US20170315127A1 - Treatment of metastatic prostate cancer - Google Patents

Treatment of metastatic prostate cancer Download PDF

Info

Publication number
US20170315127A1
US20170315127A1 US15/134,228 US201615134228A US2017315127A1 US 20170315127 A1 US20170315127 A1 US 20170315127A1 US 201615134228 A US201615134228 A US 201615134228A US 2017315127 A1 US2017315127 A1 US 2017315127A1
Authority
US
United States
Prior art keywords
prostate cancer
inhibitor
enzalutamide
cells
niclosamide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US15/134,228
Other languages
English (en)
Inventor
Allen C. Gao
Chengfei Liu
Wei Lou
Chong-xian Pan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Veterans Affairs
Original Assignee
University of California San Diego UCSD
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of California San Diego UCSD filed Critical University of California San Diego UCSD
Priority to US15/134,228 priority Critical patent/US20170315127A1/en
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GAO, Allen C., LIU, CHENGFEI, LOU, WEI
Assigned to THE REGENTS OF THE UNIVERSITY OF CALIFORNIA reassignment THE REGENTS OF THE UNIVERSITY OF CALIFORNIA ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: PAN, CHONG-XIAN
Publication of US20170315127A1 publication Critical patent/US20170315127A1/en
Assigned to U.S. Government represented by the Department of veterans Affairs reassignment U.S. Government represented by the Department of veterans Affairs ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
Priority to US16/654,847 priority patent/US11215617B2/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • A61K31/275—Nitriles; Isonitriles
    • A61K31/277—Nitriles; Isonitriles having a ring, e.g. verapamil
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4166—1,3-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. phenytoin
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • 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
    • A61K31/60—Salicylic acid; Derivatives thereof
    • A61K31/609—Amides, e.g. salicylamide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6869—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of the reproductive system: ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • CRPC castration-resistant prostate cancer
  • AR splice variants are often prevalent in advanced prostate cancer tissue, e.g., tissue from a patient having CRPC and advanced prostate cancer cell lines. These AR variants are generated by aberrant splicing, e.g., alternative mRNA splicing, and premature translation termination. Truncated AR variants drive androgen independent growth. AR also mediates gene expression.
  • AR-V7 an AR variant that lacks the ligand binding domain (LBD) targeted by enzalutamide and abiraterone, is constitutively active in some prostate cancer cells. Because AR-V7 lacks the LBD, it is therefore resistant to LBD targeted treatments, e.g., enzalutamide and abiraterone.
  • AR variants have also been observed to be up-regulated in CRPC patient samples as compared to androgen sensitive patient samples. As such, AR variants are associated with prostate cancer progression and resistance to AR-targeted therapy. Certain AR variants were observed to induce ligand independent activation of ARE-driven reporters in the absence of androgen, which indicates that those variants may have a distinct transcription program as compared to full length AR. Certain AR variants may interact with the full length AR such that full length AR may mediate the activity of the AR variants.
  • Interleukin-6 is a pleiotropic cytokine that plays a central role in host defense mechanisms.
  • the biological activities of IL-6 are mediated by the IL6 receptor, which is composed of an IL6-specific receptor subunit and a signal transducer, gp130.
  • the binding of IL-6 to its receptor results in activation of several intracellular signaling cascades including JAK-STAT and MAPK pathways.
  • IL-6 production correlates with tumor progression in certain human cancers, e.g., ovarian and prostate cancers.
  • the serum levels of IL-6 are elevated in many men with advanced, hormone-refractory prostate cancer and are associated with progression and poor prognosis.
  • IL-6 also regulates the expression of genes encoding many steroidogenic enzymes involved in androgen synthesis. Autocrine IL-6 promotes androgen independent growth in prostate cancer cells. Constitutively active STAT3 is a part of the positive autocrine IL-6 loop and STAT3 activation in human tumors is observed at the invasive front of tumors adjacent to inflammatory cells, which may mean that STAT3-dependent tumorigenesis is mediated by IL-6.
  • niclosamide targets multiple signaling pathways such as STAT3, Nuclear factor-kappa B (NF- ⁇ B), Wnt/ ⁇ -catenin, and Notch pathways (Yo Y T, et al. Mol Cancer Ther; 11(8):1703-1712).
  • Niclosamide has also been reported to inhibit cancer cell metastasis and migration in vitro and in vivo (Helfman D M. J Natl Cancer Inst; 103(13):991-992; Sack U, et al. J Natl Cancer Inst; 103(13):1018-1036).
  • the present invention provides, in part, new compositions and methods for treating these patients as well as for inhibiting prostate cancer cell growth.
  • composition comprising an AR-V7 inhibitor and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the composition can be a pharmaceutical composition and comprise a pharmaceutically acceptable excipient or diluent.
  • the amount of AR-V7 inhibitor is an amount effective to enhance the therapeutic benefit of a compound selected from the group consisting of bicalutamide, enzalutamide, abiraterone, docetaxel, and combinations thereof.
  • the composition further comprises an AKR1C3 inhibitor.
  • the composition is adapted for oral administration to a patient in need of prostate cancer treatment.
  • the AR-V7 inhibitor is niclosamide.
  • the present invention provides a composition comprising an AKR1C3 inhibitor and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the composition can be a pharmaceutical composition and comprise a pharmaceutically acceptable excipient or diluent.
  • the amount of AKR1C3 inhibitor is an amount effective to enhance the therapeutic benefit of a compound selected from the group consisting of bicalutamide, enzalutamide, abiraterone, docetaxel, and combinations thereof.
  • the composition further comprises an AR-V7 inhibitor.
  • the composition is adapted for oral administration to a patient in need of prostate cancer treatment.
  • the AKR1C3 inhibitor is indomethacin.
  • a method of treating prostate cancer in a patient comprising administering to the patient an effective amount of an AR-V7 inhibitor, such as niclosamide.
  • an AR-V7 inhibitor such as niclosamide.
  • the administration of the AR-V7 inhibitor comprises oral administration.
  • a method of reducing, reversing, or resensitizing prostate cancer cell resistance to anti-androgen drugs or docetaxel comprising contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide.
  • a method of enhancing the therapeutic effects of an anti-androgen drug or docetaxel in a patient having prostate cancer comprising administering to a patient in need of an anti-androgen drug an effective amount of an AR-V7 inhibitor or an AKR1C3 inhibitor.
  • the method comprises co-administering to the patient an effective amount of the AR-V7 inhibitor and an effective amount of the AKR1C3 inhibitor.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer), docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer
  • docetaxel-resistant prostate cancer e.g., AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide
  • the AR-V7 inhibitor such as niclosamide, or the AKR1C3 inhibitor, such as indomethacin
  • the AR-V7 inhibitor, such as niclosamide, and the AKR1C3 inhibitor, such as indomethacin are co-administered orally.
  • the treating comprises reversing or reducing prostate cancer cell resistance to an anti-androgen drug or and/or resensitizing prostate cancer cells to an anti-androgen drug.
  • the anti-androgen drug is a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the treating comprises reversing or reducing prostate cancer cell resistance to docetaxel and/or resensitizing prostate cancer cells to docetaxel.
  • the administering comprises co-administering the AR-V7 inhibitor with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide and combinations thereof.
  • the administering comprises co-administering the AKR1C3 inhibitor with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the administering comprises co-administering the AR-V7 inhibitor and the AKR1C3 inhibitor.
  • the AR-V7 inhibitor is niclosamide.
  • the AKR1C3 inhibitor is indomethacin.
  • a method of inhibiting an androgen receptor (AR) variant comprising contacting an AR variant with an amount of an AR variant inhibitor.
  • the AR variant inhibitor is an AR-V7 inhibitor, such as niclosamide.
  • the present invention provides a method of inhibiting an AR variant in a cell, comprising contacting the cell with an effective amount of niclosamide.
  • the AR variant is AR-V7.
  • the inhibiting comprises inhibiting AR variant transactivation, inhibiting AR variant expression, inhibiting AR variant-induced cell migration, inhibiting AR variant-induced invasion by prostate cancer cells, inhibiting prostate cancer cell colony formation, inhibiting recruitment of an AR variant to a prostate-specific antigen (PSA) promoter, or combinations thereof.
  • the cell is a prostate cancer cell.
  • kits comprising an AR variant inhibitor and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the AR variant inhibitor is an AR-V7 inhibitor.
  • the AR-V7 inhibitor is niclosamide.
  • kits for inhibiting STAT3 comprising contacting an AR-V7 inhibitor, such as niclosamide, with STAT3.
  • an AR-V7 inhibitor such as niclosamide
  • the present invention provides a method of treating prostate cancer in a patient, the method comprising administering to the patient an effective amount of an AR-V7 inhibitor or an effective amount of an AKR1C3 inhibitor.
  • the method comprises co-administering to the patient an effective amount of the AR-V7 inhibitor and an effective amount of the AKR1C3 inhibitor.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer), docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer
  • docetaxel-resistant prostate cancer e.g., AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide
  • the treating comprises reversing or reducing prostate cancer cell resistance to an anti-androgen drug and/or resensitizing prostate cancer cells to an anti-androgen drug.
  • the anti-androgen drug is a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the treating comprises reversing or reducing prostate cancer cell resistance to docetaxel and/or resensitizing prostate cancer cells to docetaxel.
  • the administering comprises co-administering the AR-V7 inhibitor with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the administering comprises co-administering the AKR1C3 inhibitor with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the AKR1C3 inhibitor is indomethacin.
  • the AR-V7 inhibitor is niclosamide.
  • the AR-V7 inhibitor such as niclosamide, or the AKR1C3 inhibitor, such as indomethacin
  • the AR-V7 inhibitor, such as niclosamide, and the AKR1C3 inhibitor, such as indomethacin are co-administered orally.
  • the present invention provides a method of reducing or reversing prostate cancer cell resistance to a prostate cancer drug, comprising contacting prostate cancer cells with an AR-V7 inhibitor or an AKR1C3 inhibitor.
  • the method comprises contacting prostate cancer cells with an AR-V7 inhibitor and an AKR1C3 inhibitor.
  • the prostate cancer drug is an anti-androgen drug.
  • the anti-androgen drug is a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the prostate cancer drug is docetaxel.
  • the AKR1C3 inhibitor is indomethacin.
  • the AR-V7 inhibitor is niclosamide.
  • the reducing or reversing prostate cancer cell resistance to the prostate cancer drug comprises resensitizing the prostate cancer cells to the prostate cancer drug.
  • the reducing or reversing prostate cancer cell resistance to the prostate cancer drug is in a patient having prostate cancer.
  • the AR-V7 inhibitor or the AKR1C3 inhibitor are contacted with the prostate cancer cells by orally administering the AR-V7 or the AKR1C3 inhibitor to the patient.
  • the AR-V7 inhibitor and the AKR1C3 inhibitor are co-administered orally.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer), docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer
  • docetaxel-resistant prostate cancer e.g., AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide
  • the present invention provides a method of enhancing the therapeutic effects of a prostate cancer drug in a patient having prostate cancer, comprising administering to a patient in need of prostate cancer treatment an effective amount of an AR-V7 inhibitor or an effective amount of an AKR1C3 inhibitor.
  • the method comprises co-administering to the patient in need of prostate cancer treatment an effective amount of AR-V7 inhibitor and an effective amount of an AKR1C3 inhibitor.
  • the prostate cancer drug that is enhanced is selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the prostate cancer drug that is enhanced is an anti-androgen drug.
  • the anti-androgen drug is selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the AKR1C3 inhibitor is indomethacin.
  • the AR-V7 inhibitor is niclosamide.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer), docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer
  • docetaxel-resistant prostate cancer e.g., AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide
  • the AR-V7 inhibitor such as niclosamide, or the AKR1C3 inhibitor, such as indomethacin
  • the AR-V7 inhibitor, such as niclosamide, and the AKR1C3 inhibitor, such as indomethacin are co-administered orally.
  • the present invention provides a kit comprising an AR-V7 inhibitor or an AKR1C3 inhibitor, and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the kit comprises a label describing a method of administering the AR-V7 inhibitor.
  • the label can describe oral administration of the AR-V7 inhibitor.
  • the kit comprises a label describing a method of administering the AKR1C3 inhibitor.
  • the label can describe oral administration of the AKR1C3 inhibitor.
  • the AKR1C3 inhibitor is indomethacin.
  • the AR-V7 inhibitor is niclosamide. In some embodiments, the AR-V7 inhibitor or the AKR1C3 inhibitor are adapted for oral administration to a patient in need of prostate cancer treatment. In some embodiments, both the AR-V7 inhibitor and the AKR1C3 inhibitor are adapted for oral administration to a patient in need of prostate cancer treatment.
  • the present invention provides a composition comprising an AR-V7 inhibitor and an AKR1C3 inhibitor.
  • the composition further comprises an anti-androgen drug.
  • the anti-androgen drug can be a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the composition further comprises a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the AR-V7 inhibitor and AKR1C3 inhibitor are present in an amount effective to enhance the therapeutic benefit of one or more of the foregoing other compounds.
  • the AKR1C3 inhibitor is indomethacin.
  • the AR-V7 inhibitor is niclosamide.
  • the composition is adapted for oral administration. In some cases, the composition further comprises a pharmaceutically acceptable excipient.
  • the present invention provides a method of treating prostate cancer, the method comprising administering an AR-V7 inhibitor to a patient having prostate cancer, and sequentially or simultaneously administering an AKR1C3 inhibitor to the patient.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer), docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • the AR-V7 inhibitor and AKR1C3 inhibitor are in the same composition. In some embodiments, the AR-V7 inhibitor and the AKR1C3 inhibitor are in different compositions.
  • the method further comprises administering an anti-androgen drug.
  • the anti-androgen drug can be a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and combinations thereof.
  • the method further comprises administering to the patient a drug selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the AR-V7 inhibitor or the AKR1C3 inhibitor is administered orally. In some embodiments, the AR-V7 inhibitor and the AKR1C3 inhibitor are co-administered orally. In some embodiments, the AR-V7 inhibitor is niclosamide. In some embodiments, the AKR1C3 inhibitor is indomethacin.
  • the present invention provides a method of inhibiting an aldo-keto reductase 1C3 (AKR1C3) in a cell, comprising contacting the cell with an effective amount of indomethacin.
  • the inhibiting comprises inhibiting AKR1C3 expression, inhibiting AKR1C3 activity, or combinations thereof.
  • the cell is a prostate cancer cell.
  • FIGS. 1A-1H show that AR-V7 is constitutively active in prostate cancer cells.
  • FIG. 1A shows the results of LNCaP, C4-2, CWR22rv1, and VCaP cells that were cultured in CS-FBS condition for 3 days. AR-V7 mRNA level were analyzed by qRT-PCR. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 1B shows the results of LNCaP, C4-2, CWR22rv1 and VCaP cells that were cultured in CS-FBS condition for 3 days. Whole cell protein was extracted and immunoblotted with antibody indicated.
  • FIG. 1A shows the results of LNCaP, C4-2, CWR22rv1, and VCaP cells that were cultured in CS-FBS condition for 3 days. Whole cell protein was extracted and immunoblotted with antibody indicated.
  • FIG. 1A shows the results of LNCaP, C4-2, CWR22rv1, and VCaP cells that were
  • FIG. 1C shows the results of C4-2 cells that were cultured in CS-FBS condition and transiently transfected with EGFP-AR-V7 plasmid for 3 days. The fluorescence intensity of individual cells were observed under microscope.
  • FIG. 1D shows PC3 cells that were cultured in CS-FBS condition and transiently transfected with WT-AR or AR-V7 plasmid for 3 days followed by treatment with 10 nM DHT for another 24 hours. Whole cell lysates were subjected to luciferase assay.
  • FIG. 1E shows the results of LNCaP cells that were cultured in CS-FBS condition and transiently transfected with vector or AR-V7 plasmid for 3 days followed by treatment with 10 nM DHT for another 24 hours. Whole cell lysates were subjected to luciferase assay.
  • FIG. 1F shows the results of C4-2 neo and C4-2 AR-V7 stable clones that were cultured in CS-FBS condition. AR-V7 mRNA level was determined by qRT-PCR, the AR-V7 expression was detected by western blot (inside panel).
  • FIG. 1G shows the results of C4-2 neo and C4-2 AR-V7 stable clones that were cultured in CS-FBS condition for 3 days.
  • PSA mRNA levels were determined by qRT-PCR.
  • FIG. 1H shows PSA protein levels as determined by PSA ELISA after culturing in CS-FBS for 3 days. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIGS. 2A-2D show that niclosamide inhibited prostate cancer cell growth and induced cell apoptosis.
  • FIG. 2A C4-2 neo, C4-2 AR-V7, CWR22rv1 and PZ-HPV7 cells were treated with 0.5 ⁇ M niclosamide in media containing FBS. Cell numbers were counted and cell survival rate was calculated after 48 hours.
  • FIG. 2B C4-2 neo, C4-2 AR-V7, CWR22rv1 and PZ-HPV7 cells were treated with 0.5 ⁇ M niclosamide in media containing FBS. Apoptosis was detected by Cell death ELISA after 48 hours.
  • FIG. 2A C4-2 neo, C4-2 AR-V7, CWR22rv1 and PZ-HPV7 cells were treated with 0.5 ⁇ M niclosamide in media containing FBS. Apoptosis was detected by Cell death ELISA after 48 hours.
  • FIG. 2A C4
  • FIG. 2C CWR22rv1 cells were treated with 0, 0.5 ⁇ M or 1.0 ⁇ M niclosamide and clonogenic assays were performed.
  • FIG. 2D colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate. Nicolosamide inhibited colony formation in a dose dependent manner. *P ⁇ 0.05.
  • FIGS. 3A-3E show that niclosamide inhibited AR-V7 protein expression through enhancing protein degradation.
  • FIG. 3A CWR22rv1 cells were treated with 0 ⁇ M, 0.5 ⁇ M or 1.0 ⁇ M niclosamide in RPMI 1640 media containing 10% FBS condition overnight and the whole cell lysates were immunoblotted with the indicated antibodies.
  • FIG. 3B CWR22rv1 cells were treated with 1.0 ⁇ M niclosamide in RPMI 1640 media containing 10% FBS condition, whole cell lysates were extracted at different time point and immunoblotted with the indicated antibodies.
  • FIG. 3A CWR22rv1 cells were treated with 0 ⁇ M, 0.5 ⁇ M or 1.0 ⁇ M niclosamide in RPMI 1640 media containing 10% FBS condition overnight and the whole cell lysates were immunoblotted with the indicated antibodies.
  • FIG. 3B CWR22rv1 cells were treated with 1.0 ⁇ M niclosamide in
  • FIG. 3C CWR22rv1 cells were treated with 0 ⁇ M, 0.5 ⁇ M or 1.0 ⁇ M niclosamide in RPMI 1640 media containing 10% FBS condition overnight, total RNAs were extracted and AR or AR-V7 mRNA levels were analyzed by qRT-PCR.
  • FIG. 3D the protein synthesis inhibitor 50 ⁇ g/mL cycloheximide (CHX) was added with or without 2 ⁇ M niclosamide (Nic) at time 0 hour. At specified time points, cells were harvested, and the levels of AR-V7 protein were measured by Western blot using antibodies specific against AR-V7.
  • FIG. 3E illustrates the effect of MG132 on niclosamide-induced AR protein degradation.
  • MG132 (5 ⁇ mol/L) was added to CWR22rv1 cells together with cycloheximide (50 ⁇ g/mL) in the presence and absence of 2 ⁇ M niclosamide.
  • the cell lysates were prepared at 8 h.
  • AR-V7 protein levels were determined by Western blot analysis using antibodies specifically against AR-V7 and tubulin as a control.
  • FIGS. 4A-4D show that niclosamide inhibits AR-V7 transcription activity.
  • FIG. 4A 293-AR-V7-PSA luciferase promoter stable clones were treated with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight in media containing 10% FBS or 10% CS-FBS, whole cell lysates were subjected to luciferase assay.
  • FIG. 4A 293-AR-V7-PSA luciferase promoter stable clones were treated with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight in media containing 10% FBS or 10% CS-FBS, whole cell lysates were subjected to luciferase assay.
  • FIG. 4A 293-AR-V7-PSA luciferase promoter stable clones were treated with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight in media containing 10% F
  • LNCaP cells were co-transfected with PSA luciferase promoter with AR-V7 plasmid in CS-FBS condition for 24 hours, followed by treatment with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight, whole cell lysates were subjected to luciferase assay.
  • FIG. 4B LNCaP cells were co-transfected with PSA luciferase promoter with AR-V7 plasmid in CS-FBS condition for 24 hours, followed by treatment with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight, whole cell lysates were subjected to luciferase assay.
  • FIG. 4C C4-2 AR-V7 stable cells were transfected with PSA luciferase promoter in CS-FBS condition for 24 hours, followed by treatment with 0.5 ⁇ M, 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide with or without 1 nM DHT overnight, whole cell lysates were subjected to luciferase assay.
  • FIG. 4D C4-2 neo and C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days, then treated with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight, the supernatants were collected and subjected to PSA ELISA. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIG. 5A-5H show that C4-2B cells that were chronically treated with enzalutamide express AR variants and are sensitive to niclosamide.
  • FIG. 5A C4-2B parental cells were treated with 20 ⁇ M enzalutamide in RPMI 1640 media containing 10% FBS condition, total cell numbers were counted on different days indicated.
  • FIG. 5B C4-2B MR cells were treated with 20 ⁇ M enzalutamide in RPMI 1640 media containing 10% FBS condition, total cell numbers were counted on different days indicated.
  • FIG. 5A-5H show that C4-2B cells that were chronically treated with enzalutamide express AR variants and are sensitive to niclosamide.
  • FIG. 5A C4-2B parental cells were treated with 20 ⁇ M enzalutamide in RPMI 1640 media containing 10% FBS condition, total cell numbers were counted on different days indicated.
  • FIG. 5B C4-2B MR cells were treated with 20 ⁇ M en
