nucleic acid molecules.
  • complementary nucleic acid molecules include an antisense compound and a target nucleic acid.
  • “Inhibiting KRAS” means reducing expression of KRAS mRNA and/or protein levels in the presence of a KRAS antisense compound, including a KRAS antisense oligonucleotide, as compared to expression of KRAS mRNA and/or protein levels in the absence of a KRAS antisense compound, such as an antisense oligonucleotide.
  • “Individual” means a human or non-human animal selected for treatment or therapy.
  • Internucleoside linkage refers to the chemical bond between nucleosides.
  • KRAS mutant cancer means a cancer in which a mutation has been detected in the tumour which will lead to the activation of KRAS. This includes mutations in the KRAS gene that change the amino acids at glycine 12, glycine 13 and glutamine 61.
  • Linked nucleosides means adjacent nucleosides which are bonded together.
  • Modified internucleoside linkage refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
  • Modified nucleobase refers to any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil.
  • An "unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U).
  • Modified nucleotide means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
  • a “modified nucleoside” means a nucleoside having, independently, a modified sugar moiety or modified nucleobase.
  • Modified oligonucleotide means an oligonucleotide comprising a modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
  • Modified sugar refers to a substitution or change from a natural sugar.
  • Naturally occurring internucleoside linkage means a 3' to 5' phosphodiester linkage.
  • Nucleic acid refers to molecules composed of monomeric nucleotides.
  • a nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
  • RNA ribonucleic acids
  • DNA deoxyribonucleic acids
  • siRNA small interfering ribonucleic acids
  • miRNA microRNAs
  • Nucleobase means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
  • Nucleobase sequence means the order of contiguous nucleobases independent of any sugar, linkage, or nucleobase modification.
  • Nucleoside means a nucleobase linked to a sugar.
  • Nucleotide means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
  • Oligomer means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
  • Oligonucleotide means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
  • “Overall survival” means the length of time from the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive.
  • the overall survival figure is typically determined as an average from an appropriately sized clinical trial.
  • Parenteral administration means administration through injection (e.g., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous
  • intramuscular administration intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebro ventricular
  • Peptide means a molecule formed by linking at least two amino acids by amide bonds.
  • Peptide refers to polypeptides and proteins.
  • “Pharmaceutical composition” means a mixture of substances suitable for administering to an individual.
  • a pharmaceutical composition may comprise one or more active pharmaceutical agents and a sterile aqueous solution.
  • a pharmaceutical composition shows activity in free uptake assay in certain cell lines.
  • Phosphorothioate linkage means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom.
  • a portion means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.
  • Prevent refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to indefinitely. Prevent also means reducing risk of developing a disease, disorder, or condition.
  • progression free survival means the length of time during and after the treatment of a disease, such as cancer, that a patient lives with the disease but it does not get worse.
  • the progression free survival figure is typically determined as an average from an appropriately sized clinical trial.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • responsive in the context of therapy is meant susceptible to treatment.
  • KRAS nucleic acid means any nucleic acid encoding KRAS.
  • a KRAS nucleic acid includes a DNA sequence encoding KRAS, an RNA sequence transcribed from DNA encoding KRAS (including genomic DNA comprising introns and exons), and an mRNA sequence encoding KRAS.
  • KRAS mRNA means an mRNA encoding a KRAS protein.
  • Single-stranded oligonucleotide means an oligonucleotide which is not hybridized to a complementary strand.
  • Specifically hybridizable refers to an antisense compound having a sufficient degree of complementarity (pairing between nucleobases) between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.
  • Subject is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • Synergy or “synergize” refers to an effect of a combination that is greater than additive of the effects of each component alone at the same doses.
  • Targeting or “targeted” means directed at. With respect to an antibody it refers to the ability to bind to the reference protein. With respect to an antisense compound it refers to they ability to specifically hybridize to a target nucleic acid and induce a desired effect.
  • Target nucleic acid refers to a nucleic acid capable of being targeted by antisense compounds.
  • Target segment means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted.
  • 5' target site refers to the 5 '-most nucleotide of a target segment.
  • 3' target site refers to the 3 '-most nucleotide of a target segment.
  • “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual.
  • Treating refers to administering a pharmaceutical composition to reduce or ameliorate a disease, disorder, or condition and/or any symptom associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • Unmodified nucleotide means a nucleotide composed of naturally occuring nucleobases, sugar moieties, and internucleoside linkages.
  • an unmodified nucleotide is an RNA nucleotide (i.e. ⁇ -D-ribonucleosides) or a DNA nucleotide (i.e. ⁇ -D-deoxyribonucleoside).
  • determining is used interchangeably herein with “assaying,” “measuring,” and the like. Where a quantitative determination is intended, the phrase “determining an amount” of an analyte and the like is used. Where a qualitative and/or quantitative determination is intended, the phrase
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • a “purified” or “biologically pure” protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography.
  • purified can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel.
