WO2014002007A1 - Procédé de prédiction ou de suivi de la réponse à des inhibiteurs des igf-1r et des ir, faisant appel à des biomarqueurs - Google Patents

Procédé de prédiction ou de suivi de la réponse à des inhibiteurs des igf-1r et des ir, faisant appel à des biomarqueurs Download PDF

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WO2014002007A1
WO2014002007A1 PCT/IB2013/055208 IB2013055208W WO2014002007A1 WO 2014002007 A1 WO2014002007 A1 WO 2014002007A1 IB 2013055208 W IB2013055208 W IB 2013055208W WO 2014002007 A1 WO2014002007 A1 WO 2014002007A1
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cancer
ser
compound
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ala
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Prabha Beerchandra MISHRA
Aurelio LOBO
Muralidhara Padigaru
Shashank ROHATAGI
Abhijit Roychowdhury
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Piramal Enterprises Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a method of monitoring response of a subject having cancer to treatment with a compound which is an Insulin Like Growth Factor- 1 Receptor (IGF-IR) and Insulin Receptor (IR) inhibitor.
  • the method of the present invention comprises detecting gene signature with at least one biomarker selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample.
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • IGF-IR signaling controls proliferation, differentiation, growth and cell survival in many tissues [PLoS genetics, 2012, 8(3), el002609, doi: 10.1371/journal.pgen.1002609].
  • the Insulin-like Growth Factor (IGF) family and the IGF-IR play an important role in cancer.
  • IGF-IR is a receptor tyrosine kinase (RTK) that functions as a heterotetramer and is structurally related to the Insulin Receptor (IR) with 60% homology in its amino acid sequence.
  • RTK receptor tyrosine kinase
  • IGF-IR signaling pathway Deregulation of the IGF- IR signaling pathway is involved in many malignancies including colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian and endometrial cancer as well as sarcomas.
  • the intricate and complex IGF-IR signaling pathway provides many opportunities for therapeutic intervention and several novel therapeutics aimed at IGF-IR are being developed [Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 14-30].
  • growth hormone releasing hormone antagonists e.g. JV-1-38
  • growth hormone receptor antagonists e.g. pegvisomant, (Pfizer)
  • IGF-IR antibodies e.g. CP-751,871, (Pfizer)
  • AVE1642 Sanofi-Aventis/EM164
  • IMC-A12 ImClone LLC
  • SCH-717454 Merck
  • BIIB022 Biogen circa
  • AMG 479 Amgen
  • IGF-IR tyrosine kinase inhibitors e.g.
  • BMS-536942 (Bristol-Myers Squibb), BMS-554417 (Bristol-Myers Squibb), NVP-AEW541(Novartis), NVP-ADW742 (Novartis), AG1024 (Calbiochem-EMD Biosciences), OSI-906 (OSI Pharmaceuticals), potent quinolinyl-derived imidazo (l,5-a)pyrazine (PQIP, Insmed ), picropodophyllin (PPP, Axelar AB), Nordihydroguaiaretic acid (INSM-18/NDGA, Insmed) [Recent Patents on Anti-Cancer Drug Discovery, 2009, 4, 54-72].
  • A-928605 (trans-3-[2-(2-Chlorobenzyl)-lH-benzimidazol-5-yl]-l-[4-(4-morpholinyl)cyclohexyl]- lH-pyrazolo[3,4-d]pyrimidin-4-amine) (Abbott) is a dual IGF-1R and IR inhibitor which is efficacious in in vivo models against neuroblastoma, pancreatic cancer and non-small cell lung cancer (NSCLC) [Recent Patents on Anti-Cancer Drug Discovery, 2012, 7, 14-30].
  • NSCLC non-small cell lung cancer
  • biomarkers in the early clinical trials of rationally designed molecularly targeted anticancer agents has the potential to increase patients' benefit from early clinical trials, accelerate the drug development process, exploit the ability to generate information about human cancer and decrease the risk of late and costly drug attrition [Clinical Pharmacology & Therapeutics, 2009, 85, 131-133].
