WO2015145388A2 - Procédés de traitement de cancers colorectaux avec mutations en amont de la voie wnt - Google Patents

Procédés de traitement de cancers colorectaux avec mutations en amont de la voie wnt Download PDF

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WO2015145388A2
WO2015145388A2 PCT/IB2015/052245 IB2015052245W WO2015145388A2 WO 2015145388 A2 WO2015145388 A2 WO 2015145388A2 IB 2015052245 W IB2015052245 W IB 2015052245W WO 2015145388 A2 WO2015145388 A2 WO 2015145388A2
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inhibitor
colorectal cancer
treatment
wnt
braf
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PCT/IB2015/052245
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WO2015145388A3 (fr
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Linda BAGDASARIAN
Feng Cong
Savina JAEGER
Margaret Elise CLAUGHLIN
Ronald Meyer
Andrea MYERS
Michael Ross PALMER
Youzhen Wang
Stephen David WOOLFENDEN
Ana VIVANCOS
Héctor PALMER
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Novartis Ag
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators

Definitions

  • the present invention relates to a method of treating patients suffering from BRAF -mutant and BRAF wild-type colorectal cancer by using a Wnt pathway inhibitor or combinations comprising a Wnt pathway inhibitor and another inhibitor.
  • the disclosure also relates to corresponding pharmaceutical formulations, uses, methods, combinations, and related disclosure embodiments.
  • Colorectal cancer is the fourth most frequently diagnosed cancer and the second leading cause of cancer death in the United States and the European Union. I n the last 15 years, the median overall survival (OS) for patients with metastatic CRC has markedly improved from approximately 8-12 months to 18-24 months (Kopetz, S. et al., J. Clin. Oncol. 2009, 27:3677-82). The improvement is attributed to two factors: 1) the increased utilization of hepatic resection and 2) the addition of new medical therapies (irinotecan, oxaliplatin, bevacizumab, cetuximab, and pantiumumab) to the standard treatment with 5-fluorouracil plus leucovorin.
  • new medical therapies irinotecan, oxaliplatin, bevacizumab, cetuximab, and pantiumumab
  • a Wnt inhibitor, or a combination of Wnt inhibitor and either a BRAF inhibitor, or EGFR inhibitor, or both are especially useful for treatment of a patient with a BRAF V600E -mutant colorectal cancer, particularly the cancer with a mutated Wnt pathway.
  • a Wnt inhibitor or a combination of a Wnt inhibitor and an EGFR inhibitor, can be useful for the treatment of patients with microsatellite-unstable BRAF wild-type (BRAF ⁇ ) colorectal cancer with a mutated Wnt pathway, particularly upstream Wnt pathway mutation.
  • the colorectal cancer can be KRAS wild-type (KRAS WT ).
  • CRCs colorectal cancers
  • MAPK pathway activation not Wnt pathway activation, was thought to be the dominant oncogenic driver. It has been unexpectedly found that activation of the Wnt pathway has an important role in the pathogenesis of serrated CRC.
  • Molecular epidemiology data showed that patient BRAF V600E -mu an CRC samples are enriched for genetic alterations in upstream Wnt pathway regulators (e.g. RNF43, ZNRF3, RSP02 and RSP03), strongly confirming this notion.
  • RNF43 mutations are enriched in BRAF V600E CRC. But BRAF V600E CRCs are also frequently microsatellite unstable due to hypermethylation of DNA repair genes (Lubomierski, N. et al., Cancer 2005, 104:952-61). The mismatch repair deficiency in these tumors may directly contribute to RNF43 mutagenesis, as RNF43 mutations tend to be small insertions/deletions in homopolymeric tracts.
  • BRAF 7 , KRASTM 7 CRCs with RNF43 mutations may also benefit from COM POU N D A and/or COM POU N D A+cetuximab
  • a Wnt inhibitor or a combination of a Wnt inhibitor and an EGFR inhibitor, can be useful for the treatment of patients with microsatellite-unstable, BRAF wild-type (BRAF ⁇ ), KRAS wild-type (KRAS ⁇ ) colorectal cancer harboring upstream Wnt pathway mutations.
  • BRAF ⁇ microsatellite-unstable, BRAF wild-type
  • KRAS ⁇ KRAS wild-type
  • microsatellite unstable colorectal cancer is expected to be BRAF mutant.
  • BRAF wild-type colorectal cancer was found to be sensitive to a Wnt inhibitor, either alone or in combination with another drug.
  • Wnt inhibitor either alone or in combination with another drug.
  • RN F43 mutation it would be possible to treat BRAF wt colorectal cancer as long it harbors RN F43 or other WNT upstream pathway mutation.
  • ZNRF3 and RN F43 are both homologous cell surface transmembrane E3 ubiquitin ligases for Wnt receptor Frizzled it is expected that the Wnt inhibitor could be effectively applied in the treatment of colorectal cancer, BRAF V600E mutated or BRAF wt, which is ZN RF3 mutated (but not RN F43 mutated).
  • BRAF V600E mutated or BRAF wt which is ZN RF3 mutated (but not RN F43 mutated).
  • the cancer is BRAF wt, there is no need to use a BRAF inhibitor together with the Wnt inhibitor.
  • the present disclosure provides the following aspects, advantageous features and specific embodiments, respectively alone or in combination, as listed in the following items:
  • a Wnt inhibitor for use in the treatment of BRAF V600E -mutant colorectal cancer 1.
  • a Wnt inhibitor for use in the treatment of BRAF V600E -mutant colorectal cancer according to any one of items 1 to 3 in combination with a BRAF inhibitor.
  • a Wnt inhibitor for use in the treatment of microsatellite-unstable, BRAF wild-type and KRAS wild-type colorectal cancer 5.
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to
  • colorectal cancer comprises mutated RNF43, mutated RSP02 or mutated RSP03.
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to 8, wherein the colorectal cancer comprises mutated ZN RF3, RSPOl, RSP04, LGR4-6, Frizzledl-10, LRP5, LRP6, WISP3, Wise, SFRPl-3, WI F-1, DKKl, DKK4, LKBl/STKll, Keap-1 and/or N RF2, preferably ZNRF3.
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 4 to
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 4 to
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to
  • a Wnt inhibitor for use in the treatment of colorectal cancer according to any one of items 1 to 19 in a form of a pharmaceutical composition is provided.
  • a pharmaceutical combination comprising (i) a Wnt inhibitor and (ii) a BRAF inhibitor.
  • the pharmaceutical combination according to item 21 further comprising an EGFR inhibitor.
  • 23 A pharmaceutical combination comprising a Wnt inhibitor and an EGFR inhibitor.
  • colorectal cancer comprises mutated ZN RF3, RSPOl, RSP04, LGR4-6, Frizzledl-10, LRP5, LRP6, WISP3, Wise, SFRP1- 3, WI F-1, DKK1, DKK4, LKB1/STK11, Keap-1 and/or N RF2, preferably ZN RF3.