  • modifications for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • the present invention features methods of treating cancer, particularly KRAS mutant cancer (e.g., non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma and acute lymphoblastic leukemia); with an inhibitor of the MAPK pathway, such as a MEK or ERK inhibitor, for example the MEK inhibitor selumetinib, and an antisense compound targeted to KRAS, such as Ionis 651987, in a subject in need thereof.
  • MAPK pathway inhibitors e.g., non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma and acute lymphoblastic leukemia.
  • MAPK pathway inhibitors e.g., a MEK or
  • Inhibitors of various stages of the MAPK pathway such as MEK inhibitors, ERK1 and ERK2 inhibitors are known in the art.
  • MEK inhibitors such as MEK inhibitors, ERK1 and ERK2 inhibitors are known in the art.
  • One example of a MEK inhibitor is selumetinib.
  • One example of known ERK compounds are those disclosed in WO 2016/162325.
  • KRAS antisense compounds including antisense oligonucleotides
  • Antisense compound targeted to KRAS including antisense oligonucleotides, that specifically bind to and inhibit KRAS mRNA or protein expression are useful in the present invention.
  • KRAS inhibitory antisense oligonucleotides are known in the art.
  • PCT/US2016/53334 (lonis Pharmaceuticals Inc.) also discloses numerous KRAS inhibitory antisense oligonucleotides that could feature in the present invention.
  • Antisense oligonucleotides suitable for use in the embodiments provided herein include, but are not limited to, the antisense oligonucleotides described in PCT/US2016/53334. One of these, identified therein as lonis 651987 and is reproduced below.
  • the central gap segment comprising ten 2'-deoxynucleosides, flanked on both sides (in the 5' and 3' directions) by wings comprising three nucleosides each.
  • Each nucleoside in the 5 ' wing segment and each nucleoside in the 3 ' wing segment has a cET sugar modification.
  • an antisense compound provided herein is targeted to human K-Ras mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_004985.4), or human K-Ras genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT 009714.17 truncated from nucleotides 18116000 to 18166000).
  • the antisense oligonucleotide suitable for use herein include, but are not limited to, an antisense oligonucleotide having a nucleobase sequence comprising or consisting of SEQ ID No 3.
  • the antisense oligonucleotide for use in the present invention comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 1447 to 1462 of SEQ ID NO: l .
  • the antisense oligonucleotide is Ionis 651987 (pictured below) or a salt thereof.
  • Ionis 651987 is currently being tested in pre- clinical trials including certain lung, colon and pancreatic cancer models / cell lines.
  • an antisense oligonucleotide suitable for use herein in combination with a MAPK pathway inhibitor, such as selumetinib includes a modified oligonucleotide consisting of 16 linked nucleosides having a nucleobase sequence consisting of any one of SEQ ID NOs: 4-21.
  • the KRAS specific inhibitor is ISIS # 651530, 651987, 695785, 695823, 651555, 651587, 695980, 695995, 696018, 696044, 716600, 746275, 716655, 716772, 740179, 740191, 740201, 740223, or 740233.
  • dlO 2'-deoxynucleosides
  • dlO 2'-deoxynucleosides
  • dlO 2'-deoxynucleosides
  • a method comprising administering to a patient in need thereof a MAPK pathway inhibitor and an antisense compound targeted to KRAS.
  • the agents ((i) the MAPK pathway inhibitor; and (ii) the antisense compound targeted to KRAS)) are administered simultaneously, separately or sequentially.
  • a method of treatment that involves administering: (i) a
  • the agents are administered simultaneously, separately or sequentially.
  • a MAPK pathway inhibitor, and an antisense compound targeted to KRAS for use in the treatment of a patient in need thereof.
  • the two agents are administered simultaneously, separately or sequentially.
  • a MAPK pathway inhibitor for use in the treatment of a patient in need thereof, wherein said patient is also being treated with an antisense compound targeted to KRAS.
  • the agents are administered simultaneously, separately or sequentially.
  • an antisense compound targeted to KRAS for use in the treatment of a patient in need thereof, wherein said patient is also being treated with a MAPK pathway inhibitor.
  • the agents are administered simultaneously, separately or sequentially.
  • MAPK pathway inhibitor in the manufacture of a medicament for the treatment of a patient suffering from cancer, which patient is also being treated with an antisense compound targeted to KRAS.
  • MAPK pathway inhibitor in the manufacture of a medicament for treating a patient suffering from cancer, wherein the MAPK pathway inhibitor is administered simultaneously, separately or sequentially with an antisense compound targeted to KRAS, to a patient in need thereof.
  • an antisense compound targeted to KRAS in the manufacture of a medicament for the treatment of a patient suffering from cancer, which patient is also being treated with a MAPK pathway inhibitor.
  • an antisense compound targeted to KRAS in the manufacture of a medicament for treating a patient suffering from cancer, wherein the antisense compound targeted to KRAS is administered simultaneously, separately or sequentially with a MAPK pathway inhibitor.
