WO2020205627A1 - Utilisation de biomarqueur dans la thérapie du cancer - Google Patents

Utilisation de biomarqueur dans la thérapie du cancer Download PDF

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Publication number
WO2020205627A1
WO2020205627A1 PCT/US2020/025472 US2020025472W WO2020205627A1 WO 2020205627 A1 WO2020205627 A1 WO 2020205627A1 US 2020025472 W US2020025472 W US 2020025472W WO 2020205627 A1 WO2020205627 A1 WO 2020205627A1
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Prior art keywords
steap4
expression
cancer
level
hap
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PCT/US2020/025472
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English (en)
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Vijaya TIRUNAGARU
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Rain Therapeutics Inc.
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Publication of WO2020205627A1 publication Critical patent/WO2020205627A1/fr

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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
    • 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
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • 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

  • sequence listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is RATH-009_01WO.txt.
  • the text file is 23.3 kb, and was created on March 27, 2020, and is being submitted electronically.
  • the present disclosure relates to a method of treating or preventing a cancer in a subject in need thereof, wherein the method comprises determining the level of STEAP4 expression in cancer cells to predict whether the cancer cells would be responsive to treatment by a HAP.
  • STEAP4 has been shown to be associated with certain cancers (Gomes et al, 2012; Xue et al, 2017). STEAP4 has also been shown to be highly induced by hypoxia, i.e., low oxygen at the tissue level. Moreover, hypoxia within tumours is associated with poor prognosis of cancer patients and with treatment failure (Hunter et al, 2016), such as resistance to radiotherapy and traditional chemotherapy.
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a hypoxia-activated prodrug (HAP);
  • HAP hypoxia-activated prodrug
  • tumor cells of the subject exhibit normal or an elevated level of STEAP4 expression
  • an elevated level of expression is at least 1 times a basal level.
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the HAP is of a cancer active agent, such as a tyrosine kinase inhibitor (TKI), chemotherapeutic compound or an additional therapeutic for treating or preventing a cancer.
  • TKI tyrosine kinase inhibitor
  • the HAP is of a cancer active agent, such as a TKI or chemotherapeutic compound.
  • the HAP is of a tyrosine kinase inhibitor.
  • the HAP is of a chemotherapeutic compound.
  • the expression of STEAP4 is at least 1.1 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.2 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.3 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.4 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.5 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.6 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.7 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.8 times the basal level.
  • the expression of STEAP4 is at least 1.9 times the basal level. In some embodiments, the expression of STEAP4 is at least 2 times the basal level. In some embodiments, the expression of STEAP4 is at least 3 times the basal level. In some embodiments, the expression of STEAP4 is at least 4 times the basal level. In some embodiments, the expression of STEAP4 is at least 5 times the basal level. In some embodiments, the expression of STEAP4 is at least 6 times the basal level. In some embodiments, the expression of STEAP4 is at least 7 times the basal level. In some embodiments, the expression of STEAP4 is at least 8 times the basal level.
  • the expression of STEAP4 is at least 9 times the basal level. In some embodiments, the expression of STEAP4 is at least 10 times the basal level. In some embodiments, the expression of STEAP4 is at least 11 times the basal level. In some embodiments, the expression of STEAP4 is at least 12 times the basal level. In some embodiments, the expression of STEAP4 is at least 13 times the basal level. In some embodiments, the expression of STEAP4 is at least 14 times the basal level. In some embodiments, the expression of STEAP4 is at least 15 times the basal level. In some embodiments, the expression of STEAP4 is at least 16 times the basal level.
  • the expression of STEAP4 is at least 17 times the basal level. In some embodiments, the expression of STEAP4 is at least 18 times the basal level. In some embodiments, the expression of STEAP4 is at least 19 times the basal level. In some embodiments, the expression of STEAP4 is at least 20 times the basal level. In some embodiments, the expression of STEAP4 is at least 30 times the basal level. In some embodiments, the expression of STEAP4 is at least 40 times the basal level. In some embodiments, the expression of STEAP4 is at least 50 times the basal level. In some embodiments, the expression of STEAP4 is at least 60 times the basal level.
  • the expression of STEAP4 is at least 70 times the basal level. In some embodiments, the expression of STEAP4 is at least 80 times the basal level. In some embodiments, the expression of STEAP4 is at least 90 times the basal level. In some embodiments, the expression of STEAP4 is at least 100 times the basal level.
  • the expression of STEAP4 is at least 1-3 times the basal level. In some embodiments, the expression of STEAP4 is at least 3-6 times the basal level. In some embodiments, the expression of STEAP4 is at least 7-20 times the basal level.
  • the level of STEAP4 expression is expression of a STEAP4 gene.
  • the level of STEAP4 expression is expression of a STEAP4 - expressing target nucleic acid.
  • the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA.
  • the level of STEAP4 expression is expression of a STEAP4 protein.
  • the level of STEAP4 expression is associated with a low oxygen level.
  • the in situ hybridizing score is 1. In some embodiments, the in situ hybridizing score is 2. In some embodiments, the in situ hybridizing score is 3. In some embodiments, the in situ hybridizing score is 4.
  • the in situ hybridizing score is based on dots per cell clusters.
  • the clusters are about 1 to about 3 dots per cell. In some embodiments, the clusters are about 4 to about 9 dots per cell. In some embodiments, the clusters are about 10 to about 15 dots per cell. In some embodiments, the clusters are great than about 15 dots per cell.
  • the percentage of cells with clusters is greater than about 50%. In some embodiments, the percentage of cells with clusters is greater than about 60%. In some embodiments, the percentage of cells with clusters is greater than about 70%. In some embodiments, the percentage of cells with clusters is greater than about 80%. In some embodiments, the percentage of cells with clusters is greater than about 90%.
  • the HAP is tarloxotinib.
  • the cancer is a tumor.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in cancer cells of the subject is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • a. cancer cells are provided; b. the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR; and
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in cancer cells of the subject is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR; and c. whether the cancer would be likely to be responsive to treatment by a HAP is predicted by if the cancer cells exhibit an elevated level of STEAP4 expression, wherein an elevated level of expression is an in situ hybridizing score of 1-4 according to RNAScope.
  • FIG. 1 depicts STEAP4 and PPIB staining for Group 1, SiHa Tumor Blocks.
  • FIG. 2 depicts STEAP4 and PPIB staining for Group 2, cell tumor lines.
  • FIG. 3 depicts STEAP4 and PPIB staining for Group 3, C33A-WT, S4.
  • FIG. 4 depicts STEAP4 and PPIB staining for Group 4, cell tumor lines.
  • FIG. 5 depicts STEAP4 and PPIB staining for Group 5, tumors 70161 and 70177.
  • FIG. 6 depicts STEAP4 and PPIB staining for Group 6, human tumors.
  • FIG. 7 depicts STEAP4 and PPIB staining for Group 7, human tumors.
  • FIG. 8A depicts tarloxotinib metabolism in parental and STEAP4 overexpressing C33A xenografts grown in NIH-III mice
  • FIG. 8B depicts STEAP4 expression in xenografts by qPCR following a single dose of tarloxotinib.
  • FIG. 9 depicts STEAP4 ISH staining in C33A-WT and C33A-STEAP4 overexpressing cells.
  • FIG. 10 depicts STEAP4 ISH staining in EGFR/HER2 mutant and CLU-NRG1 xenograft tumor sections.
  • FIG. 11 depicts STEAP4 ISH scoring of various FFPE xenograft tumor sections with RNAScope ISH scoring guidelines.
  • FIG. 12 depicts tarloxotinib metabolism under anoxic conditions determined by mass spectrometry in various cell lines in vitro.
  • FIG. 13 depicts STEAP4 ISH staining and scoring of EGFR mutant (del 19 and L858R) lung cancer patient tumor FFPE sections from the primary tumor and lymph node (LN) metastasis.
  • FIG. 14 depicts STEAP4 ISH staining and scoring of EGFR exon 20 insertion mutant lung cancer patient tumor FFPE sections.
  • FIG. 15 depicts STEAP4 ISH staining of FFPE sections from EGFR mutant lung cancer patients.
  • the present disclosure relates to a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • HAP hypoxia-activated prodrug
  • the STEAP4 expression is determined by in situ hybridization.
  • the cancer is a tumor.
  • the HAP is a nitromethylaryl quaternary ammonium salt (also referred to as a NMQ prodrug).
  • the HAP is of a cancer active agent, such as a tyrosine kinase inhibitor (TKI), chemotherapeutic compound or an additional therapeutic for treating or preventing a cancer.
  • TKI tyrosine kinase inhibitor
  • the HAP is of a cancer active agent, such as a TKI or chemotherapeutic compound.
  • the HAP is of a tyrosine kinase inhibitor
  • the tyrosine kinase inhibitor is tarloxotinib, crizotinib, alectinib, ceritinib, erlotinib, dacomitinib, osimertinib, afatinib, gefitinib, rociletinib, cetuximab, icotinib, sapitinib, lapatinib, neratinib, brigatinib, poziotinib, naquotinib, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab patritumumab, abemaciclib, acalabrutinib, axitinib, baricitinib, binimetinib, bosutinib, brigatinib, cabozantini
  • the HAP is of a chemotherapeutic compound.
  • the HAP is of a metabolite of a TKI or a chemotherapeutic agent.
  • the HAP is of a metabolite of a TKI.
  • the HAP is of a metabolite of a chemotherapeutic agent.
  • HAPs Hypoxia-Activated Prodrugs
  • the HAP is tarloxotinib (RN-4000; also known as“(E)-4-((4-((3- bromo-4-chlorophenyl)amino)pyrido[3,4-d]pyrimidin-6-yl)amino)-N,N-dimethyl-N-((l-methyl- 4-nitro-lH-imidazol-5-yl)methyl)-4-oxobut-2-en-l-aminium salt (bromide)”; also referred to herein as “(2E)-4- ⁇ [4-(3-bromo-4-chloroanilino)pyrido[3,4-d]pyrimidin-6-yl]amino ⁇ -N,N- dimethyl-N-[(l-methyl-4-nitro-lH-imidazol-5-yl)methyl]-4-oxo-2-buten-l -ammonium bromide”; also referred to herein as
  • Compound A may exist as a cation or salt, for example, a bromide salt, as depicted below.
  • NMQ prodrugs and/or small molecule inhibitors including any other small molecule analogues of Compound A and/or Compound B, to treat or prevent a cancer wherein the STEAP4 expression is determined by in situ hybridization scoring or qPCR.