  • Figure I Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in HEK- IGF1R cells using Microarray.
  • Figure II Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in HEK- IGF1R cells using real time quantitative polymerase chain reaction (RTQPCR).
  • RTQPCR real time quantitative polymerase chain reaction
  • Figure III Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in A673 cells using RTQPCR.
  • Figure IV Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in Colo- 205 cells using RTQPCR.
  • Figure V Effect of Compound A on down stream targets of Ras/Raf/ERK pathway in tumor tissue obtained from Colo205 xenograft model using RTQPCR.
  • the present invention relates to a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR (Insulin Like Growth Factor- 1 Receptor) and IR (Insulin Receptor) inhibitor.
  • a compound which is an IGF-IR (Insulin Like Growth Factor- 1 Receptor) and IR (Insulin Receptor) inhibitor.
  • the present invention provides a method of monitoring the response of a subject having cancer to the treatment with a compound which is an IGF-IR and IR inhibitor; said method comprising determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample pre-therapy and post-therapy.
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • the present invention provides a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising the steps of:
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • step (b) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);
  • step (c) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c);
  • the present invention also relates to a method of testing whether the compound, which is an IGF-IR and IR inhibitor produces a therapeutic response in a subject having cancer.
  • the present invention relates to a method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprises comparing expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before administering the compound with the expression level of the said biomarker transcript in a sample after administering the said compound. Decrease in the expression level of at least one of the said biomarker transcripts after administering the compound which is an IGF-IR and IR inhibitor would help in identifying that the cancer or tumor is responsive to treatment with the compound, which is an IGF-IR and IR inhibitor.
  • the present invention provides a kit for performing the method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; wherein the said kit comprising instructions for its use.
  • Biomarker refers to a molecule or molecular species (such as a protein or gene) used to indicate or measure a biological process.
  • a biological process is a process occurring in living organisms which is regulated by many means, examples include control of gene expression, protein modification or interaction with a protein or substrate molecule. Detection and analysis of a biomarker specific to a disease can aid in the identification, diagnosis, and treatment of the disease, or act as a drug response marker for the disease.
  • the level of a particular protein found in blood may be an indicator of a specific blood-associated disorder.
  • biomarker or “drug response marker” are used interchangeably as the biomarkers are used to monitor response of subject administered with a compound, which is an IGF-IR and IR inhibitor. Biomarker can be detected at mRNA level (transcript) or at the protein level.
  • the terms “biomarker” or “biomarker transcript” are used interchangeably.
  • IGF-IR and IR inhibitor refers to small molecules which act as inhibitors of IGF-IR and IR. Inhibiting the IGF-IR and IR leads to inhibition of disorders or diseases related to IGF-IR and IR.
  • the disorder or disease mediated by IGF-IR and IR is cancer. Cancer is an abnormal growth of cells which tend to proliferate in an uncontrolled way. Deregulation of the IGF-IR signaling pathway is involved in many malignancies including colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian and endometrial cancer as well as sarcomas.
  • the IGF-IR and IR inhibitor used in accordance with method of the present invention finds use in the treatment of cancer wherein treating cancer refers to ameliorating, mitigating or delaying the onset of the effects of cancer.
  • Various IGF-IR and IR inhibitors such as BMS-536942 (Bristol-Myers Squibb), BMS- 554417 (Bristol-Myers Squibb), NVP-AEW541 (Novartis), NVP-ADW742 (Novartis), AG1024 (Calbiochem-EMD Biosciences), OSI-906 (OSI Pharmaceuticals), CP-751,871 (Pfizer), AVE1642/EM164 (Sanofi Aventis/ImmunoGen), IMC-A12 (Imclone) and the compound A (as described herein) can be used to treat different types of cancers.