  • a Wnt inhibitor for use in the treatment of BRAF V600E -mutant colorectal cancer according to any one of items 4, 6 to 20, 49 or 50, or a pharmaceutical combination according to any one of items 21, 22, 24 to 29, 31 to 36 or 39 to 50, wherein the BRAF inhibitor is (S)-methyl-l-(4-(3-(5-chloro-2-fluoro-3- (methylsulfonamido)phenyl)-l-isopropyl-lH-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate, methyl N-[(2S)-l-( ⁇ 4-[3-(5-chloro-2-fluoro-3-methanesulfonamidophenyl)-l-(propan-2-yl)-lH-pyrazol-4- yl] pyrimidin-2-yl ⁇ amino)propan-2-yl]carbamate or vemurafenib
  • a Wnt inhibitor for use in the treatment of BRAF V600E -mutant colorectal cancer according to any one of items 4, 6 to 20, 49 or 50, or a pharmaceutical combination according to any one of items 21, 22, 24 to 29, 31 to 36 or 39 to 50, wherein the BRAF inhibitor is (S)-methyl-l-(4-(3-(5-chloro-2-fluoro-3- (methylsulfonamido)phenyl)-l-isopropyl-lH-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate.
  • a method of treating a patient having colorectal cancer further defined as in any one of items 1 to 20, or 49 to 57, wherein the amount of the inhibitor administered to a patient is therapeutically effective.
  • Colorectal cancer as used herein means a neoplasm arising from the colon and/or rectum, particularly from the epithelium of the colon and/or rectum.
  • “Mutated” or “mutation” denots herein an alteration from a normal functional or wild type protein, cDNA, gene or mRNA, which results in changed activity, or loss thereof. It includes changed mutation status like for example frame-shift mutation, deletion, translocation, insertion, duplication, inversion, functional mutation; or combinations thereof. It includes also DNA modification, cDNA modification, mRNA modification, protein modification, DNA function, cDNA function, mRNA function, protein function, DNA mutation, cDNA mutation, mRNA mutation, protein mutation, or combinations thereof; preferably is DNA mutation.
  • DNA modification includes DNA alkylation or acylation.
  • methylation is a biochemical process involving the addition of a methyl group to the cytosine or adenine DNA nucleotides.
  • mRNA modification includes RNA editing, which is a biochemical process involving the change of nucleotides after they have been generated by RNA polymerase to form a sequence.
  • cDNA modification includes any modification that was made at the mRNA level will be translated into cDNA modification.
  • Protein modification includes a biochemical process involving the change of amino acids after they have been translated. Protein function is understood for proteins to carry out the duties specified by the information encoded in genes, including facilitation of signaling transduction, enzymatic reactions etc.
  • Gene expression or “expression” are both used herein interchangeably and refer to the nucleic acids or amino acids (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.
  • gene expression or “expression” denote DNA expression, mRNA expression, cDNA expression, transcription, protein transcription or protein expression.
  • Detection of gene expression can be by any appropriate method, including for example, detecting the quantity of mRNA transcribed from the gene or the quantity of cDNA produced from the reverse transcription of the mRNA transcribed from the gene or the quantity of the polypeptide or protein encoded by the gene. Transcription or translation can be determined using known techniques. For example, an amplification method such as PCR may be useful. The same principles apply to "overexpression”.
  • Control denotes herein to the sequence, parameter or level measured for comparison in a noncancerous, healthy, wild-type tissue or cell.
  • the control can be the sequence, parameter or level measured in a normal colonic epithelium.
  • a Wnt pathway in the colorectal cancer can be activated (increased Wnt signaling) or inactivated (decreased Wnt signaling) compared to a control. This refers to the increased or decreased, respectively, expression or functional effect of Wnt pathway genes, corresponding mRNA and equally to transcription or functional effect of proteins encoded by said genes or mRNA.
  • “Mutated Wnt pathway” or “upstream Wnt pathway mutation” as used herein means any mutation in a gene encoding a protein in the Wnt pathway that increases Wnt signaling in colorectal cancer relative to a control, in a Wnt ligand-dependent manner.
  • the control can be Wnt signaling in normal colonic epithelium.
  • mutations in the following genes may increase Wnt signaling in a Wnt ligand-dependent manner: ZNRF3, RSPOl, RSP04, LGR4-6, Frizzledl-10, LRP5, LRP6, WISP3, Wise, SFRP1-3, WI F-1, DKK1, DKK4,
  • “Microsatellite-unstable tumor” is a tumor that is hypermutable due to inactivation of one or more mismatch repair system genes, for example MLH1, MSH2, MSH6, and PMS2.
  • FIG. 1 RNF43 mutations and RSPO fusions co-occur with BRAF V600E mutations in patient CRC samples.
  • APC mutations show a strong tendency to be non-overlapping with RNF43 mutations and RSPO fusions.
  • JHOM-2B cell line is derived from a lower Gl adenocarcinoma.
  • A Representative images of CK7 staining (Left) or CK20 staining (Right) of a JHOM-2B tumor xenograft.
  • B DNA sequence of the fusion breakpoint between exonl of PTPRK and exon 2 of RSP03 in JHOM-2B.
  • JHOM-2B tumor cells are sensitive to COMPOUND A; however, COMPOUND A antagonizes the tumor growth inhibition of COMPOUND B plus cetuximab in vitro.
  • COMPOUND A In a foci formation assay, decreased JHOM-2B colonies are seen after treatment with COMPOUND A.
  • COMPOUND A inhibits the proliferation of JHOM-2B tumor cells in a 7-day CTG assay, with an EC 50 of 0.06 ⁇ xM.
  • C Addition of COMPOUND A to COMPOUND B+cet has an antagonistic effect on tumor growth inhibition in vitro.
  • FIG. 5 Effects of COMPOUND A, COMPOUND B, cetuximab and combination treatments on Wnt and MAPK signaling pathways in JHOM-2B tumor cells.
  • JHOM-2B tumor cells were treated with 100 nM COMPOUND A, 100 nM COMPOUND B, and/or 50 nM cetuximab for 72 hours prior to collection of protein lysates and immunoblotting.
  • FIG. 7 COMPOUND A induces cell cycle arrest in JHOM-2B tumor xenografts, as a single agent and in combination.
  • A Representative images of pHH3 staining by immunohistochemistry after 14 days of treatment (the COMPOUND A and COMPOUND A+cet treatment groups were collected after 11 days of treatment).
  • Figure 8. COMPOUND A induces mucinous differentiation of JHOM-2B tumor xenografts, as a single agent and in combination.
  • FIG. 9 The triple combination of COMPOUND A+COMPOUND B+cet is efficacious in HCOX1329 patient-derived xenografts in vivo.
  • A HCOX1329-tumor-bearing mice were treated with 10 ml/kg vehicle, p.o., BID (filled circles); 5 mg/kg COMPOUND A, p.o.
  • COMPOUND A and COMPOUND B modestly induce cell cycle arrest in HCOX1329 patient- derived tumor xenografts; additional cell cycle arrest is seen with combination treatments.
  • FIG. 11 The combinations of COMPOUND A+COMPOUND B and COMPOUND A+COMPOUND B+cet induce mucinous differentiation of HCOX1329 tumor xenografts.