  • NMQ prodrugs and/or small molecule inhibitors include, but are not limited to those disclosed in W02010104406, WO2011028135, US20120077811, and US20120202832, each of which is incorporated herein by reference in its entirety.
  • the disclosure contemplates NMQ prodrugs of quaternary nitrogen salt compounds of Formula I:
  • X is any negatively charged counterion
  • Ri is a group of the formula -(CH2)nTr, where Tr is an aromatic nitroheterocycle or an aromatic nitrocarbocycle and -(CH2)nTr acts as a reductively -activated fragmenting trigger (“reductive trigger”); and
  • n is an integer from 0 to 6;
  • R2, R3 and R4 are each independently an aliphatic or an aromatic group of a tertiary amine kinase inhibitor (R2)(R3)(R4)N, or two of R2, R3, and R4 may form an aliphatic or aromatic heterocyclic amine ring of a kinase inhibitor, or one of R2, R3 and R4 may be absent and two of R2, R3 and R4 form an aromatic heterocyclic amine ring of a kinase inhibitor.
  • the compounds are of Formula II:
  • X is any negatively charged counterion
  • Y is N or C-R.7, where R is selected from H, C1-C6 alkyl, C1-C6 alkoxy, and a group of the one of the following Formulas Ilia, Illb, and IIIc:
  • T is selected from O, NH, N(CI-C6 alkyl), and a direct link;
  • n is an integer from 0 to 6;
  • U is selected from OR10, CF3, OCF3, CN, NR11R12, pyrrolidinyl, piperidinyl, piperazinyl, Nl-methylpiperazinyl, morpholinyl, CON(Ri3)(Ri4), S02N(Ri5)(Ri6), N(Rn)CORi8,
  • R9, Rio, R11, R12, R13, R14 R15, R16, R17, R18, R19, R20, R21, R22, R23, R24 are independently selected from H and C1-C6 alkyl;
  • Z is N or C-CN
  • n is an integer from 0 to 6;
  • Ri is a group of the formula (CH2)nTr where Tr is an aromatic nitroheterocycle or aromatic nitrocarbocycle and -(CH2)nTr acts as a reductive trigger;
  • n is an integer from 0 to 6;
  • R2 and R3 are independently selected from C1-C6 alkyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, CH2CH2OH, and CFkCFkCKCi-Ce alkyl); or R2 and R3 may together form a non-aromatic carbocyclic ring or non-aromatic heterocyclic ring containing at least one heteroatom;
  • R5 is selected from an aniline, an indole, an indoline, an amine, an aminoindole, and an aminoindazole, each of which may be optionally substituted with one or more substituents selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CPs, OH, NH 2 , NO2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl) 2 , CONH2, CO(Ci-Ce alkyl), SO2NH2, and S0 2 (Ci-C 6 alkyl); and R6 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, NH(CI-C6 alkyl), N(CI-C6 alkyl)2, and a group of the following Formula IV :
  • V is selected from (CH2)k, O, NH, and N(CI-C6 alkyl);
  • k is an integer from 0 to 6
  • R.25 is selected from H and C1-C6 alkyl.
  • X is selected from halide (e.g., fluoride, chloride, bromide, iodide), methanesulfonate, trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate, lactate, citrate, and formate.
  • halide e.g., fluoride, chloride, bromide, iodide
  • methanesulfonate trifluoromethanesulfonate
  • acetate trifluoroacetate
  • tosylate lactate
  • citrate citrate
  • formate X is a halide.
  • X is selected from fluoride, chloride, bromide, and iodide.
  • Ri is a group of one of the following Formulas Va-Vq:
  • R.26 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, CF3, OCF3, F, Cl, Br, I, NO2, CN, COOH, COO(Cl-C6 alkyl), CONH2, CONH(Ci-Ce alkyl), CON(Ci-Ce alkyl) 2 , CO(Ci-Ce alkyl), SO2NH2, S0 2 NH(Ci-C 6 alkyl), S0 2 N(Ci-C 6 alkyl) 2 , S0 2 (Ci-C 6 alkyl), and a group of Formula Ilia, as defined above, where * is the point of attachment to a group of Formula V;
  • R.27 is selected from H, C1-C6 alkyl, and a group of Formula Ilia, as defined above, where * is the point of attachment to a group of Formula V;
  • R.28 is selected from H and C1-C6 alkyl.
  • Ri is a group of one of the following Formulas Vr-Vae:
  • Ri is a group of Formula Vc, where R26 is H and R27 is CFb.
  • Ri is a group of Formula Vd, where R26 is selected from H, C1-C6 alkyl (e.g., methyl), C1-C6 alkoxy (e.g., OCH3), C2-C6 alkynyl (e.g., ethynyl), CONH2, CONHMe, CF 3 , OCF 3 , Br, NO2, and CN, and R27 is selected from CH 3 , CH2CH2CONH2, and CH2CH2CN.
  • R26 is selected from H, C1-C6 alkyl (e.g., methyl), C1-C6 alkoxy (e.g., OCH3), C2-C6 alkynyl (e.g., ethynyl), CONH2, CONHMe, CF 3 , OCF 3 , Br, NO2, and CN, and R27 is selected from CH 3 , CH2CH2CONH2, and CH2CH2CN.
  • Ri is a group of Formula Vd
  • R26 is selected from H and Ci-C 3 alkyl
  • R27 is selected from H and C1-C6 alkyl.
  • R26 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2, and propyn-l-yl
  • R27 is C 1-C6 alkyl.
  • R26 is H and R27 is Ci-C 3 alkyl (e.g., methyl).
  • Ri is a group of Formula Vd, where R26 is 1-propynyl and R27 is
  • Ri is a group of Formula Vq, where R26 is selected from H, C1-C6 alkyl (e.g., methyl or ethyl) and C1-C6 alkoxy (e.g., OCFb), and R27 is CFb.
  • Ri is a group of any one of Formulas Vd (1) -Vd (7) :
  • R27 is selected from methyl, ethyl and propyl. In some embodiments R27 is methyl.
  • R2 and R 3 form a ring selected from pyrrolidinium, piperidinium, piperazinium, N 1 -methylpiperazinium, and morpholinium.
  • R5 is a group of one of the following Formulas Vla-VIg:
  • R.29, rise, R37, R39, R44, R49 and R54 are independently selected from H and C1-C6 alkyl
  • R30, R31, R32, R33, R34, R35, R38, R40, R41, R42, R43, R45, R46, R47, R48, R50, R51, R52, R53, R55, R56, R57 and R58 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci-Ce alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), N(Ci-Ce alkyl) 2 , CONH2, CO(Ci-Ce alkyl), SO2NH2, and S0 2 (Ci-C6 alkyl); and
  • W is N or C-H.
  • Y is N
  • Z is N or C-CN
  • Ri is selected from the following:
  • R26 is selected from H, C1-C6 alkyl (e.g., methyl), C1-C6 alkoxy (e.g., OCH3), C2-C6 alkynyl (e.g., ethynyl), CF3, OCF3, Br, NO2, and CN, and R27 is selected from CH3, CH2CH2CONH2, and CH2CH2CN; or (ii) R26 IS 1-propynyl and R27 is CH3;
  • R26 is selected from H, C1-C6 alkyl (e.g., methyl and ethyl) and C1-C6 alkoxy (e.g., OCH3), and R27 is CH3;
  • R2 and R3 are independently selected from C1-C6 alkyl, or together form a ring selected from pyrrolidinium, piperidinium, piperazinium, Nl-methylpiperazinium, and morpholinium; and
  • R5 is selected from the following: (a) a group of Formula Via, where * is the point of attachment, R29 is H, and R30, R31, R32 are independently selected from FI, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2 ;
  • R39 is H
  • R40 and R41 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2
  • R42 and R43 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2
  • W is N or C-H
  • Y is C-H or C-(Ci-C6 alkoxy), Z is N or C-CN; and
  • Ri is selected from the following:
  • R26 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2- Ce alkynyl, CF3, OCF3, Br, NO2, and CN, and R27 is selected from CH3, CH2CH2CONH2, and CH2CH2CN; or R26 IS 1-propynyl and R27 is CH3;
  • R26 is selected from H, C1-C6 alkyl (e.g., methyl and ethyl), and C1-C6 alkoxy (e.g., OCH3), and R27 is CH3;
  • R2 and R3 are independently selected from C1-C6 alkyl, or together form a ring selected from pyrrolidinium, piperidinium, piperazinium, Nl-methylpiperazinium, and morpholinium;
  • R5 is selected from the following:
  • R29 is H
  • R30, R31, R32 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2 ;
  • R39 is H
  • R40 and R41 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2
  • R42 and R43 are independently selected from H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH2F, CHF2, CF3, OH, NH 2 , NO2, NH(Ci-Ce alkyl), and N(Ci-Ce alkyl) 2
  • W is N or C-H
  • (c) a group of Formula VId where * is the point of attachment;
  • R39 is H; and R
  • R6 is H
  • X is any negatively charged counterion
  • n l or 2.