  • a MAPK pathway inhibitor in the manufacture of a medicament for the treatment of a patient suffering from cancer.
  • the two agents are administered simultaneously, separately or sequentially.
  • a MAPK pathway inhibitor for use in the treatment of a patient suffering from cancer, which patient is also being treated with an antisense compound targeted to KRAS.
  • a MAPK pathway inhibitor for use in the treatment of a patient suffering from cancer, wherein a MAPK pathway inhibitor is administered simultaneously, separately or sequentially with an antisense compound targeted to KRAS.
  • an antisense compound targeted to KRAS for use in the treatment of a patient suffering from cancer, which patient is also being treated with a MAPK pathway inhibitor.
  • an antisense compound targeted to KRAS for use in the treatment of a patient suffering from cancer, wherein the antisense compound targeted to KRAS is administered simultaneously, separately or sequentially with a MAPK pathway inhibitor.
  • a MAPK pathway inhibitor for use in the treatment of a patient suffering from cancer, wherein each of the MAPK pathway inhibitor and the antisense compound targeted to KRAS is administered simultaneously, separately or sequentially to the patient.
  • the MAPK pathway inhibitor is a MEK inhibitor, for example selumetinib.
  • the KRAS is human KRAS, thus the combinations and methods of treatment and the like, utilize an antisense compound targeted to human KRAS.
  • Certain aspects are drawn to a combination of an antisense compound targeted to KRAS and a MAPK pathway inhibitor.
  • a combination of an antisense compound targeted to KRAS and a MAPK pathway inhibitor comprises lonis 651987, or a salt thereof, and a MAPK pathway inhibitor.
  • such a combination comprises lonis 651987, or a salt thereof, as the antisense compound targeted to KRAS and selumetinib as the MAPK pathway inhibitor.
  • the MAPK pathway inhibitor is selumetinib.
  • the antisense compound targeted to KRAS is administered to the patient on a different schedule to the MAPK pathway inhibitor.
  • the antisense compound targeted to KRAS is administered to the patient on a weekly dosng scheule of 5 days on and two days off; and the MAPK pathway inhibitor is administered once or twice daily.
  • the antisense compound targeted to KRAS can be an antisense oligonucleotide.
  • the antisense compound targeted to KRAS can be Ionis 651987 or a salt thereof.
  • the patient has a KRAS mutant cancer.
  • the patient has a KRAS mutant cancer selected from: non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma and acute lymphoblastic leukemia.
  • NSCLC non small cell lung cancer
  • pancreatic adenocarinoma selected from: non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma and acute lymphoblastic leukemia.
  • the invention provides a method of treatment involving administering selumetinib, and Ionis 651987 or a salt thereof, to a patient identified as having a KRAS mutant cancer.
  • the KRAS mutant cancer is selected from:
  • pancreatic cancer colorectal cancer
  • NSCLC colorectal cancer
  • the antisense compound targeted to KRAS is Ionis 651987, or a salt thereof.
  • an antisense compound provided herein can target human K-Ras mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM_004985.4), or human K-Ras genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT 009714.17 truncated from nucleotides 18116000 to 18166000).
  • the antisense oligonucleotide suitable for use herein include, but are not limited to, an antisense oligonucleotide having a nucleobase sequence comprising or consisting of SEQ ID No 3.
  • the antisense oligonucleotide for use in the present invention comprises a modified oligonucleotide consisting of 12 to 30 linked nucleosides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of nucleobases 1147 to 1462 of SEQ ID NO: 1.
  • the antisense oligonucleotide is Ionis 651987 or a salt thereof.
  • Ionis 651987 is a single stranded modified oligonucleotide comprising ten linked deoxynucleosides (the gap segment), a 5 ' wing segment consisting of 3 linked nucleosides; and a 3' wing segment consisting of 3 linked nucleosides.
  • the gap segment is positioned between the 5 ' wing segment and the 3 ' wing segment and each nucleoside of each wing segment comprises a constrained ethyl nucleoside.
  • Each internucleoside linkage of the oligonucleotide is a
  • the antisense compound targeted to KRAS is a compound comprising a modified oligonucleotide consisting of 12 to 30 linked nucleosides and having a nucleobase sequence comprising at least 12 contiguous nucleobases of the nucleobase sequence of SEQ ID NO : 3.
  • the modified oligonucleotide targeting KRAS consists of a single- stranded modified oligonucleotide.
  • At least one internucleoside linkage of the modified oligonucleotide targeting KRAS is a modified internucleoside linkage.
  • each internucleoside linkage of the modified oligonucleotide targeting KRAS is a phosphorothioate internucleoside linkage.
  • At least one nucleoside of the modified oligonucleotide targeting KRAS comprises a modified sugar.
  • At least one modified sugar of the modified oligonucleotide targeting KRAS is a bicyclic sugar.
  • the bicyclic sugar of the modified oligonucleotide targeting KRAS comprises a 4'-CH 2 -0-2' bridge or a 4'-CH(CH 3 )-0-2' bridge.