  • A Representative images of Alcian blue staining after 10 days of treatment. In contrast to JHOM-2B xenografts that accumulate intra- and extracellular mucin upon treatment, treatment of HCOX1329 xenografts results in a selective increase in intracellular mucin (arrows).
  • FIG. 12 No statistically significant decrease in tumor volume is seen in T70 patient-derived xenografts treated with COMPOUND A, cetuximab, or COMPOUND A+cetuximab in vivo.
  • A T70-tumor-bearing mice were treated with 10 ml/kg vehicle, p.o., BID (filled circles); 5 mg/kg COMPOUND A, p.o. BID (open squares); 20 mg/kg cetuximab, i.p., 2x/wk (filled upside down triangles); or COMPOUND A+cetuximab (open circles) for 14 days.
  • B Scatter plot of individual tumor volumes.
  • COMPOUND A and COMPOUND A+cetuximab induce cell cycle arrest in T70 patient-derived tumor xenografts.
  • A Representative images of Ki67 staining by immunohistochemistry after 14 days of treatment.
  • FIG. 14 COMPOUND A and COMPOUND A+cetuximab induce mucinous differentiation of T70 tumor xenografts.
  • A Representative images of Alcian blue staining after 14 days of treatment.
  • a Wnt inhibitor, or a combination of Wnt inhibitor and either a BRAF inhibitor, or EGFR inhibitor, or both are especially useful for treatment of a patient with a BRAF V600E -mutant colorectal cancer and microsatellite-unstable, BRAF wild-type (BRAF ⁇ ), KRAS wild- type (KRAS WT ) colorectal cancer harboring, particularly the cancer with a mutated Wnt pathway.
  • Wnt/P-catenin signaling promotes cell survival in various cell types (Orford et al., J Cell Biol, 146:855-868 (1999); Cox et al., Genetics, 155:1725-1740 (2000); Reya et al., Immunity, 13:15-24 (2000); Satoh et al., Nat Genet, 24:245-250 (2000); Shin et al., Journal of Biological Chemistry, 274:2780-2785 (1999); Chen et al., J Cell Biol, 152:87-96 (2001); Loannidis et al., Nat Immunol, 2:691-697 (2001)).
  • a Wnt inhibitor can be any compound which targets and decreases or inhibits the Wnt pathway.
  • a Wnt inhibitor can be a compound, protein or antibody that reduces the beta-catenin target gene expression of AXIN2 by at least 50% with an EC 50 of 10 ⁇ or less, preferably less than ⁇ , in a tumor cell line with autocrine Wnt signaling (H PAF-I I).
  • Gene expression is measured against a control, which can be either gene expression of AXIN2 in an untreated cell sample, and/or against the gene expression of housekeeping genes in the treated sample, such as gene expression of for example GAPDH, HSP90, 6- actin, ACTB and 62 M.
  • the Wnt inhibitor is particularly a compound, protein or an antibody which inhibit members of the Wnt pathway, for example inhibits or reduces activity of Porcupine, RSP02, RSP03, Frizzled, LRP6 or Tankyrase.
  • the inhibitor can be for example XAV939, IWR1, IWP-1, IWP-2, JW74, JW55, Tautomycin, SB239063, SB203580, ADP-H PD, 2-[4-(4-fluorophenyl)piperazin-l-yl]-6-methylpyrimidin-4(3H)-one, PJ34, Niclosamide, Cambinol, Sulindac, 3289-8625, J01-017a, NSC668036, Filipin, IC261 or other Wnt inhibitors disclosed herein.
  • a Wnt inhibitor can especially be a compound that inhibits Frizzled or Porcupine, most preferably Porcupine.
  • the Wnt inhibitor according to the present disclosure is 2-(2',3-dimethyl-2,4'-bipyridin-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide (compound A).
  • the compound A can be prepared as described in WO2010/101849 and has a structure of formula
  • Mitogen-activated protein kinase (MAPK) hyper-activation is a common property of human cancers and is often due to activating mutations in the BRAF and RAS genes.
  • BRAF kinase domain mutations result in the production of a constitutively activated form of the protein and occur in approximately 8% of human tumors (Davies et al., 2002; Wan et al., 2004).
  • BRAF mutation stimulates extracellular signal-regulated kinase (ERK) signaling, induces proliferation and is capable of promoting transformation.
  • BRAF inhibitors are compounds, especially compounds, proteins or antibodies which target BRAF, particularly mutant BRAF, and its downstream effectors.
  • BRAF inhibitor can be selected from the group consisting of: ((S)- methyl-l-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-l-isopropyl-lH-pyrazol-4-yl)pyrimidin- 2-ylamino)propan-2-ylcarbamate;
  • the BRAF inhibitor is (S)-methyl l-(4-(3-(5-chloro-2-fluoro-3- (methylsulfonamido)phenyl)-l-isopropyl-lH-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate of formula (I I ) (compound B).
  • the BRAF inhibitors can be obtained by the processes disclosed in WO2011/025927. Where the colorectal cancer is BRAF wild-type, like for example in the microsatellite-unstable BRAF wild- type and KRAS wild-type colorectal cancer, the BRAF inhibitor may not be used in combination with the Wnt inhibitor.
  • EGFR inhibitor can be any compound that targets, decreases or inhibits the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and their mutants.
  • Such compounds which target, decrease or inhibit the activity of the epidermal growth factor receptor family are especially compounds, proteins or antibodies which inhibit members of the EGF receptor tyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or EGF related ligands, and are in particular those compounds, proteins or monoclonal antibodies generically and specifically disclosed in WO 97/02266, e.g. the compound of ex.
  • trastuzumab (HerceptinTM), cetuximab (ErbituxTM), Iressa, Tarceva, OSI-774, CI-1033, EKB-569, GW-2016, El.l, E2.4, E2.5, E6.2, E6.4, E2.ll, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidine derivatives which are disclosed in WO 03/013541, gefitinib, erlotinib, lapatinib, XL-647, HKI-272 (Neratinib), BIBW2992 (Afatinib), EKB-569 (Pelitinib), AV-412, canertinib, PF00299804, BMS 690514, HM781-36b, WZ4002, AP- 26113, panitumumab, matuzumab, pertuzumab, or (R,E)-N-(7-chloro-l-(
  • the EGFR inhibitor is cetuximab.
  • the EGFR inhibitor is (/?,f)-N-(7-chloro-l-(l-(4-(dimethylamino)but-2- enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2-yl)-2-methylisonicotinamide.
  • the EGFR inhibitor is (/?,f)-N-(7-chloro-l-(l-(4-(dimethylamino)but-2- enoyl)azepan-3-yl)-lH-benzo[d]imidazol-2-yl)-2,6-dimethylisonicotinamide.
  • the combination partners are 2-(2',3-dimethyl-2,4'-bipyridin-5-yl)-N-(5-(pyrazin-2- yl)pyridin-2-yl)acetamide, (S)-methyl l-(4-(3-(5-chloro-2-fluoro-3-(methylsulfonamido)phenyl)-l- isopropyl-lH-pyrazol-4-yl)pyrimidin-2-ylamino)propan-2-ylcarbamate and/or cetuximab.