  • FIG. 5C C4-2B parental cells and C4-2B MR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, total RNAs were extracted and AR-V1, AR-V7, AR1/2/2b, AR1/2/3/2b or AR full length mRNA level were analyzed by qRT-PCR.
  • FIG. 5D C4-2B parental cells and C4-2B MR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, whole cell lysates were immunoblotted with the indicated antibodies.
  • C4-2B parental cells and C4-2B MR cells were cultured in media containing 10% CS-FBS for 3 days, the cells were harvested for preparation of cytosolic and nuclear fractions and analyzed by Western blotting using antibodies against AR-V7, AR, polymerase II, or Tubulin. The expression of polymerase II and tubulin were used as markers for the integrity of the nuclear and cytosolic fractions, respectively.
  • FIG. 5F C4-2B MR cells were cultured in media containing 10% FBS and treated with different concentrations of enzalutamide or niclosamide as indicated, after 48 hours, total cell numbers were counted.
  • 5G C4-2B MR cells were treated with 0, 0.5 ⁇ M or 1.0 ⁇ M niclosamide and clonogenic assays were performed.
  • FIG. 5H colonies were counted and the results are presented as means ⁇ SD of 2 experiments performed in duplicate. Nicolosamide inhibited colony formation in a dose dependent manner. *P ⁇ 0.05.
  • FIGS. 6A-6G show that niclosamide enhances enzalutamide and abiraterone effects in prostate cancer cells.
  • FIG. 6A CWR22rv1 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS and cell numbers were counted after 3 and 5 days.
  • FIG. 6B CWR22rv1 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M abiraterone in media containing FBS and cell numbers were counted after 3 and 5 days. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 6C CWR22rv1 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide or 20 ⁇ M abiraterone in media containing FBS and clonogenic assays were performed.
  • FIG. 6D colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate.
  • FIG. 6E C4-2B MR cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS and cell numbers were counted after 3 and 5 days.
  • FIG. 6C CWR22rv1 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide or 20 ⁇ M abiraterone in media containing FBS and clonogenic assays were performed.
  • FIG. 6D colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate.
  • FIG. 6E C4-2B MR cells were treated
  • FIG. 6F C4-2B MR cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS and clonogenic assays were performed.
  • FIG. 6G colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate. *P ⁇ 0.05.
  • FIGS. 7A-7B show that niclosamide enhances enzalutamide and abiraterone effects through AR-V7 inhibition.
  • FIG. 7A CWR22rv1 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide or 20 ⁇ M abiraterone in media containing FBS, 48 hours later whole cell lysates were extracted and subjected to western blot.
  • FIG. 7B C4-B MR cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS, 48 hours later, whole cell lysates were extracted and subjected to western blot.
  • FIGS. 8A-8E show that 293 cells stably transfected with an AR-V7-PSA promoter luciferase construct were screened against candidate AR-V7 compounds form the Prestwick Chemical Library.
  • FIG. 8A CWR22rv1, 293 and 293 GFP AR-V7-PSA promoter luciferase stable clones were cultured in media containing 10% FBS for 2 days. Whole cell lysates were prepared and subjected to Western blot analysis using antibodies as indicated.
  • FIG. 8A CWR22rv1, 293 and 293 GFP AR-V7-PSA promoter luciferase stable clones were cultured in media containing 10% FBS for 2 days.
  • Whole cell lysates were prepared and subjected to Western blot analysis using antibodies as indicated.
  • FIG. 8B 293 GFP AR-V7-PSA promoter luciferase stable clones were cultured in media containing 10% FBS for 2 days, the fluorescence intensity of individual cells were observed under microscope, the arrow shows an example of AR-V7 protein expression.
  • FIG. 8C 293 neo and 293 GFP AR-V7-PSA promoter luciferase stable clones were cultured in media containing 10% FBS for 2 days, total RNA was isolated, AR full length and AR-V7 mRNA levels were determined by qRT-PCR.
  • FIG. 8D shows a schematic of the compound screening procedure. *P ⁇ 0.05.
  • FIGS. 9A-9B show that the recruitment of AR-V7 to PSA promoter cannot be interrupted by enzalutamide.
  • FIG. 9A LNCaP cells were co-transfected PSA-E/P-luc or AREI/II-luc with AR V7 plasmid in CS-FBS condition for 3 days followed by treatment with 10 nM DHT with or without 20 ⁇ M enzalutamide overnight, whole cell lysates were subjected to luciferase assay.
  • FIG. 9A LNCaP cells were co-transfected PSA-E/P-luc or AREI/II-luc with AR V7 plasmid in CS-FBS condition for 3 days followed by treatment with 10 nM DHT with or without 20 ⁇ M enzalutamide overnight, whole cell lysates were subjected to luciferase assay.
  • FIG. 9A LNCaP cells were co-transfected PSA-E/P-luc or ARE
  • C4-2 neo and C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days followed by treatment with 10 nM DHT with or without 20 ⁇ M enzalutamide overnight, whole cell lysates were subjected to ChIP assay. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIGS. 10A-10B show that AR-V7 mediates cell growth in CWR22rv1 cells.
  • FIG. 10A CWR22rv1 cells were transiently transfected with control siRNA, AR exon7 siRNA or AR V7 siRNA in CS-FBS condition, cell numbers were counted on different days.
  • FIG. 10B CWR22rv1 cells were transient transfected with control siRNA, AR exon7 siRNA or AR-V7 siRNA in CS-FBS condition for 3 days, the knock down effects were examined by western blot. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIGS. 11A-11B show that AR-V7 induced abiraterone resistance in CWR22rv1 cells.
  • FIG. 11A CWR22rv1, C4-2B and LNCaP cells were cultured in RPMI 1640 media containing 10% FBS condition, then treated with different doses of abiraterone, total cell numbers were counted and cell survival rate was calculated after 48 hours.
  • FIG. 11B CWR22rv1 cells were transient transfected with control siRNA, AR exon7 siRNA, AR-V7 siRNA or AR exon7 siRNA plus AR-V7 siRNA in FBS, then treated with 10 ⁇ M abiraterone, total cell numbers were counted and cell survival rate were calculated after 48 hours. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIG. 12 shows a schematic of a drug screening procedure used to identify niclosamide as an AR-V7 inhibitor.
  • FIG. 13 shows a plot that demonstrates that niclosamide inhibits AR-V7 transactivation.
  • FIG. 14 shows a plot that demonstrates that niclosamide but not bicalutamide inhibits AR-V7 transactivation.
  • FIG. 15 shows a blot that demonstrates that niclosamide inhibits AR and AR-V7 expression.
  • FIG. 16 illustrates niclosamide inhibition of cell migration and invasion in prostate cancer cells.
  • FIG. 17 illustrates niclosamide inhibition of cell invasion in prostate cancer cells.
  • FIG. 18 illustrates niclosamide inhibition of colony formation in prostate cancer cells.
  • FIG. 19 shows that niclosamide inhibited C4-2B MR cell growth in a dose dependent manner.
  • FIG. 20 shows that combination treatments inhibit recruitment of AR to PSA promoter.
  • FIG. 21 shows that combination treatment inhibited C4-2B, C4-2B TAXR, C4-2B TAXR+MR, and C4-2B AbiR cell growth.
  • FIG. 22 shows that niclosamide enhanced the effects of enzalutamide in C4-2B MR cells.
  • FIG. 23 shows that niclosamide enhanced the effects of abiraterone in C4-2B AbiR cells.
  • FIG. 24 shows the results of incubating LNCaP-s17 cells with DMSO, enzalutamide, niclosamide, and combinations of enzalutamide and niclosamide.
  • FIG. 25 shows the results of incubating LNCaP-STAT3C cells with DMSO, enzalutamide, niclosamide, and combinations of enzalutamide and niclosamide.
  • FIGS. 26A-26D show that overexpression of IL-6 increases LNCaP cell resistance to enzalutamide.
  • FIG. 26A LNCaP cells stably expressing IL6 (LNCaP-S17) and control LNCaP cells (LNCaP-neo) were treated with different doses of enzalutamide in media containing complete FBS and cell numbers were counted after 48 h. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 26B LNCaP-neo and LNCaP-S17 cells were treated with 0 or 20 ⁇ M enzalutamide and clonogenic assays were performed.
  • FIG. 26C colonies were counted and presented as means ⁇ SD of 2 experiments performed in duplicate.
  • LNCaP-S17 cells formed higher numbers of colonies compared to LNCaP-neo cells when treated with enzalutamide.
  • FIG. 26D LNCaP-IL6+ cells (continuous culture in medium containing 10% FBS and 5 ng/mL IL-6) and control LNCaP cells were treated with 0, 10 or 20 ⁇ M enzalutamide in media containing complete FBS and cell numbers were counted after 48 hours. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIGS. 27A-27F show that autocrine IL-6 constitutively activates STAT3 pathway and enhances androgen receptor transactivation in prostate cancer cells.
  • FIG. 27A prostate cancer cells with autocrine IL-6 exhibit constitutive activation of STAT3.
  • LNCaP cells and LNCaP-s17 cells were cultured in FBS condition for 24 hours and the lysates were immunoblotted with the indicated antibodies.
  • LNCaP-s17 cells express high levels of c-Myc ( FIG. 27B ), Survivin ( FIG. 27C ), IL-6 mRNA ( FIG. 27D ) and protein ( FIG. 27E ) compared to LNCaP-neo cells.
  • FIG. 27F recruitment of AR to AREs in PSA promoter was analyzed by ChIP assay, LNCaP-S17 and LNCaP-STAT3C cells enhanced recruitment of AR to PSA promoter compared to LNCaP-neo cells.
  • FIGS. 28A-28D show that downregulation of STAT3 expression restores the sensitivity of LNCaP-s17 cells to enzalutamide treatment.
  • FIG. 28A downregulation of STAT3 expression increased sensitivity of LNCaP-S17 cells to enzalutamide.
  • LNCaP-s17 cells were transfected with siRNAs specific to STAT3 or control siRNA and were treated with 0 or 20 ⁇ M enzalutamide. Cell numbers were counted after 3 days.
  • FIG. 28B whole cell lysates were collected and subjected to Western blot. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 28A downregulation of STAT3 expression increased sensitivity of LNCaP-S17 cells to enzalutamide.
  • LNCaP-s17 cells were transfected with siRNAs specific to STAT3 or control siRNA and were treated with 0 or 20 ⁇ M enzalutamide. Cell numbers were counted after 3 days.
  • FIG. 28C LNCaP-neo and LNCaP-STAT3C cells were treated with DMSO, 10 or 20 ⁇ M enzalutamide in media containing FBS for 48 hours and cell numbers were counted. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 28D recruitment of AR to AREs in PSA promoter was analyzed by ChIP assay. LNCaP-neo, LNCaP-S17 and LNCaP-STAT3C cells were cultured in FBS condition with or without 20 ⁇ M enzalutamide overnight, cross-linked with 1% formaldehyde and the resultant DNA-protein complexes were subjected to ChIP assay. *P ⁇ 0.05
  • FIGS. 29A-29F show a novel STAT3 inhibitor, niclosamide, that reverses enzalutamide resistance in prostate cancer cells.
  • FIG. 29A depicts the chemical structure of niclosamide.
  • FIG. 29B niclosamide inhibited constitutively active STAT3 in a dose dependent manner in LNCaP-s17 cells.
  • FIG. 29C LNCaP-s17 cells were treated with 0.25 ⁇ M, 0.5 ⁇ M niclosamide or 10 ⁇ M AG490 combined with or without 20 ⁇ M enzalutamide in media containing FBS, cell numbers were counted after 48 h.
  • FIG. 29D apoptosis was analyzed by Cell death ELISA.
  • FIG. 29E LNCaP-s17 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS and cell numbers were counted after 2, 4 and 7 days. Results are presented as means ⁇ SD of 3 experiments performed in duplicate.
  • FIG. 29F colony formation ability was examined by clonogenic assay, LNCaP-s17 cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide, equal numbers of cells were plated in 10 cm plates, the colonies were stained with 0.5% crystal violet after 3 weeks, and colony numbers were counted.
  • FIG. 30 shows that C4-2 neo and C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days, followed by treatment with 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight. Whole cell lysates were immunoblotted with the indicated antibodies.
  • FIG. 31 shows C4-2 AR-V7 cells that were cultured in CS-FBS condition for 3 days, followed by treatment with 0.5 ⁇ M, 1.0 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight.
  • Whole cell lysates were subjected to ChIP assay. Results are presented as means ⁇ SD of 3 experiments performed in duplicate. *P ⁇ 0.05.
  • FIG. 32 shows that niclosamide increased AR-V7 protein degradation as compared to the untreated control cells. Relative amounts of AR-V7 protein are plotted on semilog scale. The amount of AR-V7 protein is normalized to 100% at time 0, the dashed line indicates 50% half-life.
  • FIG. 33 shows CWR22rv1, C4-2R-V7, and C4-2 neo cells that were cultured with 0, 0.5, and 1.0 ⁇ M niclosamide.
  • Niclosamide is shown in this figure to significantly inhibit clonogenic ability of prostate cancer cells in a dose dependent manner.
  • FIG. 34 shows a plot of colony numbers versus niclosamide concentration for CWR22rv1, C4-2R-V7, and C4-2 neo cells that were cultured with 0, 0.5, and 1.0 ⁇ M niclosamide. Colonies were counted and the results are presented as means ⁇ SD of 2 experiments performed in duplicate.
  • FIG. 35 shows that niclosamide but not enzalutamide inhibited colony formation, and also colony size, in C4-2B MR cells.
  • C4-2B MR cells were treated with DMSO, 10 ⁇ M or 20 ⁇ M enzalutamide, 0.5 ⁇ M or 1.0 ⁇ M niclosamide and clonogenic assays were performed.
  • FIG. 36 shows a plot of colony numbers versus concentrations of enzalutamide, niclosamide, and control DMSO, which shows that niclosamide but not enzalutamide inhibited colony formation and colony size in C4-2B MR cells. Colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate. Niclosamide inhibited colony formation in a dose dependent manner. *P ⁇ 0.05.
  • FIG. 37 shows the results of a clonogenic assay for CWR22rv1 cells treated with niclosamide with or without enzalutamide for 48 hours.
  • CWR22rv1 cells or C4-2B MR cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide in media containing FBS and clonogenic assays were performed.
  • FIG. 38 shows the results of a clonogenic assay as a plot of colony numbers for CWR22rv1 cells treated with DMSO, enzalutamide, niclosamide, or a combination of niclosamide and enzalutamide.
  • CWR22rv1 and C4-2B MR colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate.
  • FIG. 39 shows a plot of tumor volume versus time (days) for an in vivo study using xenografts generated from CWR22rv1 cells treated with vehicle, enzalutamide, niclosamide, or a combination thereof.
  • Mice bearing CWR22rv1 xenografts were treated with vehicle control, enzalutamide, niclosamide or a combination thereof for 3 weeks, tumor volumes were measured twice every week and the tumors were collected. *P ⁇ 0.05.
  • FIGS. 40A, 40B, 40C, and 40D show the effects of niclosamide on tumor growth of CWR22Rv1 xenograft model (niclosamide via oral administration).
  • CWR22Rv1 cells (4 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of male SCID mice.
  • Tumor-bearing mice (tumor volume around 50-75 mm 3 ) were treated 5 days per week as follows: Control: (5% PGE8000 in H 2 O p.o Bid), Niclosamide (200 mg/kg p.o Bid).
  • Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width2/2. Tumor tissues were harvested after 3 weeks of treatment. *P ⁇ 0.05.
  • FIGS. 41A-41B show the combination of niclosamide with bicalutamide in CWR22Rv1 cells in vitro.
  • FIG. 41A niclosamide in combination with bicalutamide inhibits tumor growth in vitro in a dose-dependent manner.
  • FIG. 41B niclosamide in combination with bicalutamide inhibits tumor growth in vitro in a time-dependent manner. *P ⁇ 0.05.
  • FIGS. 42A, 42B, 42C, and 42D show a CWR22Rv1 xenograft model of niclosamide combined with bicalutamide.
  • CWR22Rv1 cells (4 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of male SCID mice.
  • Tumor-bearing mice (tumor volume around 50-75 mm 3 ) were treated 5 days per week as follows: Control: (0.5% weight/volume (w/v) Methocel A4M p.o and 5% Tween 80 and 5% ethanol in PBS, i.p.), Bicalutamide (25 mg/kg p.o), Niclosamide (25 mg/kg i.p.) and Combination (25 mg/kg Bicalutamide p.o+25 mg/kg Niclosamide i.p.). Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width2/2. Tumor tissues were harvested after 3 weeks of treatment. *P ⁇ 0.05.
  • FIGS. 43A-43D show that C4-2B MDVR cells are resistant to enzalutamide in vitro and in vivo.
  • FIG. 43A C4-2B parental and C4-2B MDVR cells were treated with different concentrations of enzalutamide for 48 hours, total cell numbers were counted and cell survival rate was calculated.
  • FIG. 43B the clonogenic ability of C4-2B parental and C4-2B MDVR cells treated with 10 ⁇ M or 20 ⁇ M enzalutamide was analyzed. Enzalutamide significantly inhibited clonogenic ability of C4-2B parental cells.
  • FIG. 43A C4-2B parental and C4-2B MDVR cells were treated with different concentrations of enzalutamide for 48 hours, total cell numbers were counted and cell survival rate was calculated.
  • FIG. 43B the clonogenic ability of C4-2B parental and C4-2B MDVR cells treated with 10 ⁇ M or 20 ⁇ M enzalutamide was analyzed. Enzaluta
  • FIG. 43C C4-2B parental and C4-2B MDVR cells were injected orthotopically into the prostates of SCID mice and treated with 25 mg/Kg enzalutamide or vehicle control. Tumors were harvested and weighed at 3 weeks.
  • FIG. 43D LNCaP, LN-95 and CWR22Rv1 cells were treated with different concentrations of enzalutamide for 2 days, total cell numbers were counted and cell survival rate (%) was calculated. *p ⁇ 0.05.
  • FIGS. 44A-44B show that the intracrine androgen synthesis pathway is activated in enzalutamide resistant prostate cancer cells.
  • FIG. 44A the expression of transcripts encoding genes involved in steroid hormone biosynthesis was analyzed by gene set enrichment. Genes that were regulated 1.3 fold between C4-2B parental cells and C4-2B MDVR cells were enriched and heat map was generated by the Subio platform.
  • FIG. 44A the expression of transcripts encoding genes involved in steroid hormone biosynthesis was analyzed by gene set enrichment. Genes that were regulated 1.3 fold between C4-2B parental cells and C4-2B MDVR cells were enriched and heat map was generated by the Subio platform.
  • C4-2B parental cells and C4-2B MDVR cells were cultured in RPMI 1640 media containing 10% FBS for 3 days, total RNAs were extracted and CYP17A1, HSD3B1, HSD3B2, HSD17B3, SRD5A1, AKR1C1/2 or AKR1C3 mRNA levels were analyzed by qRT-PCR. AKR1C3, HSD3B and CYP17A1 protein levels were examined by western blot (right panel).
  • FIGS. 45A-45D show that AKR1C3 is highly expressed in metastatic prostate tumors and enzalutamide resistant xenografts.
  • FIG. 45A C4-2B parental, C4-2B MDVR, VCaP, CWR22Rv1, LNCaP and LN-95 cells were harvested and whole lysates were subjected to Western blotting.
  • FIG. 45B AKR1C3 expression level was analyzed by IHC staining in C4-2B parental, C4-2B MDVR and CWR22Rv1 xenografts.
  • FIG. 45C gene expression analysis using the Oncomine database showing the relative expression levels of AKR1C3 in two datasets comparing normal prostate tissue and prostate cancer.
  • FIG. 45D gene expression analysis using the Oncomine database showed that AKR1C3 expression was correlated with prostate cancer progression and recurrence in two independent datasets (Glinsky and Singh prostate).
  • FIGS. 46A-46D show that intracrine androgens are up regulated in enzalutamide resistant prostate cancer cells.
  • FIG. 46A C4-2B parental and C4-2B MDVR cells were cultured in serum free and phenol red free RPMI1640 medium for 5 days, and levels of steroids in the cell extracts were analyzed by LC-MS. Representative testosterone and estradiol chromatograms generated by MassLynx 4.1 software are shown.
  • FIG. 46B the difference between levels of androgen metabolites in C4-2B parental and C4-2B MDVR cells was analyzed and quantified.
  • FIG. 46C the represented steroid metabolites between C4-2B parental and C4-2B MDVR cells were quantified.
  • FIG. 46D the androgen metabolism pathway was up regulated in enzalutamide resistant prostate cancer cells.
  • FIGS. 47A-47F show that AKR1C3 confers resistance to enzalutamide in prostate cancer cells.
  • FIG. 47A CWR22Rv1 cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), following treatment with 20 ⁇ M enzalutamide and cell numbers were determined on 3 days.
  • FIG. 47B C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), following treatment with 20 ⁇ M enzalutamide and cell numbers were determined on 3 days.
  • FIG. 47A CWR22Rv1 cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), following treatment with 20 ⁇ M enzalutamide and cell numbers were determined on 3 days.
  • FIG. 47B C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3
  • FIG. 47C CWR22Rv1 or C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA (#561 and #694), cells were collected on 3 or 5 days, and whole cell lysates were subjected to Western blotting.
  • FIG. 47D LNCaP-neo or LNCaP-AKR1C3 cells were treated with different concentrations of enzalutamide for 2 days, total cell numbers were counted and cell survival rate (%) was calculated.
  • FIG. 47D LNCaP-neo or LNCaP-AKR1C3 cells were treated with different concentrations of enzalutamide for 2 days, total cell numbers were counted and cell survival rate (%) was calculated.
  • FIG. 47E LNCaP-neo or LNCaP-AKR1C3 cells were treated with 10 ⁇ M enzalutamide and a clonogenic assay was performed, the colony size pictures were taken under a microscope.
  • FIG. 47F colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate. *p ⁇ 0.05.
  • Enza Enzalutamide.
  • FIGS. 48A-48D show that indomethacin, an inhibitor of AKR1C3 activity, overcomes enzalutamide resistance.
  • FIG. 48A CWR22Rv1 cells were treated with 10 ⁇ M or 20 ⁇ M indomethacin with or without 20 ⁇ M enzalutamide for 2 days, total cell numbers were counted (left), and a clonogenic assay was performed, the colony size pictures were taken under a microscope (middle). Colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in duplicate (right).
  • FIG. 48A CWR22Rv1 cells were treated with 10 ⁇ M or 20 ⁇ M indomethacin with or without 20 ⁇ M enzalutamide for 2 days, total cell numbers were counted (left), and a clonogenic assay was performed, the colony size pictures were taken under a microscope (middle). Colonies were counted and results are presented as means ⁇ SD of 2 experiments performed in
  • FIG. 48B mice bearing CWR22Rv1 xenografts were treated with vehicle control, enzalutamide (25 mg/Kg p.o), Indomethacin (3 mg/Kg i.p) or their combination for 3 weeks, tumor volumes were measured twice weekly and the tumors were collected and weighed.
  • FIG. 48D IHC staining of Ki67 and H/E staining in each group was performed and quantified. * p ⁇ 0.05.
  • Enza Enzalutamide.
  • Indocin Indomethacin.
  • prostate cancer cell growth, survival, or drug resistance is mediated by expression or activity of androgen receptor (AR) variants, such as AR-V7.
  • AR androgen receptor
  • the present invention provides an inhibitor of AR variant expression or activity.
  • niclosamide is a novel AR variant inhibitor which inhibits prostate cancer cell growth and induces apoptosis.
  • Niclosamide can inhibit drug-resistant prostate cancer cell or tumor growth, or survival.
  • niclosamide can inhibit anti-androgen (e.g., enzalutamide) resistant prostate cancer cell or tumor growth, or survival.
  • niclosamide can exhibit synergistic effects with prostate cancer drugs.
  • niclosamide exhibits synergistic effects with anti-androgen compounds, such as enzalutamide, and can resensitize treatment resistant prostate cancer cells to prostate cancer therapies, such as anti-androgen (e.g., enzalutamide) therapy.
  • anti-androgen e.g., enzalutamide
  • AR-V7 inhibitors such as niclosamide, are effective and orally bioavailable drugs either as monotherapy or in combination with current prostate cancer therapies, including anti-androgen therapies, for treatment of advanced stage prostate cancer, e.g., metastatic prostate cancer.
  • prostate cancer cell growth, survival, or drug resistance is mediated by expression or activity of the enzyme aldo-keto reductase 1C3 (AKR1C3).
  • the present invention provides an inhibitor of AKR1C3 expression or activity.