  • Tumor refers to a group of cells that are cancerous in origin and grow uncontrollably. Tumors from various types of cancers such as colorectal, breast, pancreatic, lung, head and neck, prostate, renal, ovarian are a few examples of tumors that can be treated with compounds which are IGF-IR and IR inhibitors.
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • DNA means a polymer composed of deoxyribonucleotides
  • Oligonucleotide means a polymer composed of either DNA or RNA, and used as probes to find a complementary sequence of DNA or RNA.
  • Protein or polypeptide The terms “protein” or “polypeptide” are used interchangeably. They refer to a chain of two or more amino acids, which are linked together with peptide or amide bonds, regardless of post-translational modification (e.g., glycosylation or phosphorylation).
  • Sample The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, containing one or more components of interest.
  • Samples include, but are not limited to, biological samples obtained from natural biological sources, such as whole blood, cells or tissues.
  • the samples may be derived from a tissue biopsy or another clinical procedure, and may include tumor tissue or cells extracted from subjects including mammals or cancer patients or cells from an in vitro culture.
  • the sample may be in the form of an explant or xenograft.
  • the tissue of the present invention may be surrogate tissue, i.e. any tissue that can be used as a substitute or replacement for tumor tissue in monitoring biological responses.
  • the surrogate tissue may be non-proliferating peripheral mononuclear cells or proliferating cells, such as buccal mucosa tissue cells.
  • the surrogate tissue may be a blood sample obtained from a subject.
  • subject may refer to any mammal but may be selected either from a human or an experimental animal such as a mouse or a rat. Preferably, the mammal is human.
  • the terms "subject or subjects” and “patient or patients” may be used interchangeably.
  • Pre-therapy denotes determining the expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before either contacting the cancer cell from cell culture or from tumor tissue from a subject with a compound which is an inhibitor of IGF-IR and IR, or before administering the compound which is an inhibitor of IGF-IR and IR to a subject.
  • Post-therapy denotes determining the expression level of the biomarker transcript in a sample after either contacting the cancer cell from cell culture or from tumor tissue from a subject with a compound which is an inhibitor of IGF-IR and IR, or after administering the compound which is an inhibitor of IGF-IR and IR to a subject.
  • Tissue, cells or xenografts extracted from mammals having tumors are capable of use with the methods of the present invention.
  • the methods involve in vitro analysis of cells, a number of different cell lines can be used. Examples include, without limitation, HEK-IGF1R, A673 and Colo-205 cell lines.
  • the present invention provides a method of monitoring the response of a subject having cancer to the treatment with a compound which is an IGF-IR and IR inhibitor; said method comprising determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample pre-therapy and post-therapy.
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • the present invention provides a method of monitoring response of a subject having cancer to treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising the steps of: (a) determining the expression level of at least one of the biomarker transcripts selected from the group consisting of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample obtained from the subject before administration of the compound, which is an IGF-IR and IR inhibitor;
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • step (c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);
  • step (d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c);
  • the cancer is selected from the group consisting of: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.
  • the compound which is an IGF-IR and IR inhibitor is administered in vivo to an experimental animal having a tumor, or to a subject having cancer.
  • the present invention relates to method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprises comparing the expression level of at least one of the biomarker transcripts selected from the group of EGR-1, DUSP-5 or FOS in a sample before treatment with the compound with the expression level of the said biomarker in a sample after treatment with the said compound.
  • the method of identifying response of a cancer cell or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor comprises contacting the compound to a cancer cell or a tumor tissue from a subject, ex vivo, for example a xenograft or explant.
  • the method of identifying response of a tumor comprises contacting the tumor tissue or cancer cells with the compound, which is an IGF-IR and IR inhibitor.
  • the level of at least one of the biomarkers selected from the group of EGR-1, DUSP-5 and FOS is measured in a tumor tissue extracted from the subject having cancer wherein the subject is administered with a compound which is an IGF-IR and IR inhibitor.
  • the cancer cell is from the cancer selected from the group consisting of: astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non-small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.