  • the combination partners are 2-(2',3-dimethyl-2,4'-bipyridin-5-yl)-N-(5- (pyrazin-2-yl)pyridin-2-yl)acetamide, (S)-methyl l-(4-(3-(5-chloro-2-fluoro-3-
  • the present disclosure embodiments also include pharmaceutically acceptable salts of the compounds useful according to the disclosure described herein.
  • pharmaceutically acceptable salt refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
  • the salt is sulphate salt, or bisulphate salt.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • treating comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease.
  • treatment can be the diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer.
  • the term “treat” also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • protecting is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject, e.g., a mammal or human.
  • the compounds useful according to the disclosure can also be present as tautomers, N-oxides or solvates, e.g. hydrates. All these variants, as well as any single one thereof or combination of two or more to less than all such variants, are encompassed and to be read herein where a compound included in the inventive combination products, e.g. a Wnt inhibitor and/or a BRAF inhibitor, is mentioned.
  • the present disclosure relates to a pharmaceutical combination, especially a pharmaceutical combination product, comprising the mentioned combination partners and at least one pharmaceutically acceptable carrier.
  • “Pharmaceutical combination” refers to use, application or formulations of the separate partners with or without, preferably with, instructions for combined use or to combination products.
  • the combination partners may thus administered entirely separately or be entirely separate pharmaceutical dosage forms.
  • the combination partners may be pharmaceutical compositions that are also sold independently of each other and where just instructions for their combined use are provided in the package equipment, e.g. leaflet or the like, or in other information e.g. provided to physicians and medical staff (e.g. oral communications, communications in writing or the like), for simultaneous or sequential use for being jointly active, especially as defined below.
  • the effect is synergistic.
  • the selected combination partner are to be administered to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration and/or at the same time.
  • pharmaceutical combination as used herein thus means a pharmaceutical product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients (which may also be combined).
  • the compounds of the present disclosure can be used as a fixed or non-fixed combination.
  • the term "fixed combination” means that the active ingredients, e.g. a Wnt inhibitor and a BRAF inhibitor, are both administered to a patient simultaneously in the form of a single entity or dosage.
  • the active ingredients are present in one dosage form, e.g. in one tablet or in one capsule.
  • non-fixed combination means that the active ingredients are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient.
  • cocktail therapy e.g. the administration of three or more active ingredients.
  • non-fixed combination thus defines especially administration, use, composition or formulation in the sense that the combination partners (i) Wnt inhibitor and either (ii) BRAF inhibitor or (iii) EGFR inhibitor, or both (and if present further one or more co-agents); for example (i) Wnt inhibitor and EGFR inhibitor alone; as defined herein can be dosed independently of each other or by use of different fixed combinations with distinguished amounts of the combination partners, i.e.
  • the combination partners may also be used as entirely separate pharmaceutical dosage forms or pharmaceutical formulations that are also sold independently of each other and just instructions of the possibility of their combined use is or are provided in the package equipment, e.g. leaflet or the like, or in other information e.g. provided to physicians and medical staff.
  • the independent formulations or the parts of the formulation, product, or composition can then, e.g. be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts.
  • the time intervals are chosen such that the effect on the treated disease in the combined use of the parts is larger than the effect which would be obtained by use of only any one of the combination partners (i), (ii) and (iii), thus being jointly active.
  • the total amounts of the combination partners to be administered in the combined preparation can be varied, e.g. in order to cope with the needs of a patient sub-population to be treated or the needs of the single patient which different needs can be due to age, sex, body weight, etc. of the patients.
  • the combination partners, a Wnt inhibitor, a BRAF inhibitor and a EGFR inhibitor in any disclosed embodiment are preferably formulated or used to be jointly (prophylactically or especially
  • beneficial effect e.g. a mutual enhancing of the effect of the combination partners, in particular a synergism, e.g. a more than additive effect, additional advantageous effects (e.g. a further therapeutic effect not found for any of the single compounds), less side effects, a combined therapeutic effect in a non-effective dosage of one, both or all three combination partners, and very preferably a clear synergism of the combination partners.
  • beneficial effect e.g. a mutual enhancing of the effect of the combination partners, in particular a synergism, e.g. a more than additive effect, additional advantageous effects (e.g. a further therapeutic effect not found for any of the single compounds), less side effects, a combined therapeutic effect in a non-effective dosage of one, both or all three combination partners, and very preferably a clear synergism of the combination partners.
  • the term "jointly (therapeutically) active” may mean that the compounds may be given separately or sequentially (in a chronically staggered manner, especially a sequence-specific manner) in such time intervals that they preferably, in the warm-blooded animal, especially human, to be treated, and still show a (preferably synergistic) interaction (joint therapeutic effect).
  • a joint therapeutic effect can, inter alia, be determined by following the blood levels, showing that both compounds are present in the blood of the human to be treated at least during certain time intervals, but this is not to exclude the case where the compounds are jointly active although they are not present in blood simultaneously.
  • the present disclosure thus pertains to a combination product for simultaneous or sequential use, such as a combined preparation or a pharmaceutical fixed combination, or a combination of such preparation and combination.
  • the compounds useful according to the disclosure may be manufactured and/or formulated by the same or different manufacturers.
  • the combination partners may be brought together into a combination therapy: (a) prior to release of the combination product to physicians (e.g. in the case of a kit comprising the compound of the disclosure and the other therapeutic agent); (b) by the physician themselves (or under the guidance of a physician) shortly before administration; (c) in the patient themselves, e.g. during sequential administration of the compound of the disclosure and the other therapeutic agent.
  • the combination partners form ing a corresponding combination according to the disclosure may be mixed to form a fixed pharmaceutical composition or they may be administered separately or pairwise (i.e. before, simultaneously with or after the other drug substance(s)).
  • pharmaceutically effective preferably relates to an amount that is effective against the progression of a disease or disorder as disclosed herein.
  • a Wnt inhibitor, the combinations according to the present disclosure, pharmaceutical compositions or combination product of the present disclosure is especially suitable for treatment of a patient suffering from a proliferative disorder, in particular a solid tumor, BRAF V600E -mutant colorectal cancer, which can be microsatellite-unstable, or microsatellite-unstable BRAF wild-type and KRAS wild-type colorectal cancer.
  • a Wnt pathway in the BRAF V600E -mutant colorectal cancer or microsatellite- unstable BRAF wild-type and KRAS wild-type colorectal cancer is activated compared to a control.
  • AXIN2 expression in the colorectal cancer is higher compared to a control.
  • the cancer comprises mutated RNF43, mutated RSP02 or mutated RSP03, particularly mutated RNF43. Because RNF43 and ZN RF3 are two homologous Zinc/RI NG finger cell surface transmembrane E3 ubiquitin ligases for Wnt receptor Frizzled that inhibit the cell surface levels of Wnt receptor complex composing Frizzled and LRP6, a Wnt inhibitor can also be used in ZN FR43 mutated colorectal cancer.
  • the cancer can alternatively or in addition comprise RSPO fusion.