  • Y is C-R7, where R7 is a group of Formula Illb; Z is N or C-CN;
  • Ri is selected from the following:
  • R 26 is selected from H, C1-C6 alkyl, C1-C6 alkoxy, C2- Ce alkynyl, CF3, OCF3, Br, N0 2 , and CN
  • R 27 is selected from CFb, CH 2 CH 2 CONH 2 , and CFhCFhCN; or R 26 i s 1-propynyl; and R 27 is CH3;
  • RM is selected from H, C1-C6 alkyl (e.g., methyl and ethyl) and C1-C6 alkoxy (e.g., OCH3); and R 27 is CH3;
  • R 2 and R3 are independently selected from C1-C6 alkyl, or together form a ring selected from pyrrolidinium, piperidinium, piperazinium, Nl-methylpiperazinium, and morpholinium;
  • R5 is selected from the following:
  • R39 is H
  • R40 and R41 are independently selected from H, C1-C6 alkyl, C 2 -C6 alkenyl, C 2 -Ce alkynyl, Ci-Ce alkoxy, F, Cl, Br, I, CN, CH 2 F, CHF 2 , CF3, OH, NH 2 , N0 2 , NH(CI-C6 alkyl), and N(Ci-C6 alkyl) 2
  • R42 and R43 are independently selected from H, C1-C6 alkyl, C 2 -C6 alkenyl, C 2 -C6 alkynyl, C1-C6 alkoxy, F, Cl, Br, I, CN, CH 2 F, CHF 2 , CF3, OH, NH 2 , N0 2 , NH(CI-C6 alkyl), and N(CI-C6 alkyl)
  • R6 is H
  • the compounds are of Formula VII:
  • R.59 is H
  • R.60 is (3-chlorobenzyl)oxy- and R.6i is chloro;
  • R.60 is 2-pyridinylmethoxy and R6i is chloro;
  • R6O and R6i are both chloro
  • R6O is chloro and R6i is bromo
  • R6O and R6i are both bromo
  • R6O is fluoro and R6i is ethynyl
  • R6O is chloro and R6i is ethynyl
  • R6O is bromo and R6i is ethynyl
  • R6O is 2-pyridinylmethoxy and R6i is fluoro
  • R6O is 2-pyridinylmethoxy and R6i is bromo
  • R59, R60 and R6i is selected from benzyloxy, 3-chlorobenzyloxy, and 2- pyridinylmethoxy, and when at least one of R59, R60 and R6i is not benzyloxy, 3- chlorobenzyloxy or 2-pyridinylmethoxy, each of the others is independently selected from H, halogen, and C2-C4 alkynyl, with the proviso that when one of R59, R60 and R6i is benzyloxy or 2-pyridinylmethoxy, the other two of R59, R60 and R6i are not H;
  • the compound of Formula VII is a compound according to Formula VIII:
  • R.62 is H, and either
  • R.63 is (3-chlorobenzyl)oxy- and R.64 is chloro;
  • R63 is 2-pyridinylmethoxy and R64 is chloro
  • R63 is chloro and R64 is bromo
  • R63 is fluoro and R64 is ethynyl
  • R63 is chloro and R64 is ethynyl
  • R63 is bromo and R64 is ethynyl
  • R63 is 2-pyridinylmethoxy and R64 is fluoro;
  • R63 is 2-pyridinylmethoxy and R64 is bromo.
  • the compound of Formula VII is selected from the group consisting of:
  • the compounds are of Formula IX:
  • R59, R.60 and R.61 are as defined for Formula VII, R.65 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2, and propyn- 1-yl, and R66 is C1-C6 alkyl.
  • the compounds are of Formula X:
  • R59, R60 and R6i are as defined for Formula VII and R67 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2, and propyn-l-yl.
  • the compounds are of Formula XI:
  • R62, R63 and R64 are as defined for Formula VIII and R68 is selected from H, methyl, ethyl, trifluoromethyl, -CN, -CONH2, and propyn-l-yl.
  • X is selected from halide (e.g., fluoride, chloride, bromide, iodide), methanesulfonate, trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate, lactate, citrate and formate.
  • halide e.g., fluoride, chloride, bromide, iodide
  • methanesulfonate e.g., methanesulfonate, trifluoromethanesulfonate, acetate, trifluoroacetate, tosylate, lactate, citrate and formate.
  • the compounds are selected from the group consisting of: (2£)-4-[(4- ⁇ 3-chloro-4-[(3-chlorobenzyl)oxy]anilino ⁇ pyrido[3,4-cf]pyrimi din-6- yl)ami no
  • the compounds are selected from the group consisting of:
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the terms“alkyl”,“alkenyl”,“alkynyl” and“alkoxy” include both straight chain and branched chain groups, and unsubstituted and substituted groups.
  • the optional substituents may include, without limitation, halogen, C1-C6 alkoxy, CN, OH, NH2, NO2, NH(CI-C6 alkyl), N(Ci- Ce alkyl) 2 , CONH2, CO(Ci-Ce alkyl), SO2NH2 and S0 2 (Ci-C6 alkyl).
  • quademisable nitrogen means a fully substituted nitrogen of sufficient basicity (or nucleophilicity) to react with an electrophilic group such as an a-methyl halide/mesylate/tosylate or triflate to provide a quaternary ammonium salt of the nitrogen.
  • aromatic nitroheterocycle means an aromatic heterocyclic moiety substituted at any ring position by one or more nitro (NO2) groups.
  • the aromatic heterocyclic moiety may be a monocyclic or bi cyclic ring containing 4 to 12 atoms of which at least one atom is chosen from nitrogen, sulphur or oxygen.
  • the aromatic heterocyclic moiety may be carbon or nitrogen linked.
  • the aromatic heterocyclic moiety may additionally be substituted by one or more additional substituents at any available ring carbon or heteroatom.
  • the substituents may include, but are not limited to the groups as defined for R26 in Formula V.
  • aromatic nitrocarbocycle means a benzene moiety substituted at any position by one or more nitro (NO2) groups.
  • two adjacent ring carbon atoms may optionally be linked to form a fused carbocyclic or heterocyclic ring.
  • the benzene moiety (and optional fused ring) may additionally be substituted by one or more additional substituents at any available carbon or heteroatom.
  • the substituents may include, but are not limited to, the groups as defined for R26 in Formula V.
  • pharmaceutically acceptable means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary as well as human pharmaceutical use.
  • Such salts include: acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, methanesulfonic acid, maleic acid, tartaric acid, citric acid and the like; and salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminium ion; or coordinates with an organic or inorganic base.
  • Acceptable organic bases include ethanolamine, diethanolamine, N-methylglucamine, triethanolamine and the like.
  • Acceptable inorganic bases include aluminium hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and
  • prodrug refers to a compound that, after administration, is metabolised or otherwise converted to a biologically active or more active compound (or drug) with respect to at least one property.
  • a prodrug, relative to the drug is modified chemically in a manner that renders it, relative to the drug, less active or inactive, but the chemical modification is such that the corresponding drug is generated by metabolic or other biological processes after the prodrug is administered.
  • a prodrug may have, relative to the active drug, altered metabolic stability or transport characteristics, fewer side effects or lower toxicity, or improved flavour (for example, see the reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392, incorporated herein by reference).
  • a prodrug may be synthesized using reactants other than the corresponding drug.
  • hypooxic refers to a concentration of oxygen in tissue that is significantly lower the normal physiological concentration of oxygen in healthy well perfused tissue, in particular oxygen tensions below approximately 1% (10,000 parts per million oxygen; 7.6 mmHg).
  • anoxia or“anoxic conditions” refers to an absence (or near absence) of oxygen in tissue, and in particular oxygen tensions below approximately 1 parts per million oxygen.
  • the terms“treat”,“treating”, and“treatment” as used herein refer to an action that occurs while an individual is suffering from the specified cancer, which reduces the severity of the cancer or the symptoms of the cancer, or retards or slows the progression of the cancer.
  • “treatment” may refer to a 5%, 10%, 25%, 50%, or 100% decrease in the rate of progress of a tumour.
  • “treatment” may refer to a 5%, 10%, 25%, 50%, or 100% decrease in determined tumour burden (i.e., the number of cancerous cells present in the subject, or the size of the tumour).
  • “treatment” may refer to a 5%, 10%, 25%, 50%, or 100% decrease in the physical symptoms of a cancer.
  • “treatment” may refer to a 5%, 10%, 25%, 50%, or 100% increase in the general health of the subject, as determined by any suitable means, such as cell counts, assay results, or other suitable means.
  • prevention refers to any method to partially or completely prevent or delay the onset of one or more symptoms or features of a disease, disorder, and/or condition. Prevention treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition.
  • the cancer may be any suitable cancer, including those as further defined herein.
  • the term“individual” or“subject” as used interchangeably herein refers to any mammalian animal including a human being. Suitably, the subject is a human being.
  • tumour cells may refer to the step of obtaining cells of the subject ( e.g . , by way of biopsy or otherwise), or may refer to the step of receiving a sample of tumour cells which has previously been obtained from the subject.
  • the tumour cells may comprise a sample.
  • the sample comprises a biological sample and can be, for instance, a cell, a cell culture, a tissue, or a biological fluid.
  • the biological sample may comprise a tumour cell biopsy, a plurality of samples from a clinical trial, or the like.
  • the sample can be a crude sample, or can be purified to various degrees prior to storage, processing, or measurement.
  • the term“determining” as used herein generally refers to any form of measurement, and includes determining if the Six-Transmembrane Epithelial Antigen of Prostate 4 (STEAP4) protein or mRNA is present or not.
  • the term“determining” includes both quantitative and/or qualitative determination.
  • the STEAP4 expression levels may be determined by any suitable method known to those skilled in the art, including those as further defined herein.
  • the expression“elevated level of STEAP4 expression” is further defined herein.
  • the terms “determining”, “measuring”, “evaluating”,“assessing” and“assaying” are used interchangeably herein.
  • the term“predict” or“predicting” generally means to determine or tell in advance.
  • the term“predict” can mean that the likelihood of the outcome of the cancer treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.
  • a predictive method may also be described as a prognostic method.
  • “likelihood”,“likely to”, and similar generally refers to an increase in the probability of an event.
  • “likelihood”,“likely to”, and similar when used in reference to responsiveness to cancer therapy generally contemplates an increased probability that the subject will exhibit a reduction in the severity of cancer or the symptoms of cancer or the retardation or slowing of the cancer progression.
  • the term“likelihood”,“likely to”, and similar when used in reference to responsiveness to cancer therapy can also generally mean the increase of indicators, such as mRNA or protein expression, that may evidence an increase in cancer treatment.
  • hypoxia-activated prodrug and HAP refer to any suitable HAP, including those as further defined herein. Suitable administration methods for HAPs are further described herein.
  • a therapeutically effective amount of a HAP refers to an amount of the HAP, alone or in combination with other therapies, which is sufficient to treat a cancer.
  • a therapeutically effective amount of a compound refers to the amount of the compound that, when administered, is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of the cancer.
  • the term also refers to the amount of the compound that is sufficient to elicit a biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.
  • a biological molecule e.g., a protein, enzyme, RNA, or DNA
  • a therapeutically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the cancer.
  • the term encompasses an amount of the compound that improves overall therapy, reduces, or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent.
  • the term“level” refers to the amount, accumulation, or rate of a biomarker molecule.
  • a level can be represented, for example, by the amount or the rate of synthesis of a messenger RNA (mRNA) encoded by a gene, the amount or the rate of synthesis of a polypeptide or protein encoded by a gene, the amount or the rate of synthesis of a biological molecule accumulated in a cell or biological fluid, or the biological activity of a biological molecule in a cell or biological fluid.
  • the term“level” refers to an absolute amount of a molecule in a sample or to a relative amount of the molecule, determined under steady-state or non-steady-state conditions.
  • the terms“basal level” refers to levels of the gene expressed in hosts or cell culture that would be found in a normal adult without a disease or disorder (e.g., the level expressed in a subject without cancer).