  • astrocytoma basal or squamous cell carcinoma
  • brain cancer gliobastoma
  • bladder cancer breast
  • the IGF-IR and IR inhibitor can be contacted with tumor tissue or cancer cells in vivo, in vitro or ex vivo using known methods.
  • the cancer cells in culture or tumor tissues can be exposed to the compound, which is an IGF-IR and IR inhibitor over a time period ranging from 3 to 12 hours.
  • the experimental animals can be exposed to plurality of concentrations of the compound, which is an IGF-IR and IR inhibitor.
  • the experimental animals can be treated with the concentrations of the compound, which is an IGF-IR and IR inhibitor ranging from 10 to 400 mg/kg.
  • prolonged administration of the inhibitor can be achieved by treating the experimental animals continuously on consecutive days.
  • the compound which is an IGF-IR and IR inhibitor is selected from: a compound of formula I (as described herein), BMS-536924 (Bristol-Myers Squibb), BMS- 554417 (Bristol-Myers Squibb), BVP51004 (Biovitrium), NVP-ADW 742 (Novartis), NVP- AEW541 (Novartis), Tyrphostin AG1024 (tyrosine kinase inhibitor), A-928605 (Abbott), AG1024 (Calbiochem-EMD biosciences), OSI-906 (OSI Pharmaceuticals), quinolinyl-derived imidazo (l,5-a)pyrazine PQIP (OSI Pharmaceuticals), picropodophyllin PPP(AXL-1717) (Karolinska Institute/Biovitrium), CP-751871 (Pfizer), IMC-A12 (Imclone), AMG-479 (Amgen), AMG-655 (Amgen),
  • R 1 is halogen
  • R 2 is C(0)OR 3 and R 3 is H or Q-C3 alkyl; or a pharmaceutically acceptable salt thereof.
  • the compounds of formula I are described in PCT Appln. No. PCT/US 12/34188; which is incorporated herein by reference.
  • the compound of formula I is (S)-ethyl 4-(2-carbamoyl -5-chloro-3-(2- (phenoxymethyl) morpholinosulfonyl)-lH-indol-7-ylamino) piperidine-l-carboxylate methane sulfonate (hereinafter referred to as Compound A).
  • the compound is a crystalline form of (S)-ethyl 4-(2-carbamoyl-5-chloro-3-(2-(phenoxymethyl) morpholinosulfonyl)-lH-indol-7-ylamino) piperidine-l-carboxylate methane sulfonate (crystalline form of Compound A) disclosed in PCT Appln. PCT/IB2012/051967 incorporated herein by reference.
  • RNA extracted from tumor tissue, cells or xenografts is used for real time quantitative polymerase chain reaction (RTQ-PCR) analysis.
  • the biomarker EGR- 1 in a sample e.g. cancer cells in culture or a xenograft mouse model is down regulated when the sample is treated with a compound which is an IGF-IR and IR inhibitor.
  • the length of the EGR- 1 polynucleotide that can be used in the methods of the present invention is 3136 base pair (bp) (accession no. NM_001964.2) and the length of the corresponding protein sequence is 543 amino acids.
  • the DNA sequence (SEQ ID NO: 1) of EGR-1 is:
  • EGR-1 Polypeptide encodes the following amino acid sequence: EGR-1 Polypeptide (SEQ ID NO: 2):
  • Pro Pro lie Thr Tyr Thr Gly Arg Phe Ser Leu Glu Pro Ala Pro Asn
  • DUSP-5 polynucleotide that can be used in the methods of the present invention is 2545 bp
  • the DNA sequence (SEQ ID NO: 3) of DUSP-5 is:
  • DUSP-5 Polypeptide encodes the following amino acid sequence: DUSP-5 Polypeptide (SEQ ID NO: 4):
  • the biomarker FOS in a sample e.g. cancer cells in culture or a xenograft mouse model
  • a compound which is an IGF-IR and IR inhibitor is down regulated when the sample is treated with a compound which is an IGF-IR and IR inhibitor.