  • indomethacin is an AKR1C3 inhibitor which can inhibit prostate cancer cell growth or survival, or induce apoptosis.
  • AKR1C3 inhibitors can inhibit drug-resistant prostate cancer cell or tumor growth, or survival.
  • AKR1C3 inhibitors can inhibit anti-androgen (e.g., enzalutamide) resistant prostate cancer cell or tumor growth, or survival.
  • AKR1C3 inhibitors can exhibit synergistic effects with other prostate cancer therapies.
  • AKR1C3 inhibitors can exhibit synergistic effects with anti-androgen compounds, such as enzalutamide, and can resensitize treatment resistant prostate cancer cells to anti-androgen (e.g., enzalutamide) therapy.
  • anti-androgen compounds such as enzalutamide
  • AKR1C3 inhibitors such as indomethacin
  • the present invention provides new compositions and methods for treating prostate cancer, e.g., anti-androgen drug-resistant prostate cancer, castration-resistant prostate cancer, or combinations thereof.
  • These new compositions include, but are not limited to, pharmaceutical compositions that include an AR-V7 inhibitor, such as niclosamide, and/or an AKR1C3 inhibitor, such as indomethacin.
  • These new methods include, but are not limited to, methods of administering effective amounts of an AR-V7 inhibitor, such as niclosamide, and/or an AKR1C3 inhibitor, such as indomethacin, to treat patients having prostate cancer.
  • the present invention also provides methods of inhibiting androgen receptor variant expression, e.g.
  • AR-V7 and methods of killing of cells expressing AR-V7.
  • the present invention also provides methods of inhibiting AKR1C3 expression or activity, and methods of killing of cells expressing AKR1C3.
  • the present invention also provides methods of enhancing the efficacy of prostate cancer drugs, including anti-androgen prostate cancer drugs.
  • an effective amount includes a dosage sufficient to produce a desired result with respect to the indicated disorder, condition, or mental state.
  • the desired result may comprise a subjective or objective improvement in the recipient of the dosage.
  • an effective amount of an AR-V7 inhibitor (e.g., niclosamide) or an AKR1C3 inhibitor (e.g., indomethacin) includes an amount sufficient to alleviate the signs, symptoms, or causes of prostate cancer, e.g. CRPC.
  • an effective amount can be an amount that slows or reverses tumor growth, increases mean time of survival, inhibits tumor progression or metastasis, or resensitizes a prostate cancer cell to a prostate cancer drug to which it has become or is resistant.
  • an effective amount of an AR-V7 inhibitor e.g., niclosamide
  • an AKR1C3 inhibitor e.g., indomethacin
  • an effective amount of an AR-V7 inhibitor includes an amount sufficient to cause a substantial improvement in a subject having prostate cancer when administered to the subject.
  • the amount will vary with the type of prostate cancer being treated, the stage of advancement of the prostate cancer, the type and concentration of composition applied, and the amounts of anti-androgens drugs that are also administered to the subject.
  • an effective amount of an AR-V7 inhibitor e.g., niclosamide
  • an AKR1C3 inhibitor e.g., indomethacin
  • an effective amount of an AR-V7 inhibitor can include an amount that is effective in enhancing the prostate cancer therapeutic activity of drugs such as enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the term “treating” includes, but is not limited to, methods and manipulations to produce beneficial changes in a recipient's health status, e.g., a patient's prostate cancer status.
  • the changes can be either subjective or objective and can relate to features such as symptoms or signs of the prostate cancer being treated. For example, if the patient notes decreased pain, then successful treatment of pain has occurred. For example, if a decrease in the amount of swelling has occurred, then a beneficial treatment of inflammation has occurred.
  • the clinician notes objective changes such as reducing the number of prostate cancer cells, the growth of the prostate cancer cells, the size of prostate cancer tumors, or the resistance of the prostate cancer cells to another prostate cancer drug, then treatment of prostate cancer has also been beneficial.
  • Treating also includes administering an AR-V7 inhibitor (e.g., niclosamide) or an AKR1C3 inhibitor (e.g., indomethacin) to a patient having prostate cancer.
  • an AR-V7 inhibitor e.g., niclosamide
  • an AKR1C3 inhibitor e.g., indomethacin
  • administering includes activities associated with providing a patient an amount of a compound described herein, e.g., an AR-V7 inhibitor (e.g., niclosamide) or an AKR1C3 inhibitor (e.g., indomethacin).
  • Administering includes providing unit dosages of compositions set forth herein to a patient in need thereof.
  • Administering includes providing effect amounts of compounds, e.g., an AR-V7 inhibitor (e.g., niclosamide) or an AKR1C3 inhibitor (e.g., indomethacin), for specified period of time, e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days, or in a specified sequence, e.g., administration of an AR-V7 inhibitor (e.g., niclosamide) or an AKR1C3 inhibitor (e.g., indomethacin) followed by the administration of a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof, or vice versa.
  • an AR-V7 inhibitor e.g., niclosamide
  • an AKR1C3 inhibitor e.g., indomethacin
  • co-administering includes sequential or simultaneous administration of two or more structurally different compounds.
  • two or more structurally different pharmaceutically active compounds can be co-administered by administering a pharmaceutical composition adapted for oral administration that contains two or more structurally different active pharmaceutically active compounds.
  • two or more structurally different compounds can be co-administered by administering one compound and then administering the other compound.
  • the co-administered compounds are administered by the same route.
  • the co-administered compounds are administered via different routes.
  • one compound can be administered orally, and the other compound can be administered, e.g., sequentially or simultaneously, via intravenous or intraperitoneal injection.
  • the phrase “advanced stage prostate cancer” or “advanced prostate cancer” includes a class of prostate cancers that has progressed beyond early stages of the disease. Typically, advanced stage prostate cancers are associated with a poor prognosis.
  • Types of advanced stage prostate cancers include, but are not limited to, metastatic prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like), hormone refractory prostate cancer, castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, docetaxel-resistant prostate cancer, AR-V7-induced drug-resistant prostate cancer such as AR-V7-induced anti-androgen-resistant prostate cancer (e.g., AR-V7-induced enzalutamide-resistant prostate cancer), AKR1C3-induced drug-resistant prostate cancer such as AKR1C3-induced anti-androgen-resistant prostate cancer (e.g., AKR1C3-induced enzalutamide-resistant prostate cancer), and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer,
  • the advanced stage prostate cancers do not generally respond, or are resistant, to treatment with one or more of the following conventional prostate cancer therapies: enzalutamide, arbiraterone, bicalutamide, and docetaxel.
  • Compounds, compositions, and methods of the present invention are provided for treating prostate cancer, such as advanced stage prostate cancer, including any one or more (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) of the types of advanced stage prostate cancers disclosed herein.
  • the phrase “ameliorating the symptoms of prostate cancer” includes alleviating or improving the symptoms or condition of a patient having prostate cancer. Ameliorating the symptoms includes reducing the pain or discomfort associated with prostate cancer. Ameliorating the symptoms also includes reducing the markers of prostate cancer, e.g., reducing the number of prostate cancer cells or reducing the size of prostate cancer tumors.
  • enhancing the therapeutic effects includes any of a number of subjective or objective factors indicating a beneficial response or improvement of the condition being treated as discussed herein.
  • enhancing the therapeutic effects of an anti-androgen drug e.g., enzalutamide, abiraterone, or bicalutamide
  • docetaxel includes resensitizing anti-androgen drug or docetaxel resistant prostate cancer to anti-androgen or docetaxel therapy.
  • enhancing the therapeutic effects of an anti-androgen drug or docetaxel includes altering anti-androgen drug or docetaxel resistant prostate cancer cells so that the cells are not resistant to anti-androgen drugs or docetaxel.
  • enhancing the therapeutic effects of an anti-androgen drug or docetaxel includes additively or synergistically improving or increasing the activity of the anti-androgen drug or docetaxel.
  • the enhancement includes a one-fold, two-fold, three-fold, five-fold, ten-fold, twenty-fold, fifty-fold, hundred-fold, or thousand-fold increase in the therapeutic activity of an anti-androgen drug or docetaxel used to treat prostate cancer.
  • the phrase “reversing prostate cancer cell resistance” includes altering or modifying a prostate cancer cell that is resistant to anti-androgen drug or docetaxel therapy so that the cell is no longer resistant to anti-androgen drug or docetaxel therapy.
  • prostate cancer cell resistance includes increasing the therapeutic activity of an anti-androgen drug or docetaxel towards prostate cancer cells that are, or previously were, resistant to anti-androgen drug or docetaxel therapy.
  • the phrase “resensitizing prostate cancer cell resistance” includes inducing sensitization towards anti-androgen drug or docetaxel therapy in prostate cancer cells which are resistant to anti-androgen drug or docetaxel therapy. Sensitization as used herein includes inducing the ability of a prostate cancer cell to be effectively treated with anti-androgen drugs or docetaxel. Sensitization also includes reducing the dosage required to achieve a beneficial effect with anti-androgen drug or docetaxel therapy.
  • anti-androgen drug includes anti-androgen compounds that alter the androgen pathway by blocking the androgen receptors, competing for binding sites on the cell's surface, or affecting or mediating androgen production.
  • Anti-androgens are useful for treating several diseases including, but not limited to, prostate cancer.
  • Anti-androgens include, but are not limited to, enzalutamide, abiraterone, and bicalutamide.
  • AR androgen receptor
  • AREs Androgen Response Elements
  • AR variant includes a splice variant of full-length AR.
  • Various AR variants are known. See, Guo et al., Cancer Res. 2009 Mar. 15; 69(6):2305-13.
  • Exemplary AR variants include, but are not limited to, variants lacking a functional ligand binding domain (LBD).
  • LBD functional ligand binding domain
  • An example of an AR variant that lacks an LBD is AR-V7.
  • AR-V7 includes androgen receptor splice variant 7, a contituitively active variant of an AR that lacks a functional ligand binding domain (LBD). See, e.g., Hu et al., Cancer Research, 69(1):16-22 (2009).
  • the term “individual,” “subject,” or “patient” typically includes humans, but also includes other animals such as, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • “Pharmaceutically acceptable” or “therapeutically acceptable” includes a substance which does not interfere with the effectiveness or the biological activity of the active ingredients and which is not toxic to the hosts in the amounts used, and which hosts may be either humans or animals to which it is to be administered.
  • the present invention relates to AR-V7 inhibitors.
  • inhibition of AR-V7 can resensitize drug-resistant prostate cancer cells to prostate cancer drugs selected from the group consisting of docetaxel, an anti-androgen drug (e.g., bicalutamide, enzalutamide, and arbiraterone), and combinations thereof.
  • an anti-androgen drug e.g., bicalutamide, enzalutamide, and arbiraterone
  • inhibition of AR-V7 can enhance the effectiveness of prostate cancer drugs selected from the group consisting of docetaxel, an anti-androgen drug (e.g., bicalutamide, enzalutamide, and arbiraterone), and combinations thereof.
  • AR-V7 inhibitors can be administered in combination with AKR1C3 inhibitors.
  • AR-V7 inhibitors include, but are not limited to, compounds that inhibit AR-V7 transcription, translation, stability, or activity. Inhibition of AR-V7 activity can include inhibition of recruitment of AR-V7 to Androgen Response Elements (AREs). In some embodiments, inhibition of AR-V7 activity can include inhibition of recruitment of AR-V7 to the PSA promoter. In some embodiments, inhibition of AR-V7 activity can include inhibition of AR-V7-induced activation of the PSA promoter. In some embodiments, inhibition of AR-V7 activity can include inhibition of AR-V7-induced PSA production. For example, inhibition of AR-V7 can include inhibition of production of PSA in the absence of DHT.
  • AREs Androgen Response Elements
  • the present invention also relates to compositions that include an AR-V7 inhibitor and a compound selected from the group consisting of enzalutamide (Xtandi), abiraterone (Zytiga), docetaxel (Taxotere), bicalutamide (Casodex, Cosudex, Calutide, Kalumid), indomethacin, and combinations thereof.
  • Exemplary AR-V7 inhibitors include, but are not limited to, nucleic acid-based inhibitors.
  • nucleic acid based inhibitors of AR-V7 can include, but are not limited to, short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) that target AR-V7 mRNA.
  • nucleic acid analogs can be utilized to target AR-V7 transcription, splicing, or translation.
  • Such nucleic acid analogs include, but are not limited to, antisense phosphorodiamidate morpholino oligonucleotides, locked nucleic acids, or peptide nucleic acids.
  • exemplary AR-V7 inhibitors include, but are not limited to, LY294002, Wortmanin, and AKT inhibitor II. See, e.g., Mediwala et al., Prostate, 2013 Feb. 15; 73(3):267-77.
  • AR-V7 inhibitors can include niclosamide.
  • the present invention provides compositions and methods for administering an AR-V7 inhibitor, such as niclosamide, to inhibit AR variant activity, such as AR-V7 transcription activity.
  • an AR-V7 inhibitor such as niclosamide
  • the present invention also provides compositions and methods for reducing the recruitment of AR variants, such as AR-V7, to the PSA promoter.
  • the present invention also provides compositions and methods for administering an AR-V7 inhibitor, such as niclosamide, to inhibit prostate cancer cell growth, or induce prostate cancer cell death, by targeting AR variant signaling.
  • the present invention also provides compositions and methods for administering an AR-V7 inhibitor, such as niclosamide, to inhibit prostate cancer cell growth, or induce prostate cancer cell death, by overcoming drug resistance, e.g., enzalutamide, bicalutamide, or abiraterone-resistance, in prostate cancer cells.
  • the present invention further provides compositions comprising an AR-V7 inhibitor, such as niclosamide, alone or in combination with prostate cancer therapies, such as anti-androgen therapies, enzalutamide-based therapies, bicalutamide-based therapies, abiraterone-based therapies, docetaxel-based therapies, indomethacin-based therapies, or combinations thereof.
  • the present invention also provides such compositions which are tailored for patients having advanced stage prostate cancer, such as drug-resistant prostate cancer, metastatic prostate cancer, castration-resistant prostate cancer, or combinations thereof.
  • the present invention additionally provides compositions including an AR-V7 inhibitor, such as niclosamide, which are tailored for patients with prostate cancer that is resistant to known prostate cancer treatments such as enzalutamide, bicalutamide, abiraterone, or docetaxel.
  • the present invention additionally provides compositions containing an AR-V7 inhibitor, such as niclosamide, that provide an unexpected synergistic or additive effect on prostate cancer cells when used in combination with enzalutamide, bicalutamide, abiraterone, docetaxel, indomethacin, or combinations thereof.
  • the present invention provides compositions and methods for inhibiting AR-V7 protein expression by protein degradation via the proteasome dependent pathway. Based on the assays set forth herein, AR variants such as AR-V7 were observed to induce enzalutamide and abiraterone resistance in prostate cancer cells. Furthermore, the compositions including an AR-V7 inhibitor, such as niclosamide, as well as the methods set forth herein are suitable for inhibiting AR activity, such as AR-V7 transcription activity. The compositions including an AR-V7 inhibitor, such as niclosamide, as well as the methods set forth herein are suitable for reducing AR variant, e.g., AR-V7, recruitment to the prostate specific antigen (PSA) promoter. By inhibiting AR-V7, for example, the compositions and methods set forth herein are suitable for treating enzalutamide- or abiraterone-resistant prostate cancer, or a combination thereof.
  • PSA prostate specific antigen
  • the present invention provides a composition including an AR-V7 inhibitor, such as niclosamide, and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, indomethacin, and combinations thereof.
  • an AR-V7 inhibitor such as niclosamide
  • the amount of an AR-V7 inhibitor is an amount effective to enhance the therapeutic benefit of a compound selected from the group consisting of bicalutamide, enzalutamide, abiraterone, docetaxel, and indomethacin.
  • the compound is enzalutamide.
  • the compound is abiraterone.
  • the compound is docetaxel.
  • the compound is bicalutamide.
  • the compound is indomethacin.
  • the composition includes both an AR-V7 inhibitor, such as niclosamide, and enzalutamide. In some other compositions described herein, the composition includes an AR-V7 inhibitor, such as niclosamide, and abiraterone. In some compositions described herein, the composition includes an AR-V7 inhibitor, such as niclosamide, and docetaxel. In yet other compositions described herein, the composition includes an AR-V7 inhibitor, such as niclosamide, and bicalutamide. In yet other compositions described herein, the composition includes both an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • an AR-V7 inhibitor such as niclosamide
  • abiraterone such as niclosamide
  • the composition includes an AR-V7 inhibitor, such as niclosamide, and docetaxel.
  • the composition includes an AR-V7 inhibitor, such as niclosamide, and bicalutamide.
  • niclosamide is a Food and Drug Administration (FDA) approved drug effective against human tapeworms.
  • FDA Food and Drug Administration
  • the chemical structure of niclosamide is set forth below:
  • niclosamide is (5-chloro-N-2chloro-4-nitro-phenyl)-2-hydroxybenzamide.
  • niclosamide includes analogs and derivatives thereof, including pro-drugs that are converted to niclosamide or an analog or derivative thereof in a subject to which it is administered. See, e.g., Ulrike Sack et al., J. Natl. Cancer Inst ., July 6; 103(13):1018-36 (2011); and International Patent Publication No. WO 2012/143377.
  • Exemplary niclosamide analogs include, but are not limited to, compounds 50643-F/1; 69360-X/1; 82020-K/D2; 111338-C/1; 164315-M/2; and 306664-O/1 as described in Sack et al., 2011.
  • niclosamide also includes salts, such as pharmaceutically acceptable salts, of niclosamide.
  • Niclosamide salts can include, but are not limited to, niclosamide ethanolamine salt, niclosamide methanol solvate, and niclosamide hydrate.
  • niclosamide also includes nanoparticle packaged niclosamide formulations, such as those described in Ye et al., Drug Dev. Ind. Pharm ., September 10: 1-9 (2014).
  • the present invention also relates to compositions containing inhibitors of aldo-keto reductase 1C3 (AKR1C3), an enzyme that catalyzes the conversion of the weak androgen precursors 4-androstene-3,17-dione ( ⁇ 4 -AD) and 5 ⁇ -androstane-3,17-dione to the potent androgens testosterone and 5 ⁇ -dihydrotestosterone (DHT), respectively.
  • AKR1C3 activation can confer resistance to therapeutic treatments commonly employed against prostate cancer cells.
  • AKR1C3 activation can confer resistance to bicalutamide, enzalutamide, or abiraterone.
  • inhibitors of AKR1C3 transcription, translation, protein stability, or enzymatic activity can reduce, reverse, or eliminate drug resistance in prostate cancer cells.
  • Exemplary AKR1C3 inhibitors include, but are not limited to, nucleic acid-based inhibitors.
  • nucleic acid based inhibitors of AKR1C3 include, but are not limited to, short hairpin RNAs (shRNAs) or small interfering RNAs (siRNAs) that target AKR1C3 mRNA.
  • nucleic acid analogs can be utilized to target AKR1C3 transcription, splicing, or translation.
  • Such nucleic acid analogs include, but are not limited to, antisense phosphorodiamidate morpholino oligonucleotides, locked nucleic acids, or peptide nucleic acids.
  • the inhibitor of AKR1C3 is indomethacin.
  • Indomethacin is a Food and Drug Administration (FDA) approved Non-Steroidal Anti-Inflammatory Drug (NSAID) that can reduce fever, pain, stiffness, and swelling by inhibition of prostaglandin production.
  • FDA Food and Drug Administration
  • NSAID Non-Steroidal Anti-Inflammatory Drug
  • Indomethacin is also an inhibitor of androgen biosynthesis.
  • indomethacin inhibits AKR1C3.
  • indomethacin also known as indocin or indometacin, is set forth below:
  • indomethacin The chemical name of indomethacin is 2- ⁇ 1-[(4-Chlorophenyl)carbonyl]-5-methoxy-2-methyl-1H-indol-3-yl ⁇ acetic acid.
  • indomethacin includes analogs and derivatives thereof, including pro-drugs that are converted to indomethacin or an analog or derivative thereof in a subject to which it is administered.
  • the indomethacin derivatives can include any of the derivatives described in Liedtke et al., J Med Chem., 2013 Mar. 28; 56(6):2429-46.
  • Additional exemplary AKR1C3 inhibitors include, but are not limited to, ASP9521; 4-methyl(de-dimethylamine)-tetracycline; baccharin; stylopine; flufenamic acid; 3-((4′-(trifluoromethyl)phenyl)amino)benzoic acid; SN33638; N-(4-chlorobenzoyl)-melatonin; 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid, 3-(4′-(nitronaphthalen-1-amino))benzoic acid, and salts, analogs, or derivatives thereof.
  • Exemplary AKR1C3 inhibitors can further include, but are not limited to, any of the AKR1C3 inhibitor compounds described in Khanim et al., Br J Cancer. 2014 Mar. 18; 110(6):1506-16; Liedtke et al., J Med Chem. 2013 Mar. 28; 56(6):2429-46; Adeniji et al., Endocr. Rev. 2012, 33, SAT-537; Adeniji et al., J Med Chem. 2012 Mar. 8; 55(5):2311-23; Adeniji et al., Bioorg Med Chem Lett. 2011 Mar. 1; 21(5):1464-8; Chen et al., Bioorg Med Chem Lett.
  • the present invention also relates to compositions that include an AKR1C3 inhibitor, such as indomethacin or a derivative thereof, and a compound selected from the group consisting of enzalutamide (Xtandi), abiraterone (Zytiga), docetaxel (Taxotere), bicalutamide (Casodex, Cosudex, Calutide, Kalumid), niclosamide, and combinations thereof.
  • an AKR1C3 inhibitor such as indomethacin or a derivative thereof
  • the present invention also provides compositions and methods for administering an AKR1C3 inhibitor, such as indomethacin, to inhibit prostate cancer cell growth or induce prostate cancer cell death.
  • the present invention also provides compositions and methods for administering indomethicin to inhibit prostate cancer cell growth by overcoming or reducing bicalutamide, enzalutamide, or abiraterone-resistance in prostate cancer cells.
  • the present invention further provides compositions of indomethacin, alone or in combination with current anti-androgen therapies.
  • the present invention also provides such compositions which are tailored for patients having advanced stage prostate cancer, such as drug-resistant prostate cancer, metastatic prostate cancer, castration-resistant prostate cancer, or combinations thereof.
  • the present invention additionally provides compositions including an AKR1C3 inhibitor such as indomethacin which are tailored for patients with prostate cancer that is resistant to known prostate cancer treatments such as enzalutamide, abiraterone, or docetaxel.