  • the modified sugar of the modified oligonucleotide targeting KRAS comprises a 2'-0(CH 2 ) 2 -OCH 3 group or a 2'-0-CH 3 group.
  • at least one nucleoside of the modified oligonucleotide targeting KRAS comprises a modified nucleobase.
  • the said modified nucleobase is a 5-methylcytosine.
  • the modified oligonucleotide targeting KRAS comprises:
  • a 5 '-wing consisting of 1 to 5 linked nucleosides
  • a 3 '-wing consisting of 1 to 5 linked nucleosides
  • a gap between the 5 '-wing and the 3 '-wing consisting of 8 to 12 linked 2'-deoxynucleosides; and wherein at least one of the 5 '-wing and the 3 '-wing comprises at least one bicyclic nucleoside or 2'- substituted nucleoside.
  • the modified oligonucleotide targeting KRAS comprises Ionis
  • the modified oligonucleotide targeting KRAS consists of Ionis 651987 or a salt thereof.
  • the antisense compound targeting KRAS has a nucleobase sequence that, when written in the 5' to 3 ' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
  • antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase.
  • a nucleoside is a base-sugar combination.
  • the nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety.
  • Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar.
  • Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
  • Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
  • Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
  • RNA and DNA The naturally occuring internucleoside linkage of RNA and DNA is a 3' to 5'
  • Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
  • Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom.
  • Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
  • antisense compounds targeted to a KRAS nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified
  • internucleoside linkages are phosphorothioate linkages.
  • each internucleoside linkages are phosphorothioate linkages.
  • internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
  • Antisense compounds provided herein can optionally contain one or more nucleosides wherein the sugar group has been modified.
  • Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds.
  • nucleosides comprise a chemically modified ribofuranose ring moiety.
  • BNA bicyclic nucleic acids
  • Examples of chemically modified sugars include, 2'-F-5'-methyl substituted nucleoside ⁇ see, PCT International Application WO 2008/101157 for other disclosed 5', 2'-bis substituted nucleosides), replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'- position ⁇ see, published U.S. Patent Application US2005/0130923), or, alternatively, 5 '-substitution of a BNA ⁇ see, PCT International Application WO 2007/134181 , wherein LNA is substituted with, for example, a 5'-methyl or a 5'-vinyl group).
  • nucleosides having modified sugar moieties include, without limitation, nucleosides comprising 5 * -vinyl, 5 * -methyl (R or S), 4 * -S, 2 * -F, 2 * -OCH 3 , and 2 * -0(CH 2 )20CH 3 substituent groups.
  • Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides.
  • Typical conjugate groups include cholesterol moieties and lipid moieties.
  • Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
  • the MAPK pathway inhibitor and the antisense compound targeted to a KRAS nucleic acid can be utilized in pharmaceutical compositions by combining the compound(s) with a suitable pharmaceutically acceptable diluent or carrier.
  • a pharmaceutically acceptable diluent includes phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • Suitable examples of carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Co., A.R Gennaro edit., 1985.
  • compositions comprising the MAPK pathway inhibitor and/or the antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
  • the two agents are formulated separately.
  • a pharmaceutical composition comprising a MAPK pathway inhibitor and an antisense compound targeted to KRAS and one or more
  • the agents of the present invention may be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative.
  • the compositions may take such forms as solutions, suspensions, or emulsions in oily or aqueous vehicles, for example solutions in aqueous polyethylene glycol.
  • oily or nonaqueous carriers, diluents, or vehicles include propylene glycol, polyethylene glycol, vegetable oils (e.g., olive oil), and injectable organic esters (e.g. ethyl oleate).
  • compositions may also contain other formulatory agents such as wetting, emulsifying or suspending, preserving, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • a suitable vehicle e.g., sterile, pyrogen-free water.
  • the MAPK pathway inhibitor and/or the antisense compound targeted to KRAS may alternatively be formulated as solid dosage forms, such as tablets and include one or more pharmacetucially-acceptable excipients, such as diluents, carriers and/or lubricants.
  • the treatment is administered according to each component's preferred dosing schedules.
  • selumetinib is conventiently dosed twice daily, orally.
  • lonis 651987 is conveniently dosed daily, or in a weekly repeating pattern, for example 5 days of dosing, followed by two days without dosing. Administration is conveniently by intravenous infusion.
  • the method results in an increase in overall survival (e.g., an increase of weeks, months or years) as compared to the administration of either the MAPK pathway inhibitor or the lonis 651987 alone.
  • the increase in survival is more than about 4-6 weeks, 1-2 months, 3-4 months, 5-7 months, 6-8 months, or 9-12 months.
  • a patient presenting with a cancer/tumor is administered: (i) a MAPK pathway inhibitor; and, (ii) an antisense compound targeted to KRAS; in pharmaceutically effective amounts.
  • a patient presenting with a cancer/tumor is administered: (i) selumetinib; and (ii) lonis 651987; in pharmaceutically effective amounts.