  • the term“responsiveness” or“responsive” when used in reference to a treatment refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease, disorder, or condition being treated.
  • the term“increased responsiveness” when used in reference to a treatment of a cell or a subject refers to an increase in the effectiveness in lessening or decreasing the symptoms of the disease when measured using any methods known in the art.
  • the increase in the effectiveness is at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, or at least about 50%.
  • STEAP Six-Transmembrane Epithelial Antigen of Prostate
  • the STEAP4 protein also known as STAMP2 or TIARP, is a metalloreductase that reduces iron and copper ions.
  • STEAP4 has equivalent activity under either physiological or acidic pH (pH5.5 - 7.5).
  • the full length nucleotide sequence encoding the STEAP4 protein i.e., the STEAP 4 gene
  • the full length amino acid sequence of the STEAP4 protein are known in the art (see, e.g., NCBI Gene ID: 79689, NCBI Acc. No. NM_024636, NCBI Acc. No. NM 001205315.1, NCBI Acc. No. NM_001205316.1, and UniProt Acc. No. Q687X5).
  • Isoform 1 is 459 amino acids in length and is shown in SEQ ID NO: 1. This isoform 1 is encoded by two variants: variant 1 and variant 2.
  • Variant 1 is the predominant nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO:2.
  • Variant 2 is an alternative nucleotide sequence encoding isoform 1 of the STEAP4 protein, and is shown in SEQ ID NO: 3.
  • Isoform 2 of the STEAP4 protein is 283 amino acids long, and is shown in SEQ ID NO:4.
  • Variant 3 is a nucleotide sequence encoding this shorter isoform of STEAP4 protein (isoform 2) and is shown in SEQ ID NO: 5.
  • reference to the“STEAP4”,“STEAP4 protein”, or similar refers to isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), or a protein encoded by variant 3 (SEQ ID NO:5), and/or to any other variant thereof.
  • Variants contemplated within the scope of the present disclosure include protein variants which are substantially homologous to a native STEAP4 protein.
  • substantially homologous refers to a protein having one or more naturally or non-naturally occurring amino acid deletions, insertions, or substitutions (e.g., derivatives, homologs, and fragments), as compared to the amino acid sequence of a native STEAP4 protein.
  • the amino acid sequence of a STEAP4 variant may be at least about 40% identical, at least about 50% identical, at least about 60% identical, at least about 70% identical, at least about 80% identical, at least about 90% identical, or at least about 95% identical to a native STEAP4 protein.
  • a“native STEAP4 protein” refers to the STEAP4 proteins which are found in nature and are not manipulated by man, and includes isoform 1 (SEQ ID NO: l), isoform 2 (SEQ ID NO:4), a protein encoded by variant 1 (SEQ ID NO:2), a protein encoded by variant 2 (SEQ ID NO:3), and a protein encoded by variant 3 (SEQ ID NO:5).
  • variant 1 SEQ ID NO: l
  • SEQ ID NO:4 isoform 1
  • variant 2 SEQ ID NO:3
  • variant 3 protein encoded by variant 3
  • Variants contemplated within the scope of the present disclosure also include proteins encoded by polynucleotide variants which have substantial sequence similarity or sequence identity to a native STEAP4 gene.
  • the polynucleotide sequence of a STEAP4 variant may have at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% sequence similarity or sequence identity with a native STEAP4 gene.
  • a“native STEAP4 gene” refers to genes: (a) which are found in nature and are not manipulated by man, and encode STEAP4 proteins; and (b) which encode STEAP4 proteins that are found in nature and are not manipulated by man; and includes the gene having the polynucleotide sequence encoding isoform 1 (SEQ ID NO: l), the gene having the polynucleotide sequence encoding isoform 2 (SEQ ID NO:4), the gene with the polynucleotide sequence of variant 1 (SEQ ID NO:2), the gene with the polynucleotide sequence of variant 2 (SEQ ID NO:3), and the gene with the polynucleotide sequence of variant 3 (SEQ ID NO:5).
  • High levels of STEAP4 expression are associated with certain cancers.
  • the inventors when STEAP4 is highly expressed in a cancer, the inventors have surprisingly found that STEAP4 can catalyse conversion of an administered drug into its active form, such as a reaction leading to release of a drug payload. Accordingly, in some embodiments, high STEAP4 expression leads to release of the drug payload in HAPs.
  • the high STEAP4 expression is associated with hypoxic metabolism.
  • the high STEAP4 expression is associated with a hypoxic environment.
  • the high STEAP4 expression is associated with a hypoxic tumour.
  • the high STEAP4 expression is not associated with low oxygen levels.
  • STEAP4 when STEAP4 is present in hypoxic tumour environments, STEAP4 catalyses one-electron reduction of cell-excluded quaternary ammonium salt HAPs, leading to their fragmentation selectively in pathophysiologically hypoxic tumour tissues, releasing the active drug which can then cross the cell wall and kill the cancer cell. [0130] Without wishing to be bound by theory, it is thought that STEAP4 is located on the plasma membrane. As such, in some embodiments, STEAP4 reduces the HAP extracellularly, forming a molecule that is capable of diffusing into the cell (e.g., a reduced form of the molecule).
  • STEAP4 is on the plasma membrane and reduces the HAP extracellularly, at which point the charged molecule undergoes fragmentation and diffuses into the cell to inhibit EGFR. In some embodiments, the charged HAP molecule undergoes fragmentation under hypoxic conditions. In some embodiments the charged HAP molecule undergoes fragmentation in the presence of elevated levels of STEAP4. In some embodiments, the HAP is Compound A. In some embodiments, the HAP is Compound B. In some embodiments, the HAP is selected from the group consisting of Compound A and Compound B.
  • NMQ prodrugs used in the disclosed methods function by releasing an active molecule having undergone reduction in the extracellular medium
  • other HAPs for use in the disclosed methods do not fragment and release an active molecule, but instead are effective because the reduced form of the molecule is able to penetrate the cell membrane and then cause cell death.
  • the HAPs may have a limiting or low rate of membrane penetration such that extracelluclar metabolism by STEAP4 will contribute a significant proportion of total cellular metabolism and such that intracellular reductases will contribute a less significant proportion of total cellular metabolism.
  • One such class of HAPs is the nitrophenyl mustards.
  • HAPs with a net neutral charge may be hydrophilic in nature, e.g., may have a low partition coefficient, which can result in a limiting rate of cell membrane penetration.
  • extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
  • HAPs with a low pKa may be protonated at physiologically relevant pH range and thus carry a net positive charge, resulting in a low partition coefficient, which can result in a limiting rate of cell membrane penetration.
  • extracellular metabolism by STEAP4 contributes a significant proportion of total cellular metabolism, and the remainder of HAP cellular metabolism is due to intracellular reductases.
  • the inventors surprisingly discovered, in part, a previously unidentified role played by the enzyme STEAP4 in hypoxic tumour environments in metabolising HAPs, including cell-excluded HAPs, at the cell surface, having the effect of releasing the active drug from the prodrug. This can enable the active drug to cross the cell membrane and deliver its payload to the intracellular targets and thus kill the cancer cell.
  • the inventors have also shown, for example, that this activity appears to be unique to the STEAP4 reductase enzyme, and is not shared with the other STEAP proteins or with other reductases.
  • ISH In Situ Hybridization
  • Nucleic acids if preserved adequately within a histologic specimen, can be detected through the application of a complementary strand of nucleic acid (e.g., mRNA) to which a reporter molecule is attached.
  • mRNA complementary strand of nucleic acid
  • ISH is used to map and order genes and other DNA and RNA sequences to their location on chromosomes and within nuclei.
  • STEAP4 expression levels may be determined by ISH.
  • STEAP4 expression levels may be determined by assessing the level of STEAP4 gene expression, by assessing the level of STEAP4 protein itself, or by assessing a biomarker in order to determine STEAP4 expression levels.
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a hypoxia-activated prodrug (HAP).
  • HAP hypoxia-activated prodrug
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a hypoxia-activated prodrug (HAP), wherein the tumor cells of the subject exhibit normal or an elevated level of STEAP4 expression.
  • HAP hypoxia-activated prodrug
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a hypoxia-activated prodrug (HAP);
  • HAP hypoxia-activated prodrug
  • tumor cells of the subject exhibit normal or an elevated level of STEAP4 expression
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising determining the level of STEAP4 expression in cancer cells of the subject by in situ hybridization scoring or qPCR.
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising providing cancer cells to be tested with ISH.
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the present disclosure provides a method of treating or preventing a cancer in a subject in need thereof, the method comprising:
  • the subject is a mammal. In some embodiments, the mammal is human.
  • the expression of STEAP4 is at least 1.1 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.2 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.3 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.4 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.5 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.6 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.7 times the basal level. In some embodiments, the expression of STEAP4 is at least 1.8 times the basal level.
  • the expression of STEAP4 is at least 1.9 times the basal level. In some embodiments, the expression of STEAP4 is at least 2 times the basal level. In some embodiments, the expression of STEAP4 is at least 3 times the basal level. In some embodiments, the expression of STEAP4 is at least 4 times the basal level. In some embodiments, the expression of STEAP4 is at least 5 times the basal level. In some embodiments, the expression of STEAP4 is at least 6 times the basal level. In some embodiments, the expression of STEAP4 is at least 7 times the basal level. In some embodiments, the expression of STEAP4 is at least 8 times the basal level.
  • the expression of STEAP4 is at least 9 times the basal level. In some embodiments, the expression of STEAP4 is at least 10 times the basal level. In some embodiments, the expression of STEAP4 is at least 11 times the basal level. In some embodiments, the expression of STEAP4 is at least 12 times the basal level. In some embodiments, the expression of STEAP4 is at least 13 times the basal level. In some embodiments, the expression of STEAP4 is at least 14 times the basal level. In some embodiments, the expression of STEAP4 is at least 15 times the basal level. In some embodiments, the expression of STEAP4 is at least 16 times the basal level.
  • the expression of STEAP4 is at least 17 times the basal level. In some embodiments, the expression of STEAP4 is at least 18 times the basal level. In some embodiments, the expression of STEAP4 is at least 19 times the basal level. In some embodiments, the expression of STEAP4 is at least 20 times the basal level. In some embodiments, the expression of STEAP4 is at least 30 times the basal level. In some embodiments, the expression of STEAP4 is at least 40 times the basal level. In some embodiments, the expression of STEAP4 is at least 50 times the basal level. In some embodiments, the expression of STEAP4 is at least 60 times the basal level.
  • the expression of STEAP4 is at least 70 times the basal level. In some embodiments, the expression of STEAP4 is at least 80 times the basal level. In some embodiments, the expression of STEAP4 is at least 90 times the basal level. In some embodiments, the expression of STEAP4 is at least 100 times the basal level.
  • the expression of STEAP4 is about 1.1 times the basal level. In some embodiments, the expression of STEAP4 is about 1.2 times the basal level. In some embodiments, the expression of STEAP4 is about 1.3 times the basal level. In some embodiments, the expression of STEAP4 is about 1.4 times the basal level. In some embodiments, the expression of STEAP4 is about 1.5 times the basal level. In some embodiments, the expression of STEAP4 is about 1.6 times the basal level. In some embodiments, the expression of STEAP4 is about 1.7 times the basal level. In some embodiments, the expression of STEAP4 is about 1.8 times the basal level.
  • the expression of STEAP4 is about 1.9 times the basal level. In some embodiments, the expression of STEAP4 is about 2 times the basal level. In some embodiments, the expression of STEAP4 is about 3 times the basal level. In some embodiments, the expression of STEAP4 is about 4 times the basal level. In some embodiments, the expression of STEAP4 is about 5 times the basal level. In some embodiments, the expression of STEAP4 is about 6 times the basal level. In some embodiments, the expression of STEAP4 is about 7 times the basal level. In some embodiments, the expression of STEAP4 is about 8 times the basal level. In some embodiments, the expression of STEAP4 is about 9 times the basal level.
  • the expression of STEAP4 is about 10 times the basal level. In some embodiments, the expression of STEAP4 is about 11 times the basal level. In some embodiments, the expression of STEAP4 is about 12 times the basal level. In some embodiments, the expression of STEAP4 is about 13 times the basal level. In some embodiments, the expression of STEAP4 is about 14 times the basal level. In some embodiments, the expression of STEAP4 is about 15 times the basal level. In some embodiments, the expression of STEAP4 is about 16 times the basal level. In some embodiments, the expression of STEAP4 is about 17 times the basal level. In some embodiments, the expression of STEAP4 is about 18 times the basal level.
  • the expression of STEAP4 is about 19 times the basal level. In some embodiments, the expression of STEAP4 is about 20 times the basal level. In some embodiments, the expression of STEAP4 is about 30 times the basal level. In some embodiments, the expression of STEAP4 is about 40 times the basal level. In some embodiments, the expression of STEAP4 is about 50 times the basal level. In some embodiments, the expression of STEAP4 is about 60 times the basal level. In some embodiments, the expression of STEAP4 is about 70 times the basal level. In some embodiments, the expression of STEAP4 is about 80 times the basal level. In some embodiments, the expression of STEAP4 is about 90 times the basal level. In some embodiments, the expression of STEAP4 is about 100 times the basal level.
  • the expression of STEAP4 is at least 1-3 times the basal level. In some embodiments, the expression of STEAP4 is at least 3-6 times the basal level. In some embodiments, the expression of STEAP4 is at least 7-20 times the basal level.
  • the expression of STEAP4 is about 1-3 times the basal level. In some embodiments, the expression of STEAP4 is about 3-6 times the basal level. In some embodiments, the expression of STEAP4 is about 7-20 times the basal level.
  • the level of STEAP4 expression is expression of a STEAP4 gene.
  • the level of STEAP4 expression is expression of a STEAP4- expressing target nucleic acid.
  • the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA.
  • the level of STEAP4 expression is expression of a STEAP4 protein.
  • the level of STEAP4 expression is associated with a low oxygen level.
  • the in situ hybridizing score is 1-4. In some embodiments, the in situ hybridizing score is 2-4. In some embodiments, the in situ hybridizing score is 3-4. In some embodiments, the in situ hybridizing score is 1-3. In some embodiments, the in situ hybridizing score is 2-3. In some embodiments, the in situ hybridizing score is 1-2.
  • the in situ hybridizing score is 1. In some embodiments, the in situ hybridizing score is 2. In some embodiments, the in situ hybridizing score is 3. In some embodiments, the in situ hybridizing score is 4.
  • the in situ hybridizing score is based on dots per cell clusters.
  • the clusters are about 1 to about 3 dots per cell. In some embodiments, the clusters are about 4 to about 9 dots per cell. In some embodiments, the clusters are about 10 to about 15 dots per cell. In some embodiments, the clusters are great than about 15 dots per cell.
  • the percentage of cells with clusters is greater than about 50%. In some embodiments, the percentage of cells with clusters is greater than about 60%. In some embodiments, the percentage of cells with clusters is greater than about 70%. In some embodiments, the percentage of cells with clusters is greater than about 80%. In some embodiments, the percentage of cells with clusters is greater than about 90%.
  • STEAP4 expression is elevated.
  • an elevated level of STEAP4 expression is measured by comparison of basal level.
  • an elevated level of STEAP4 expression is at least 1 times a basal level.
  • an elevated level of STEAP4 expression is measured by an in situ hybridizing score (e.g., of 1-4 according to RNAScope).
  • the prediction of an individual that is likely to be responsive to treatment by a HAP may be made by comparing the determined STEAP4 expression levels in the tumour cells to a reference level of STEAP4 expression.
  • the present disclosure provides a method of predicting the responsiveness of a subject with cancer to treatment with a HAP, the method comprising:
  • the present disclosure provides a method of predicting the responsiveness of a subject with cancer to treatment with tarloxotinib, the method comprising:
  • the present disclosure provides a method of predicting the responsiveness of a subject with cancer to treatment with a HAP, the method comprising:
  • the present disclosure provides a method of predicting the responsiveness of a subject with cancer to treatment with tarloxotinib, the method comprising:
  • the present disclosure provides a method of predicting the responsiveness of a subject with cancer to treatment with a HAP, the method comprising:
  • the present disclosure provides a method of predicting the responsiveness of an individual with cancer to treatment with tarloxotinib, the method comprising:
  • the method comprises administration of the HAP to the subject. In some embodiments, the method further comprises administration of the HAP to the individual if the individual is predicted to be likely to be responsive to the treatment. In some embodiments, the HAP is administered in a therapeutically effective amount. In some embodiments, the HAP comprises an NMQ prodrug and/or a nitrophenyl mustard. In some embodiments, the HAP is Compound A. In some embodiments, the HAP is Compound B. [0176] In one aspect, the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in cancer cells of the subject is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides use of a HAP in the manufacture of a medicament for treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in cancer cells of the subject is determined by in situ hybridization scoring or qPCR; and b. whether the cancer would be likely to be responsive to treatment by a HAP is predicted by if the cancer cells exhibit an elevated level of STEAP4 expression, wherein an elevated level of expression is at least 1 times a basal level.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • the present disclosure provides a HAP for use in treating or preventing a cancer, wherein:
  • a. cancer cells are provided;
  • the level of STEAP4 expression in the cancer cells is determined by in situ hybridization scoring or qPCR.
  • cancer and“cancerous” as used herein may refer to any cancer or cancerous condition and refers to the physiological condition in mammals characterised by unregulated cell growth or a malignant tumour.
  • Suitable cancers include, but are not limited to, prostate cancer (including but not limited to neuroendocrine prostate cancer/NEPC), lung cancer (including but not limited to lung adenocarcinoma, lung squamous cell cancer, small cell lung cancer, non-small cell lung cancer/NSCLC, and mesothelioma), breast cancer, esophageal cancer, head and neck cancer, upper aerodigestive tract cancer, neuroblastoma, cancer of the brain (including but not limited to glioma), cancer of the kidney, leukemia, chronic lymphocytic leukemia (CLL), monoclonal B cell lymphocytosis (MBL), childhood B-cell acute lymphobastic leukemia (B-ALL), chronic myeloid leukemia (CML), T-cell acute lymphobastic leukemia (T-ALL), sarcoma (including but not limited to Ewings sarcoma), pancreatic cancer, gastric cancer (also known as stomach cancer), desmoplastic small-
  • the cancer is a prostate cancer. In some embodiments, the cancer is a neuroendocrine prostate cancer (NEPC). In some embodiments, the cancer is a lung cancer. In some embodiments, the cancer is a lung adenocarcinoma. In some embodiments, the cancer is a lung squamous cell cancer. In some embodiments, the cancer is a small cell lung cancer. In some embodiments, the cancer is a non-small cell lung cancer (NSCLC). In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a esophageal cancer. In some embodiments, the cancer is a head and neck cancer.
  • NEPC neuroendocrine prostate cancer
  • the cancer is a lung cancer. In some embodiments, the cancer is a lung adenocarcinoma. In some embodiments, the cancer is a lung squamous cell cancer. In some embodiments, the cancer is a small cell lung
  • the cancer is a upper aerodigestive tract cancer. In some embodiments, the cancer is a neuroblastoma. In some embodiments, the cancer is a cancer of the brain. In some embodiments, the cancer is a glioma). In some embodiments, the cancer is a cancer of the kidney. In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is a chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a monoclonal B cell lymphocytosis (MBL). In some embodiments, the cancer is a childhood B-cell acute lymphobastic leukemia (B-ALL). In some embodiments, the cancer is a chronic myeloid leukemia (CML).
  • CML chronic myeloid leukemia
  • the cancer is a T-cell acute lymphobastic leukemia (T-ALL).
  • T-ALL T-cell acute lymphobastic leukemia
  • the cancer is a sarcoma.
  • the cancer is a Ewings sarcoma.
  • the cancer is a pancreatic cancer.
  • the cancer is a gastric cancer (also known as stomach cancer).
  • the cancer is a desmoplastic small-round-cell tumour (DESM).
  • the cancer is a uterine cancer.
  • the cancer is a uterine carcinosarcoma (UCS).
  • AML acute myeloid leukemia
  • the cancer is a B-cell acute lymphobastic leukemia (B-ALL).
  • B-ALL B-cell acute lymphobastic leukemia
  • the cancer is a liver cancer.
  • the cancer is a bladder cancer.
  • the cancer is a cancer of the urinary tract.
  • the cancer is a childhood acute myeloid leukemia (AML).
  • the cancer is a renal cancer.
  • the cancer is a colorectal cancer.
  • the cancer is a cervical cancer.
  • the cancer is a multiple myeloma.
  • the cancer is a endometrial cancer.
  • the cancer is a ovarian cancer.
  • the cancer is a lymphoma. In some embodiments, the cancer is a diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is a Hodgkin’s lymphoma. In some embodiments, the cancer is a non-Hodgkin’s lymphoma. In some embodiments, the cancer is a Burkitt lymphoma. In some embodiments, the cancer is a glioblastoma. In some embodiments, the cancer is a medulloblastoma. In some embodiments, the cancer is a melanoma. In some embodiments, the cancer is a cutaneous T-cell lymphoma (CTCL).
  • CTCL cutaneous T-cell lymphoma
  • the cancer is a astrocytoma. In some embodiments, the cancer is a bile duct cancer. In some embodiments, the cancer is a cholangiocarcinoma. In some embodiments, the cancer is a osteosarcoma. In some embodiments, the cancer is a meningioma. In some embodiments, the cancer is a thyroid cancer. In some embodiments, the cancer is a soft tissue sarcoma. In some embodiments, the cancer is a and chondrosarcoma.
  • the cancer is selected from the group consisting of prostate cancer, lung cancer (including but not limited to lung adenocarcinoma, lung squamous cell cancer, and mesothelioma), breast cancer, esophageal cancer, gastric cancer, cervical cancer, squamous cell carcinoma of the cervix, squamous cell carcinoma of the head and neck, liver cancer, neuroblastoma, chronic myeloid leukemia, and sarcoma.
  • the cancer is a prostate cancer.
  • the cancer is a lung cancer.
  • the cancer is a lung adenocarcinoma.
  • the cancer is a lung squamous cell cancer. In some embodiments, the cancer is a mesothelioma. In some embodiments, the cancer is a breast cancer. In some embodiments, the cancer is a esophageal cancer. In some embodiments, the cancer is a gastric cancer. In some embodiments, the cancer is a cervical cancer. In some embodiments, the cancer is a squamous cell carcinoma of the cervix. In some embodiments, the cancer is a squamous cell carcinoma of the head and neck. In some embodiments, the cancer is a liver cancer. In some embodiments, the cancer is a neuroblastoma. In some embodiments, the cancer is a chronic myeloid leukemia.
  • the cancer is a sarcoma.
  • the combinations disclosed herein are administered to treat cancer.
  • the cancer to be treated comprises lung cancer.
  • the lung cancer comprises non-small cell lung cancer.
  • the cancer comprises gastric cancer.
  • the cancer comprises breast cancer.
  • the cancer comprises head and neck squamous cell carcinoma (HNSCC).
  • the cancer comprises gastric/gastroesophageal (GE) junction cancer.
  • the cancer comprises oesophageal cancer.
  • the cancer comprises salivary cancer.
  • the cancer comprises ovarian cancer.
  • the cancer comprises endometrial cancer. In some embodiments, the cancer comprises uterine cancer. In some embodiments, the cancer comprises pancreatic cancer. In some embodiments, the cancer comprises biliary tract cancer. In some embodiments, the cancer comprises bladder cancer. In some embodiments, the cancer comprises colorectal cancer. In some embodiments, the cancer comprises renal cancer. In some embodiments, the cancer comprises brain and/or spinal cord cancer (glioblastoma). In some embodiments, the cancer comprises lymphoma, e.g., primary central nervous system lymphoma. In some embodiments, the cancer comprises leukaemia, e.g., acute lymphoblastic leukaemia.
  • the cancer is selected from the group of lung cancer, gastric cancer, breast cancer, HNSCC, GE junction cancer, oesophageal cancer, salivary cancer, ovarian cancer, endometrial cancer, uterine cancer, prostate cancer, pancreatic cancer, colon cancer, biliary tract cancer, bladder cancer, colorectal, renal, glioblastoma, mesothelioma, adenocarcinoma, lymphoma, and leukaemia.
  • the cancer is selected from bone cancer, lung cancer, breast cancer, cancer of the head and neck, prostate cancer, pancreatic cancer, skin cancer, uterine cancer, ovarian cancer, cancer of the urethra, cancer of the adrenal gland, cancer of the small intestine, cancer of the kidney, cancer of the bladder, cancers of the brain, colorectal cancer, oesophageal cancer, gastric cancer, anal cancer, liver cancer, thyroid cancer, ocular cancer, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland and testicular cancer.
  • the cancer is selected from chronic or acute leukaemia, acute myeloid leukaemia, chronic myeloid leukaemia, acute lymphoblastic leukaemia, chronic lymphocytic leukaemia, carcinoma of the cervix, carcinoma of the vulva, carcinoma of the vagina, Hodgkin’s Disease, brain stem glioma, melanoma, Merkel cell carcinoma, Urothelial carcinoma, lymphomas, gliomas, meningiomas, pituitary adenomas, nerve sheath tumours, and retinoblastoma, and non-small cell lung cancer.
  • the cancer is non-small cell lung cancer. [0193] In some embodiments, the cancer is prostate cancer.
  • the cancer is cervical carcinoma.
  • the cancer is breast cancer.
  • the cancer is brain cancer.
  • the cancer is glioblastoma.
  • the cancer is spinal cord cancer.
  • the cancer is a solid tumour.
  • Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas).
  • the solid tumour can be sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, oesophageal adenocarcinoma, oesophageal squamous cell carcinoma, squamous cell carcinoma of the head and neck (HNSCC), oral carcinoma, gastric carcinoma, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,
  • the solid tumour is malignant melanoma, adrenal carcinoma, breast carcinoma, renal cell cancer, carcinoma of the pancreas, non-small-cell lung carcinoma (NSCLC) or carcinoma of unknown primary.
  • NSCLC non-small-cell lung carcinoma
  • the solid tumour is breast cancer, ovarian cancer, brain cancer, gastric cancer, oesophageal cancer prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, and thyroid cancer.
  • the solid tumour is selected from the groups consisting of carcinoma, melanoma, sarcoma, or chronic granulomatous disease.
  • HAP Hypoxia-Activated Prodrug
  • HAP Any route of administration of a HAP may be used.
  • an HAP can be administered by oral, parenteral, intravenous, transdermal, intramuscular, rectal, sublingual, mucosal, nasal, or other means.
  • the HAP is administered intravenously administration or intraperitoneally administration or is formulated for intravenous administration or for intraperitoneal administration.
  • the dosage amount of HAP will depend on the specific HAP used, the type of cancer being treated, and any optional additional agents concurrently administered to the patient.
  • Typical dosage amounts comprise an amount of from about 0.001 to about 2500 mg/m 2 .
  • suitable dosage amounts comprises from about 0.001, 0.01, 0.1, 1, 2, 5, 7.5, 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150, 200, 250, or 300 to about 10, 12.5, 15, 17.5, 20, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2250, or 2500 mg.
  • the dosage amount comprises Compound A in an amount of about 10 mg/m 2 to about 270 mg/m 2 , suitably about 110 mg/m 2 to about 170 mg/m 2 , more suitably about 140 mg/m 2 to about 160 mg/m 2 , most suitably about 150 mg/m 2 .
  • the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 200 mg/kg of body weight of a subject. In some embodiments, the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, from about 0.1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject, or from about
  • the dosage amount of HAP is from about 1 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject, from about 1 mg/kg of body weight of a subject to about 10 mg/kg of body weight of a subject, or from about 1 mg/kg of body weight of a subject to about 5 mg/kg of body weight of a subject.
  • the dosage amount of HAP is from about 10 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject, from about 10 mg/kg of body weight of a subject to about 20 mg/kg of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/kg of body weight of a subject.
  • the dosage amount of HAP is from about 20 mg/kg of body weight of a subject to about 100 mg/kg of body weight of a subject, from about 20 mg/kg of body weight of a subject to about 50 mg/kg of body weight of a subject, or from about 20 mg/kg of body weight of a subject to about 25 mg/kg of body weight of a subject.
  • the HAP is Compound A.
  • the HAP is Compound B.
  • the dosage amount of HAP is from about 0.1 mg/kg of body weight of a subject to about 300 mg/m 2 of body weight of a subject. In some embodiments, the dosage amount of HAP is from about 0.1 mg/m 2 of body weight of a subject to about 200 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 25 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 20 mg/m 2 of body weight of a subject, from about 0.1 mg/m 2 of body weight of a subject to about 15
  • the dosage amount of HAP is from about 1 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, about 1 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about 25 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about 20 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about 15 mg/m 2 of body weight of a subject, from about 1 mg/m 2 of body weight of a subject to about 10 mg/m 2 of body weight of a subject, or from about mg/m 2 of body weight of a subject to about 5 mg/m 2 of body weight of a subject.
  • the dosage amount of HAP is from about 10 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 25 mg/m 2 of body weight of a subject, from about 10 mg/m 2 of body weight of a subject to about 20 mg/m 2 of body weight of a subject, or from about 10 mg/kg of body weight of a subject to about 15 mg/m 2 of body weight of a subject.
  • the dosage amount of HAP is from about 20 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 20 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 20 mg/m 2 of body weight of a subject to about 50 mg/m 2 of body weight of a subject, or from about 20 mg/m 2 of body weight of a subject to about 25 mg/m 2 of body weight of a subject.
  • the dosage amount of HAP is from about 50 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, from about 50 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject, from about 50 mg/m 2 of body weight of a subject to about 80 mg/m 2 of body weight of a subject, or from about 50 mg/m 2 of body weight of a subject to about 75 mg/m 2 of body weight of a subject.
  • the dosage amount of HAP is from about 75 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, or from about 75 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject. In some embodiments, the dosage amount of HAP is from about 80 mg/m 2 of body weight of a subject to about 150 mg/m 2 of body weight of a subject, or from about 80 mg/m 2 of body weight of a subject to about 100 mg/m 2 of body weight of a subject.
  • the dosage amount of HAP is from about 0.1 mg/m 2 , about 1 mg/m 2 , about 5 mg/m 2 , about 10 mg/m 2 , about 15 mg/m 2 , about 20 mg/m 2 , about 25 mg/m 2 , about 30 mg/m 2 , about 40 mg/m 2 , about 50 mg/m 2 , about 60 mg/m 2 , about 70 mg/m 2 , about 75 mg/m 2 , about 80 mg/m 2 , about 100 mg/m 2 , about 150 mg/m 2 , about 200 mg/m 2 , or about 300 mg/m 2 .
  • the HAP is Compound A.
  • the HAP is Compound B.
  • the HAP is administered, or is formulated for administration, by intravenous or intraperitoneal administration.
  • the HAP is administered, or is formulated for administration by at least one route selected from the group consisting of inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal, intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, intratracheal, optic, intraocular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical.
  • the HAP is administered orally, parenterally, rectally, topically, intravenously, intramuscularly, subcutaneously, or intraperitoneally.
  • the HAP is administered intravenously.
  • the HAP is administered at a frequency of at least once per day, at least once per week or at least once per month.
  • the HAP may be administered for a period of 1-3 weeks or up to at least 30 weeks or up, at least 1 day or up to at least 150 days. In some embodiments, longer periods of administration are employed.
  • the dosage amount comprises Compound A in an amount of about 140 mg/m 2 to about 160 mg/m 2 , more suitably about 150 mg/m 2 , where the dosage is administered once per week.
  • the dosage is administered intravenously over a period of approximately one hour. In other embodiments, the dosage is administered intravenously over a period of two to four hours.
  • the HAPs is administered to the patient in dosages that range from one to five times per day or more. In some embodiments, the HAP is administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the HAPs contemplated herein varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the present disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the ahending physical taking all other factors about the patient into account.
  • the amount of HAP dosed per day may be administered, in non limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days.
  • a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.
  • the HAP is administered once, twice, three times, four times, five times, or six times a day. In some embodiments, the HAP is administered once, twice, three times, four times, five times, or six times a week. In some embodiments, the HAP is administered every week, every two weeks, every three weeks, every four weeks, every five weeks, or every six weeks.
  • the administration of the HAPs contemplated herein is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e.. a“drug holiday”).
  • the length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the disease or disorder, to a level at which the improved disease is retained.
  • patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection
  • compositions provided herein can also contain one of more pharmaceutically acceptable excipients. See, e.g., Rowe et al, Handbook of Pharmaceutical Excipients, 4th Ed. (2003), entirety of which is incorporated herein by reference.
  • the HAPs contemplated herein are formulated in a pharmaceutical composition using one or more pharmaceutically acceptable excipients or carriers.
  • the pharmaceutical compositions contemplated herein comprise a therapeutically effective amount of a combination contemplated herein and a pharmaceutically acceptable carrier.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal and the like.
  • isotonic agents for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
  • Suitable administration methods for a HAP compounds can be found in PCT/NZ2010/000040, PCT/NZ2010/000174, and PCT/US2015/063806, each of which is incorporated by reference herein in its entirety.
  • Additional dosage forms include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389; 5,582,837; and 5,007,790. Additional dosage forms also include dosage forms as described in U.S. Patent Applications Nos. 20030147952; 20030104062; 20030104053; 20030044466; 20030039688; and 20020051820.
  • Further dosage forms include dosage forms as described in PCT Applications Nos. WO 03/35041 ; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO 01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO 90/11757.
  • the HAP may be administered in combination with other agents, including but not limited to a further HAP, a chemotherapeutic agent, a radiotherapeutic agent, and/or an immunotherapeutic agent.
  • the HAP may comprise Compound A and may be administered in combination with another tyrosine kinase inhibitor selected from the group consisting of crizotinib, alectinib, ceritinib, erlotinib, dacomitinib, osimertinib, afatinib, gefitinib, rociletinib, cetuximab, icotinib, sapitinib, lapatinib, neratinib, brigatinib, poziotinib, naquotinib, TAS-121, panitumumab, nimotuzumab, catumaxomab, duligotuzumab patritumumab, abemaciclib, acalabrutinib, axitinib, baricitinib, binimetinib, bosutinib, brigatinib
  • the HAPs contemplated herein, or a salt or solvate thereof can be used concurrently or in combination with one or more agents known to be useful in treating or preventing cancer, such as the cancers described herein.
  • Non-limiting examples of additional anti proliferative agents contemplated include, but are not limited to, compounds listed on the cancer chemotherapy drug regimens in the 14 th Edition of the Merck Index (2006), which is hereby incorporated by reference, such as asparaginase, bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin, etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide, irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine, mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone, prednisone, procarba
  • Additional anti-proliferative agents include other molecular targeted agents that modulate parallel pathways such as MEK 1/2 inhibitors, AKT inhibitors and mTOR inhibitors, monoclonal antibodies (such as Cetuximab), oxaliplatin, gemcitabine, gefmitib, taxotere, ara A, ara C, herceptin, BCNU, CCNU, DTIC, and actinomycin D. Still further anti-proliferative agents include but are not limited to those compounds acknowledged to be used in the treatment of neoplastic diseases in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Eleventh Edition), editor Molinoff et al, publ.
  • a HAP is disclosed herein is co-administered with at least one additional agent. In some embodiments, a HAP is disclosed herein is is co-formulated with at least one additional agent.
  • the formulations of the present application may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.
  • sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period.
  • the period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.
  • the compounds may be formulated with a suitable polymer or hydrophobic material that provides sustained release properties to the compounds.
  • the compounds for use in the methods disclosed herein may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.
  • the compounds contemplated herein are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.
  • delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that may, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.
  • pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.
  • immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.
  • short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration.
  • rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.
  • the disclosed methods are based at least in part on the surprising discovery by the inventors that in some embodiments, the cell surface reductase STEAP4 plays a role in metabolising HAPs and therefore that individuals with elevated STEAP4 expression may be identified as those likely to response to treatment by a HAP. It is believed that this subgroup of individuals (i.e., individuals with elevated levels of STEAP4 expression as a subgroup of all individuals) may be more responsive to treatment by a HAP due to their elevated levels of STEAP4 expression. Accordingly, in some embodiments, the identification of individuals with elevated STEAP4 expression and subsequent treatment of those individuals may permit the HAP to be administered at a low dosage.
  • a“low dosage” may refer to a dosage that is lower than the dosage conventionally employed.
  • the HAP may be administered at a dosage that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 35%, 50%, 60%, or more lower than the conventionally employed HAP dosage.
  • a conventionally employed HAP dosage may be the dosage approved for administration by medical regulatory authorities in a particular jurisdiction, or may be the dosage employed in previous clinical studies, or elsewhere.
  • the identification of individuals with elevated STEAP4 expression levels will enable treatment by a HAP to be targeted to cancer cells and thus avoid or minimise delivery of the HAP at other sites in the body where the cytotoxic metabolite of the HAP may have undesirable effects. Accordingly, in some embodiments, the identification of individuals with elevated STEAP4 expression and subsequent treatment of those individuals may permit the HAP to be administered at a high dosage.
  • a“high dosage” may refer to a dosage that is higher than the dosage conventionally employed.
  • the HAP may be administered at a dosage that is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 35%, 50%, 60%, or more higher than the conventionally employed HAP dosage.
  • a conventionally employed HAP dosage may be the dosage approved for administration by medical regulatory authorities in a particular jurisdiction, or may be the dosage employed in previous clinical studies, or elsewhere. [0233] It is also believed that the identification of individuals with elevated STEAP4 expression levels will enable treatment of those individuals with fewer or reduced side effects.
  • fewer or reduced side effects refers to a smaller number or a smaller magnitude of side effects compared to those experienced in a group of individuals receiving HAP treatment who have not been stratified to identify those with elevated levels of STEAP4 expression (e.g., a group comprising individuals with elevated levels of STEAP4 expression and individuals without elevated levels of STEAP4 expression), and/or compared to one or more individuals who does not exhibit elevated levels of STEAP4 expression.
  • the side effects that may be reduced in number or in magnitude include, but are not limited to, hearing loss, muscle cramping, diarrhoea, skin rash, nausea, and vomiting.
  • the phrase“elevated STEAP4 expression levels” and similar may refer generally to an absolute level of STEAP4 expression which is considered to be elevated, or a level of STEAP4 expression which is elevated relative to (e.g., higher or greater than) a comparative level (e.g., a reference level or a level in a control sample).
  • the term“expression” and similar terms as used herein refers to a transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one or more of the two nucleic acid strands of the gene.
  • the term“expressed” or“expression” as used herein also refers to the translation from the RNA molecules to give a protein, a polypeptide, or a portion thereof.
  • the phrase“elevated STEAP4 expression levels” refers to an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, 300%, 500%, or more STEAP4 mRNA level relative to a comparative level (e.g., reference level or level in a control sample) of STEAP4 mRNA level or an increase of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, 300%, 500%, or more STEAP4 protein level relative to a comparative level (e.g., reference level or level in a control sample) of STEAP4 protein.
  • a comparative level e.g., reference level or level in a control sample
  • the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample is one that a treatment decision is made based on whether an individual has an expression level of STEAP4 (e.g., in their tumour cells) that is elevated as compared to the reference level of STEAP4 expression or as compared to the level of STEAP4 expression in a control sample.
  • An individual who has a level of STEAP4 expression that is elevated as compared to the reference level of STEAP4 expression or as compared to a level of STEAP4 expression in a control sample has a different probability of responsiveness to the treatment than an individual who has a level of STEAP4 expression the same as or lower than the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample.
  • the reference level of STEAP4 expression and/or the level of STEAP4 expression in a control sample is determined simultaneously with the determination of the level of STEAP4 expression in the tumour cells of the subject or in the sample of tumour cells from the subject. In some embodiments, the reference level of STEAP4 expression level and/or the level of STEAP4 expression in a control sample is determined independently from the level of STEAP4 expression in the tumour cells of the subject or in the sample of tumour cells from the subject.
  • the reference level of STEAP4 expression may suitably be determined in a sample of non tumour cells from the same individual, and/or in a sample of non-tumour cells from a different individual (including from a different individual who does not have cancer), and/or in a sample of non-tumour cells from a group of individuals (including from one or more individuals who do not have cancer).
  • control sample may suitably comprise a sample of non-tumour cells from the same individual, and/or a sample of non-tumour cells from a different individual (including from a different individual who does not have cancer), and/or a sample of non-tumour cells from a group of individuals (including from one or more individuals who do not have cancer).
  • the reference level of STEAP4 expression is predetermined. In some embodiments, the level of STEAP4 expression in the control sample is predetermined.
  • the phrase“elevated STEAP4 expression levels” refers to a STEAP4 expression level which is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more, higher relative to the expession of a reference gene, such as a house-keeping gene.
  • the reference gene may comprise glyceraldehyde 3-phosphate dehydrogenase ( GAPDH) and/or hypoxanthine phosphoribosyl transferase ( HPRT ).
  • the phrase“elevated STEAP4 expression levels” refers to a rate of reduction of Fe 3+ to Fe 2+ that is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 100%, 150%, 200%, or more relative to a comparative level (e.g., reference level or level in a control sample).
  • a comparative level e.g., reference level or level in a control sample.
  • the rate may be measured by measuring formation of the Fe 2+ -ferrozine complex.
  • the phrase“elevated STEAP4 expression levels” refers to a rate of production of Compound B above 100 picomole/hr/10 6 cells under anoxic conditions ( ⁇ 1 ppm oxygen). More suitably, the phrase“elevated STEAP4 expression levels” refers to a rate of production of Compound B above 200 picomole/hr/10 6 cells under anoxic conditions ( ⁇ 1 ppm oxygen).
  • the present disclosure also provides kits for performing the disclosed methods.
  • the present disclosure provides a kit useful for predicting whether an individual with cancer is likely to be responsive to treatment with a HAP.
  • kits for detecting the STEAP4 mRNA biomarkers can be prepared.
  • the kits can include, for example, a probe or probe set comprising oligonucleotides that can bind to the mRNA biomarker(s) of interest for a given disease, compound, or other parameter.
  • the probe may be complementary to at least 20, 50, 100, 200, 350, or more basis of STEAP4 mRNA. Washing solutions, reagents for performing a hybridisation assay, mRNA isolation or purification means, detection means, as well as positive and negative controls can also be included.
  • the kit can also include instructions for using the components of the kit.
  • the kit can be tailored for in-home use, clinical use, or research use.
  • kits for detecting STEAP4 protein levels can be prepared.
  • the kits can include, for example, a dipstick coated with an antibody that recognises the protein, washing solutions, reagents for performing the assay, protein isolation or purification means, detection means, as well as positive and negative controls.
  • the kit can also include instructions for using the components of the kit.
  • the kit can be tailored for in-home use, clinical use, or research use.
  • the kit comprises a solid support, and a means for detecting the protein expression of STEAP4 mRNA in a sample (e.g., a biological sample).
  • a sample e.g., a biological sample
  • a kit can employ, for instance, a dipstick a membrane, a chip, a disk, a test strip, a filter, a microsphere, a slide, a multiwell plate, or an optical filter.
  • the kits described here may be employed in the methods described herein.
  • kits described here may be employed in the methods of the present disclosure.
  • the mRNA ISH assay was developed and validated for STEAP4 in FFPE tissues, using both FFPE cell lines and FFPE tissues as both positive and negative controls.
  • STEAP4 mRNA ISH staining and scoring was performed on a small cohort of EFGR and HER2 mutated FFPE patient specimens from Japan.
  • the assay utilized the ACD LSx Brown Kit. Validation for the STEAP4 for mRNA ISH assay was performed on the Leica BOND Rx autostainer using FFPE sections. The standard workflow for mRNA ISH staining was followed.
  • RNAscope 2.5 LSx PPIB probe was used as a positive control.
  • the protocol used was the RNAscope 2.5 LSx DapB ISH with an additional fifteen minute exposure with the AMP 5 DapB step.
  • the protocol used was the RNAscope 2.5 LSx at 95 degrees centigrade and exposed to epitope retrieval 2 (high pH).
  • the enzyme protease reagent was used for pretreatment using Leica protocol RNAscope 2.5 LSx Enzyme. Hybridization was extended by twenty-five minutes at 42 °C, using Leica protocol RNAscope 2.5 LSx Hybridization. Following staining, slides were cleared and dehydrated on an automated Tissue-Tek Prisma platform and cover slipped using a Tissue-Tek Film cover slipper.
  • Controls Both positive and negative FFPE tissues were used for the STEAP4 mRNA ISH assay. These specimens included C33A-WT (negative control) and C33A-S4 (positive control). All STEAP4 mRNA ISH staining runs included both positive and negative control slides to ensure consistent and accurate staining.
  • RNAscope BROWN kit was used for STEAP4 staining.
  • Imaging All stained slides were evaluated using an Olympus BX43 phase contrast microscope. Digital images were acquired using an Olympus DP71 camera attachment and Olympus cellSens Entry software version 1.9. Images were stored as TIFF files. Table 3 outlines the specimen groups which were stained using the STEAP4 mRNA ISH assay.
  • Specimens identified in Group 1 are xenografts. The specimens were stained for STEAP4, PPIB and DapB. Scoring data was as per ACD’s scoring guidelines as identified above.
  • SiHa WT and STEAP4 K/O tumor tissues demonstrated strong PPIB staining, indicating the specimens were adequate for mRNA ISH analysis.
  • Evaluation of STEAP4 staining revealed strong expression in both the SiHa WT and STEAP4 K/O specimens.
  • SiHa STEAP4 KO tumor sections were identified as negative controls.
  • SiHa STEAP4 KO tumor sections are the KO generated by CRISPR.
  • ACDBIO evaluation of the sequence revealed RNAScope probes still bind to the mRNA sequence of STEAP4 that are still intact (FIG. 1).
  • Group 3 tumors were developed in a mouse model and derived from overexpression of STEAP4 in a cell line. Specimens were in two groups, including wild type (WT) and overexpressed (S4). Following mRNA ISH staining for STEAP4, PPIB, and DapB, the specimens were scored (Table 6).
  • Scoring data for Group 3 demonstrated all tissues were adequate based on PPIB expression. A separation in groups was determined for STEAP4 expression wherein WT specimens were negative and C33A STEAP4 specimens demonstrated high expression. C33A WT and C33A STEAP4 were identified as negative and positive controls, respectively, to be used as in-run controls for all subsequent STEAP4 mRNA ISH staining (FIG. 3).
  • Tumors generated in mice were developed and tumor blocks were cut. Sections of tumors were placed on charged glass slides. Four sections were provided for each tumor. Tumors were either from control animals or those treated with drug. All specimens were stained for STEAP4, PPIB and DapB accroding to the mRNA ISH protocol. Specimens were scored and the data shown below in Table 7.
  • Tumor blocks specimens were cut from FFPE tumor blocks on a microtome at 5 microns and placed on charged glass slides. Following mRNA ISH staining for STEAP4, PPIB, and DapB, the specimens were scored (Table 8).
  • Scoring data reveals PPIB expression to be variable from 1-4 indicating variability in the adequacy of the tissue. Low PPIB expression may be a result of a variety of factors. Specimens in Group 7 are likely to be from older tissue blocks including 07B18145, 17B4233 and B94830. STEAP4 expression was variable (0-4) (FIG. 7). In-run controls were scored and demonstrated accurate staining.
  • the STEAP4 mRNA ISH assay was developed to enhance both probe hybridization time, as well as an increase in AMP 5 time.
  • the optimized STEAP4 mRNA ISH assay can be used to evaluate a large number of specimens.
  • PPIB analysis is used to ensure the adequacy of specimens prior to analysis of STEAP4 mRNA ISH. Variability in specimens can be due to variation in tissue processing and embedding, as well as specimen ageing. Specimens demonstrated variable STEAP4 expression.
  • Appropriate positive and negative controls for STEAP4 C33A S4, C33A WT were identified. The controls were used for each staining run as in-run controls to ensure accurate and reproducible staining quality.
  • ISH In Situ Hybridization
  • Slides were stained on a Leica Bond RX autostainer using the RNAScope 2.5 LSx Brown Reagent kit (#322700) with the RNAscope 2.5 LSx STEAP4 probe (#542158).
  • the RNAscope 2.5 LSx PPIB probe (#313908) was used as a positive control.
  • the protocol for Example 1 was used for Example 2 mRNA ISH detection, the protocol used was the RNAscope 2.5 LSx DAB ISH with an additional fifteen minute exposure with the AMP 5 DAB step. For heat pretreatment, the protocol used was the RNAscope 2.5 LSx at 95 degrees centigrade and exposed to epitope retrieval 2 (high pH).
  • the enzyme protease was used for pretreatment using Leica protocol RNAscope 2.5 LSx Enzyme. Hybridization was extended by twenty-five minutes at 42 degrees centigrade, using Leica protocol RNAscope 2.5 LSx Hybridization. Following staining, slides were cleared and dehydrated on an automated Tissue-Tek Prisma platform and cover slipped using a Tissue-Tek Film cover slipper.
  • Tarloxotinib metabolism was measured in an isogenic pair of parental (WT) and STEAP4 overexpressing C33A xenografts.
  • C33A cells have low endogenous STEAP4 expression.
  • Tarloxotinib metabolism in STEAP4 overexpressing xenografts increased relative to parental xenografts.
  • Tarloxotinib metabolism in parental and STEAP4 overexpressing C33A xenografts grown in NIH-III mice is shown in Fig. 8A. Tumors were harvested 3 hours following a single dose (48 mg/kg, i.p.) of tarloxotinib and STEAP4 expression in xenografts was determined by qPCR (Fig. 8B).
  • STEAP4 ISH staining in situ across a panel of xenografts was characterized and validated in comparison to C33A-WT and C33A-STEAP4 overexpressing cells (Figs. 9A-9B).

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Abstract

Dans certains aspects, la présente invention concerne un procédé de traitement ou de prévention d'un cancer chez un sujet en ayant besoin, le procédé comprenant la détermination du niveau d'expression de STEAP4 dans des cellules cancéreuses pour prédire si les cellules cancéreuses sont sensibles au traitement par un HAP.
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WO2010127399A1 (fr) * 2009-05-06 2010-11-11 Walter And Eliza Hall Institute Of Medical Research Profils d'expression génétique et leurs utilisations
WO2019125184A1 (fr) * 2017-12-19 2019-06-27 Auckland Uniservices Limited Utilisation de biomarqueur en thérapie du cancer

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010127399A1 (fr) * 2009-05-06 2010-11-11 Walter And Eliza Hall Institute Of Medical Research Profils d'expression génétique et leurs utilisations
WO2019125184A1 (fr) * 2017-12-19 2019-06-27 Auckland Uniservices Limited Utilisation de biomarqueur en thérapie du cancer

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CEREN G KORKMAZ; KEMAL S KORKMAZ; PIOTR KURYS; CEM ELBI; LING WANG; TOVE I KLOKK; CLARA HAMMARSTROM; GUNHILD TROEN; AUD SVINDLAND;: "Molecular cloning and characterization of STAMP2, an androgen-regulated six transmembrane protein that is overexpressed in prostate cancer", ONCOGENE, vol. 24, no. 31, 9 May 2005 (2005-05-09), pages 4934 - 4945, XP055300813, ISSN: 0950-9232, DOI: 10.1038/sj.onc.1208677 *
COURTNEY M. ANDERSON, ZHANG BINGQING, MILLER MELANIE, BUTKO EMERALD, WU XINGYONG, LAVER THOMAS, KERNAG CASEY, KIM JEFFREY, LUO YUL: "Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues", JOURNAL OF CELLULAR BIOCHEMISTRY, vol. 117, no. 10, 18 May 2016 (2016-05-18), pages 2201 - 2208, XP055744644, ISSN: 0730-2312, DOI: 10.1002/jcb.25606 *
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