  • the present invention additionally provides compositions containing an AK1RC3 inhibitor, such as indomethacin, that provide an unexpected synergistic or additive effect on prostate cancer cells when used in combination with enzalutamide, bicalutamide, abiraterone, docetaxel, niclosamide, or combinations thereof.
  • the present invention provides a composition including an AKR1C3 inhibitor, such as indomethacin, and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, niclosamide, and combinations thereof.
  • an AKR1C3 inhibitor such as indomethacin
  • the amount of AKR1C3 inhibitor is an amount effective to enhance the therapeutic benefit of a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, niclosamide, and docetaxel.
  • the compound is enzalutamide.
  • the compound is abiraterone.
  • the compound is docetaxel.
  • the compound is bicalutamide.
  • the compound is niclosamide.
  • the composition includes both an AKR1C3 inhibitor, such as indomethacin, and enzalutamide. In some other compositions described herein, the composition includes both an AKR1C3 inhibitor, such as indomethacin, and abiraterone. In some compositions described herein, the composition includes, both an AKR1C3 inhibitor, such as indomethacin, and docetaxel. In yet other compositions described herein, the composition includes both an AKR1C3 inhibitor, such as indomethacin, and bicalutamide. In yet other compositions described herein, the composition includes both an AKR1C3 inhibitor, such as indomethacin, and an AR-V7 inhibitor, such as niclosamide.
  • an AKR1C3 inhibitor such as indomethacin
  • an AR-V7 inhibitor such as niclosamide.
  • composition may further include a pharmaceutically acceptable excipient or diluent.
  • compositions of the present invention encompass compositions made by admixing an AR-V7 inhibitor, such as niclosamide, and a pharmaceutically acceptable carrier and/or excipient or diluent. Such compositions are suitable for pharmaceutical use in an animal or human.
  • compositions of the present invention also encompass compositions made by admixing an AKR1C3 inhibitor, such as indomethacin, and a pharmaceutically acceptable carrier and/or excipient or diluent. Such compositions are suitable for pharmaceutical use in an animal or human.
  • compositions of the present invention may be prepared by any of the methods well-known in the art of pharmacy.
  • Pharmaceutically acceptable carriers suitable for use with the present invention include any of the standard pharmaceutical carriers, buffers and excipients, including phosphate-buffered saline solution, water, and emulsions (such as an oil/water or water/oil emulsion), and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, 19th ed. 1995). Preferred pharmaceutical carriers depend upon the intended mode of administration of the active agent.
  • the pharmaceutical compositions of the present invention can include drug, e.g., enzalutamide, abiraterone, docetaxel, bicalutamide, an AKR1C3 inhibitor (e.g., indomethacin), or an AR-V7 inhibitor (e.g., niclosamide), or any pharmaceutically acceptable salts thereof, as an active ingredient and a pharmaceutically acceptable carrier and/or excipient or diluent.
  • the pharmaceutical composition can include an AKR1C3 inhibitor, such as indomethacin, and an AR-V7 inhibitor, such as niclosamide.
  • a pharmaceutical composition may optionally contain other therapeutic ingredients.
  • the compounds of the present invention can be combined as the active ingredient in intimate admixture with a suitable pharmaceutical carrier and/or excipient according to conventional pharmaceutical compounding techniques. Any carrier and/or excipient suitable for the form of preparation desired for administration is contemplated for use with the compounds disclosed herein.
  • compositions include compositions suitable for topical, parenteral, pulmonary, nasal, rectal, or oral administration.
  • the most suitable route of administration in any given case will depend in part on the nature and severity of the prostate cancer condition and also optionally the stage of the prostate cancer.
  • compositions suitable for systemic (enteral or parenteral) administration include oral, rectal, sublingual, or sublabial administration.
  • the compositions may be administered via a syringe or intravenously.
  • compositions for pulmonary administration include, but are not limited to, dry powder compositions consisting of the powder of a compound described herein, or a salt thereof, and the powder of a suitable carrier and/or lubricant.
  • the compositions for pulmonary administration can be inhaled from any suitable dry powder inhaler device known to a person skilled in the art.
  • compositions for systemic administration include, but are not limited to, dry powder compositions consisting of the composition as set forth herein and the powder of a suitable carrier and/or excipient.
  • the compositions for systemic administration can be represented by, but not limited to, tablets, capsules, pills, syrups, solutions, and suspensions.
  • the present invention provides compositions further including a pharmaceutical surfactant. In other embodiments, the present invention provides compositions further including a cryoprotectant. In some embodiments, the cryoprotectant is selected from the group consisting of glucose, sucrose, trehalose, lactose, sodium glutamate, PVP, HP ⁇ CD, CD, glycerol, maltose, mannitol, and saccharose.
  • the present invention provides a pharmaceutical composition including an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and a pharmaceutically acceptable excipient.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • the pharmaceutically acceptable excipient includes a salt or a diluent.
  • the present invention provides compositions including an effective amount of an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the composition is formulated for oral administration or intravenous administration and includes an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and at least one member selected from the group consisting of an aqueous solution and a buffer solution.
  • the composition can include both an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in Remington: The Science and Practice of Pharmacy, 21st Ed., University of the Sciences in Philadelphia, Lippencott Williams & Wilkins (2005).
  • Controlled release parenteral formulations of the compositions of the present invention can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Polymers can be used for ion-controlled release of compositions of the present invention.
  • Various degradable and nondegradable polymeric matrices for use in controlled drug delivery are known in the art (Langer R., Accounts Chem. Res., 26:537-542 (1993)).
  • the block copolymer, polaxamer 407 exists as a viscous yet mobile liquid at low temperatures but forms a semisolid gel at body temperature. It has shown to be an effective vehicle for formulation and sustained delivery of recombinant interleukin 2 and urease (Johnston et al., Pharm. Res., 9:425-434 (1992); and Pec et al., J. Parent. Sci. Tech., 44(2):58 65 (1990)).
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins (Ijntema et al., Int. J. Pharm., 112:215-224 (1994)).
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug (Betageri et al., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)).
  • Numerous additional systems for controlled delivery of therapeutic proteins are known. See, e.g., U.S. Pat. Nos.
  • the present invention provides a method of treating prostate cancer in a patient (e.g., advanced stage prostate cancer), wherein the method comprises administering to the patient an effective amount of an AR-V7 inhibitor, such as niclosamide, an AKR1C3 inhibitor, such as indomethacin, or a combination thereof.
  • an AR-V7 inhibitor such as niclosamide, an AKR1C3 inhibitor, such as indomethacin, or a combination thereof.
  • the prostate cancer is advanced stage prostate cancer, such as any one or more of the types of advanced staged prostate cancers disclosed herein.
  • the prostate cancer is drug resistant.
  • the prostate cancer is anti-androgen drug resistant.
  • the prostate cancer is metastatic.
  • the prostate cancer is metastatic and drug resistant (e.g., anti-androgen drug resistant). In some of these embodiments, the prostate cancer is castration resistant. In some of these embodiments, the prostate cancer is metastatic and castration resistant. In some of these embodiments, the prostate cancer is enzalutamide resistant. In some of these embodiments, the prostate cancer is enzalutamide and arbiraterone resistant. In some of these embodiments, the prostate cancer is enzalutamide, arbiraterone, and bicalutamide resistant. In some of these embodiments, the prostate cancer is docetaxel resistant. In some of these embodiments, the prostate cancer is enzalutamide, arbiraterone, bicalutamide, and docetaxel resistant.
  • the prostate cancer is enzalutamide, arbiraterone, bicalutamide, and docetaxel resistant.
  • the treating comprises inhibiting prostate cancer cell growth; inhibiting prostate cancer cell migration; inhibiting prostate cancer cell invasion; ameliorating the symptoms of prostate cancer; reducing the size of a prostate cancer tumor; reducing the number of prostate cancer tumors; reducing the number of prostate cancer cells; inducing prostate cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death; or enhancing the therapeutic effects of a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the treating comprises inhibiting prostate cancer cell growth. In some methods of treating prostate cancer, described herein, the treating comprises inhibiting prostate cancer cell migration. In some methods of treating prostate cancer, described herein, the treating comprises inhibiting prostate cancer cell invasion. In some methods of treating prostate cancer, described herein, the treating comprises ameliorating the symptoms of prostate cancer. In some methods of treating prostate cancer, described herein, the treating comprises reducing the size of a prostate cancer tumor. In some methods of treating prostate cancer, described herein, the treating comprises reducing the number of prostate cancer tumors. In some methods of treating prostate cancer, described herein, the treating comprises reducing the number of prostate cancer cells. In some methods of treating prostate cancer, described herein, the treating comprises inducing prostate cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death.
  • the treating comprises enhancing the therapeutic effects of a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the treating comprises enhancing the therapeutic effects of enzalutamide.
  • the treating comprises enhancing the therapeutic effects of abiraterone.
  • the treating comprises enhancing the therapeutic effects of docetaxel.
  • the treating comprises enhancing the therapeutic effects of bicalutamide.
  • the enhancement can be synergistic or additive.
  • the treating comprises reversing, or reducing prostate cancer cell resistance to anti-androgen drugs. In certain embodiments of the methods set forth herein, the treating comprises resensitizing prostate cancer cells to anti-androgen drugs.
  • the anti-androgen drug is a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof. In certain embodiments, the anti-androgen drug is enzalutamide.
  • the treating may comprise reversing prostate cancer cell resistance to enzalutamide; reducing prostate cancer cell resistance to enzalutamide; or resensitizing prostate cancer cells to enzalutamide.
  • the treating comprises reversing prostate cancer cell resistance to enzalutamide.
  • the treating comprises reducing prostate cancer cell resistance to enzalutamide.
  • the treating comprises resensitizing prostate cancer cells to enzalutamide.
  • the treating comprises reversing prostate cancer cell resistance to docetaxel. In some other embodiments, the treating comprises reducing prostate cancer cell resistance to docetaxel. In yet other embodiments, the treating comprises resensitizing prostate cancer cells to docetaxel.
  • the treating comprises reversing prostate cancer cell resistance to abiraterone. In some other embodiments set forth herein, the treating comprises reducing prostate cancer cell resistance to abiraterone. In yet other embodiments set forth herein, the treating comprises resensitizing prostate cancer cells to abiraterone.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer, anti-androgen-resistant prostate cancer, bicalutamide resistant prostate cancer, enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, docetaxel-resistant prostate cancer, AR-V7- or AKR1C3-induced drug-resistant prostate cancer, AR-V7- or AKR1C3-induced anti-androgen drug-resistant prostate cancer, AR-V7- or AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • the prostate cancer is castration-resistant prostate cancer. In other embodiments, the prostate cancer is metastatic castration-resistant prostate cancer. In still other embodiments, the prostate cancer is advanced stage prostate cancer. In other embodiments, the prostate cancer is anti-androgen-resistant prostate cancer. In some other embodiments, the prostate cancer is enzalutamide-resistant prostate cancer. In yet other embodiments, the prostate cancer is abiraterone-resistant prostate cancer. In some other embodiments, the prostate cancer is docetaxel-resistant prostate cancer. In certain other embodiments, the prostate cancer is AR-V7- or AKR1C3-induced enzalutamide-resistant prostate cancer. In some embodiments, the prostate cancer is a combination of one, two, three, four, five, six, seven, eight, nine, ten, or more of the foregoing types of prostate cancer.
  • the methods of treating prostate cancer may comprise co-administering an AR-V7 inhibitor, such as niclosamide, with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, an AKR1C3 inhibitor, such as indomethacin, and combinations thereof.
  • the methods of treating prostate cancer may additionally or alternatively comprise co-administering an AKR1C3 inhibitor, such as indomethacin, with a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, niclosamide, and combinations thereof.
  • Some embodiments of the present invention include methods for treating enzalutamide-resistant and abiraterone-resistant prostate cancer that comprise administering an AR-V7 inhibitor, such as niclosamide, an AKR1C3 inhibitor, such as indomethacin, or a combination thereof, to a patient having enzalutamide-resistant or abiraterone-resistant prostate cancer.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • the present invention provides a method for inhibiting androgen receptor splice variants in a cell, wherein the method comprises contacting the cell or the androgen receptor splice variant with an amount of an AR variant inhibitor, such as niclosamide.
  • an AR variant inhibitor such as niclosamide.
  • the variants are AR-V7 splice variants.
  • the amount is an effective amount.
  • the cell is a prostate cancer cell, such as a castration-resistant prostate cancer cell, an anti-androgen-resistant (e.g., enzalutamide-resistant) prostate cancer cell, or a combination thereof.
  • the present invention provides a method for inhibiting STAT3 activation in a cell, wherein the method comprises contacting the cell or STAT3 with an amount of an AR-V7 inhibitor.
  • the amount is an effective amount.
  • the AR-V7 inhibitor is niclosamide or an analog or derivative thereof.
  • the cell is a prostate cancer cell, such as a castration-resistant prostate cancer cell, an anti-androgen-resistant (e.g., enzalutamide-resistant) prostate cancer cell, or a combination thereof.
  • the present invention provides a method for inhibiting IL-6 signalling in a cell, wherein the method comprises contacting the cell or STAT3 with an amount of an AR-V7 inhibitor.
  • the amount is an effective amount.
  • the AR-V7 inhibitor is niclosamide or an analog or derivative thereof.
  • the cell is a prostate cancer cell, such as a castration-resistant prostate cancer cell, an anti-androgen-resistant (e.g., enzalutamide-resistant) prostate cancer cell, or a combination thereof.
  • the present invention provides a method for inhibiting AKR1C3 in a cell, wherein the method comprises contacting the cell or AKR1C3 with an amount of an AKR1C3 inhibitor. In some embodiments, the amount is an effective amount. In some embodiments, the AKR1C3 inhibitor is indomethacin or an analog or derivative thereof.
  • the cell is a prostate cancer cell, such as a castration-resistant prostate cancer cell, an anti-androgen-resistant (e.g., enzalutamide-resistant) prostate cancer cell, or a combination thereof.
  • the present invention provides a method for inhibiting prostate cancer cell growth, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the present invention provides a method for inhibiting prostate cancer cell migration, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the present invention provides a method for inhibiting prostate cancer cell invasion, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the present invention provides a method for reversing prostate cancer cell resistance to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for reversing prostate cancer cell resistance to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for reversing prostate cancer cell resistance to enzalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for reversing prostate cancer cell resistance to enzalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for reversing prostate cancer cell double resistance to enzalutamide and abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for reversing prostate cancer cell double resistance to enzalutamide and abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for resensitizing prostate cancer cells to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to enzalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for resensitizing prostate cancer cells to enzalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides for a method for resensitizing prostate cancer cells to abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to enzalutimide and abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for resensitizing prostate cancer cells to enzalutimide and abiraterone, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to bicalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for resensitizing prostate cancer cells to bicalutamide, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for resensitizing prostate cancer cells to docetaxel, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for resensitizing prostate cancer cells to docetaxel, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the amount is an effective amount.
  • the present invention provides a method for reducing, reversing, or resensitizing prostate cancer cell resistance to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for reducing, reversing, or resensitizing prostate cancer cell resistance to anti-androgen drugs, wherein the method comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the anti-androgen drug may be a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the reducing, reversing, or resensitizing prostate cancer cell resistance to anti-androgen drugs is in a patient having prostate cancer.
  • the present invention also provides a method for enhancing the therapeutic effects of an anti-androgen drug in a patient having prostate cancer, wherein the method comprises administering to a patient in need of an anti-androgen drug an effective amount of an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention also provides a method for enhancing the therapeutic effects of an anti-androgen drug in a patient having prostate cancer, wherein the method comprises administering to a patient in need of an anti-androgen drug an effective amount of an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for enhancing the therapeutic effects of enzalutamide, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of enzalutamide in combination with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the method comprises co-administering to the patient an effective amount of enzalutamide in combination with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for enhancing the therapeutic effects of docetaxel, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of docetaxel in combination with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the method comprises co-administering to the patient an effective amount of docetaxel in combination with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for enhancing the therapeutic effects of abiraterone, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of abiraterone in combination with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the method comprises co-administering to the patient an effective amount of abiraterone in combination with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for enhancing the therapeutic effects of bicalutamide, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of bicalutamide in combination with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the method comprises co-administering to the patient an effective amount of bicalutamide in combination with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a method for enhancing the therapeutic effects of an AR-V7 inhibitor, such as niclosamide, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of an AR-V7 inhibitor, such as niclosamide, in combination with an AKR1C3 inhibitor, such as indomethacin.
  • an AR-V7 inhibitor such as niclosamide
  • the present invention provides a method of enhancing the therapeutic effects of an AR-V7 inhibitor, such as niclosamide, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of an AR-V7 inhibitor, such as niclosamide, in combination with a drug selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide, and combinations thereof.
  • an AR-V7 inhibitor such as niclosamide
  • the present invention provides a method for enhancing the therapeutic effects of an AKR1C3 inhibitor, such as indomethacin, in a patient having prostate cancer, the method comprising co-administering to the patient an effective amount of the AKR1C3 inhibitor in combination with a drug selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide, and combinations thereof.
  • an AKR1C3 inhibitor such as indomethacin
  • the present invention provides a method for inhibiting an androgen receptor (AR) variant, comprising contacting an AR variant with an amount of an AR-V7 inhibitor, such as niclosamide.
  • an AR-V7 inhibitor such as niclosamide.
  • the AR variant is AR-V7.
  • the present invention provides a method for inhibiting AR transactivation, inhibiting AR expression, inhibiting AR-cell migration, inhibiting AR-cell invasion in prostate cancer cells, inhibiting prostate cancer cell colony formation, and inhibiting recruitment of an AR variant to a prostate-specific antigen (PSA) promoter.
  • this method of inhibition comprises contacting prostate cancer cells with an AR-V7 inhibitor, such as niclosamide, and optionally one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the present invention also provides a method for inhibiting AR full length and AR-V7 expression, wherein the method comprises contacting an AR or a prostate cancer cell with an AR-V7 inhibitor, such as niclosamide.
  • the present invention further provides a method for inhibiting AR full length and AR-V7 expression, wherein the method comprises contacting a prostate cancer cell with an AR-V7 inhibitor, such as niclosamide.
  • the present invention provides a method for inhibiting enzalutamide/abiraterone-resistant CRPC cell growth, migration or invasion, wherein the method comprises contacting a prostate cancer cell with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin.
  • the method comprises contacting a prostate cancer cell with an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the prostate cancer cell is a CRPC cell.
  • the present invention provides a method for synergistically enhancing enzalutamide/abiraterone effects for treating prostate cancer, wherein the method comprises administering an effective amount of an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, to a patient having prostate cancer.