  • the patient is administered additional follow-on doses.
  • follow-on doses can be administered at various time intervals depending on the patient's age, weight, clinical assessment, tumor burden, and/or other factors, including the judgment of the attending physician.
  • At least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, or at least fifteen doses or more of each agent can be administered to the patient.
  • the MAPK pathway inhibitor is administered over a two-week treatment period, over a four-week treatment period, over a six -week treatment period, over an eight- week treatment period, over a twelve -week treatment period, over a twenty-four-week treatment period, or over a one -year or more treatment period.
  • Ionis 651987 is administered over a two-week treatment period, a four- week treatment period, over an eight- week treatment period, over a twelve -week treatment period, over a twenty- four-week treatment period, or over a one-year or more treatment period.
  • Ionis 651987 is administered 4 times in the first 11 days, then weekly from day 15.
  • KRAS to be administered to an individual patient may depend on various parameters such as the patient's age, weight, clinical assessment, tumor burden and/or other factors, including the judgment of the attending physician.
  • the amount of the MAPK pathway inhibitor, and antisense compound targeted to a KRAS nucleic acid (e.g. Ionis 651987) to be administered to the patient may be determined from comprehensive clinical trials of the agents in the patient population.
  • the antisense compound targeted to KRAS can be administered by intravenous infusion or by subcutaneous injection.
  • the administration is by intravenous infusion.
  • the MAPK pathway inhibitor is administered orally, for example as a tablet or capsule.
  • Effective treatment with a combination of a MAPK pathway inhibitor and an antisense compound targeted to KRAS includes, for example, reducing the rate of progression of the cancer, retardation or stabilization of tumor or metastatic growth, tumor shrinkage, and/or tumor regression, either at the site of a primary tumor, or in one or more metastases.
  • the reduction or retardation of tumor growth can be statistically significant.
  • a reduction in tumor growth can be measured by comparison to the growth of patient's tumor at baseline, against an expected tumor growth, against an expected tumor growth based on a large patient population, or against the tumor growth of a control population.
  • the methods of the invention increase survival.
  • Clinical response to administration of a MAPK pathway inhibitor such as a MEK or ER inhibitor and an antisense compound targeted to KRAS (such as lonis 651987) can be assessed using diagnostic techniques known to clinicians, including but not limited to magnetic resonance imaging (MRI) scan, x-radiographic imaging, computed tomographic (CT) scan, flow cytometry or fluorescence-activated cell sorter (FACS) analysis, histology, gross pathology, and blood chemistry, including but not limited to changes detectable by ELISA, RIA, and chromatography.
  • MRI magnetic resonance imaging
  • CT computed tomographic
  • FACS fluorescence-activated cell sorter
  • the methods provided herein can decrease or retard cancer tumor growth. In some cases the reduction or retardation can be statistically significant.
  • a reduction in tumor growth can be measured by comparison to the growth of patient's tumor at baseline, against an expected tumor growth, against an expected tumor growth based on a large patient population, or against the tumor growth of a control population.
  • administering the dose of an antisense compound targeted to KRAS and a MAPK pathway inhibitor reduces tumor size or tumor volume in the subject. In certain embodiments, administering the dose of the antisense compound and a MAPK pathway inhibitor prolongs survival of the subject. In certain embodiments, administering the dose of the antisense compound targeted to a KRAS and a MAPK pathway inhibitor, treats cancer, such as non small cell lung cancer (NSCLC), pancreatic adenocarinoma, colorectal adenocarcinoma, uterine endometrial cancer, cholangiocarcinoma, multiple myeloma or acute lymphoblastic leukemia, in the subject. In certain embodiments, the method is effective to treat cancer and acceptably tolerable in a subject. In certain embodiments, a tumor response is measured using the Immune-related Response
  • a tumor response is measured using the Response
  • a tumor response is detectable at week 7 or thereafter, such as at week 13, at week 25, at week 41, at week 52.
  • a patient achieves disease control (DC).
  • Disease control can be a complete response (CR), partial response (PR), or stable disease (SD).
  • CR complete response
  • a “partial response” refers to a decrease in tumor burden > 30% relative to baseline. Confirmation can be obtained using a consecutive repeat assessment at least 4 weeks from the date of first documentation.
  • “Stable disease” indicates a decrease in tumor burden of less than about 30% relative to baseline cannot be established and a 20% or greater increase compared to nadir cannot be established.
  • administration of the immunomodulatory agent and the antisense compound targeted to KRAS can increase progression-free survival (PFS).
  • PFS progression-free survival
  • administration of the immunomodulatory agent and the antisense compound targeted to KRAS can increase overall survival (OS).
  • administration of selumetinib and Ionis 651987 can increase progression-free survival (PFS).
  • PFS progression-free survival
  • administration of selumetinib and Ionis 651987 can increase overall survival (OS).
  • the patient has previously received treatment with at least one chemotherapeutic agent. In some embodiments, the patient has previously received treatment with at least two chemotherapeutic agents.