  • the length of the FOS polynucleotide that can be used in the methods of the present invention is 2158 bp (accession no. NM_005252.3) and the length of the corresponding protein sequence is 380 amino acids.
  • the DNA sequence (SEQ ID NO: 5) of FOS is:
  • FOS Polypeptide (SEQ ID NO: 6):
  • the present invention also relates to a method for testing whether a compound which is an IGF- IR and IR inhibitor produces a therapeutic response in a subject having cancer; said method comprising the steps of:
  • EGR-1 Early Growth Response-1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • step (c) determining the expression level of at least one of the said biomarker transcripts in the sample obtained in step (b);
  • step (d) comparing the expression level of at least one of the said biomarker transcripts as determined in step (a) with the expression level determined in step (c);
  • the present invention provides a method of determining response of a cancer or a tumor to the treatment with a compound, which is an IGF-IR and IR inhibitor; said method comprising comparing expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response- 1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample before treatment with said compound with the expression level of the said biomarker transcript in a sample after treatment with the said compound.
  • EGR-1 Early Growth Response- 1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • the IGF-IR and IR inhibitor used in the method of determining response is the compound of Formula I.
  • the IGF-IR and IR inhibitor used in the method of determining response is compound A.
  • the biomarker transcript used in the method of determining response is Early Growth Response- 1 (EGR-1).
  • biomarker transcript used in the method of determining response is Dual Specificity Phosphatase-5 (DUSP-5).
  • the biomarker transcript used in the method of determining response is FBJ murine osteosarcoma viral oncogene homolog (FOS).
  • the present invention provides a kit for performing the method of the present invention, comprising:
  • (b) means for measuring expression level of at least one of the biomarker transcripts selected from the group of Early Growth Response-1 (EGR-1), Dual Specificity Phosphatase-5 (DUSP-5) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in a sample; ;
  • EGR-1 Early Growth Response-1
  • DUSP-5 Dual Specificity Phosphatase-5
  • FOS FBJ murine osteosarcoma viral oncogene homolog
  • the means for measuring the expression level of the biomarker transcripts can be microarray or RTQ-PCR.
  • expression level of the biomarker transcripts are measured as described herein, particularly, in reference to the examples but not limited thereto.
  • the sample used in a method of the present invention is a tumor tissue extracted from the subject having cancer.
  • the tumor tissue is from a tumor selected from the group consisting of astrocytoma, basal or squamous cell carcinoma, brain cancer, gliobastoma, bladder cancer, breast cancer, colon carcinoma, colorectal cancer, chrondrosarcoma, cervical cancer, adrenal cancer, choriocarcinoma, esophageal cancer, endometrial carcinoma, erythroleukemia, Ewing's sarcoma, gastrointestinal cancer, head and neck cancer, hepatoma, glioma, hepatocellular carcinoma, leukemia, leiomyona, melanoma, non- small cell lung cancer, neural cancer, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, rhabdomyosarcoma, small cell lung cancer, thymona, thyroid cancer, testicular cancer or osteosarcoma.
  • the sample is a surrogate tissue.
  • the surrogate tissue can be a blood sample of the subject having cancer which comprises serum or plasma.
  • HEK-IGF1R cells Human Embryonic Kidney cells over expressed for IGF1R obtained from Merck were treated with Compound A (20mM stock was prepared in DMSO; final concentration in the assay was 1 ⁇ ) for 3, 6 and 12 hours.
  • the cells treated with the compound A are referred to as “treated cells” and the cells not treated with the Compound A are referred to as "untreated cells”.
  • Treated and untreated cells were either stimulated with ⁇ g/mL of IGF1 or were left unstimulated for another half an hour duration.
  • RNA was isolated and subjected to microarray using alumina platform. Data was obtained after image quantification and analyzed using Genespring (GX 10.0. Agilent Technologies, USA).