  • the method comprises administering an effective amount of an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin, to a patient having prostate cancer.
  • the present invention also provides a method for inhibiting recruitment of AR to a PSA promoter, the method comprises contacting AR with an AR-V7 inhibitor, such as niclosamide.
  • an AR-V7 inhibitor such as niclosamide.
  • the AR-V7 inhibitor is administered orally, alone or in combination with enzalutamide, abiraterone, an AKR1C3 inhibitor, or combinations thereof.
  • the AKR1C3 inhibitor is administered orally, alone or in combination with enzalutamide, abiraterone, an AR-V7 inhibitor such as niclosamide, or combinations thereof.
  • the AKR1C3 inhibitor is administered orally in combination with an AR-V7 inhibitor, such as niclosamide.
  • the present invention also provides a method for synergistically or additively enhanced anti-androgen (e.g., bicalutamide, enzalutamide, or abiraterone) effects in prostate cancer cells, wherein the method comprises contacting a prostate cancer cell with an AR-V7 inhibitor, such as niclosamide, or an AKR1C3 inhibitor, such as indomethacin, and optionally also one or more anti-androgen drugs such as bicalutamide, enzalutamide, or abiraterone.
  • the prostate cancer cells are resistant to one or more compounds selected from the group consisting of enzalutamide, abiraterone, docetaxel, and bicalutamide.
  • the compound is enzalutamide.
  • the compound is abiraterone.
  • the compound is docetaxel.
  • the compound is bicalutamide.
  • the present invention also provides a method for inhibiting STAT3, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide).
  • an AR-V7 inhibitor e.g., niclosamide
  • the present invention also provides a method for reversing STAT3-mediated enzalutamide resistance in prostate cancer cells, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide).
  • an AR-V7 inhibitor e.g., niclosamide
  • the present invention provides a method for inhibiting STAT3, wherein the method comprises administering an AR-V7 inhibitor (e.g., niclosamide) to a patient having prostate cancer.
  • the present invention provides a method for inhibiting STAT3, wherein the method comprises administering an AR-V7 inhibitor (e.g., niclosamide) to a patient having (e.g., enzalutamide resistant) prostate cancer.
  • the present invention provides a method for inhibiting STAT3, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide).
  • an AR-V7 inhibitor e.g., niclosamide
  • the contacting is in, on, or near prostate cancer cells.
  • the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide) and enzalutamide.
  • the present invention provides a method for inhibiting prostate cancer cell growth, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide). In certain embodiments, the contacting is in, on, or near prostate cancer cells. In some embodiments, the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide) and enzalutamide.
  • an AR-V7 inhibitor e.g., niclosamide
  • enzalutamide e.g., niclosamide
  • the present invention provides a method for inducing prostate cancer cell apoptosis, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide).
  • the contacting is in, on, or near prostate cancer cells.
  • the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide) enzalutamide.
  • the present invention provides a method for inhibiting prostate cancer cell colony formation, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide). In certain embodiments, the contacting is in, on, or near prostate cancer cells. In some embodiments, the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide) and enzalutamide.
  • an AR-V7 inhibitor e.g., niclosamide
  • enzalutamide e.g., niclosamide
  • the present invention provides a method for reversing enzalutamide resistance though inhibition of STAT3 expression, wherein the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide).
  • an AR-V7 inhibitor e.g., niclosamide
  • the contacting is in, on, or near prostate cancer cells.
  • the method comprises contacting STAT3 with an AR-V7 inhibitor (e.g., niclosamide) and enzalutamide.
  • an AR-V7 inhibitor such as niclosamide, is administered to a patient having prostate cancer.
  • an AR-V7 inhibitor such as niclosamide, is administered in combination with enzalutamide.
  • an AR-V7 inhibitor such as niclosamide, is administered in combination with abiraterone.
  • an AR-V7 inhibitor such as niclosamide
  • niclosamide is adminstered in combination with both enzalutamide and abiraterone.
  • an AR-V7 inhibitor such as niclosamide, is adminstered in combination with docetaxel.
  • an AR-V7 inhibitor such as niclosamide
  • niclosamide is adminstered in combination with both enzalutamide and docetaxel.
  • an AR-V7 inhibitor such as niclosamide
  • niclosamide is adminstered in combination with both abiraterone and docetaxel.
  • the methods comprise first administering an AR-V7 inhibitor, such as niclosamide, to a patient having prostate cancer, and then administering enzalutamide to the patient.
  • the methods comprise first administering enzalutamide to a patient having prostate cancer, and then administering an AR-V7 inhibitor, such as niclosamide, to the patient.
  • the methods of administration comprise administering an AR-V7 inhibitor, such as niclosamide, alone or in combination with enzalutamide to a patient in need thereof. In some other embodiments of the present invention, the methods of administration comprise administering an AR-V7 inhibitor, such as niclosamide, alone or in combination with abiraterone to a patient in need thereof. In yet other embodiments of the present invention, the methods comprise administering an AR-V7 inhibitor, such as niclosamide, alone or in combination with docetaxel to a patient in need thereof. In still other embodiments of the present invention, the methods comprise administering an AR-V7 inhibitor, such as niclosamide, alone or in combination with bicalutamide to a patient in need thereof.
  • the present invention provides a method of delivering an effective amount of an AR-V7 inhibitor, such as niclosamide, to a patient having prostate cancer.
  • an AR-V7 inhibitor such as niclosamide
  • the AR-V7 inhibitor (e.g., niclosamide) formulations of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament.
  • a pharmaceutical composition or medicament can be administered to a subject in need thereof, e.g. a patient having prostate cancer.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like), docetaxel-resistant prostate cancer, AR-V7-induced anti-androgen-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like
  • docetaxel-resistant prostate cancer e.g., AR-V7-induced anti-androgen-resistant prostate cancer such as AR-V7-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • an AKR1C3 inhibitor such as indomethacin, is administered to a patient having prostate cancer.
  • an AKR1C3 inhibitor such as indomethacin, is administered in combination with enzalutamide.
  • an AKR1C3 inhibitor such as indomethacin, is administered in combination with abiraterone.
  • an AKR1C3 inhibitor such as indomethacin, is adminstered in combination with both enzalutamide and abiraterone.
  • an AKR1C3 inhibitor such as indomethacin
  • docetaxel In some other embodiments, an AKR1C3 inhibitor, such as indomethacin, is administered in combination with an AR-V7 inhibitor, such as niclosamide.
  • an AKR1C3 inhibitor such as indomethacin, is adminstered in combination with both enzalutamide and docetaxel.
  • an AKR1C3 inhibitor such as indomethacin, is adminstered in combination with both abiraterone and docetaxel.
  • the methods comprise first administering an AKR1C3 inhibitor, such as indomethacin, to a patient having prostate cancer, and then administering enzalutamide to the patient.
  • the methods comprise first administering enzalutamide to a patient having prostate cancer, and then administering an AKR1C3 inhibitor, such as indomethacin, to the patient.
  • the methods of administration comprise administering an AKR1C3 inhibitor, such as indomethacin, alone or in combination with enzalutamide to a patient in need thereof.
  • the methods of administration comprise administering an AKR1C3 inhibitor, such as indomethacin, alone or in combination with abiraterone to a patient in need thereof.
  • the methods comprise administering an AKR1C3 inhibitor, such as indomethacin, alone or in combination with docetaxel to a patient in need thereof.
  • the methods comprise administering an AKR1C3 inhibitor, such as indomethacin, alone or in combination with bicalutamide to a patient in need thereof.
  • the present invention provides a method of delivering an effective amount of an AKR1C3 inhibitor, such as indomethacin, to a patient having prostate cancer.
  • an AKR1C3 inhibitor such as indomethacin
  • the AKR1C3 inhibitor (e.g., indomethacin) formulations of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament.
  • a pharmaceutical composition or medicament can be administered to a subject in need thereof, e.g. a patient having prostate cancer.
  • the prostate cancer is selected from the group consisting of castration-resistant prostate cancer, metastatic castration-resistant prostate cancer, advanced stage prostate cancer, drug-resistant prostate cancer such as anti-androgen-resistant prostate cancer (e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like), docetaxel-resistant prostate cancer, AKR1C3-induced anti-androgen-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • anti-androgen-resistant prostate cancer e.g., enzalutamide-resistant prostate cancer, abiraterone-resistant prostate cancer, bicalutamide-resistant prostate cancer, and the like
  • docetaxel-resistant prostate cancer e.g., AKR1C3-induced anti-androgen-resistant prostate cancer such as AKR1C3-induced enzalutamide-resistant prostate cancer, and combinations thereof.
  • compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in “Remington's Pharmaceutical Sciences” by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, intravenously, parenterally, or rectally.
  • Typical formulations for topical administration include creams, ointments, sprays, lotions, and patches.
  • the pharmaceutical composition can, however, be formulated for any type of administration, e.g., intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices.
  • Formulation for administration by inhalation e.g., aerosol
  • oral or rectal administration is also contemplated.
  • Suitable formulations for transdermal application include an effective amount of one or more compounds described herein, optionally with a carrier.
  • Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host.
  • transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
  • Matrix transdermal formulations may also be used.
  • a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient.
  • the present invention provides tablets and gelatin capsules comprising an AKR1C3 inhibitor, such as indomethacin, and/or an AR-V7 inhibitor, such as niclosamide, alone or in combination with other compounds such as anti-androgen drugs and/or docetaxel, or a dried solid powder of these drugs, together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum, stearic acid, magnesium or calcium salt, metallic stea
  • Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid.
  • the preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound(s).
  • compositions and formulations set forth herein can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
  • Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers.
  • the active ingredient(s) can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
  • a suitable vehicle for example, sterile pyrogen-free water
  • they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient(s).
  • compositions of the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound(s) and a suitable powder base, for example, lactose or starch.
  • compositions set forth herein can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.
  • the active ingredient(s) can be formulated as a depot preparation.
  • Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • one or more of the compounds described herein can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • a pharmaceutical composition or medicament of the present invention can comprise (i) an effective amount of an AR-V7 inhibitor, such as niclosamide, and (ii) optionally a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, and combinations thereof.
  • a pharmaceutical composition or medicament of the present invention can additionally or alternatively comprise (i) an effective amount of an AKR1C3 inhibitor such as indomethacin, and (ii) optionally a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, and combinations thereof.
  • the therapeutic agent(s) may be used individually, sequentially, or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, a compound of the present invention, etc.). Administration may be by the same or different route of administration or together in the same pharmaceutical formulation.
  • compositions or medicaments can be administered to a subject at a therapeutically effective dose to prevent, treat, resensitize, or control prostate cancer as described herein.
  • the pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.
  • a unit dosage for oral administration to a mammal of about 50 to about 70 kg may contain between about 5 and about 500 mg, about 25-200 mg, about 100 and about 1000 mg, about 200 and about 2000 mg, about 500 and about 5000 mg, or between about 1000 and about 2000 mg of the active ingredient.
  • a unit dosage for oral administration to a mammal of about 50 to about 70 kg may contain about 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,250 mg, 1,500 mg, 2,000 mg, 2,500 mg, 3,000 mg, or more of the active ingredient.
  • a dosage of the active compound(s) of the present invention is a dosage that is sufficient to achieve the desired effect.
  • Optimal dosing schedules can be calculated from measurements of active agent accumulation in the body of a subject. In general, dosage may be given once or more of daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.
  • Optimum dosages, toxicity, and therapeutic efficacy of the compositions of the present invention may vary depending on the relative potency of the administered composition and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD 50 /ED 50 .
  • Agents that exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects.
  • Optimal dosing schedules can be calculated from measurements of active ingredient accumulation in the body of a subject. In general, dosage is from about 1 ng to about 1,000 mg per kg of body weight and may be given once or more daily, weekly, monthly, or yearly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. One of skill in the art will be able to determine optimal dosing for administration of AR-V7 inhibitors, such as niclosamide, or AKR1C3 inhibitors, such as indomethacin, to a human being following established protocols known in the art and the disclosure herein.
  • AR-V7 inhibitors such as niclosamide, or AKR1C3 inhibitors, such as indomethacin
  • the data obtained from, for example, animal studies can be used to formulate a dosage range for use in humans.
  • the dosage of compounds of the present invention lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage can vary within this range depending upon the dosage form employed and the route of administration.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC).
  • HPLC high performance liquid chromatography
  • the dose equivalent of a chimeric protein, preferably a composition is from about 1 ng/kg to about 100 mg/kg for a typical subject.
  • a single oral dose of 5 mg/kg niclosamide in rats can generate a maximal plasma concentration of 1.08 ⁇ mol/mL
  • a typical composition of the present invention for oral or intravenous administration can be about 0.1 to about 10 mg of active ingredient per patient per day; about 1 to about 100 mg per patient per day; about 25 to about 200 mg per patient per day; about 50 to about 500 mg per patient per day; about 100 to about 1000 mg per patient per day; or about 1000 to about 2000 mg per patient per day.
  • Exemplary dosages include, but are not limited to, about 10 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1,000 mg, 1,250 mg, 1,500 mg, 2,000 mg, 2,500 mg, 3,000 mg, or more of the active ingredient per patient per day.
  • Exemplary doses of the compositions described herein include milligram or microgram amounts of the composition per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a composition depend upon the potency of the composition with respect to the desired effect to be achieved. When one or more of these compositions is to be administered to a mammal, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained.
  • the specific dose level for any particular mammal subject will depend upon a variety of factors including the activity of the specific composition employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.
  • a pharmaceutical composition or medicament of the present invention is administered, e.g., in a daily dose in the range from about 1 mg of compound per kg of subject weight (1 mg/kg) to about 1 g/kg.
  • the dose is a dose in the range of about 5 mg/kg to about 500 mg/kg.
  • the dose is about 10 mg/kg to about 250 mg/kg.
  • the dose is about 25 mg/kg to about 150 mg/kg.
  • a preferred dose is about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 25, 30, 40, or 50 mg/kg.
  • the daily dose can be administered once per day or divided into subdoses and administered in multiple doses, e.g., twice, three times, or four times per day.
  • compositions described herein may be administered in different amounts and at different times.
  • certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or malignant condition, previous treatments, the general health and/or age of the subject, and other diseases present.
  • treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or, preferably, can include a series of treatments.
  • compositions set forth herein may be administered for at least three consecutive days, often for at least five consecutive days, more often for at least ten, and sometimes for 20, 30, 40 or more consecutive days. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every other day, every third day, or, if higher dose ranges are employed and tolerated by the subject, once a week.
  • the AR-V7 inhibitor niclosamide is orally administered.
  • the niclosamide is orally administered to a subject (e.g., an adult human) at a daily dose of approximately 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 1,250; 1,500; 1,750; 2,000; 2,500; 3,000; 3,500; 4,000; 4,500; 5,000; or more mg of niclosamide per day.
  • the niclosamide is orally administered to a subject (e.g., an adult human) at a daily dose of between 1,000 and 2,000 mg per day.
  • the AKR1C3 inhibitor indomethacin is orally administered.
  • the indomethacin is orally administered to a subject (e.g., an adult human) at a daily dose of approximately 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 500, or more mg of indomethacin per day.
  • the indomethacin is orally administered to a subject (e.g., an adult human) at a daily dose of between 25 and 200 mg per day.
  • the indomethacin and the niclosamide are orally co-administered.
  • the indomethacin can be co-administered at a daily oral dose of between 25 and 200 mg per day with niclosamide at a daily oral dose of between 1000 and 2000 mg per day.
  • the methods comprise sequentially administering an AR-V7 inhibitor, such as niclosamide, followed by a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and docetaxel. In some embodiments, the methods comprise sequentially administering a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and docetaxel followed by an AR-V7 inhibitor, such as niclosamide.
  • an AR-V7 inhibitor such as niclosamide
  • the methods comprise administering an AR-V7 inhibitor, such as niclosamide, in combination with a compound selected from the group consisting of bicalutamide, enzalutamide, abiraterone, and docetaxel.
  • an AR-V7 inhibitor such as niclosamide
  • the amount of bicalutamide, enzalutamide, aberiterarone, or docetaxel is less than the amount of bicalutamide, enzalutamide, aberiterarone, or docetaxel that would be administered to a patient having prostate cancer than would be adminstered if an AR-V7 inhibitor, such as niclosamide, were not adminstered to the patient.
  • the methods comprise sequentially administering an AKR1C3 inhibitor, such as indomethacin, followed by a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and docetaxel.
  • the methods comprise sequentially administering a compound selected from the group consisting of enzalutamide, abiraterone, bicalutamide, and docetaxel followed by an AKR1C3 inhibitor, such as indomethacin.
  • the methods comprise administering an AKR1C3 inhibitor, such as indomethacin, in combination with a compound selected from the group consisting of bicalutamide, enzalutamide, abiraterone, and docetaxel.
  • an AKR1C3 inhibitor such as indomethacin
  • the amount of bicalutamide, enzalutamide, aberiterarone, or docetaxel is less than the amount of bicalutamide, enzalutamide, aberiterarone, or docetaxel that would be administered to a patient having prostate cancer than would be adminstered if the AKR1C3 inhibitor were not adminstered to the patient.
  • a pharmaceutical composition or medicament is administered to a patient at a therapeutically effective dose to prevent, treat, or control prostate cancer.
  • the pharmaceutical composition or medicament is administered to a patient in an amount sufficient to elicit an effective therapeutic or diagnostic response in the patient.
  • An effective therapeutic or diagnostic response is a response that at least partially arrests or slows the symptoms or complications of prostate cancer. An amount adequate to accomplish this is defined as “therapeutically effective dose.”
  • an efficacious or effective amount of an composition is determined by first administering a low dose or small amount of the composition, and then incrementally increasing the administered dose or dosages, adding a second or third medication as needed, until a desired effect of is observed in the treated subject with minimal or no toxic side effects.
  • compositions are administered depending on the dosage and frequency as required and tolerated by the patient.
  • the composition should provide a sufficient quantity of the compositions of this invention to effectively treat the patient.
  • the dose is sufficient to treat or ameliorate symptoms or signs of disease without producing unacceptable toxicity to the patient.
  • kits, systems, and compositions can be prepared according to the present invention, depending upon the intended user of the kit and system and the particular needs of the user.
  • the present invention provides a kit that includes an AR-V7 inhibitor, such as niclosamide, and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the kit includes an AR-V7 inhibitor, such as niclosamide, and enzalutamide.
  • the kit includes an AR-V7 inhibitor, such as niclosamide, and abiraterone.
  • the kit includes an AR-V7 inhibitor, such as niclosamide, and docetaxel. In some other embodiments, the kit includes an AR-V7 inhibitor, such as niclosamide, and bicalutamide. In some other embodiment, the kit includes an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin.
  • the present invention provides a kit that includes an AKR1C3 inhibitor, such as indomethacin, and a compound selected from the group consisting of enzalutamide, abiraterone, docetaxel, bicalutamide, and combinations thereof.
  • the kit includes the AKR1C3 inhibitor, such as indomethacin, and enzalutamide.
  • the kit includes the AKR1C3 inhibitor, such as indomethacin, and abiraterone.
  • the kit includes the AKR1C3 inhibitor, such as indomethacin, and docetaxel.
  • the kit includes the AKR1C3 inhibitor, such as indomethacin, and bicalutamide. In other embodiments, the kit includes the AKR1C3 inhibitor, such as indomethacin, and an AR-V7 inhibitor, such as niclosamide.