  • a therapeutically effective amount of a compound of the present disclosure refers to an amount of the compound of the present disclosure that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
  • a therapeutically effective amount refers to the amount of the compound of the present disclosure that, when administered to a subject, is effective to (1) at least partially alleviating, inhibiting, preventing and/or ameliorating a condition, or a disorder or a disease (i) mediated by a Wnt pathway, or mediated by BRAF activity and/or EGFR activity, or (ii) characterized by activity (normal or abnormal) of Wnt pathway, EGFR and/or BRAF; or (2) reducing or inhibiting the activity of Wnt pathway, EGFR and/or of BRAF.
  • the term "subject" refers to an animal. Typically the animal is a mammal. A subject also refers to for example, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In certain embodiments, the subject is a primate. In yet other embodiments, the subject is a human.
  • primates e.g., humans
  • the subject is a primate.
  • the subject is a human.
  • Wnt inhibitor can be for example administered in unit dosage of about 1-5000 mg of active ingredient(s) for a subject of about 50-70 kg, or about lmg - 3g or about 1-250 mg or about 1-150 mg or about 0.5- 100 mg, or about 1-50 mg of active ingredients.
  • the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof, is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
  • BRAF inhibitor of the present disclosure can be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art.
  • a therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 30mg/kg per body weight.
  • An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5mg to about 2000mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form.
  • Suitable unit dosage forms for oral administration comprise from about 1 to 500mg active ingredient.
  • an indicated daily dosage in the larger mammal e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form.
  • Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.
  • a therapeutic amount or dose of the compounds of the present disclosure may range from about 0.1 mg/kg to about 500 mg/kg, alternatively from about 1 to about 50 mg/kg.
  • treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses (such as two, three, or four times daily).
  • Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.
  • the dosage of the active ingredient to be applied to a warm-blooded animal depends upon a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed.
  • a physician, clinician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Optimal precision in achieving concentration of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
  • carrier or “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289- 1329). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
  • the pharmaceutical combination product according to the disclosure (as fixed combination, or as kit, e.g. as combination of a fixed combination and individual formulations for one or both combination partners or as kit of individual formulations of the combination partners) comprises the combination of the present disclosure and one or more pharmaceutically acceptable carrier materials (carriers, excipi- ents).
  • the pharmaceutical combination or the combination partners constituting it can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc.
  • the combination products of the present disclosure can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions).
  • the combination products and/or their combination partners can be subjected to conventional pharmaceutical operations such as sterilization and/or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifers and buffers, etc.
  • the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more commonly known carriers, e.g. one or more carriers selected from the group consisting of a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or
  • carriers e.g., one or more carriers selected from the group consisting of a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
  • lubricants e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or
  • binders e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth,
  • methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and e) absorbents, colorants, flavors and sweeteners.
  • disintegrants e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures
  • absorbents, colorants, flavors and sweeteners e.g., sodium carboxymethylcellulose and/or polyvinylpyrrolidone
  • Tablets may be either film coated or enteric coated according to methods known in the art.
  • compositions for oral administration especially include an effective amount of one or more or in case of fixed combination formulations each of the combination partners (active ingredients) in the form of tablets, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use are prepared according to any method known in the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets may contain the active ingredient(s) in admixture with nontoxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets are uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example, peanut oil, liquid paraffin or olive oil.
  • Parenteral compositions, transdermal, topical compositions and other can be prepared by known methods in the art.
  • Example 1 Co-occurrence of BRAF V600E and upstream Wnt pathway mutations in patient CRC samples
  • CRCs There are at least two distinct molecular pathways that underlie the pathogenesis of sporadic CRC: the conventional pathway and the serrated pathway. Approximately 70% of CRCs arise via the conventional pathway. These tumors are characterized by mutational activation of the Wnt pathway through inactivating mutations in APC or AXIN2 or mutations in the GSK3-target residues in beta-catenin (Jin, L.H., et al. Int. J. Cancer. 2003, 107:696-99; Korinek, V., et ai, Science. 1997, 275:1784-7; Morin, P.J., et ai, Science.
  • APC associates with AXI N, GSK-3beta and casein kinase 1 to form the beta-catenin destruction complex. This complex phosphorylates beta-catenin, resulting in its ubiquitylation and subsequent degradation by the proteasome (Polakis, P. Curr. Biol. 2002, 12:R499-R501).
  • TNF/LEF T-cell factor/lymphoid enhancer factor
  • Serrated pathway CRCs contain either BRAF or KRAS mutations, and lack APC mutations (Bettington, M., et al., Histopathol. 2013, 62:367-86).
  • Wnt pathway activation was identified as an important step in the progression from intestinal hyperplasia to carcinoma (Rad, R., et al., Cancer Cell. 2013, 24:15-29).
  • BRAF-mutant CRCs tend to have minimal chromosomal instability and exhibit hypermethylation of CpG islands, which can decrease the expression of DNA repair genes and cell cycle regulators.
  • the mutant BRAF inhibitor vemurafenib achieves a response rate of approximately 80% in BRAF V600E -mutant melanoma
  • the response rate in BRAF V600E -mutant CRC is approximately 5% (Kopetz, S. et al. J. Clin. Oncol, abstr. 2010, 28:3534; Corcoran, R.B., et al., J. Clin. Oncol. Suppl. abstr. 2012, 3528).
  • RSPOl-4 secreted glycoproteins R-spondins 1-4
  • RSPOs bind to LGR4-6 receptors and the transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, forming a ternary complex (Chen, P-H., et al., Genes Dev. 2013, 27:1345-50; Xie, Y., et al., EMBO Rep. 2013, 14:1120-6; Zebisch, M., et al., Nature Com. 2013, 4:1-12).
  • RNF43/ZNRF3 antagonize Wnt signaling by promoting the turnover of the Wnt receptors, Frizzled and LRP6 (Hao, H.X., et al., Nature. 2012, 485:195-200; Koo, B.K., et al., Nature. 2012, 488:665-9). Binding of RSPO induces the endocytosis of RNF43/ZNRF3, thereby increasing levels of membrane-bound Frizzled and LRP6 and enhancing Wnt signaling.
  • RSPO fusions that elevate expression of RSP02 or RSP03 have been identified in colon cancer (Seshagiri, S., et al., Nature. 2012, 488:660-4), and inactivating mutations in RNF43 have been reported in mucinous tumors of the pancreas, mucinous ovarian carcinoma, and
  • BRAF -mutant CRC resection specimens Forty-five BRAF -mutant CRC resection specimens were purchased from Asterand, PLC. The FFPE blocks were sent to Foundation Medicine, Inc. for genetic analysis using the T5b NGS panel. Complete characterization was received, and BRAF/Wnt pathway genetic mutation annotation was performed manually. Tabularization and graphical representation was performed using EXCEL.
  • Threshold setting was automatically set by ABI7900 software (SDS2.4), and all samples were normalized to a UBC control gene. Samples with expression higher than 30 Ct's in the fusion assays are considered positive for the corresponding RSPO fusion.
  • the frequency of RSPO fusions may also be enriched in the BRAF V600E -mutant CRCs, with a frequency of RSP02 plus RSP03 fusions of 13.3% in BRAF V600E -mutant CRC samples versus 2% when 430 unselected CRC samples in the TCGA database are analyzed with a trial version of OmicSoft.
  • This molecular epidemiology data strongly suggests that Wnt pathway activation, via genetic alterations in upstream Wnt pathway regulators, is important for BRAF V600E -mutant CRC tumorigenesis.
  • Example 2 Efficacy of a Wnt pathway inhibitor and combinations comprising (i) a Wnt pathway inhibitor, (ii) a mutant BRAF inhibitor, and/or (iii) an EGFR inhibitor in a BRAF V600E ;RSPO3 fusion + lower Gl adenocarcinoma cell line model and a patient-derived BRAF V600E ;RNF43-mutant CRC xenograft model
  • Genomic DNA of J HOM-2B cells was isolated .
  • the DNA fragment containing PTPRK-RSP03 fusion was amplified by PCR using HH3 and HH4 primers, and analyzed by electrophoresis. DNA sequence of PCR fragment was determined by Sanger sequencing. Sequence of HH3 and HH4 primers are following. HH3: AAACTCGGCATGGATACGAC. HH4: G CTTCATG CC AATTCTTTCC .
  • JHOM-2B cells were cultured in DMEM:F-12 (ATCC, Cat# 30-2006), supplemented with 10% fetal bovine serum (5% C02 at 37°C).
  • JHOM-2B cells For foci formation assay, approximately 12,000 JHOM-2B cells were seeded in a 6-well tissue culture plate with 2 mL of growth media. After overnight culture for cell attachment, media was replaced with fresh growth media containing 1 ⁇ of COM POUND A and/or DMSO. When cell colonies reached a desirable size (24 days), cells were fixed with 10% (vol/vol) buffered formalin and stained with crystal violet solution. After several washes, the plate was dried and imaged.
  • Cell proliferation and EC 50 values were determined through the use of CellTiter-Glo ® (CTG), a method based on luminescent detection of mitochondrial ATP (Promega, Cat# G7573).
  • CCG CellTiter-Glo ®
  • CTG was determined at day 0 and day 7 of treatment as follows: 100 ⁇ of the CTG reagent was added to each well of the 96 well plates, and placed on a shaker for 30-45 minutes at room temperature. Luminescence was measured using the Perkin Elmer-VictorTM X4. Raw luminescent readings were entered into an Excel spread sheet that was formulated to determine the EC 50 value for each PDAC cell lines (day 7 - day 0).
  • the combination assay was set-up in the same manner as the growth assay, with several exceptions.
  • Compound was added in an 8 x 8 dose-matrix fashion, with COMPOUND A dilutions added to one axis, and COMPOUND B added to the other axis.
  • a CTG assay was performed on the plates after 7 days of compound treatment.
  • Raw luminescent readings were entered into an Excel spread sheet that was formulated to determine inhibition values, which were then entered into an Excel spread sheet that was used uploaded to the Chalice Analyzer server.
  • the final inhibition grid was visualized using the Chalice Analyzer Client program.
  • JHOM-2B cells were plated onto 10 cm tissue culture dishes at approximately 1.5 e+6 cells per dish, and treated with COMPOUND A (100 nM) or DMSO for 72 hours.
  • Total RNA from COMPOUND A treated and untreated JHOM-2B cells was extracted using the RNeasy Plus Mini Kit (Qiagen, Cat# 74134) and reverse transcribed with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat# 4368814), according to the manufacture's protocol. Gene transcript expression levels were determined using the Applied Biosystems 7900HT Fast Real-time PCR System.
  • Quantitative real-time polymerase chain reactions were performed in 12 ⁇ reactions, consisting of 0.6 ⁇ of 20x TaqMan ® Gene Expression Assay, 6 ⁇ TaqMan ® 2x Universal PCR Master Mix (Applied Biosystems, Cat# 4304437), 0.4 ⁇ DNase/RNase free distilled water and 5 ⁇ cDNA template (1 ng/ ⁇ ). Thermocycling conditions were as follows: 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. All experimental reactions were carried out in quadruplicate. Gene expression was quantified using the ⁇ 2 cycle threshold method, with 18s serving as an internal control.
  • Applied Biosystems TaqMan ® Gene Expression Assay reagents used were as follows: AXIN2 (Hs00610344_ml), CCND1 (CyclinDl) (Hs0076553_ml), LGR5 (Hs00969418_ml), MYC (Hs00153408_ml) and 18s (4319413E). Statistical analysis was performed using a Student's t-test.
  • JHOM-2B cells were plated onto 8-10 cm tissue culture dishes at approximately 1.5 e+6 cells per dish, and treated for 72 hours under the following conditions: DMSO; COM POUND A (100 nM); NVP- COMPOUND B-NX-12 (COMPOUND B) (100 nM); cetuximab (50 nM); COMPOUND A (100 nM) +
  • COMPOUND B (100 nM); COMPOUND A (100 nM) + cetuximab (50 nM); COMPOUND B (100 nM) + cetuximab (50 nM); and COMPOUND A (100 nM) + COMPOUND B (100 nM) + cetuximab (50 nM).
  • Phospho-LRP6 Cell Signaling Technology (CST), Cat# 2568, 2% milk
  • LRP6 CST, Cat# 3395, 2 % milk
  • Phospho-EGFR Tyrl068
  • CST Cell Signaling Technology
  • Phospho-EGFR Tyrl068
  • CST Cell Signaling Technology
  • EGFR CST, Cat#2232 , 5 % BSA
  • Phospho-p44/42 MAPK Erkl/2)(Thr202/Tyr204)
  • p44/42 MAPK Erkl/2
  • Cleaved Caspase-8 CST, Cat# 9496, 5 % BSA
  • c-Myc Abeam, Cat# ab32072, 2% milk
  • p21/Wafl/Cipl CST, Cat# 2947, 5% milk
  • ⁇ -Myc Abeam,
  • Blocking reagents were prepared fresh in phosphate buffered saline 0.1% (vol/vol) Tween-20 (PBS-T), and membranes were blocked for 1 hr on a shaker at room temperature.
  • Primary antibodies were added at 1:1000 dilutions (p44/42 MAPK (Erkl/2) 1:5000, and ⁇ -Tubulin 1:10,000), and incubated overnight with gentle rocking at 4° C. Immunoblots were washed 3 times, 5 min each with PBS-T, and secondary antibody was added at 1:10,000 dilution into PBS-T milk for 1 hour on a shaker at room temperature. After several washes, enhanced chemiluminescence (ECL) reactions were performed according to manufacture recommendations (SuperSignal ® West Dura Extended Duration Substrate; Thermo Scientific, Cat# 34076).
  • IHC was performed on the Ventana DISCOVERY XT automated I HC platform (Tuscon, AZ), using optimized protocols described below.
  • FFPE slides were cut at 3.5 ⁇ thickness, attached to charged slides (Thermo-Scientific Colormark Plus Cat # CM-5951PLUS BLUE), baked at 60°C for 1 hour and loaded on the DISCOVERY XT Staining System. Slides were deparaffinized using Ventana EZ Prep (Cat # 950- 100). Antigen retrival was performed using Ventana Cell Conditioning #1 reagent (Cat # 950-124).
  • a rabbit monoclonal antibody produced by immunizing animals with a synthetic peptide corresponding to residues surrounding C-terminus of human keratin 7 protein (Catalog # M3522, Lot# 121022C) was obtained from Spring Bioscience (Pleasanton, CA.). The antibody was stored at 4°C until use.
  • Primary antibody was diluted at a concentration of 1:100 in DAKO Cytomation Antibody Diluent (Cat # S0809), manually applied during the antibody titration step in 100 ⁇ volume, and incubated for 60 minutes at 37°C. Subsequently, incubation with Ventana OmniMap prediluted HRP-conjugated anti-rabbit antibody (Cat # 760-4311) was performed for 4 minutes.
  • a rabbit monoclonal antibody produced by immunizing animals with a synthetic peptide corresponding to residues surrounding C-terminus of human keratin 20 protein (Catalog # M3332, Lot# 121218CD) was obtained from Spring Bioscience (Pleasanton, CA.). The antibody was stored at 4°C until use.
  • a rabbit polyclonal antibody produced by immunizing animals with a synthetic peptide corresponding to residues surrounding SerlO of phospho-Histone H3 was obtained from Cell Signaling Technology (Danvers, MA). The antibody was stored at -20°C until use.
  • the optimal IHC protocol included Heat and Ventana "Standard- Cell Conditioning 1" setting for antigen retrieval.
  • a rabbit polyclonal antibody produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ki67 was obtained from Cell Signaling
  • the antibody was stored at -20°C until use.
  • the optimal I HC protocol included Ventana "Standard- Cell Conditioning 1" setting for antigen retrieval.
  • Primary antibody was diluted at a concentration of 1:100 in DAKO Cytomation Antibody Diluent (Cat # S0809), manually applied during the antibody titration step in 100 ⁇ volume, and incubated for 60 minutes at RT.
  • FFPE slides were cut at 3.5 ⁇ thickness, attached to charged slides (Thermo-Scientific Colormark Plus Cat # CM-5951PLUS BLUE), baked at 60°C for 1 hour. Slides were then placed in a rack and
  • Alcian Blue pH2.5 staining kit (Cat# k066) was obtained from Poly Scientific R&D Corp (Bayshore, NY). Alcian Blue pH 1.0 (Cat# s2412) was obtained and used in place of Alcian Blue pH2.5. Slides were transferred from distilled water and placed in 3% aqueous acidic acid for 3 minutes RT. Slides were then directly placed in Alcian Blue pHl.O for 30 minutes.
  • slides are washed in running tap water for 10 minutes, rinsed in distilled water, and placed in 0.1% Nuclear Fast Red Kernechtrot for 5 minutes. Slides were removed and then washed in running water for 1 minute. After wash slides are dehydrated in 95% alcohol, 100% alcohol, and cleared in xylene, 2 changes each. Slides were then mounted in xylene based mounting media.
  • Necrotic regions and stroma were manually excluded from HCOX1329 xenograft Ki67 image analysis, and the tumor tissues were segmented using TissuemorphDP module. Staining was quantified as percent blue pixels for Alcian blue, percent positive pixels for pHH3, or percent positive nuclei for Ki67. Unpaired t tests were used to determine statistical significance. Symbols used: *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ns, not significant.
  • JHOM-2B human lower Gl tumor cells were obtained from RIKEN (Japan) and a working stock was frozen at passage 14. The line was shown to be free of Mycoplasma sp. and murine viruses in the IMPACT VIII PCR assay panel (RADIL, University of Missouri, Columbia, MO). These cells were maintained in
  • DMEM HAM's F12 1:1 (ATCC #30-2006) plus 10% FBS (Omega Scientific Inc. #FB-09) at 37°C in a humidified atmosphere containing 5% carbon dioxide.
  • JHOM-2B cells were harvested at 95-100% confluency, washed, and resuspended in a 1:1 mixture of cold DPBS and MatrigelTM (Becton-Dickinson #354234) at a concentration of 1 X 108 cells/1.5 ml. Finally, 1x107 cells in a total volume of 150 ⁇ . were implanted subcutaneously into the right axillary region of female nu/nu mice.
  • HCOX1329 is a human primary xenograft model derived from a poorly differentiated colonic adenocarcinoma, which was implanted and passaged. HCOX1329 contains a Sff ⁇ F ⁇ mutation and several mutations/variants in RNF43 (P192fs, G659fs, and C272R) (Korn, J., et a ⁇ ., Manuscript in preparation).
  • mice Female nu/nu mice were implanted subcutaneously with 3x3x3mm tumor fragment containing 50% phenol red-free matrigel (BD Biosciences) in DMEM.
  • mice were anesthetized with continuous flow of 1.5-3.5% isoflurane/oxygen mixture using the integrated multi chambers anesthesia center (I MCAC).
  • I MCAC integrated multi chambers anesthesia center
  • mice with average tumor volume of 200 mm 3 were randomized into 8 experimental groups on day 21 (JHOM-2B) or day 17 (HCOX1329) post implantation. Tumors were measured with digital calipers twice a week during active dosing period. Tumor volumes were calculated using the ellipsoid formula: (length x width 2 )/2. Body weights were recorded twice a week at the time of tumor measurement. After 11 days (Groups 2 and 6) or 14 days of treatment, the dosing of mice harboring JHOM-2B xenografts was terminated, and animals euthanized. After 10 days of treatment, the dosing of mice harboring
  • HCOX1329 xenografts was terminated, and animals euthanized. Plasma and tumors were collected at 7hr post last dose for pharmacokinetic (PK) and pharmacodynamic (PD) analysis. All data were expressed as mean ⁇ standard error of the mean (SEM). Delta tumor volume and body weight were used for statistical analysis. Between group comparisons were carried out using the Kruskal-Wallis ANOVA followed by a post hoc Dunn's test. For all statistical evaluations, the level of significance was set at p ⁇ 0.05. Significance compared to the vehicle control group is reported unless otherwise stated. The standard protocols used in pharmacology studies are not pre-powered to demonstrate statistically significant superiority of a combination over the respective single agent treatment. The statistical power is often limited by potent single agent response and/or model variability.
  • JHOM-2B is a human cancer cell line that is reported to be derived from a mucinous ovarian carcinoma (Riken Bioresource Center). However, it can be extremely challenging to clinically differentiate a mucinous tumor of the ovary from a colorectal or appendiceal tumor, because all of these tumor types disseminate widely throughout the abdomen. Recently, it has become clear that the expression pattern of CK7 and CK20 is very useful in determining the site of origin of such tumors (Vang, R., USCAP presentation. 2013). JHOM-2B xenografts have rare cells that stain positively for CK7, whereas CK20 is diffusely positive (Fig. 2A). This expression pattern is consistent with lower Gl, not ovarian, origin.
  • JHOM-2B contains a BRAF V600E mutation (Barretina, J., et al., Nature. 2012, 492:603-7), which has not been reported in appendiceal cancer (Shen, J. P., et al., J. Clin. Oncol. 2012, suppl 34; abstr 397), strongly suggesting the JHOM-2B is of colorectal origin.
  • a survey for genetic alterations in upstream Wnt pathway regulators revealed that JHOM-2B also harbors a fusion between exonl of PTPRK and exon2 of RSP03 (Fig. 2B).
  • HCOX1329 is a human primary xenograft model derived from a poorly differentiated colonic adenocarcinoma. HCOX1329 contains a Sff ⁇ F ⁇ mutation and several mutations/variants in RNF43 (P192fs, G659fs, and C272R) (Korn, J., et al., Manuscript in prepartation).
  • JHOM-2B tumor cells are sensitive to treatment with COMPOUND A in vitro, as assessed by both a foci formation assay (Fig. 3A) and a 7-day CTG assay (Fig. 3B), with an EC 50 of approximately 0.06 ⁇ .
  • Treatment with the triple combination of COM POU N D A+COM POU ND B+cet caused regression (T/TO -26%), which although statistically not superior to COM POUN D B+cet, showed a trend toward greater anti-tumor activity (Fig. 6).
  • Treatment with COM POU ND A as a single agent or in combination decreased cell proliferation (Fig. 7) and induced mucinous differentiation of J HOM-2B tumor cells (Fig. 8).
  • the accumulation of mucin in xenografts treated with COM POUN D A may affect tumor volume
  • HCOX1329 is also microsatellite unstable (I HC of the patient's tumor sample at the time of diagnosis was reported as M LH1, negative; PMS2, negative; MSH2, positive; MSH6, positive) and harbors two mutations/variants in ZNRF3 (H537R and E383fs).
  • Example 3 Phase 1, open-label, dose escalation and dose expansion study, to evaluate the safety, pharmacokinetics, and activity of the porcupine inhibitor COMPOUND A in patients with BRAF V600E - mutant CRC harboring upstream Wnt pathway mutations
  • Dose expansion cohort Additional molecular pre-selection for RNF43 mutations or RSPO fusions
  • Example 4 Phase lb, open label, multicenter, dose escalation and expansion study, to evaluate the safety, pharmacokinetics, and activity of the porcupine inhibitor COMPOUND A in combination with the BRAF inhibitor COMPOUND B and the EGFR inhibitor cetuximab in patients with BRAF V600E -mutant CRC harboring upstream Wnt pathway mutations Inclusion criteria:
  • Dose expansion cohort Additional molecular pre-selection for RNF43 mutations or RSPO fusions
  • Dose escalation cohorts Prior treatment with chemotherapy permitted; prior treatment with RAF inhibitors and EGFR monoclonal antibodies permitted.
  • Dose expansion cohort Prior treatment with chemotherapy permitted; prior treatment with RAF inhibitors and EGFR monoclonal antibodies not allowed.
  • Example 5 Efficacy of a Wnt pathway inhibitor and combinations comprising (i) a Wnt pathway inhibitor, (ii) a BRAF inhibitor, and/or (iii) an EGFR inhibitor in additional patient-derived,
  • Example 6 Decreased cell proliferation and mucinous differentiation induced by a Wnt pathway inhibitor and a combination comprising (i) a Wnt pathway inhibitor and (ii) an EGFR inhibitor in a patient-derived microsatellite-unstable, RNF43 and ZNRF3-mutant CRC xenograft model that is wild type for BRAF and KRAS
  • T70 is a human primary xenograft model derived from a poorly differentiated colonic adenocarcinoma, which was implanted and passaged in nu/nu mice.
  • T70 is microsatellite unstable, BRAF 7 , KRAS 7 , and harbors mutations/variants in RNF43 (W200L, E278delE) and ZNRF3 (Q226*,C230fs) (as determined by Haloplex custom cancer panel).
  • Female nu/nu mice were implanted subcutaneously with 100,000 tumor cells containing 50% phenol red-free matrigel (BD Biosciences) in DMEM.
  • mice were anesthetized with continuous flow of 1.5-3.5% isoflurane/oxygen mixture using the integrated multi chambers anesthesia center (I MCAC).
  • I MCAC integrated multi chambers anesthesia center
  • a 12G trocar was used to take tumor cells with 0.1 ml of the diluted matrigel and implant them subcutaneously into the right axillary region of nude mice, and this was repeated in the left axillary region.
  • mice with average tumor volume of 200 mm 3 were randomized into 4 experimental groups on day 21 post implantation. Tumors were measured with digital calipers twice a week during active dosing period. Tumor volumes were calculated using the ellipsoid formula: (length x width 2 )/2. Body weights were recorded twice a week at the time of tumor measurement. After 14 days the dosing of mice harboring T70 xenografts was terminated, and animals euthanized. Plasma and tumors were collected at 7hr post last dose for pharmacokinetic (PK) and pharmacodynamic (PD) analysis.
  • PK pharmacokinetic
  • PD pharmacodynamic
  • Example 7 Phase 1, open-label, dose escalation and dose expansion study, to evaluate the safety, pharmacokinetics, and activity of the porcupine inhibitor COMPOUND A in patients with
  • Dose expansion cohort Additional molecular pre-selection for RNF43 mutations, RSPO fusions, or other upstream Wnt pathway mutations
  • Example 8 Phase lb, open label, multicenter, dose escalation and expansion study, to evaluate the safety, pharmacokinetics, and activity of the porcupine inhibitor COMPOUND A in combination with the EGFR inhibitor cetuximab in patients with microsatellite-unstable, BRA ", KRAS WT CRC harboring upstream Wnt pathway mutations
  • Dose expansion cohort Additional molecular pre- selection for RNF43 mutations, RSPO fusions, or other upstream Wnt pathway mutations

Abstract

La présente invention concerne un procédé de traitement de patients souffrant d'un cancer colorectal, plus spécifiquement un mutant de BRAF V600E ou BRAF type sauvage, à l'exception de KRAS de type sauvage et d'un cancer colorectal à instabilité microsatellitaire à l'aide d'un inhibiteur de la voie Wnt ou d'une combinaison comprenant un inhibiteur de la voie Wnt et un inhibiteur de BRAF et/ou inhibiteur d'EGFR. L'invention concerne en outre des formulations pharmaceutiques, des utilisations, des procédés, des combinaisons, et des modes de réalisation associés.
PCT/IB2015/052245 2014-03-27 2015-03-26 Procédés de traitement de cancers colorectaux avec mutations en amont de la voie wnt WO2015145388A2 (fr)

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WO2017221142A1 (fr) * 2016-06-22 2017-12-28 Novartis Ag Inhibiteurs de wnt destinés à être utilisés dans le traitement de la fibrose
WO2019245460A1 (fr) * 2018-06-21 2019-12-26 National University Of Singapore Stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase e3 rnf43

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WO2017221142A1 (fr) * 2016-06-22 2017-12-28 Novartis Ag Inhibiteurs de wnt destinés à être utilisés dans le traitement de la fibrose
CN109415342A (zh) * 2016-06-22 2019-03-01 诺华股份有限公司 用于治疗纤维化的wnt抑制剂
AU2017281980B2 (en) * 2016-06-22 2020-05-21 Novartis Ag Wnt inhibitors for use in the treatment of fibrosis
WO2019245460A1 (fr) * 2018-06-21 2019-12-26 National University Of Singapore Stratification de patients cancéreux hébergeant des mutations oncogènes dans l'ubiquitine ligase e3 rnf43

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