  • RNA samples were washed in nuclease free water and homogenized in 800 ⁇ lysis buffer with 8 ⁇ ⁇ -mercaptoethanol (Nucleopore RNASure Mini kit, Qiagen GmbH, Germany). The tubes were then centrifuged at 10,000g for 1 minute. This supernatant was used to obtain total RNA. The final elution was carried out in 32uL nuclease free water. The quality and quantity of the RNA was determined using a UV- visible spectrophotometer (NanoDrop ND-1000 spectrophotometer, NanoDrop products, USA). The 260/280 & the 260/230 ratios for all the samples used in the study were within 1.8 - 2.1, indicating highly pure total RNA species. The RNA samples were stored at -80°C until further use.
  • Microarray was performed using the Illumina Sentrix BeadChip® HumanHT-12 Expression BeadChip containing approximately 48,000 probes.
  • HEK-IGFIR cells not treated with the Compound A showed over expression of EGR1, FOS and DUSP5 at mRNA level after stimulation with IGF1.
  • HEK-IGFIR cells treated with the Compound A showed significant inhibition (down regulation) of transcript level of the biomarkers EGR1, FOS and DUSP5 at mRNA level at all the time points i.e. at 3, 6 and 12 hours.
  • HEK-IGFIR cells were treated with the following compounds: (i) 0.1 and 1 ⁇ of Compound A (stock of 20mM was prepared in DMSO); (ii) 0.1 and 1 ⁇ of OSI-906 (Linsitinib, Active Biochem catalogue no. A- 1058) (stock of 20mM was prepared in DMSO) and (iii) 0.1 and 1 ⁇ of BMS-754807 (Active Biochem, catalogue no. A-1013) (stock of 20mM was prepared in DMSO) for 3, 6 and 12 hours.
  • OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors. Cells not treated with the said compounds are referred to as untreated cells.
  • Treated and untreated cells were either stimulated with ⁇ g/mL of IGFl or were left unstimulated for another half an hour duration.
  • Stimulated untreated cells are vehicle (DMSO) control cells.
  • RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.
  • biomarker transcripts EGR-1, DUSP-5 and FOS
  • RTQ-PCR Three biomarker transcripts (EGR-1, DUSP-5 and FOS) from IGF1R signaling pathway were analyzed by RTQ-PCR to measure the molecular response of the Compound A, OST906 and BMS-754807 in HEK-IGFIR cells.
  • the primer sequences used in the RTQ-PCR for the said transcripts are listed below:
  • EGR-1 Forward Primer acctcatacc catcccctgt 20 (SEQ ID NO: 7) Reverse Primer tgtcctggga gaaaaggttg 20 (SEQ ID NO: 8)
  • Reverse Primer cctgcttcac caccttcttg a 21 (SEQ ID NO: 14) cDNA synthesized from total RNA isolated was used as the template. All the PCR reactions were performed using the QuantiFast SYBR Green PCR Kit (Qiagen GmbH, InVitrogen Corporation, USA). Each PCR reaction contained IX master mix, 1 ⁇ L ⁇ of the diluted cDNA, and 250 nM of forward and reverse primers designed to yield 80 to 125-bp amplicons. Following the initial 3 minutes enzyme activation at 95°C a PCR reaction was carried out through 40 cycles (95°C for 10 seconds and 60°C for 30 seconds).
  • the cycling threshold (C t ) value for each of the transcript was calculated after normalization of the data using data for housekeeping gene and the fold difference in the expression level of the transcripts was calculated. Data is represented graphically as the relative fold change (log 2 scale) of transcript levels as compared to cells treated with vehicle (DMSO) control.
  • A673 cells were treated with the following compounds: (i) 0.1 and 1 ⁇ of Compound A (20mM stock was prepared in DMSO); (ii) 0.1 and 1 ⁇ of OSI-906 (Linsitinib, Active Biochem catalogue no. A- 1058) (20mM stock was prepared in DMSO) and (iii) 0.1 and 1 ⁇ of BMS-754807 (Active Biochem, catalogue no. A- 1013) (20mM stock was prepared in DMSO) for 3, 6 and 12 hours.
  • OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors.
  • Treated and untreated cells were either stimulated with ⁇ g/mL of IGF1 or were left unstimulated for another half an hour duration.
  • Stimulated untreated cells are vehicle (DMSO) control cells.
  • RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.
  • Gene expression was calculated by the 2 " ⁇ method (METHODS, 2001, 25, 402-408) and then expressed on log 2 scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.
  • Colo-205 cells (colorectal adenocarcinoma cells; ATCC number: CCL-222) were treated with the following compounds: (i) 0.1 and 1 ⁇ of Compound A (20mM stock was prepared in DMSO); (ii) 0.1 and 1 ⁇ of OSI-906 (Linsitinib, Active Biochem catalogue no. A-1058) (20mM stock was prepared in DMSO) and (iii) 0.1 and 1 ⁇ of BMS-754807 (Active Biochem, catalogue no. A-1013) (20mM stock was prepared in DMSO) for 3, 6 and 12 hours.
  • OSI-906 and BMS-754807 are IGF-1R tyrosine kinase inhibitors.
  • Treated and untreated cells were either stimulated with ⁇ g/mL of IGF1 or were left unstimulated for another half an hour duration.
  • Stimulated untreated cells are vehicle (DMSO) control cells.
  • RNA was isolated according to procedure provided in Example 1 and utilized for RTQPCR assay.
  • Gene expression was calculated by the 2 " ⁇ method (METHODS, 2001, 25, 402-408) and then expressed on log 2 scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.
  • Colo-205 cells were harvested and re-suspended in saline at 6 x 10 6 cells per 0.2 mL per mouse volume and injected to severe combined immunodeficient (SCID) mice on right flank. Animals were observed for the tumors till the tumors size attained a diameter of -150-200 mm mm and were randomized into control and treatment group.
  • Treatment group received various doses of Compound A (10, 30, 100, 200, 400 mg/kg) or 50 mg/kg OSI-906 (Linsitinib, Active Biochem catalogue no. A-1058) in water by oral administration route for 3 days. On day 3 after 2 hours of treatment for the day, treated and untreated animals were either stimulated with of IGF1 or were left unstimulated for another half an hour duration. After sacrificing the animals, the tumors were stored and RNA was isolated and subjected to RTQPCR assay.
  • Gene expression was calculated by the 2 " ⁇ method (METHODS, 2001, 25, 402-408) and then expressed on log 2 scale. A change in 2-fold or more is considered significant in either direction. Values for the stimulated control group are expressed as fold change over the unstimulated control and for the groups treated with the said compounds are expressed as fold change over the stimulated control.
  • results obtained are depicted in Figure V.
  • Analysis of mRNA level of biomarker transcripts EGR1, FOS and DUSP5 in tumors isolated from animals not treated with Compound A or OSI-906 showed over expression of said biomarker transcripts in comparison to unstimulated control.
  • the expression of the transcripts EGRl, FOS and DUSP5 in tumors of animals treated with Compound A or OST906 are as follows:
  • SEQIDNO: 10 aggtaagcca tgcagatggt
  • SEQIDNO: 11 cgtgccagac atggacctat
  • SEQIDNO: 12 gtgaagagaa ggaagacg
  • SEQIDNO: 13 tgtgtccgtc gtggatctga
  • SEQIDNO: 14 cctgcttcac caccttcttg a

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Abstract

La présente invention concerne un procédé de suivi de la réponse d'un sujet souffrant d'un cancer à un traitement impliquant un composé qui est un inhibiteur du récepteur du facteur de croissance 1 analogue à l'insuline (IGF-1R) et du récepteur de l'insuline (IR). Le procédé de la présente invention consiste à comparer le niveau d'expression d'au moins un biomarqueur choisi dans le groupe constitué des EGR-1 (Early Growth Response-1), DUSP-5 (Dual Specificity Phosphatase-5) et FOS (FBJ murine osteosarcoma viral oncongene homolog) dans un échantillon prélevé chez le sujet souffrant du cancer avant et après l'administration dudit composé. Une baisse du niveau d'expression d'au moins l'un desdits biomarqueurs après l'administration du composé permet de prédire que le sujet va répondre favorablement au traitement par ledit composé.
PCT/IB2013/055208 2012-06-26 2013-06-25 Procédé de prédiction ou de suivi de la réponse à des inhibiteurs des igf-1r et des ir, faisant appel à des biomarqueurs WO2014002007A1 (fr)

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Cited By (6)

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CN111122873A (zh) * 2019-12-30 2020-05-08 厦门大学附属中山医院 维甲酸受体反应蛋白1作为生物标记物在慢性肾病中的应用
CN111679074A (zh) * 2020-07-11 2020-09-18 成都益安博生物技术有限公司 一种前列腺癌的外周血tcr标志物及其检测试剂盒和应用
CN111693702A (zh) * 2020-07-11 2020-09-22 成都益安博生物技术有限公司 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用
CN111812325A (zh) * 2020-07-11 2020-10-23 成都益安博生物技术有限公司 一种乳腺癌的外周血tcr标志物及其检测试剂盒和应用
CN113365630A (zh) * 2018-12-21 2021-09-07 库拉肿瘤学公司 用于鳞状细胞癌的疗法
CN113884684A (zh) * 2021-09-10 2022-01-04 上海交通大学医学院 一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法

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WO2008144345A2 (fr) * 2007-05-17 2008-11-27 Bristol-Myers Squibb Company Biomarqueurs et procédés pour déterminer la sensibilité de modulateurs de récepteur de facteur de croissance de type 1 semblable à l'insuline
US20100196889A1 (en) * 2006-11-13 2010-08-05 Bankaitis-Davis Danute M Gene Expression Profiling for Identification, Monitoring and Treatment of Colorectal Cancer
WO2012145471A1 (fr) * 2011-04-21 2012-10-26 Merck Sharp & Dohme Corp. Inhibiteurs du récepteur du facteur de croissance 1 analogue à l'insuline

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US20100196889A1 (en) * 2006-11-13 2010-08-05 Bankaitis-Davis Danute M Gene Expression Profiling for Identification, Monitoring and Treatment of Colorectal Cancer
WO2008144345A2 (fr) * 2007-05-17 2008-11-27 Bristol-Myers Squibb Company Biomarqueurs et procédés pour déterminer la sensibilité de modulateurs de récepteur de facteur de croissance de type 1 semblable à l'insuline
WO2012145471A1 (fr) * 2011-04-21 2012-10-26 Merck Sharp & Dohme Corp. Inhibiteurs du récepteur du facteur de croissance 1 analogue à l'insuline

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113365630A (zh) * 2018-12-21 2021-09-07 库拉肿瘤学公司 用于鳞状细胞癌的疗法
CN111122873A (zh) * 2019-12-30 2020-05-08 厦门大学附属中山医院 维甲酸受体反应蛋白1作为生物标记物在慢性肾病中的应用
CN111679074A (zh) * 2020-07-11 2020-09-18 成都益安博生物技术有限公司 一种前列腺癌的外周血tcr标志物及其检测试剂盒和应用
CN111693702A (zh) * 2020-07-11 2020-09-22 成都益安博生物技术有限公司 一种黑色素瘤的外周血tcr标志物及其检测试剂盒和应用
CN111812325A (zh) * 2020-07-11 2020-10-23 成都益安博生物技术有限公司 一种乳腺癌的外周血tcr标志物及其检测试剂盒和应用
CN113884684A (zh) * 2021-09-10 2022-01-04 上海交通大学医学院 一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法
CN113884684B (zh) * 2021-09-10 2023-09-15 上海依赛洛森生物医药有限公司 一种活动性结核病多组学整合标志物、试剂盒及检测模型的构建方法

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