  • kits described herein include a label describing a method of administering an AR-V7 inhibitor, such as niclosamide.
  • the label may describe oral administration of an AR-V7 inhibitor, such as niclosamide.
  • kits described herein include a label describing a method of administering an AKR1C3 inhibitor, such as indomethacin.
  • the label may describe oral administration of an AKR1C3 inhibitor, such as indomethacin.
  • kits described herein include a label describing a method of preventing, treating, or controlling prostate cancer, e.g., castration-resistant prostate cancer or anti-androgen-resistant prostate cancer.
  • compositions of the present invention including but not limited to compositions including an AR-V7 inhibitor, such as niclosamide, or compositions including an AKR1C3 inhibitor, such as indomethacin, may, if desired, be presented in a bottle, jar, vial, ampoule, tube, or other container-closure system approved by the Food and Drug Administration (FDA) or other regulatory body, which may provide one or more dosages containing the active ingredient.
  • the package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, the notice indicating approval by the agency.
  • the kit may include a formulation or composition as taught herein, a container closure system including the formulation or a dosage unit form including the formulation, and a notice or instructions describing a method of use as taught herein.
  • the kit includes a container which is compartmentalized for holding the various elements of a formulation (e.g., the dry ingredients and the liquid ingredients) or composition, instructions for making the formulation or composition, and instructions for preventing, treating, or controlling prostate cancer, e.g., castration-resistant prostate cancer, anti-androgen-resistant prostate cancer, or a combination thereof.
  • the kit may include the pharmaceutical preparation in dehydrated or dry form, with instructions for its rehydration (or reconstitution) and administration.
  • Kits with unit doses of the active composition e.g. in oral, rectal, transdermal, or injectable doses (e.g., for intramuscular, intravenous, or subcutaneous injection), are provided.
  • injectable doses e.g., for intramuscular, intravenous, or subcutaneous injection
  • the containers containing the unit doses will be an informational package insert describing the use and attendant benefits of the composition in preventing, treating, or controlling prostate cancer, e.g., castration-resistant prostate cancer, anti-androgen-resistant prostate cancer, or a combination thereof.
  • Suitable active compositions and unit doses are those described herein.
  • Some embodiments of the present invention include packages that include an AR-V7 inhibitor, such as niclosamide, packaged together with a compound selected from the group consisting of bicalutamide, enzalutamide, aberiterarone, docetaxel, and combinations thereof.
  • Some embodiments of the present invention include packages that include an AKR1C3 inhibitor, such as indomethacin, packaged together with a compound selected from the group consisting of bicalutamide, enzalutamide, aberiterarone, docetaxel, and combinations thereof.
  • Some embodiments of the present invention include packages that include an AR-V7 inhibitor, such as niclosamide, and an AKR1C3 inhibitor, such as indomethacin, packaged together with a compound selected from the group consisting of bicalutamide, enzalutamide, aberiterarone, docetaxel, and combinations thereof.
  • an AR-V7 inhibitor such as niclosamide
  • an AKR1C3 inhibitor such as indomethacin
  • the deletion of LBD results in constitutive activation of the AR in prostate cancer cells.
  • AR-V7 mRNA expression in different cell lines was detected as shown in FIG. 1A .
  • CWR22rv1 and VCaP cells expressed significantly higher AR-V7 than LNCaP and C4-2 cells; the expression level of AR-V7 in CWR22rv1 cells was 25 times higher than in LNCaP and C4-2 cells, whereas VCaP cells exhibited a 15 fold increase in comparison.
  • the results were also confirmed by Western blot, as shown in FIG. 1B , in which CWR22rv1 and VCaP cells expressed higher protein expression levels of AR variants, especially AR-V7, than LNCaP and C4-2 cells.
  • AR-V7 has been shown constitutively active in prostate cancer cells. To confirm these results, EGFP-AR-V7 was transiently transfected into C4-2 cells, and 48 hours later, as shown in FIG. 1C , AR-V7 was only expressed in the nucleus of C4-2 cells suggesting that AR-V7 which lacks the classic NLS (nuclear location sequence) is still capable of being translocated to the nucleus by mechanisms currently unknown. Next, the transcription activity of AR-V7 in the PC3 cell line was confirmed, which is void of AR, and LNCaP cells, which express mutant AR (T877A). As shown in FIGS.
  • AR-V7 was constitutively activated in both PC3 and LNCaP cells and cannot be interrupted by androgen.
  • AR-V7 was stably transfected into C4-2 cells, as shown in FIG. 1F .
  • C4-2 AR-V7 stable clone expressed significantly higher AR-V7 mRNA compared with C4-2 neo cells.
  • the AR-V7 functionally increased PSA mRNA and protein expression in C4-2 cells ( FIGS. 1G and 1H ). Collectively, these data confirmed AR-V7 is constitutively active in prostate cancer cells.
  • LNCaP, C4-2, C4-2B, CWR22rv1, DU145, and 293 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 0.1 mg/ml streptomycin.
  • VCaP cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 0.1 mg/ml streptomycin.
  • C4-2-neo and C4-2 AR-V7 cells were stably transfected with pc DNA3.1 or containing AR-V7 plasmid and maintained in 300 ⁇ g/mL G418 RPMI1640 medium.
  • 293-AR-V7-PSA-E/P-LUC cells were stably transfected with AR-V7 plasmid and PSA-E/P-LUC reporter plasmid and maintained in 300 ⁇ g/mL G418 RPMI1640 medium.
  • LNCaP-neo LNCaP cells stably expressing pcDNA3.1 control vector
  • LNCaP-S17 LNCaP cells stably expressing IL-6
  • LNCaP-STAT3C cells LNCaP cells stably expressing constitutively active STAT3
  • STAT 3 enhances the growth of LNCaP human prostate cancer cells in intact and castrated male nude mice .
  • Human recombinant IL-6 was obtained from R&D Systems (Minneapolis, Minn.).
  • C4-2B cells were incubated with increasing concentrations of enzalutamide (5 ⁇ M ⁇ 40 ⁇ M) over 12 months in FBS and stored for further analysis.
  • the resistant cells were isolated and referred to as C4-2B MR (i.e., C4-2B enzalutamide resistant).
  • Parental C4-2B cells were passaged alongside the enzalutamide treated cells as an appropriate control.
  • C4-2B MR cells were maintained in 20 ⁇ M enzalutamide containing medium. Nicolosamide was purchased from Sigma.
  • siRNA small interfering RNA
  • cells were seeded at a density of 1 ⁇ 10 5 cells per well in 12-well plates or 3 ⁇ 10 5 cells per well in 6-well plates and transfected with siRNA (Dharmacon) targeting the AR Exon7 sequence UCAAGGAACUCGAUCGUAU (SEQ ID NO: 1) or AR-V7 sequence GUAGUUGUAAGUAUCAUGA (SEQ ID NO: 2) or siRNA targeting STAT3 (Cell signaling #6582) using lipofectamine-RNAiMAX (invitrogen).
  • siRNA Dharmacon
  • Cells were transiently transfected with indicated expression plasmids, e.g., AR-V7 or EGFP-AR-V7, using Attractene (QIAGEN).
  • DNA-AR protein complexes were cross-linked inside the cells by the addition of 1% formaldehyde.
  • Whole-cell extracts were prepared by sonication, and an aliquot of the cross-linked DNA-protein complexes was immunoprecipitated by incubation with the AR-specific antibody (AR-441 obtained from Santa Cruz Biotechnology) overnight at 4° C. with rotation.
  • Chromatin-antibody complexes were isolated from solution by incubation with protein A/G agarose beads for 1 hour at 4° C. with rotation.
  • the bound DNA-protein complexes were washed and eluted from beads with elution buffer (1% SDS and 0.1 mol/L NaHCO3), crosslinking was reversed, and DNA was extracted.
  • elution buffer 1% SDS and 0.1 mol/L NaHCO3
  • crosslinking was reversed, and DNA was extracted.
  • the resulting chromatin preparations were analyzed by PCR using primers spanning either the proximal or the distal enhancer AREs of the PSA promoter. Isotype-matched IgG was used as control.
  • LNCaP, C4-2 or 293 cells were transfected with pGL3-PSA6.0-Luc, pGL3-AREI/II-Luc reporters along with AR-V7, for example as indicated in the figures, in FBS or CS-FBS condition.
  • Cell lysates were subjected to luciferase assays with the Luciferase Assay System (Promega).
  • CWR22rv1 cells, C4-2B MR or C4-2B AbiR cells were seeded on 12-well plates at a density of 0.5 ⁇ 10 5 cells/well in RPMI 1640 media containing 10% FBS and treated 0.25 ⁇ M niclosamide with 20 ⁇ M enzalutamide or 20 ⁇ M abiraterone in media containing FBS. Total cell numbers were counted after 2, 4, and 7 days.
  • LNCaP-neo, LNCaP-517, LNCaP-IL6+ or LNCaP-STAT3C cells were seeded in 12-well plates at a density of 1 ⁇ 10 5 cells/well in RPMI 1640 media containing 10% FBS. The cells were treated as indicated and total cell numbers were counted.
  • C4-2 neo, C4-2 AR-V7, CWR22rv1 or C4-2B MR cells were treated with DMSO, 0.5 ⁇ M or 1.0 ⁇ M niclosamide in media containing 10% complete FBS.
  • CWR22rv1 cells or C4-2B MR cells were treated with 0.25 ⁇ M niclosamide with or without 20 ⁇ M enzalutamide.
  • Cells were plated at equal density in 100 mm dishes for 14 days; the medium was changed every 7 days. The colonies were rinsed with PBS before staining with 0.5% crystal violet/4% formaldehyde for 30 min and the number of colonies was counted.
  • LNCaP-neo cells or LNCaP-S17 stable clone cells were treated with DMSO or enzalutamide in media containing 10% complete FBS. Cells were plated at equal density (1000 cells/dish) in 100 mm dishes for 14 days. The colonies were rinsed with PBS before staining with 0.5% crystal violet/4% formaldehyde for 30 min and the number of colonies was counted.
  • C4-2 neo, C4-2 AR-V7, CWR22rv1 or pzHPV7 cells were seeded on 12-well plates (1 ⁇ 10 5 cells/well) in RPMI 1640 media containing 10% FBS and treated with DMSO or 0.5 ⁇ M niclosamide for 48 hours. Mono- and oligonucleosomes in the cytoplasmic fraction were measured by the Cell Death Detection ELISA kit (Roche, Cat. NO. 11544675001) according to the manufacturer's instructions. Floating and attached cells were collected and homogenized in 400 ⁇ L of incubation buffer. The wells were coated with antihistone antibodies and incubated with the lysates, horseradish peroxidase-conjugated anti-DNA antibodies, and the substrate. Absorbance was measured at 405 nm.
  • RNAs were extracted using TriZOL reagent (Invitrogen). cDNAs were prepared after digestion with RNase-free RQ1 DNase (Promega). The cDNAs were subjected to real-time reverse transcription-PCR (RT-PCR) using Sso Fast Eva Green Supermix (Bio-Rad) according to the manufacturer's instructions or as described in Liu C, et al. Andrographolide targets androgen receptor pathway in castrate - resistant prostate cancer , Genes Cancer; 2: 151-9. Each reaction was normalized by co-amplification of actin. Triplicates of samples were run on default settings of Bio-Rad CFX-96 real-time cycler.
  • PSA levels were measured in the culture supernatants using ELISA (United Biotech, Inc., Mountain View, Calif.) according to the manufacturer's instructions.
  • IL-6 secretion 50 ⁇ l of cell culture supernatants were used to determine levels of IL-6 secretion. Secretion of IL-6 by LNCaP-neo and LNCaP-s17 cells was determined by ELISA according to the manufacturer's protocol (eBioscience, San Diego, Calif.).
  • the Prestwick Chemical Library® contains 1200 small molecules (FDA, EMEA and other agencies).
  • the active compounds were selected for their high chemical and pharmacological diversity as well as for their known bioavailability and safety in humans.
  • the present assay identified FDA-approved drugs that could target AR-V7 activity.
  • the drug screening was conducted using luciferase activity assay to determine the AR-V7 activity 24 hours after treated with the library's compound.
  • 293 cell line were used which were AR void. 293 cells were stable transfected with EGFP-AR-V7 plasmid and PSA-E/P-luciferase reporter plasmid, and stable clones were selected by G418, as shown in FIGS. 8A-8E and FIG. 12 .
  • 293 AR-V7-PSA-luc stable clones more greatly expressed AR-V7 protein compared with 293 neo cells.
  • CWR22rv1 cells were used as a positive control.
  • the nuclear translocation was examined under the fluorescence microscope.
  • FIG. 8B 293 AR-V7-PSA-luc stable clone highly expressed AR-V7 in the nucleus.
  • the AR-V7 expression was also confirmed by qRT-PCR, where 293 AR-V7-PSA-luc stable clones expressed 140 fold higher AR-V7 mRNA level compared to 293 neo cells although both cells don't express full length AR ( FIG. 8C ).
  • luciferase assay was again performed and the compounds which significantly inhibited cell growth were excluded on the first round of screening. After the second round, high toxicity compounds were diluted further to screen again, and from this array of criteria, niclosamide was selected as a drug of interest.
  • C4-2 neo, C4-2 AR-V7, CWR22rv1 and pz-HPV7 cells were treated with DMSO or 0.5 ⁇ M niclosamide for 48 hours, as shown in FIG. 2A .
  • 0.5 ⁇ M niclosamide significantly inhibited cell growth in prostate cancer cells and had little effects on pz-HPV7 normal prostate epithelial cells.
  • cell death ELISA was performed, as shown in FIG. 2B .
  • 0.5 ⁇ M niclosamide significantly induced cell apoptosis in prostate cancer cells and had little effect on pz-HPV7 cells.
  • niclosamide significantly inhibited prostate cancer cells clonogenic ability in a dose dependent manner which confirmed niclosamide has the great potential to be a candidate of prostate cancer therapy.
  • niclosamide significantly inhibited clonogenic ability of prostate cancer cells in a dose dependent manner. As shown in FIGS. 37 and 38 , niclosamide significantly inhibits colony formation in C42B MR cells. Moreover, niclosamide significantly enhances enzalutamide inhibition of colony formation in C42B MR and CWR22rv1 cells. These results revealed that niclosamide inhibited prostate cancer cell growth and induced cell apoptosis with minimal effects on normal prostate epithelial cells.
  • CWR22rv1 cells were transiently transfected with AR exon7 siRNA or AR-V7 siRNA in CS-FBS condition.
  • Cell numbers were counted on subsequent days, as shown in FIG. 10A , and knocked-down full length AR had moderate growth inhibition effects on CWR22rv1 cells while knocked-down AR-V7 significantly inhibited cell growth.
  • the knock down effects were confirmed by Western blot ( FIG. 10B ) suggesting AR-V7 function is important to prostate cancer cell growth and targeting AR-V7 is an effective strategy to treat CRPC patients.
  • niclosamide affects AR-V7 expression
  • CWR22rv1 cells which express high endogenous AR-V7
  • niclosamide inhibited AR-V7 protein in a dose dependent manner.
  • 0.5 ⁇ M niclosamide significantly inhibited AR-V7 expression but had little effects on full length AR (AR FL) expression, which suggested that niclosamide has a greater effect on truncated AR inhibition.
  • Niclosamide also inhibited AR-V7 protein expression in a time dependent manner ( FIG. 3B ).
  • niclosamide decreases AR-V7 protein expression
  • the effects of niclosamide on AR-V7 expression were determined at the transcriptional level. As shown in FIG. 3C , niclosamide did not affect AR-V7 or full length AR mRNA level, suggesting that niclosamide did not affect AR-V7 expression at the transcriptional level.
  • the effect of niclosamide on AR-V7 protein degradation was examined after new protein synthesis was blocked by cycloheximide as a potential mechanism for down regulation of AR-V7 protein level.
  • the protein synthesis inhibitor cycloheximide 50 ⁇ g/mL was added with or without 2 ⁇ M niclosamide at time 0 hour.
  • This assay confirms that enzalutamide cannot inhibit AR-V7 transcriptional activity and also cannot reduce recruitment of AR-V7 to the PSA promoter in prostate cancer cells ( FIG. 9 ).
  • niclosamide has an effect on AR-V7 transcriptional activity
  • the 293 AR-V7-PSA-E/P-LUC cell system was used, as shown in FIG. 4A , and niclosamide significantly inhibited AR-V7 transcription activity while enzalutamide had no effect.
  • LNCaP cells were transiently transfected with AR-V7 following treatment with niclosamide or enzalutamide with or without DHT overnight, as shown in FIG. 4B .
  • niclosamide To further dissect the mechanism of AR-V7 inhibition effects by niclosamide, a ChIP assay was performed. C4-2 AR-V7 cells were cultured in CS-FBS condition for 3 days, following treatment with 1 ⁇ M niclosamide or 20 ⁇ M enzalutamide overnight; whole cell lysis was subjected to ChIP assay, as shown in FIG. 4D , and niclosamide significantly reduced recruitment of AR-V7 to PSA promoter while enzalutamide had no effect. Collectively, the results confirmed niclosamide has the potential to be a new AR-V7 inhibitor on both the protein and mRNA level.
  • AR variants have been shown to be one of the possible mechanisms to induce enzalutamide resistance, but to date, there is little evidence that enzalutamide induces AR variants in prostate cancer cells or patients.
  • C4-2B cells were chronically treated with enzalutamide in FBS condition, as shown in FIGS. 5A and 5B .
  • C4-2B MR (C4-2B enzalutamide resistant) cells exhibited more resistance than C4-2B parental cells.
  • FIG. 5C we examined the AR variants level in C4-2B parental and C4-2B MR cells, as shown in FIG. 5C .
  • C4-2B MR cells express higher AR variant mRNA level than C4-2B parental cells, including AR-V1, AR-V7, AR1/2/2b and AR1/2/3/2b. The results were also confirmed by Western blot, as shown in FIG. 5D .
  • C4-2B MR cells expressed significantly higher AR-V7 in the nucleus compared to C4-2B parental cells. Full length AR was also up-regulated in C4-2B MR cells which suggested overexpression of AR signaling may be an important mechanisms of enzalutamide resistance. Knocked down AR-V7 significantly reversed enzalutamide resistance in C4-2B MR cells ( FIG. 5E ), suggesting AR-V7 is the major driver of enzalutamide resistance in C4-2B MR cells.
  • C4-2B MR cells were resistant to enzalutamide but significantly inhibited by niclosamide in a dose dependent manner.
  • the results were also confirmed by clonogenic assay, as shown in FIG. 5G , where 0.5 ⁇ M niclosamide significantly inhibited C4-2B MR cells colony formation and colony size.
  • niclosamide but not enzalutamide significantly inhibited colony formation by C4-2B MR cells.
  • niclosamide but not enzalutamide significantly inhibited the colony size of the C4-2B MR cells.
  • combination treatment with niclosamide and enzalutamide significantly inhibits cell growth in C4-2B, C4-2B MR, CB-2B TAXR, and C4-2B TAXR-MR cells.
  • niclosamide enhances the effect of enzalutamide on growth or viability of C4-2B and C4-2B MR cells.
  • niclosamide enhances the effect of arbiraterone on growth or viability of C4-2B and C4-2B AbiR cells.
  • AR variants have shown induced enzalutamide resistance in CWR22rv1 cells.
  • This assay confirmed AR-V7 dominated cell growth in CWR22rv1 cells instead of full length AR, and CWR22rv1 cells were more resistant than LNCaP and C4-2B cells treated by abiraterone ( FIG. 11A ).
  • AR-V7 specific siRNA or AR exon 7 siRNA were transiently transfected into CWR22rv1 cells following treatment with 10 ⁇ M abiraterone for 48 hours, as shown in FIG. 11B .
  • AR-V7 level in the CWR22rv1 cells treated with niclosamide and enzalutamide was examined, as shown in FIGS. 7A and 15 .
  • Single treatments with niclosamide reduced AR-V7 expression but when combined with enzalutamide or abiraterone the inhibition effects were significantly enhanced, suggesting inhibited AR-V7 expression could be a direct therapy strategy for enzalutamide or abiraterone resistance.
  • the results were also confirmed in C4-2B MR cells ( FIG. 7B ).
  • the results suggested niclosamide as a novel AR-V7 inhibitor could be a potent drug for advanced prostate cancer patients, especially to those who are resistant to enzalutamide or abiraterone.
  • Sensitivity of prostate cancer cells to enzalutamide was tested using cell growth assays and clonogenic assays. Quantitative reverse transcription-PCR, ELISA and Western blotting were performed to detect expression levels of IL-6, c-Myc, survivin and AR. Expression of STAT3 was downregulated using siRNA specific to STAT3. ChIP assay was performed to examine recruitment of AR to the PSA promoter.
  • the STAT3 inhibitor niclosamide reversed enzalutamide resistance in prostate cancer cells, while combination treatment with enzalutamide and niclosamide significantly inhibited cell growth, induced cell apoptosis and inhibited colony formation.
  • the autocrine IL-6 pathway induces enzalutamide resistance in prostate cancer cells via the constitutive activation of STAT3.
  • LNCaP-neo, LNCaP-s17 or LNCaP-STAT3C cells were treated as indicated.
  • DNA-AR protein complexes were cross-linked inside the cells by the addition of 1% formaldehyde.
  • Whole-cell extracts were prepared by sonication, and an aliquot of the cross-linked DNA-protein complexes was immunoprecipitated by incubation with the AR-specific antibody (AR-C19; Santa Cruz Biotechnology) overnight at 4° C. with rotation.
  • Chromatin-antibody complexes were isolated from solution by incubation with protein A/G agarose beads for 1 hour at 4° C. with rotation.
  • the bound DNA-protein complexes were washed and eluted from beads with elution buffer (1% SDS and 0.1 mol/L NaHCO3), cross links were reversed, and DNA was extracted.
  • elution buffer 1% SDS and 0.1 mol/L NaHCO3
  • cross links were reversed, and DNA was extracted.
  • the resulting chromatin preparations were analyzed by PCR using primers spanning either the proximal or the distal enhancer AREs of the PSA promoter. Isotype-matched IgG was used as control.
  • LNCaP-s17 LNCaP-s17 cells
  • LNCaP-s17 LNCaP-s17 cells
  • This assay tested whether expression of IL-6 affects the response of prostate cancer cells to enzalutamide, LNCaP-s17 cells were treated with increasing doses of enzalutamide and cell numbers were counted.
  • LNCaP-neo cells were highly sensitive to enzalutamide treatment compared to LNCaPs17 cells.
  • Enzalutamide at a concentration of 5 ⁇ M reduced the growth of LNCaP-neo cells by more than 30%, while it had almost no effect on the growth of LNCaP-s17 cells. Even at a higher concentration of enzalutamide (40 ⁇ M), the growth of LNCaP-s17 cells was only reduced by about 30% compared to almost 60% reduction in LNCaP-neo cells.
  • LNCaP-neo cells and LNCaP-s17 cells were treated with 20 ⁇ M enzalutamide and clonogenic ability was determined. As shown in FIGS. 26A-26C , the colony formation ability was significantly inhibited in LNCaP-neo cells treated with 20 ⁇ M enzalutamide, while LNCaP-s17 cells continued to grow and form colonies.
  • LNCaP-IL6+ cells LNCaP cells expressing IL-6 by long-term culture of LNCaP cells in media containing IL-6, were treated with 10 ⁇ M and 20 ⁇ M enzalutamide in media containing complete FBS for 48 hours.
  • enzalutamide significantly inhibited growth of LNCaP cells.
  • enzalutamide had little effect on the growth of LNCaP-IL6+ cells.
  • LNCaP-s17 cells express constitutively activated STAT3 (STAT3 phosphorylated at Tyr705) and express higher levels of AR, c-Myc, survivin, and Bcl-2 proteins than LNCaP-neo cells. Consistent with the protein levels, LNCaP-s17 cells express higher levels of c-Myc and survivin mRNA than LNCaP-neo cells ( FIGS. 27B and 27C ).
  • LNCaP-s17 cells expressed higher levels of IL-6 mRNA and protein than LNCaP-neo cells ( FIGS. 27D and 27E ).
  • This assay also determined whether constitutively active STAT3 increases the recruitment of AR to the ARE sites.
  • ChIP assay was performed in LNCaP, LNCaP-s17 and LNCaP-STAT3C cells. As shown in FIG. 27F , both LNCaP-s17 cells and LNCaP-STAT3C cells showed enhanced recruitment of AR to both the proximal binding site (AREI/II) and the distal enhancer binding site (AREIII) of the PSA promoter compared to LNCaP-neo cells.
  • AREI/II proximal binding site
  • AREIII distal enhancer binding site
  • LNCaP-neo and LNCaP-s17 cells were transfected with siRNA specific to STAT3.
  • FIG. 28A knockdown of STAT3 expression in LNCaP-s17 cells resensitized the cells to enzalutamide treatment.