  • the chemotherapeutic agent can be, for example, and without limitation, Vemurafenib, Gefitinib, Erlotinib, Afatinib, Cetuximab, Carboplatin, Bevacizumab, and/or Pemetrexed.
  • the cancer is refractory or resistant to at least one chemotherapeutic agent. In some embodiments, the cancer is refractory or resistant to at least two chemotherapeutic agents.
  • the cancer can be refractory or resistant to one or more of, for example, and without limitation, Vemurafenib, Gefitinib, Erlotinib, Afatinib, Cetuximab, Carboplatin, Bevacizumab, and/or Pemetrexed.
  • the patient has an Eastern Cooperative Oncology Group (ECOG) (Oken MM, et al. Am. J. Clin. Oncol. 5: 649-55 (1982)) performance status of 0, 1, or 2 prior to the administration of selumetinib, and Ionis 651987.
  • ECOG Eastern Cooperative Oncology Group
  • Treatment of a patient with a cancer using both (i) a MAPK pathway inhibitor, such as selumetinib; and (ii) an antisense compound targeted to KRAS, such as lonis 651987, (i.e., co- therapy) as provided herein can result in an additive and/or synergistic effect.
  • the term "synergistic" refers to a combination of therapies which is more effective than the additive effects of the single therapies.
  • a synergistic effect of a combination of therapies may permit the use of lower dosages of one or more of the therapeutic agents and/or less frequent administration of said therapeutic agents to a patient with cancer.
  • the ability to utilize lower dosages of therapeutic agents and/or to administer said therapies less frequently reduces the toxicity associated with the administration of said therapies to a subject without reducing the efficacy of said therapies in the treatment of a cancer.
  • a synergistic effect can result in improved efficacy of therapeutic agents in the management, treatment, or amelioration of a cancer.
  • the synergistic effect of a combination of therapeutic agents can avoid or reduce adverse or unwanted side effects associated with the use of either single therapy.
  • the synergistic effect of a combination of therapeutic agents may also manifest itself as a reduction in tumor mass (or tumor regression).
  • the synergistic effect of a combination of therapeutic agents may also manifest itself as a sustained reduction in tumor growth rate.
  • Treatment of a patient with a cancer using both (i) a MAPK pathway inhibitor, such as a MEK inhibitor, for example selumetinib; and (ii) an antisense compound targeted to KRAS, such as lonis 651987, (i.e., co-therapy) as provided herein can result in a reduction in the MAPK pathway reactivation caused by feedback mechanisms after administration of a MAPK pathway inhibitor, such as a MEK inhibitor.
  • a MAPK pathway inhibitor such as a MEK inhibitor, for example selumetinib
  • an antisense compound targeted to KRAS such as lonis 651987
  • the MAPK pathway inhibitor (such as selumetinib, can be optionally included in the same pharmaceutical composition as the antisense compound targeted to KRAS (such as lonis 651987), or may be included in a separate pharmaceutical composition.
  • the pharmaceutical composition comprising the MAPK pathway inhibitor is suitable for administration prior to, simultaneously with, or following administration of the pharmaceutical composition comprising the antisense compound targeted to KRAS.
  • the MAPK pathway inhibitor is administered at overlapping times as the antisense compound targeted to KRAS in a separate composition.
  • kits for treating cancer comprising: (i) an antisense compound targeted to KRAS (such as lonis 651987); and, (ii) MAPK pathway inhibitor such as selumetinib.
  • the kit includes a therapeutic composition comprising the MAPK pathway inhibitor such as selumetinib, and the antisense compound targeted to KRAS (such as lonis 651987), each in unit dosage form.
  • the kit comprises a sterile container which contains one or more therapeutic compositions; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • a sterile container which contains one or more therapeutic compositions; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding
  • the kit further comprises instructions for administering the MAPK pathway inhibitor such as selumetinib, and the antisense compound targeted to KRAS (e.g. lonis 651987) to a subject having a cancer.
  • the instructions include at least one of the following: description of the therapeutic agent(s); dosage schedule and administration for treatment or prevention of cancer or symptoms thereof; precautions; warnings; indications; counter- indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • the antisense compound targeted to KRAS is, or can be, lonis 651987.
  • Figure 1 A Effect of pre-treatment with 651987 on selumetinib induced pMEKl /2.
  • Antisense oligonucleotide (ASO) treatment was for 72 hours and selumetinib treatment for 24 hours.
  • selumetinib was added 48 hours post-dosing with 651987 for the final 24 hours of incubation.
  • Figure IB Western analysis of MAPK signalling and apoptotic markers in lysates from NCI- H358 cells following 72 hours treatment with 5 ⁇ CTRL ASO (549148) or 651987 in combination for the final 24 hours with a dose response of selumetinib.
  • Figure 1C Effect of dose responses of selumetinib in combination with CTRL ASO (549148) or 651987 on colony formation in soft agar of NCI-H358 (KRAS G12C) or PC9 (KRAS wild type) cells.
  • Figure ID Effect of dose responses of selumetinib in combination with CTRL ASO (549148) or 651987 on colony formation in soft agar of NCI-H2122 (KRAS G12C mutant), A549 (KRAS G12S mutant) or NCI-H1437 (KRAS wild type) cells.
  • Example 1 The effect of combining a KRAS ASO and MAPK pathway inhibitor on growth and signalling of cancer cells in vitro
  • KRAS ASO in combination with the MEK1/2 inhibitor selumetinib was assessed in cancer cells expressing mutant KRAS (NCI-H358, NCI-H2122 and A549) or wild type KRAS (PC9 and NCI-H1437).
  • the impact of the treatment on down-sream effector pathway signalling was assessed by Western blot analysis and the impact on proliferation by 3D growth assays.
  • NCI-H358 cells pre-treatment with 651987 for 48 hours limited selumetinib induced pathway reactivation and phosphorylation of MEK1/2 at serine 218/222, which was not observed in the KRAS wild type PC9 cells ( Figure 1 A).
  • the combination treatment in NCI-H358 cells was also more effective at inhibiting down-stream signalling and inducing pro-apoptotic/apoptotic markers than the monotherapy treatments ( Figure IB).
  • the selumetinib and 651987 combination showed enhanced inhibition of cell growth in colony formation assays compared with either single-agent treatment in NCI-H358 KRAS mutant cells and no combination effect was seen with a control ASO ( Figure 1C).
  • KRAS ASO in combination with the MEK1/2 inhibitor selumetinib on proliferation of NSCLC cells was additionally assessed in NCI-H2122 (KRAS G12C), A549 (KRAS G12S) or NCI- H1437 (KRAS wild type) cells.
  • Dose response of the selumetinib and AZD4785 combination showed an enhanced inhibition of cell growth in colony formation assays compared with either single-agent treatment in the KRAS mutant cells and no effect was seen with a control ASO ( Figure ID).
  • STR short-tandem repeat analysis
  • NCI-H358 and PC9 cells were treated with CTRL ASO (549148) or 651987 for 72 hours and with selumetinib for 24 hours.
  • selumetinib was added 48 hours post-dosing with antisense for the final 24 hours of incubation.
  • signalling endpoint For analysis of signalling endpoint by Western blot, cells were washed once in cold PBS and lysed in RIPA buffer supplemented with PhosSTOP Phosphatase Inhibitor Cocktail Tablets and cOmplete Protease Inhibitor Cocktail Tablets, Roche (Basel, Switzerland).
  • 3D colony assays Colony assays were performed in 96 well plates. Cells were seeded in 75 ⁇ of 0.3% agar onto a 50 ⁇ 1 1% agar layer in 10% RPMI-1640 growth media at 2000 cells/well. The agar layers were then covered with 50 ⁇ 1 of media containing treatment taking into account the entire volume of agar and media.
  • Colonies were grown for 7 days (PC9 and NCI-H1437) or 10 days (NCI-H358, NCI-H2122 and A549) and colony formation assessed by scanning on a GelCount scanner (Oxford Optronix, Abingdon, UK) counting colonies >40 ⁇ in diameter for NCI-H358, NCI-H2122 and A549 cells and >70 ⁇ in diameter for PC9 and NCI-H1437 cells.
  • NCI-H358 cells were authenticated via the AstraZeneca (AZ) Cell Bank using DNA fingerprinting short tandem repeat (STR) assays. All revived cells were used within 12 passages, and cultured for less than 2 months.
  • AZ AstraZeneca
  • STR DNA fingerprinting short tandem repeat
  • Cells were grown in RPMI-1640 media, 10% FCS, 2mmol/L glutamine at 37°C/7.5% C02 and maintained by split ratio of 1 :5 + 1 : 10 twice weekly.
  • the cells were harvested from T225 tissue culture flasks by a 2- to 5 -minute treatment with 0.05% trypsin (Invitrogen) in EDTA solution followed by re- suspension in required media. Cells were centrifuged at lOOOrpm for 5 minutes and were washed once in phosphate buffered saline (Invitrogen). Only single-cell suspensions of greater than 90% viability, as determined by trypan blue exclusion, were used for injection.
  • trypsin Invitrogen
  • Pathogen-free male athymic nude mice (Hsd:Athymic Nude- oxni - Harlan, UK) were housed in pathogen-free conditions in groups of 3. The animals were maintained in rooms under controlled conditions of temperature (19-23°C), humidity (55 ⁇ 10%), photoperiod (12 hours light/12 hours dark) and air exchange. Animals were housed in individual vented cages, with food and water provided ad libitum. The facilities have been approved by the Home Office Licence and meet all current regulations and standards of the UK. The mice were used between the ages of 8 and 12 weeks in accordance with institutional guidelines.
  • mice On day zero, 3xlO A 6 cells in 50% Matrigel (BD Bioscience #354234) were injected s.c. on the left flank of the animals in a volume of O.lmL / mouse. On day 3, mice were randomized into control and treatment groups of 12 (average tumour volume was approximately 0.1 cm3).
  • mice in the treatment groups were dosed with AZD4785 subcutaneously at 50mg/kg with a dose volume of 0. lml/lOg for 5 - 7 weeks with a schedule of 5 days on 2 days off per week or AZD6244 at 12.5mg/kg PO twice daily in the schedules listed in table 1.
  • Mice in the control groups received either phosphate buffered saline (Invitrogen) or Control KRAS ASO Gen 2.5 at 50mg/kg. Both control groups were dosed subcutaneously for 5 - 7 weeks with a schedule of 5 days on 2 days off per week.
  • Tumours were measured two times weekly by calliper and volume calculated using elliptical formula (pi/6 width _ width _ length) animal bodyweight and tumour condition were also recorded twice weekly for the duration of the study. 24 hours after the last dose, the animals were humanely sacrificed by C0 2 and tumour samples collected. Half the tumour was snap-frozen in liquid nitrogen and stored at -80°C for
  • %TGI Tumour growth inhibition
  • Tumour regression was calculated as the percentage reduction in tumour volume from baseline (pre- treatment) value: % Regression 1 ⁇ 4 (1 _ RTV) 100 % where RTV 1 ⁇ 4 Geometric Mean Relative.
  • RNA samples were diluted to 5 ng/ ⁇ or 0.05 ng/ ⁇ (for 18S rRNA) in nuclease-free water (Ambion AM9937) and 4 ⁇ per 10 ⁇ reaction were used together with 0.5 ⁇ of 20x ABI Assay Probes FAM MGB (Taqman, Life Technologies), 5 ⁇ of 2x Reaction Buffer and 0.1 ⁇ of RT enzyme (Qiagen One Step RT PCR Kit 210215). Reactions were run in triplicates.
  • qRT PCR were performed on Roche Lichtcycler 480 or Applied Biosystems QuantStudio using the following protocol: (1) 50°C 30 mins, (2) 95°C 15mins, 40 cycles of (3) 95°C 15 sec and 60°C lmin. On run completion, automatic threshold was set and Ct values were generated. Gene expression was normalised to the house keeper gene and percent inhibition (or fold change) determined relative to PBS control. Statistically significant changes were determined using ANOVA analysis.
  • lonis 549148 is a control oligonucleotide that does not target K-Ras and was included in each experiment as a negative control; lonis 549148 is a 3-10-3 cEt gapmer, GGCTACTACGCCGTCA, designated herein as SEQ ID NO: 25).
  • Antisense oligonucleotides were designed targeting a K-Ras nucleic acid and were tested for their effects on K-Ras mRNA in vitro. The antisense oligonucleotides were tested in a series of experiments that had similar culture conditions. Cultured SKOV3 cells at a density of 20,000 cells per well were transfected 5 using electroporation with 2,500 nM antisense oligonucleotide. After a treatment period of approximately 24 hours, RNA was isolated from the cells and K-Ras mRNA levels were measured by quantitative real-time PCR.
  • Human primer probe set RTS246 (forward sequence CCCAGGTGCGGGAGAGA, designated herein as SEQ ID NO: 22; reverse sequence GCTGTATCGTCAAGGCACTCTTG; designated herein as SEQ ID NO: 23; probe sequence CTTGTGGTAGTTGGAGCTGGTGGCGTAG, designated herein as SEQ ID NO: 10 24) was used to measure mRNA levels.
  • K-Ras mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. Results are presented as percent inhibition of K-Ras, relative to untreated control cells. As used herein, a value of '0' indicates that treatment with the antisense oligonucleotide did not inhibit mRNA levels.
  • the newly designed chimeric antisense oligonucleotides in the Tables below were designed as 3-10-3 15 cEt gapmers.
  • the gapmers are 16 nucleosides in length, wherein the central gap segment comprises of ten 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising three nucleosides each.
  • Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a cEt sugar modification.
  • “Start site” indicates the 5 '-most nucleoside to which the gapmer is targeted in the human gene sequence.
  • “Stop site” indicates the 3 '-most nucleoside to which the gapmer is targeted human gene sequence.
  • the gapmer listed in the Table below is targeted to either a human K-Ras mRNA, designated herein as SEQ ID NO: 1 (GENBANK Accession No. NM 004985.4), the human K-Ras genomic sequence, designated herein as SEQ ID NO: 2 (the complement of GENBANK Accession No. NT 009714.17 truncated from 5 nucleotides 18116000 to 18166000).

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Abstract

La présente invention concerne des méthodes de traitement de cancer par un inhibiteur de voie MAPK, tel que le sélumétinib, et un composé antisens ciblant KRAS chez un sujet en ayant besoin.
PCT/EP2018/053463 2017-02-13 2018-02-12 Combinaison d'un inhibiteur de voie mapk et d'un composé antisens ciblant kras WO2018146316A1 (fr)

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