  • the knock down effects were confirmed by Western blotting using p-STAT3 (Tyr705) and STAT3 antibodies ( FIG. 28B ).
  • LNCaP cells expressing constitutively active STAT3 (STAT3C) exhibited increased resistance to enzalutamide ( FIG. 28C ).
  • enzalutamide One of the mechanisms of action of enzalutamide is to inhibit the recruitment of the AR to AREs in promoters of target genes.
  • ChIP assay was performed. As shown in FIG. 28D , enzalutamide significantly inhibited recruitment of the AR to AREs in the PSA promoter (both proximal binding site and distal binding site) in LNCaP-neo cells. In contrast, enzalutamide had little effect on the recruitment of the AR to the AREs in LNCaP-s17 cells and LNCaP-STAT3C cells.
  • This assay tested whether a small non-peptide drug, niclosamide ( FIG. 29A ), which has been shown previously to be able to inhibit STAT3 expression in DU145 cells, is able to reverse enzalutamide resistance in prostate cancer cells.
  • LNCaP-s17 cells were treated with 20 ⁇ M enzalutamide and 0.25 ⁇ M niclosamide individually or in combination for different time points.
  • the growth of LNCaP-s17 cells was significantly inhibited by enzalutamide in the presence of niclosamide, while treatment with either enzalutamide or niclosamide alone had little effect on cell growth ( FIG. 29E ).
  • the clonogenic ability of LNCaP-s17 cells was significantly inhibited by enzalutamide in the presence of niclosamide ( FIG. 29F ).
  • This example shows that the IL6-STAT3 axis is involved in the development of resistance to enzalutamide in prostate cancer.
  • this example shows that targeting the IL-6-STAT3 axis may be a potential therapeutic strategy in patients resistant to enzalutamide.
  • niclosamide inhibits cell growth, migration, invasion, and colony formation in prostate cancer cells. The effect appears to be dose dependent. As shown in FIG. 24 , niclosamide inhibits cell migration or invasion in LNCap-s17 cells, and the effect is enhanced when niclosamide is used in combination with enzalutamide. As shown in FIG. 25 , niclosamide inhibits cell migration or invasion in LNCap-Stat3C cells, and the effect is enhanced when niclosamide is used in combination with enzalutamide.
  • This assay tested whether niclosamide overcomes enzalutamide resistance of prostate cancer in vivo.
  • xenografts generated from CWR22rv1 cells were treated with vehicle, enzalutamide, niclosamide or their combination for 3 weeks.
  • CWR22rv1 cells (3 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of 6-7 week male SCID mice.
  • Tumor-bearing mice (tumor volume around 50-100 mm 3 ) were randomized into four groups and treated as follows: (1) vehicle control (5% Tween 80 and 5% ethanol in PBS, i.p.), (2) enzalutamide (25 mg/kg, p.o.), (3) niclosamide (25 mg/kg, i.p.), (4) enzalutamide (25 mg/kg, p.o.)+niclosamide (25 mg/kg, i.p.).
  • Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width 2 /2. Tumor tissues were harvested after 3 weeks of treatment.
  • CWR22rv1 cells that were resistant to enzalutamide treatment showed tumor volumes comparable to those in the vehicle treated control group.
  • Niclosamide alone decreased the tumor volume while the combination of niclosamide and enzalutamide synergistically decreased CWR22rv1 tumors.
  • This assay indicates that niclosamide can overcome enzalutamide resistance and restore sensitivity of CWR22rv1 xenografts to enzalutamide in vivo.
  • these results demonstrate that niclosamide can improve enzalutamide treatment and overcome enzalutamide resistance.
  • CWR22Rv1 cells (4 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of male SCID mice, tumor-bearing mice (tumor volume around 50-75 mm 3 ) were treated 5 days per week as follows: Control: (5% PGE8000 in H2O p.o B.I.D.), niclosamide (200 mg/kg p.o B.I.D.). Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width2/2. Tumor tissues were harvested after 3 weeks of treatment. The results are depicted in FIGS. 40A-40D . As shown in FIGS.
  • niclosamide significantly inhibited Rv1 xenograft tumor growth when administered orally.
  • the dosage of niclosamide was well-tolerated as illustrated by maintenance of body weight in comparison to control mice that did not receive niclosamide.
  • niclosamide significantly enhanced bicalutamide effects in a dose ( FIG. 41A ) and time ( FIG. 41B ) dependent manner in CWR22Rv1 cells in vitro.
  • Niclosamide also enhanced bicalutamide treatment of CWR22Rv1 cells in vivo in a mouse xenograft model.
  • CWR22Rv1 cells (4 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of male SCID mice.
  • Tumor-bearing mice (tumor volume around 50-75 mm 3 ) were treated 5 days per week as follows: Control: (0.5% weight/volume (w/v) Methocel A4M p.o and 5% Tween 80 and 5% ethanol in PBS, i.p.), Bicalutamide (25 mg/kg p.o), Niclosamide (25 mg/kg i.p.) and Combination (25 mg/kg Bicalutamide p.o+25 mg/kg Niclosamide i.p.).
  • FIGS. 42A-42D Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width2/2. Tumor tissues were harvested after 3 weeks of treatment. The results are depicted in FIGS. 42A-42D .
  • niclosamide significantly enhanced bicalutamide effects on CWR22Rv1 cells in vivo in a mouse xenograft model.
  • the dosage of niclosamide, whether alone or in combination with bicalutamide, was well-tolerated as illustrated by maintenance of body weight in comparison to control mice that did not receive niclosamide.
  • PCa prostate cancer
  • CRPC castrate-resistant prostate cancer
  • Enzalutamide a second-generation antiandrogen, was recently approved for the treatment of castration resistant prostate cancer (CRPC) in patients. Despite these advances that provide temporary respite, resistance to enzalutamide occurs frequently.
  • AR variants expression (Antonarakis et al., 2014; Li et al., 2013; Liu et al., 2014a), IL6-STAT3-AR axis activation (Liu et al., 2014b), AR F876L mutation (Joseph et al., 2013; Korpal et al., 2013) and glucocorticoid receptor (GR) overexpression (Arora et al., 2013; Isikbay et al., 2014).
  • Intratumoral androgen biosynthesis has been well characterized as a mechanism of CRPC (Cai et al., 2011; Ishizaki et al., 2013; Locke et al., 2008; Mohler et al., 2011), but its role in enzalutamide resistance is yet to be understood.
  • Clinical reports have shown that patients treated with enzalutamide have elevated testosterone levels in the bone marrow (Efstathiou et al., 2014; Efstathiou E, 2011).
  • a cascade of enzymes is involved in the biosynthesis of intratumoral androgens, including CYP17A1, HSD3B and AKR1C3.
  • Aldo-keto reductase family 1 member C3 (AKR1C3) is a multi-functional enzyme and is one of the most important genes involved in androgen synthesis and metabolism. AKR1C3 facilitates the conversion of weak androgens androstenedione (A′ dione) and 5 ⁇ -androstanedione (5 ⁇ -dione) to the more active androgens testosterone and DHT respectively (Bauman et al., 2006; Labrie et al., 1997).
  • C4-2B MDVR enzalutamide resistant prostate cancer cells
  • LNCaP cells are sensitive to enzalutamide, while CWR22Rv1 and LN-95 cells are resistant to enzalutamide treatment, consistent with previously published studies (Dehm et al., 2008; Hu et al., 2013; Nadiminty et al., 2013).
  • Intratumoral androgen biosynthesis has been well characterized as a mechanism of CRPC (Cai et al., 2011; Fankhauser et al., 2014; Ishizaki et al., 2013; Locke et al., 2008; Mohler et al., 2011), but its role in enzalutamide resistance has been unclear.
  • microarray analysis was performed on enzalutamide resistant C4-2B-MDVR cells and enzalutamide sensitive C4-2B parental cells. Expression of transcripts encoding for steroid hormone biosynthesis was analyzed by gene set enrichment.
  • CYP17A1, HSD3B1, HSD3B2, HSD17B3, SRD5A1, AKR1C1/2 and AKR1C3 mRNA levels were measured using specific primers by qRT-PCR. As shown in FIG. 44B left, the levels of mRNA expression were consistent with the microarray data. The results were also confirmed by western blot, as shown in FIG. 44B right, C4-2B MDVR cells express significantly higher levels of AKR1C3, HSD3B and CYP17A1 proteins compared to C4-2B parental cells. These results show that androgen synthesis signaling is upregulated in enzalutamide resistant prostate cancer cells.
  • AKR1C3 is Highly Expressed in Metastatic and Recurrent Prostate Cancer and Enzalutamide Resistant Prostate Xenograft Tumors
  • AKR1C3 was found to be up regulated by more than 16-fold in enzalutamide resistant C4-2B MDVR cells compared to the C4-2B parental cells. AKR1C3 expression was examined in the following different prostate cancer cell lines: VCaP, CWR22Rv1, LNCaP, LN-95, C4-2B and C4-2B-MDVR cells. C4-2B MDVR, CWR22Rv1, and LN-95 cells are resistant to enzalutamide while C4-2B and LNCaP cells are sensitive to enzalutamide. As shown in FIG.
  • C4-2B MDVR, VCaP, CWR22Rv1 and LN-95 cells all express significantly higher levels of AKR1C3; C4-2B MDVR, CWR22Rv1 and LN-95 cells express higher levels of HSD3B; C4-2B MDVR and LN-95 cells also expressed higher levels of CYP17A1.
  • AKR1C3 expression was also examined in tumor xenografts by IHC, as shown in FIG. 45B , C4-2B MDVR and CWR22Rv1 tumors express higher levels of AKR1C3 compared to C4-2B parental tumors.
  • AKR1C3 was highly expressed in late stage prostate cancer and in enzalutamide resistant prostate cancer xenografts.
  • AKR1C3 (also named 17 ⁇ HSD5) is one of the most important genes involved in androgen synthesis and metabolism. AKR1C3 facilitates the conversion of weak androgens androstenedione (A′ dione) and 5 ⁇ -androstanedione (5 ⁇ -dione) to the more active androgens, testosterone and DHT respectively.
  • A′ dione weak androgens androstenedione
  • 5 ⁇ -dione 5 ⁇ -androstanedione
  • C4-2B parental and C4-2B MDVR cells were cultured in serum free and phenol red free medium for 5 days, and steroid metabolites were extracted from 50 ⁇ 10 6 cells and subjected to LC-MS analysis.
  • C4-2B MDVR cells synthesize extremely high levels of testosterone (131.025 vs. 0.15 pg/50 million cells), dihydrotestosterone (17.55 vs. 0 pg/50 million cells), and DHEA (72.075 vs. 0 pg/50 million cells), compared to C4-2B parental cells. Intriguingly, the active estrogen metabolite estradiol was significantly reduced (82.725 vs.
  • AKR1C3 is upregulated in enzalutamide resistant prostate cancer cells and in late stage prostate cancer patients. It was found that AKR1C3 was sufficient to confer resistance to enzalutamide in prostate cancer cells.
  • CWR22Rv1 cells or C4-2B MDVR cells were transiently transfected with control shRNA or AKR1C3 shRNA following treatment with enzalutamide for three days. As shown in FIGS.
  • CWR22Rv1 and C4-2B MDVR cells are resistant to enzalutamide, while knock down of AKR1C3 expression by two independent shRNAs (#561 and #694) restored their sensitivity to enzalutamide.
  • the down regulation of AKR1C3 by shRNA was confirmed by western blot ( FIG. 47C ).
  • LNCaP cells stably expressing AKR1C3 LNCaP-AKR1C3 were also generated to test whether exogenous expression of AKR1C3 induces enzalutamide resistance.
  • LNCaP-AKR1C3 and LNCaP-neo vector control cells were treated with different concentrations of enzalutamide for 48 hours and cell numbers were counted. As shown in FIG. 47D , LNCaP-AKR1C3 cells exhibited greater resistance to enzalutamide than LNCaP-neo cells. These results were also confirmed by clonogenic ability assay. LNCaP-AKR1C3 cells showed significantly more clonogenic ability than the control LNCaP-neo cells in response to enzalutamide treatment ( FIGS. 47E and 47F ).
  • Indomethacin a non-steroidal anti-inflammatory drug (NSAID) used for reducing fever, pain and inflammation, has been shown to be able to inhibit AKR1C3 activity (Cai et al., 2011; Flanagan et al., 2012; Liedtke et al., 2013).
  • NSAID non-steroidal anti-inflammatory drug
  • indomethacin was used to hinder AKR1C3 activation and the effects on the response of PCa cells to enzalutamide treatment were examined in vitro and in vivo. As shown in FIG.
  • indomethacin did not have an effect on CWR22Rv1 cell growth at 10 ⁇ M but inhibited cell growth marginally at 20 ⁇ M.
  • combination of indomethacin with enzalutamide significantly inhibited the growth of enzalutamide resistant CWR22Rv1 cells.
  • the results were also confirmed by clonogenic assay.
  • FIG. 48A right combination of indomethacin with enzalutamide significantly inhibited colony numbers and reduced colony size in CWR22Rv1 cells. Similar results were also obtained in C4-2B MDVR cells ( FIG. 48B ).
  • CWR22Rv1 xenograft model was used. As shown in FIG. 48C , while CWR22Rv1 tumors were resistant to enzalutamide treatment, indomethacin significantly inhibited tumor growth. Combination of indomethacin with enzalutamide further inhibited tumor growth of CWR22Rv1 xenografts. Immunohistochemical staining of Ki67 showed that cell proliferation was significantly inhibited by indomethacin, and further inhibited by the combination treatment ( FIG. 48D ).
  • the second generation androgen antagonist enzalutamide represents an improvement in therapy options for late stage metastatic CRPC (Scher et al., 2010; Scher et al., 2012).
  • the initial responders develop resistance inevitably.
  • the potential mechanisms associated with enzalutamide resistance have been the focus of intense investigation.
  • AKR1C3 activation and elevated intracrine androgens were identified as potential mechanisms contributing to enzalutamide resistance. This study demonstrates that AKR1C3 is overexpressed in enzalutamide resistant prostate cancer cells.
  • AKR1C3 overexpression of AKR1C3 confers resistance to enzalutamide, while down regulation of AKR1C3 sensitizes PCa cells to enzalutamide treatment.
  • overexpression of AKR1C3 is demonstrated in clinical metastatic prostate cancer and correlated with disease progression. It is also demonstrated that intracrine steroids including androgens are elevated in enzalutamide resistant cells, possibly through increased expression of steroidogenic enzymes such as AKR1C3. It is further demonstrated that indomethacin, a potent inhibitor of AKR1C3, can be used to overcome enzalutamide resistance.
  • Intracrine androgen biosynthesis has been well characterized as a mechanism of CRPC (Cai et al., 2011; Fankhauser et al., 2014; Ishizaki et al., 2013; Locke et al., 2008; Mohler et al., 2011).
  • Many enzymes are involved in androgen synthesis, including CYP17A1, AKR1C3 and HSD3B.
  • CYP17A1 can be inhibited by abiraterone in clinical treatments (de Bono et al., 2011; Ryan et al., 2013).
  • AKR1C3 is a steroidogenic enzyme involved in steroid biosynthesis and mediates the last step of testosterone biosynthesis from androstenedione.
  • AKR1C3 It catalyzes conversion of steroids and modulates trans-activation of steroid receptors. Elevated expression of AKR1C3 has been associated with PCa progression and aggressiveness (Stanbrough et al., 2006; Wako et al., 2008). AKR1C3 has also been identified as an AR co-activator (Yepuru et al., 2013). In this study, gene enrichment analysis was used to compare enzalutamide resistant cells to enzalutamide sensitive cells. It was found that the steroid biosynthesis genes were highly enriched in C4-2B MDVR cells.
  • AKR1C3, HSD3B and CYP17A1 were up regulated in enzalutamide resistant cells.
  • AKR1C3 was highly expressed compared to C4-2B or LNCaP cells, suggesting that AKR1C3 plays a pivotal role in enzalutamide resistance.
  • steroid levels in C4-2B parental and C4-2B MDVR cells were determined by Liquid Chromatography-Mass Spectrometry (LC-MS).
  • Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) used for reducing fever, pain and inflammation.
  • NSAID non-steroidal anti-inflammatory drug
  • studies revealed indomethacin might have the potential to increase the sensitivity of cancer cells to anticancer agents, such as that of human melanoma cells to TRAIL-induced apoptosis (Tse et al., 2013), and of colon cancer cells to cisplatin (Brunelli et al., 2012).
  • Indomethacin also has the ability to inhibit PSA and ERG protein expression and decreased testosterone and DHT levels in relapsed VCaP xenograft tumors (Cai et al., 2011).
  • inhibition of AKR1C3 enzyme activity by indomethacin restored enzalutamide sensitivity in enzalutamide resistant prostate cancer cells both in vitro and in vivo.
  • the combination of indomethacin and enzalutamide resulted in significantly greater inhibition of enzalutamide-resistant tumor growth.
  • the data described herein demonstrate that inhibition of AKR1C3 can restore anti-tumor effects in patients resistant to enzalutamide.
  • Inhibition of AKR1C3 by shRNA or indomethacin overcomes resistance to enzalutamide.
  • the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth.
  • Targeting AKR1C3 thus provides an effective treatment strategy for patients resistant to enzalutamide.
  • LNCaP, CWR22Rv1, VCaP and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, Va.). All experiments with cell lines were performed within 6 months of receipt from ATCC or resuscitation after cryopreservation. ATCC uses Short Tandem Repeat (STR) profiling for testing and authentication of cell lines.
  • C4-2B cells were kindly provided and authenticated by Dr. Leland Chung, Cedars-Sinai Medical Center, Los Angeles, Calif.
  • LN-95 cells were kindly provided and authenticated by Dr. Joel Nelson, University of Pittsburgh, Pa.
  • LNCaP-neo and LNCaP-AKR1C3 cells were generated by stable transfection of LNCaP cells with either empty vector pcDNA3.1 or pcDNA3.1 encoding AKR1C3 and were maintained in RPMI1640 medium containing 300 ⁇ g/mL G418.
  • AKR1C3 shRNA TRCN0000026561 and TRCN0000025694 were purchased from Sigma.
  • C4-2B MDVR C4-2B enzalutamide resistant Cells resistant to enzalutamide were referred to as C4-2B MDVR (C4-2B enzalutamide resistant) as described previously (Liu et al., 2014a). All cells were maintained at 37° C. in a humidified incubator with 5% carbon dioxide.
  • C4-2B parental and C4-2B MDVR cells were cultured in serum- and phenol red-free RPMI1640 medium for 5 days, then cells were suspended in 4 mL of a 1:1 water/methanol mixture. The suspension was homogenized, and the resulting homogenate was cooled on ice. The precipitated material was removed by centrifuging at high speed for 5 min, and the supernatant was removed and evaporated in a SpeedVac (Labconco Inc.) followed by lyophilizer (Labconco Inc.).
  • the residue was suspended in 150 ⁇ L of CH3OH/H2O (1:1), filtered through a 0.2 ⁇ m ultracentrifuge filter (Millipore inc.) and subjected to UPLC/MS-MS analysis. Samples were run in duplicate during UPLC-MS/MS analysis. Samples were placed in an Acquity sample manager which was cooled to 8° C. to preserve the analytes. Pure standards were used to optimize the UPLC-MS/MS conditions prior to sample analysis. Also, the standard mixture was run before the first sample to prevent errors due to matrix effect and day-to-day instrument variations.
  • RNA quality of all samples was tested by RNA electrophoresis to ensure RNA integrity. Samples were analyzed by the Genomics Shared Resource (UC Davis Medical Center, Sacramento, Calif.) using the Affymetrix Human Gene 1.0 ST array. The data was analyzed by Subio platform and Ingenuity Pathway Analysis (IPA).
  • IPA Ingenuity Pathway Analysis
  • C4-2 parental or C4-2B MDVR cells were treated with DMSO, 10 ⁇ M or 20 ⁇ M enzalutamide in media containing 10% FBS.
  • CWR22Rv1 cells or C4-2B MDVR cells were treated with 10 ⁇ M or 20 ⁇ M indomethacin with or without 20 ⁇ M enzalutamide, cells were plated at equal density (1500 cells/dish) in 100 mm dishes for 14 days, the medium was changed every 3 days;
  • LNCaP-neo or LNCaP-AKR1C3 cells were treated with DMSO or 10 ⁇ M enzalutamide in media containing 10% complete FBS, cells were plated at equal density (10000 cells/dish) in 100 mm dishes for 28 days, the colonies were rinsed with PBS before staining with 0.5% crystal violet/4% formaldehyde for 30 min and the numbers of colonies were counted.
  • RNAs were extracted using TriZOL reagent (Invitrogen). cDNAs were prepared after digestion with RNase-free RQ1 DNase (Promega). The cDNAs were subjected to real-time reverse transcription-PCR (RT-PCR) using Sso Fast Eva Green Supermix (Bio-Rad) according to the manufacturer's instructions and as described previously (Liu et al., 2011). Each reaction was normalized by co-amplification of actin. Triplicates of samples were run on default settings of Bio-Rad CFX-96 real-time cycler.
  • RT-PCR real-time reverse transcription-PCR
  • Primers used for Real-time PCR are: AKR1C3, 5′-gagaagtaaagctttggaggtcaca-3′ (forward) and 5′-caacctgctcctcattattgtataaatga-3′ (reverse); AKR1C1/2, 5′-ggtcacttcatgcctgtcct-3′ (forward) and 5′-actctggtcgatgggaattg-3′ (reverse); HSD3B1, 5′-agaatctagaccactcttctgtccagatt-3′ (forward) and 5′-ctttgaattcaactatgtgaaggaatggaa-3′ (reverse); HSD3B2, 5′-cgggcccaactcctacaag-3′ (forward) and 5′-ttttccagaggctcttcgtcgt-3′ (reverse); CYP17A1, 5′-gggc
  • PSA levels were measured in sera from C4-2B parental or C4-2B MDVR tumor bearing mice using PSA ELISA Kit (KA0208, Abnova, Inc., Walnut, Calif.) according to the manufacturer's instructions.
  • C4-2B parental or C4-2B MDVR cells (4 million) were mixed with matrigel (1:1) and injected into the prostates of 6-7 week male SCID mice.
  • serum PSA level reached 5 ng/ml
  • mice were randomized into two groups (4 mice in each group) and treated as follows: (1) vehicle control (0.5% weight/volume (w/v) Methocel A4M p.o), (2) enzalutamide (25 mg/kg, p.o.). Tumors were monitored by PSA level. All tumor tissues were harvested after 3 weeks of treatment.
  • CWR22Rv1 cells (4 million) were mixed with matrigel (1:1) and injected subcutaneously into the flanks of 6-7 week male SCID mice.
  • Tumor-bearing mice (tumor volume around 50-100 mm3) were randomized into four groups (5 mice in each group) and treated as follows: (1) vehicle control (5% Tween 80 and 5% ethanol in PBS, i.p.), (2) enzalutamide (25 mg/kg, p.o.), (3) indomethacin (3 mg/kg, i.p.), (4) enzalutamide (25 mg/kg, p.o.)+indomethacin (3 mg/kg, i.p.). Tumors were measured using calipers twice a week and tumor volumes were calculated using length ⁇ width2/2. Tumor tissues were harvested after 3 weeks of treatment.
  • Tumors were fixed by formalin and paraffin embedded tissue blocks were dewaxed, rehydrated, and blocked for endogenous peroxidase activity.
  • Antigen retrieving was performed in sodium citrate buffer (0.01 mol/L, pH 6.0) in a microwave oven at 1,000 W for 3 min and then at 100 W for 20 min. Nonspecific antibody binding was blocked by incubating with 10% fetal bovine serum in PBS for 30 min at room temperature. Slides were then incubated with anti-Ki-67 (at 1:500; NeoMarker), anti-AKR1C3 (at 1:100; Sigma) at 4° C. overnight.
  • AR-full length (SEQ ID NO: 4) 5′-AAG CCA GAG CTG TGC AGA TGA (SEQ ID NO: 5) 3′-TGT CCT GCA GCC ACT GGT TC AR-V1: (SEQ ID NO: 6) 5′-AAC AGA AGT ACC TGT GCG CC (SEQ ID NO: 7) 3′-TGA GAC TCC AAA CAC CCT CA AR-V7: (SEQ ID NO: 8) 5′-AAC AGA AGT ACC TGT GCG CC (SEQ ID NO: 9) 3′-TCA GGG TCT GGT CAT TTT GA AR V1/2/2b: (SEQ ID NO: 10) 5′-TGG ATG GAT AGC TAC TCC GG (SEQ ID NO: 11) 3′-GTT CAT TCT GAA AAA TCC TTC AGC AR1/2/3/2b: (SEQ ID NO: 12) 5′-AAC AGA AGT ACC TGT GCG CC (SEQ ID
  • PSA promoter AREI/II- (SEQ ID NO: 16) 5′-CCTAGATGAAGTCTCCATGAGCTACA AREI/II- (SEQ ID NO: 17) 3′-GGGAGGGAGAGCTAGCACTTG (PROXIMAL)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Urology & Nephrology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Genetics & Genomics (AREA)
  • Physics & Mathematics (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Reproductive Health (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Endocrinology (AREA)
US15/134,228 2013-10-28 2016-04-20 Treatment of metastatic prostate cancer Abandoned US20170315127A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US15/134,228 US20170315127A1 (en) 2013-10-28 2016-04-20 Treatment of metastatic prostate cancer
US16/654,847 US11215617B2 (en) 2013-10-28 2019-10-16 Treatment of metastatic prostate cancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201361896250P 2013-10-28 2013-10-28
US201361914389P 2013-12-11 2013-12-11
PCT/US2014/062455 WO2015065919A1 (en) 2013-10-28 2014-10-27 Treatment of metastatic prostate cancer
US15/134,228 US20170315127A1 (en) 2013-10-28 2016-04-20 Treatment of metastatic prostate cancer

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/062455 Continuation WO2015065919A1 (en) 2013-10-28 2014-10-27 Treatment of metastatic prostate cancer

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/654,847 Division US11215617B2 (en) 2013-10-28 2019-10-16 Treatment of metastatic prostate cancer

Publications (1)

Publication Number Publication Date
US20170315127A1 true US20170315127A1 (en) 2017-11-02

Family

ID=53004994

Family Applications (2)

Application Number Title Priority Date Filing Date
US15/134,228 Abandoned US20170315127A1 (en) 2013-10-28 2016-04-20 Treatment of metastatic prostate cancer
US16/654,847 Active US11215617B2 (en) 2013-10-28 2019-10-16 Treatment of metastatic prostate cancer

Family Applications After (1)

Application Number Title Priority Date Filing Date
US16/654,847 Active US11215617B2 (en) 2013-10-28 2019-10-16 Treatment of metastatic prostate cancer

Country Status (5)

Country Link
US (2) US20170315127A1 (enrdf_load_stackoverflow)
EP (1) EP3062808B1 (enrdf_load_stackoverflow)
JP (2) JP6548641B2 (enrdf_load_stackoverflow)
CN (1) CN105899223A (enrdf_load_stackoverflow)
WO (1) WO2015065919A1 (enrdf_load_stackoverflow)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019155416A3 (en) * 2018-02-09 2019-10-03 Kashiv Pharma Llc A stable pharmaceutical composition of poorly soluble nonsteroidal antiandrogens
WO2020046767A1 (en) * 2018-08-26 2020-03-05 Trovagene, Inc. Plk1 target phosphorylation status and treatment of cancer with plk1 inhibitors
US10722527B2 (en) 2015-04-10 2020-07-28 Capsugel Belgium Nv Abiraterone acetate lipid formulations
US11215617B2 (en) 2013-10-28 2022-01-04 The Regents Of The University Of California Treatment of metastatic prostate cancer
WO2022048492A1 (zh) * 2020-09-02 2022-03-10 深圳艾欣达伟医药科技有限公司 一种akr1c3检测方法、检测akr1c3的诊断试剂盒及其用途

Families Citing this family (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9314005B2 (en) 2009-07-01 2016-04-19 Transposagen Biopharmaceuticals, Inc. Genetically modified rat models for severe combined immunodeficiency (SCID)
WO2015179404A1 (en) * 2014-05-19 2015-11-26 The Johns Hopkins University Methods for identifying androgen receptor splice variants in subjects having castration resistant prostate cancer
WO2016179002A1 (en) * 2015-05-01 2016-11-10 The Regents Of The University Of California Compositions and methods for treatment of cancer
WO2017040864A1 (en) 2015-09-01 2017-03-09 First Wave Biopharma Methods and compositions for treating conditions associated with an abnormal inflammatory responses
CN108883084A (zh) * 2015-09-16 2018-11-23 新加坡科技研究局 氯硝柳胺在p53缺失细胞的处理中的应用
CA2996426A1 (en) * 2015-10-05 2017-04-13 Cedars-Sinai Medical Center Method of classifying and diagnosing cancer
WO2017120218A1 (en) * 2016-01-04 2017-07-13 The Regents Of The University Of Colorado, A Body Corporate Treatment of prostate cancer cells with fat oxidation inhibitors and enzalutamide
WO2017210117A1 (en) * 2016-06-02 2017-12-07 Board Of Trustees, Southern Illinois University Treatment of endometriosis and niclosamide derivatives
WO2017207702A1 (en) * 2016-06-02 2017-12-07 Sint-Augustinus Androgen receptor splice variants and androgen deprivation therapy
JP2020502188A (ja) 2016-12-19 2020-01-23 エフ・ホフマン−ラ・ロシュ・アクチェンゲゼルシャフト Ａｒ−ｖ７インヒビター
JP7300386B2 (ja) * 2017-01-19 2023-06-29 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア アンドロゲン非依存性がんを処置するための組成物および方法
WO2018183823A1 (en) * 2017-03-31 2018-10-04 Hera Testing Laboratories, Inc. Novel immunodeficient rat for modeling human cancer
RU2696288C2 (ru) * 2017-10-16 2019-08-01 Федеральное государственное бюджетное образовательное учреждение высшего образования "Кировский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Кировский ГМУ Минздрава России) Способ лечения кастрационно-устойчивого рака простаты
RU2673816C1 (ru) * 2017-10-16 2018-11-30 Федеральное государственное бюджетное образовательное учреждение высшего образования "Кировский государственный медицинский университет" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Кировский ГМУ Минздрава России) Способ лечения кастрационно-устойчивого рака простаты
CA3084474A1 (en) * 2017-11-14 2019-05-23 Institut Regional Du Cancer De Montpellier (Icm) Association of actives for treating prostate cancer
CN109836499A (zh) * 2017-11-27 2019-06-04 益善生物技术股份有限公司 抗人雄激素受体剪接变异体7的单克隆抗体及其应用
CN108187067A (zh) * 2018-01-09 2018-06-22 中国人民解放军第三0九医院 ITCNs组装体负载抗肿瘤化合物的应用
CN110693892A (zh) * 2018-07-09 2020-01-17 深圳艾欣达伟医药科技有限公司 化合物用于制备预防、治疗或改善疼痛的药物的用途
US12318386B2 (en) 2018-09-07 2025-06-03 Karyopharm Therapeutics Inc. Combination of XPO1 inhibitors and second agents for the treatment of prostate cancer
TWI816880B (zh) * 2018-09-13 2023-10-01 大陸商恒翼生物醫藥（上海）股份有限公司 治療前列腺癌之組合療法
CN109234393A (zh) * 2018-09-30 2019-01-18 上海交通大学医学院附属仁济医院 一种用于检测转移性去势抵抗性前列腺癌的基因探针组合物及试剂盒
HUP1900281A1 (hu) * 2019-08-06 2021-03-01 Toxie Europe Intelligens Kemiai Szenzorokat Kutato Monoamin-oxidáz-B gátlók prosztata karcinóma megelõzésében vagy kezelésében történõ alkalmazásra
WO2021031991A1 (zh) * 2019-08-16 2021-02-25 中国科学院分子细胞科学卓越创新中心 治疗激素依赖性疾病及减少激素依赖性疾病患者药物耐受的组合物和方法
JP7623375B2 (ja) 2019-11-18 2025-01-28 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア アンドロゲン受容体／ａｋｒ１ｃ３二重阻害剤
US20230005594A1 (en) * 2019-12-06 2023-01-05 H. Lee Moffitt Cancer Center And Research Institute, Inc. Clinical decision support for personalized adaptive prostate cancer therapy
US10980756B1 (en) 2020-03-16 2021-04-20 First Wave Bio, Inc. Methods of treatment
TW202304450A (zh) * 2021-03-31 2023-02-01 日商大鵬藥品工業股份有限公司 以雄性素受體拮抗劑進行的轉移性去勢抗性前列腺癌患者之治療
WO2022250466A1 (ko) * 2021-05-27 2022-12-01 사회복지법인 삼성생명공익재단 전립선암의 엔잘루타미드 저항성 억제용 약학 조성물 및 엔잘루타미드 저항성을 가지는 전립선암 치료용 약학 조성물
CN115721720A (zh) * 2021-08-31 2023-03-03 北京大学 Kras抑制剂与akr抑制剂联用在对抗肿瘤细胞耐药性中的应用
CN115721629B (zh) * 2022-10-31 2024-02-20 西安交通大学 多西他赛与恩杂鲁胺联合用药pH响应型铁-铜磁性纳米载药系统及其制备和应用

Family Cites Families (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US4957735A (en) 1984-06-12 1990-09-18 The University Of Tennessee Research Corporation Target-sensitive immunoliposomes- preparation and characterization
US5019369A (en) 1984-10-22 1991-05-28 Vestar, Inc. Method of targeting tumors in humans
US4902505A (en) 1986-07-30 1990-02-20 Alkermes Chimeric peptides for neuropeptide delivery through the blood-brain barrier
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5004697A (en) 1987-08-17 1991-04-02 Univ. Of Ca Cationized antibodies for delivery through the blood-brain barrier
US5055303A (en) 1989-01-31 1991-10-08 Kv Pharmaceutical Company Solid controlled release bioadherent emulsions
US5271961A (en) 1989-11-06 1993-12-21 Alkermes Controlled Therapeutics, Inc. Method for producing protein microspheres
US5188837A (en) 1989-11-13 1993-02-23 Nova Pharmaceutical Corporation Lipsopheres for controlled delivery of substances
US5268164A (en) 1990-04-23 1993-12-07 Alkermes, Inc. Increasing blood-brain barrier permeability with permeabilizer peptides
US5254342A (en) 1991-09-30 1993-10-19 University Of Southern California Compositions and methods for enhanced transepithelial and transendothelial transport or active agents
WO1993017668A1 (en) 1992-03-12 1993-09-16 Alkermes Controlled Therapeutics, Inc. Controlled release acth containing microspheres
US5534496A (en) 1992-07-07 1996-07-09 University Of Southern California Methods and compositions to enhance epithelial drug transport
US5514670A (en) 1993-08-13 1996-05-07 Pharmos Corporation Submicron emulsions for delivery of peptides
IL112778A0 (en) 1994-03-04 1995-05-26 Merck & Co Inc Substituted heterocycles, their preparation and pharmaceutical compositions containing them
US20030144312A1 (en) 2001-10-30 2003-07-31 Schoenhard Grant L. Inhibitors of ABC drug transporters in multidrug resistant cancer cells
IL166156A0 (en) * 2002-07-09 2006-01-15 Point Therapeutics Inc Boroproline compound combination therapy
KR20050029209A (ko) 2002-07-15 2005-03-24 미리어드 제네틱스, 인크. 화합물, 조성물 및 이의 사용방법
AU2003249244A1 (en) 2002-07-15 2004-02-02 Combinatorx, Incorporated Methods for the treatment of neoplasms
WO2005060951A2 (en) * 2003-12-19 2005-07-07 Bionaut Pharmaceuticals, Inc. Anti-neoplastic agents, combination therapies and related methods
US20070009533A1 (en) 2005-07-06 2007-01-11 Branimir Sikic Treatment of cancer patients exhibiting activation of the P-glycoprotein efflux pump mechanism
WO2008103920A2 (en) 2007-02-23 2008-08-28 Specigen, Inc. Targeted protein cages
GB2451875B (en) * 2007-08-15 2012-05-16 Mono Pumps Ltd Filtration system
US20090208493A1 (en) 2007-11-27 2009-08-20 Stc. Unm Compounds and methods for the selective inhibition of ABCB1, ABCC1 and ABCG2 transporters and the treatment of cancers, especially drug resistant cancers and high throughput flow cytometry assay to detect selective inhibitors
US9146238B2 (en) 2008-04-16 2015-09-29 The Johns Hopkins University Compositions and methods for treating or preventing prostate cancer and for detecting androgen receptor variants
WO2012142208A1 (en) * 2011-04-13 2012-10-18 The Trustees Of The University Of Pennsylvania Bifunctional akr1c3 inhibitors/androgen receptor modulators and methods of use thereof
WO2012143377A1 (en) * 2011-04-18 2012-10-26 Max-Delbrück-Centrum für Molekulare Medizin Niclosamide for the treatment of cancer metastasis
US9346803B2 (en) * 2011-10-17 2016-05-24 Vanderbilt University Indomethacin analogs for the treatment of castrate-resistant prostate cancer
KR101434461B1 (ko) 2011-10-21 2014-09-01 한국생명공학연구원 2-하이드록시아릴아마이드 유도체 또는 이의 약학적으로 허용가능한 염, 이의 제조방법 및 이를 유효성분으로 함유하는 암 예방 또는 치료용 약학적 조성물
WO2013142390A1 (en) 2012-03-21 2013-09-26 Gtx, Inc. Aldo-keto reductase subfamily 1c3 (akr1c3) inhibitors
JOP20200097A1 (ar) 2013-01-15 2017-06-16 Aragon Pharmaceuticals Inc معدل مستقبل أندروجين واستخداماته
EP3062808B1 (en) 2013-10-28 2021-09-29 The Regents of the University of California Treatment of metastatic prostate cancer
CN104771386A (zh) 2014-01-10 2015-07-15 中国科学院广州生物医药与健康研究院 苯甲酰胺类化合物及其应用
AU2015346076A1 (en) 2014-11-13 2017-06-15 Aeromics, Inc. Novel methods
WO2016179002A1 (en) 2015-05-01 2016-11-10 The Regents Of The University Of California Compositions and methods for treatment of cancer
CN105566147B (zh) 2015-12-07 2018-03-09 北京信德润兴科技有限公司 一种化合物及其制备方法和用途及相应的靶向给药系统和化疗药物
JP7300386B2 (ja) 2017-01-19 2023-06-29 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア アンドロゲン非依存性がんを処置するための組成物および方法

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11215617B2 (en) 2013-10-28 2022-01-04 The Regents Of The University Of California Treatment of metastatic prostate cancer
US10722527B2 (en) 2015-04-10 2020-07-28 Capsugel Belgium Nv Abiraterone acetate lipid formulations
WO2019155416A3 (en) * 2018-02-09 2019-10-03 Kashiv Pharma Llc A stable pharmaceutical composition of poorly soluble nonsteroidal antiandrogens
WO2020046767A1 (en) * 2018-08-26 2020-03-05 Trovagene, Inc. Plk1 target phosphorylation status and treatment of cancer with plk1 inhibitors
WO2022048492A1 (zh) * 2020-09-02 2022-03-10 深圳艾欣达伟医药科技有限公司 一种akr1c3检测方法、检测akr1c3的诊断试剂盒及其用途

Also Published As

Publication number Publication date
JP6548641B2 (ja) 2019-07-24
WO2015065919A1 (en) 2015-05-07
EP3062808A1 (en) 2016-09-07
JP2016540738A (ja) 2016-12-28
CN105899223A (zh) 2016-08-24
US20200041516A1 (en) 2020-02-06
US11215617B2 (en) 2022-01-04
JP2019172700A (ja) 2019-10-10
JP6974392B2 (ja) 2021-12-01
EP3062808B1 (en) 2021-09-29
EP3062808A4 (en) 2017-08-30

Similar Documents

Publication Publication Date Title
US11215617B2 (en) Treatment of metastatic prostate cancer
US10959984B2 (en) Methods for treating cancer with RORγ inhibitors
US20080085874A1 (en) Small molecule potentiator of hormonal therapy for breast cancer
CN107108686A (zh) 用于恶性肿瘤的rna干扰组合物和方法
Liu et al. Estrogen receptor β agonist enhances temozolomide sensitivity of glioma cells by inhibiting PI3K/AKT/mTOR pathway
CN106715443A (zh) Fl118母核化学结构平台产生用于治疗人类疾病的fl118衍生物的用途
Ma et al. Cucurbitacin B inhibits the translational expression of hypoxia-inducible factor-1α
JP2014509607A (ja) 前立腺癌を治療するための、抗クラステリンオリゴヌクレオチドとアンドロゲン受容体アンタゴニストとの併用
US20220233535A1 (en) Use of inhibitors of yap/taz for the treatment of cancer
US20190091220A1 (en) Targeting parp1 for treatment of tsc and cancers
JP2014509608A (ja) 前立腺癌を治療するための、抗クラステリンオリゴヌクレオチドとｈｓｐ９０阻害剤との併用
Ma et al. Targeting CD146 in combination with vorinostat for the treatment of ovarian cancer cells
Huang et al. Tetracaine hydrochloride induces cell cycle arrest in melanoma by downregulating hnRNPA1
WO2007143630A2 (en) Treatment of neurofibromatosis with hsp90 inhibitors
JP2009544679A (ja) 異常な細胞増殖に関連する疾患の治療におけるレチノイド及び小分子のＮｒｆ２アンタゴニストとしての使用
US20180153850A1 (en) Compositions and methods for treatment of cancer
US20200009088A1 (en) Compositions and methods for treating androgen-independent cancer
Kasukabe et al. Inhibition of rapamycin-induced Akt phosphorylation by cotylenin A correlates with their synergistic growth inhibition of cancer cells
US20220280590A1 (en) Use of inhibitors of yap and sox2 for the treatment of cancer
Pisano et al. Metabolic approach to the enhancement of antitumor effect of chemotherapy: a key role of acetyl-L-carnitine
HK1228747A1 (en) Treatment of metastatic prostate cancer
HK1228747B (en) Treatment of metastatic prostate cancer
US20230121867A1 (en) Compositions and methods for treating diseases and conditions by depletion of mitochondrial or genomic dna from circulation
WO2009108361A1 (en) Methods for treatment and prevention of breast cancer
US20140335077A1 (en) Compositions and Methods for the Treatment of Cancer Using IGF-IR Antagonists and MAPK/ERK Inhibitors

Legal Events

Date Code Title Description
AS Assignment

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GAO, ALLEN C.;LIU, CHENGFEI;LOU, WEI;REEL/FRAME:042210/0345

Effective date: 20160426

AS Assignment

Owner name: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA, CALIF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:PAN, CHONG-XIAN;REEL/FRAME:042935/0511

Effective date: 20170705

AS Assignment

Owner name: U.S. GOVERNMENT REPRESENTED BY THE DEPARTMENT OF V

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:THE REGENTS OF THE UNIVERSITY OF CALIFORNIA;REEL/FRAME:045385/0901

Effective date: 20180314

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION