ARIL HYDROCARBIDE (AHR) RECEIVER MODIFIERS AS NEW CANCER THERAPEUTICS

专利摘要:
aryl hydrocarbon receptor (AHR) modifiers as novel cancer therapeutics. Novel agents that modulate ahr activity are provided herein for use in therapeutic compositions and methods thereof for inhibiting cancer cell proliferation and tumor cell invasion and metastasis. the agents comprise ahr inhibitor or non-constitutive ahr agonists of formula (i) and (ii) for inhibiting cancer cell growth and parameters characterizing tumor metastases, such as tumor cell invasiveness.
公开号:BR112013002079B1
申请号:R112013002079-2
申请日:2011-07-27
公开日:2021-09-14
发明作者:David H. Sherr；Michael Pollastri；Jennifer Schlezinger；Sarah Haigh Molina；Scott Schaus；Joshua Robert Giguere
申请人:Trustees Of Boston University；Boston Medical Center Corporation；Northeastern University；
IPC主号:

专利说明:

CROSS REFERENCE TO RELATED ORDERS
[0001] The present application claims benefit under 35 USC § 119(e) of United States Provisional Patent Application Serial No.: 61/368,042, filed July 27, 2010, the contents of which are incorporated herein by reference in the its entirety. FIELD OF INVENTION
[0002] The invention relates, in general, to compositions and methods to modulate the activity of AhR. GOVERNMENT SUPPORT
[0003] The present invention was made with Government support under Contract Nos. ES011624 and CA134882, granted by the National Institutes of Health (NIH). The Government has certain rights in the invention. BACKGROUND
[0004] Cancer remains one of the most dangerous threats to human health. In the United States, cancer affects about 1.3 million new patients each year and is the second leading cause of death after heart disease, accounting for about 1 in 4 deaths. Cancer is also predicted to overtake cardiovascular disease as the number one cause of death within the next decade. Solid tumors are responsible for most of these deaths. While there have been significant advances in the medical treatment of certain cancers, the overall 5-year survival rate for all cancers has improved by only about 10% over the past 20 years. Cancers or malignant tumors metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.
[0005] In the United States, the age-adjusted incidence of breast cancer increased ~1% per year between 1940 and 1990 (27, 28) and 0.4%/year between 1987 and 2002 (29) due in part , to increased exposure to environmental carcinogens. Breast cancer is now the second most common cancer (after skin cancer) in women, with 225,000 new cases in the United States and 40,000 breast cancer-related deaths annually. It is estimated that 1 in 8 women born this year will be diagnosed with breast cancer during their lifetime (34). The total number of women diagnosed with breast cancer tends to grow significantly over the next 20 years as population demographics shift towards an older population (34). SUMMARY OF THE INVENTION
[0006] New agents that modulate AhR activity for use in therapeutic compositions and methods thereof for inhibiting cancer cell proliferation and tumor cell invasion and metastasis are described here. These compositions and methods are based on the inventors' groundbreaking discovery that pharmaceutical compositions comprising small molecule compounds of Formula (I) and (II) described herein modulate AhR activity, such as constitutive AhR activity. The inventors discovered that these small molecules can bind to the AhR and block or inhibit those functions and signaling pathways regulated by the AhR that influence cancer cell growth and proliferation and tumor cell invasiveness. The inventors have further discovered that pharmaceutical compositions comprising the small molecule compounds of Formula (I) and (II) described herein can act as either AhR inhibitors or non-constitutive AhR agonists and further provide new methods and assays to distinguish the types of AhR modulators. Accordingly, provided herein are novel therapeutic pharmaceutical compositions and methods thereof which comprise AhR inhibitors or non-constitutive AhR agonists of Formula (I) and (II) for inhibiting the growth of cancer cells and parameters that characterize tumor metastasis, such as the invasiveness of tumor cells.
[0007] Consequently, new AhR modulators are described here. In some aspects, described herein are pharmaceutical compositions comprising an aryl hydrocarbon receptor (AhR) modulator of Formula (I):
FORMULA (I) wherein: Y is C or N; X is OR1, NHR1, SR1, CH2(n)R1, halo or H; n is 0-6; Z is O, S or NH; R1 and R2 are independently H, alkyl, alkenyl, alkynyl, amino, aminosulfonyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R 3 , R 4 , R 5 and R 6 are independently absent, H, halo, alkyl, alkenyl, alkynyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; and pharmaceutically acceptable salts thereof.
In some embodiments of these aspects and all of said aspects described herein, the AhR modulator of Formula (I) is an AhR inhibitor or a non-constitutive agonist of AhR. In some such embodiments, the AhR inhibitor is CB7993113, having a chemical structure:

[0009] In some such embodiments, the non-constitutive agonist of AhR is CB7950998, having a chemical structure:

[0010] In some aspects, the pharmaceutical composition comprises an AhR modulator of Formula (Ia):
FORMULA (Ia) wherein: X' is H, alkyl, aminosulfonyl, alkoxy, amino, acyl, aryl or heteroaryl, each of which may be optionally substituted; n is 0-6; R2 is H, alkyl, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R3, R4, R5 and R6 are independently H, alkyl, acyl, halo, aryl or heteroaryl, each of which may be optionally substituted; and pharmaceutically acceptable salts thereof.
[0011] In some embodiments of the AhR inhibitor of Formula (Ia), X' is alkyl, alkoxy, amino, or aminosulfonyl; n is 0 or 1; R2 is aryl, substituted aryl, heteroaryl or substituted aryl; and R3, R4, R5 and R6 are independently H, alkoxy, alkyl or halo.
In some embodiments of these aspects and all of said aspects described herein, the AhR modulator of Formula (Ia) is an AhR inhibitor or a non-constitutive agonist of AhR.
[0013] In other aspects, provided herein are compositions comprising a compound of Formula (II):
FORMULA (II) wherein: Y is C or N; X is OR1, NHR1, SR1, CH2(n)R1, halo or H; n is 0-6; Z is O, S or NH; R1 and R2 are independently H, alkyl, alkenyl, alkynyl, amino, aminosulfonyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R 3 , R 4 , R 5 and R 6 are independently absent, H, halo, alkyl, alkenyl, alkynyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; and stereoisomers thereof.
In some embodiments of these aspects, the C at position 2 is in the R configuration and the C at position 3 is in the S configuration. In some embodiments of these aspects, the C at position 2 is in the S configuration and the C at position 3 is in the R configuration. In some embodiments of these aspects, the C at position 2 is in the R configuration and the C at position 3 is in the R configuration. position 3 is in the S configuration.
[0015] In some embodiments of these aspects, the compound is CMLD-2166, having a chemical structure:

[0016] In some aspects, provided herein are compositions comprising a compound of formula (IIa):
FORMULA (IIa) wherein: X' is H, alkyl, aminosulfonyl, alkoxy, acyl, aryl or heteroaryl, each of which may be optionally substituted; n is 0-6; R2 is H, alkyl, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R3, R4, R5 and R6 are independently H, alkyl, acyl, halo, aryl or heteroaryl, each of which may be optionally substituted; and stereoisomers thereof.
In some embodiments of these aspects and all of said aspects described herein, X' is alkyl, alkoxy, aminosulfonyl; n is 0 or 1; R2 is aryl, substituted aryl, heteroaryl or substituted aryl; and R3, R4, R5 and R6 are independently H, alkoxy, alkyl or halo.
[0018] In some aspects, provided herein are pharmaceutical compositions comprising an aryl hydrocarbon receptor (AhR) modulator of Formula (II):
FORMULA (II) wherein: Y is C or N; X is OR1, NHR1, SR1, CH2(n)R1, halo or H; n is 0-6; Z is O, S or NH; R1 and R2 are independently H, alkyl, alkenyl, alkynyl, amino, aminosulfonyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R 3 , R 4 , R 5 and R 6 are independently absent, H, halo, alkyl, alkenyl, alkynyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; stereoisomers thereof; and pharmaceutically acceptable salts thereof.
In some embodiments of these aspects and all such aspects described herein, the stereoisomers thereof comprise the C at position 2 in an R configuration and the C at position 3 in an S configuration. thereof comprise the C at the 2 position in an S configuration and the C at the 3 position in an R configuration. In some embodiments of the aspect, the stereoisomers thereof comprise the C at the 2 position in an R configuration and the C at the 3 position in an R configuration. In some embodiments of the aspect, stereoisomers thereof comprise the C at position 2 in an S configuration and the C at position 3 in an S configuration.
In some embodiments of these aspects and all of said aspects described herein, the AhR modulator of Formula (II) is an AhR inhibitor or a non-constitutive agonist of AhR.
[0021] In some embodiments of these aspects, the AhR modulator of Formula (II) is CMLD-2166, having a chemical structure:

[0022] In some aspects, provided herein are pharmaceutical compositions comprising an aryl hydrocarbon receptor (AhR) modulator of Formula (IIa):
FORMULA (IIa) wherein: X' is H, alkyl, aminosulfonyl, alkoxy, acyl, aryl or heteroaryl, each of which may be optionally substituted; n is 0-6; R2 is H, alkyl, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R3, R4, R5 and R6 are independently H, alkyl, acyl, halo, aryl or heteroaryl, each of which may be optionally substituted; stereoisomers thereof; and pharmaceutically acceptable salts thereof.
In some embodiments of these aspects of the AhR modulator of Formula (IIa), X' is alkyl, alkoxy, aminosulfonyl; n is 0 or 1; R2 is aryl, substituted aryl, heteroaryl or substituted aryl; and R3, R4, R5 and R6 are independently H, alkoxy, alkyl or halo.
In some embodiments of these aspects and all of said aspects described herein, the AhR modulator of Formula (IIa) is an AhR inhibitor or a non-constitutive agonist of AhR.
[0025] In some aspects, methods of modulating the constitutive AhR activity in an individual in need thereof are described here. Such methods comprise administering to a subject having constitutive AhR activity, a therapeutically effective amount of any of the pharmaceutical compositions comprising an Ahr modulator, such as an AhR inhibitor (e.g., CB7993113 or CMLD-2166) or a non-constitutive AhR agonist (e.g., CB7950998) of Formulas (I), (Ia), (II) or (IIa) described herein. In some embodiments of these aspects and all of the aforementioned aspects described here, the methods further comprise the step of selecting the individual who has constitutive AhR activity.
[0026] In other aspects, methods of treating a cancer or a cancerous condition by modulating AhR activity are described herein. Such methods comprise administering, to a subject having a cancer or a cancerous condition, a therapeutically effective amount of any of the pharmaceutical compositions that comprise an Ahr modulator, such as an AhR inhibitor (e.g., CB7993113 or CMLD-2166 ) or with a non-constitutive AhR agonist (e.g., CB7950998) of Formulas (I), (Ia), (II) or (IIa) described herein.
In some aspects, described herein are methods of inhibiting tumor cell invasiveness in an individual who has a cancer, a cancerous condition, or a tumor. Such methods comprise administering to a subject having a cancer or tumor, a therapeutically effective amount of any of the pharmaceutical compositions that comprise an AhR modulator, such as an AhR inhibitor (e.g., CB7993113 or CMLD-2166) or with a non-constitutive AhR agonist (e.g., CB7950998) of Formulas (I), (Ia), (II) or (IIa) described herein.
[0028] In some embodiments of these aspects and all of said aspects described herein, the methods further comprise the step of selecting the individual having a cancer, a cancerous condition or a tumor.
[0029] In some embodiments of these methods, the cancer is a breast cancer, squamous cell cancer, lung cancer, a peritoneum cancer, a hepatocellular cancer, a gastric cancer, a pancreatic cancer, a glioblastoma, a cervical cancer, an ovarian cancer, a liver cancer, a bladder cancer, a hepatoma, a colon cancer, a colorectal cancer, an endometrial or uterine carcinoma, a salivary gland carcinoma, a kidney or kidney cancer, a cancer prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, B cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, or leukemia chronic myeloblastic disease. In some such embodiments, the cancer is breast cancer.
[0030] Some embodiments of these methods may further comprise administration or treatment with one or more additional anti-cancer therapies. In some of such embodiments, the additional anti-cancer therapy comprises surgery, radiotherapy, biotherapy, immunotherapy, chemotherapy, or any combination thereof.
[0031] Some embodiments of these methods may further comprise administration or treatment with one or more anti-cancer therapeutic agents. In some such embodiments, the anti-cancer therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenesis agent, a cytotoxic agent, an anti-hormonal agent, a prodrug, or a cytokine.
Also provided herein in other aspects are pharmaceutical compositions comprising an AhR modulator such as an AhR inhibitor (eg CB7993113 or CMLD-2166) or a non-constitutive AhR agonist (eg CB7950998) of Formulas (I), (Ia), (II) or (IIa), for use in modulating constitutive AhR activity in an individual in need thereof.
In some aspects, pharmaceutical compositions comprising an AhR modulator, such as an AhR inhibitor (eg CB7993113 or CMLD-2166) or a non-constitutive AhR agonist (eg CB7950998) of Formulas (I), (Ia), (II) or (IIa), are provided for use in treating a cancer or cancerous condition by modulating AhR activity.
In some aspects, pharmaceutical compositions comprising an AhR modulator, such as an AhR inhibitor (eg CB7993113 or CMLD-2166) or a non-constitutive AhR agonist (eg CB7950998) of Formulas (I), (Ia), (II) or (IIa), are provided for use in inhibiting tumor cell invasiveness in an individual who has a cancer, a cancerous condition, or a tumor.
[0035] In some embodiments of these aspects and all of the aforementioned aspects described herein, the use further comprises the step of selecting the individual who has a cancer, a cancerous condition or a tumor. In some embodiments, the cancer is breast cancer, squamous cell cancer, lung cancer, peritoneum cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, a liver cancer, a bladder cancer, a hepatoma, a colon cancer, a colorectal cancer, an endometrial or uterine carcinoma, a salivary gland carcinoma, a kidney or kidney cancer, a prostate cancer, a cancer of the vulva, a thyroid cancer, a head and neck cancer, a B-cell lymphoma, a chronic lymphocytic leukemia (CLL), an acute lymphoblastic leukemia (ALL), a hairy cell leukemia, or a chronic myeloblastic leukemia. In some such embodiments, the cancer is breast cancer.
In some embodiments of these aspects and all of said aspects described herein, the use further comprises one or more additional anti-cancer therapies. In some embodiments, the additional anti-cancer therapy comprises surgery, radiotherapy, biotherapy, immunotherapy or chemotherapy.
In some embodiments of these aspects and all of said aspects described herein, the use further comprises one or more anti-cancer therapeutic agents. In some embodiments, the anti-cancer therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenesis agent, a cytotoxic agent, an anti-hormonal agent, a prodrug, or a cytokine.
[0038] Other aspects described herein provide novel screening methods for identifying an AhR modulator, such as an AhR inhibitor or a non-constitutive AhR agonist of Formula (I), (Ia), (II) or (IIa) . In one such aspect, a screening method for identifying an AhR modulator is provided, the method comprising: contacting a cell comprising a sequence encoding an AhR receptor operably linked to a sequence encoding a reporter molecule with a strong activator of AhR, such that the expression of the reporter molecule by the cell indicates that the AhR receptor is activated by the strong activator of AhR; contacting the cell of (a) with a test agent such that a decrease in expression of the reporter molecule in the presence of the test agent indicates that the test agent is an AhR modulator; and contacting a cell comprising a sequence encoding an AhR receptor operably linked to a sequence encoding a reporter molecule with the AhR modulator in the absence of a strong AhR activator, wherein the lack of expression of the reporter molecule by the cell indicates that the AhR modulator is an AhR inhibitor and where expression of the reporter molecule by the cell indicates that the AhR modulator is an AhR partial agonist.
In some embodiments of the aspect and all of said aspects described herein, the screening method further comprises contacting the AhR partial agonist with a tumor activity assay system comprising: (i) a tumor cell; and (ii) a mixture comprising one or more extracellular matrix components, wherein inhibition of tumor activity by the AhR partial agonist indicates that the AhR partial agonist is a non-constitutive AhR agonist and that an increase in tumor activity by AhR partial agonist indicates that the AhR partial agonist is a constitutive agonist of AhR.
[0040] In some embodiments of the screening methods described herein, the reporter is a fluorescent protein, such as GFP (Green Fluorescent Protein) or RFP (Red Fluorescent Protein). In some embodiments of the screening methods, the strong AhR activator is TCDD or BNF.
In some embodiments of the screening methods, the tumor activity being measured in the tumor activity assay system is tumor cell proliferation or tumor cell invasiveness. In some embodiments of the screening methods, the tumor cell is a human breast tumor cell. In some embodiments of the screening methods, the mixture that comprises one or more extracellular matrix components is Matrigel. In some embodiments, one or more extracellular matrix components comprise laminin and collagen. Definitions
[0042] For convenience, certain terms used in this document, in the descriptive report, examples and attached claims are collected here. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings given below. Unless otherwise specified or evident from the context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it belongs. Definitions are provided to help describe particular embodiments and are not intended to limit the claimed invention because the scope of the invention is limited only by the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0043] As used herein, the term "aryl hydrocarbon receptor (AhR) modulator" refers to an agent that causes or facilitates a qualitative or quantitative change, alteration or modification in one or more processes, mechanisms, effects, responses , functions, activities or pathways mediated by the AhR receptor. Such changes mediated by an AhR modulator, such as a non-constitutive AhR inhibitor or agonist described herein, may refer to a decrease or an increase in AhR activity or function, such as a decrease, inhibition or shift in activity. constitutive of AhR.
The terms "AhR inhibitor" or "AhR inhibitor" refer to an agent or compound that inhibits one or more signaling and effector pathways downstream of AhR, including constitutive AhR signaling, as these terms are used herein . Thus, the term AhR inhibitor refers to an agent that inhibits the expression of the AhR polypeptide or polynucleotide encoding AhR or one that binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactive, desensitizes or down-regulates the activity of the AhR polypeptide or polynucleotide encoding AhR. Such AhR inhibitors can, for example, inhibit AhR expression, e.g. AhR translation, AhR post-translational processing, AhR polypeptide stability, nuclear or cytoplasmic degradation or localization or transcription, post-transcriptional processing, stability or degradation of a polynucleotide encoding the AhR or binding to, partially or completely blocking the stimulation, DNA binding, or transcription factor activity of the AhR. An AhR inhibitor can act directly or indirectly.
An "AhR antagonist" refers to an AhR inhibitor that does not elicit a biological response per se when it specifically binds to the AhR polypeptide or polynucleotide encoding AhR, but blocks or dampens agonist-mediated or agonist-mediated responses ligand, that is, an AhR antagonist can bind but does not activate the AhR polypeptide or polynucleotide encoding the AhR and the binding interrupts the interaction, displaces an AhR agonist, and/or inhibits the function of an AhR agonist. Thus, as used here, an AhR antagonist does not function as an inducer of AhR activity when bound to AhR, that is, they function as pure inhibitors of AhR.
[0046] As used herein, the terms "non-constitutive AhR agonist", "non-constitutive AhR agonist", "non-constitutive AhR activator" or "Non-constitutive AhR activator" refer to an agent or compound that binds to AhR and hyperactive or bypass signaling and effector pathways downstream of constitutive AhR, as these terms are used herein. Such non-constitutive AhR agonists can increase expression of the AhR polypeptide or polynucleotide encoding the AhR, or bind to the AhR and to partially or totally shift constitutive AhR signaling and downstream activity such as cancer cell growth and tumor invasive properties. Consequently, a non-constitutive AhR agonist can shift constitutive AhR signaling by at least 10% or more, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more at least 90% or more, at least 95% or more, or at least 100% or completely, compared to a reference or control level in the absence of the non-constitutive AhR agonist.
The AhR agonists, non-constitutive AhR agonists, AhR inhibitors and AhR antagonists described herein may be ligands, antagonists, agonists, small chemical molecules, antibodies or antigen-binding fragments thereof, RNA molecules (ie. is, siRNA or antisense RNA) naturally-occurring and synthetic inhibitors, and the like. Assays to identify AhR agonists, non-constitutive AhR agonists, AhR inhibitors, and AhR antagonists include, for example, applying putative AhR modulating compounds to cells in the presence or absence of a polypeptide or polynucleotide and then determining the functional effects on a polypeptide or polynucleotide.
The terms "subject" and "individual" are used interchangeably herein and refer to an animal, eg, a human, that receives the small molecule AhR modulators of Formula (I) or Formula (II ) described here. For treatment of those disease states that are specific to a specific animal, such as a human being, the term "subject" refers to that specific animal. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and non-human primates. The term "subject" also includes any vertebrate animal including, but not limited to, mammals, reptiles, amphibians and fish.
The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth or proliferation, which interferes with the normal functioning of bodily organs and systems. Cancers that migrate from their places of origin and settle in vital organs can eventually lead to the subject's death through functional deterioration of Organs affected organs. Hematopoietic cancers, such as leukemia, are able to compete with the normal hematopoietic compartments in a subject, thus leading to hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately causing death. This definition includes benign and malignant cancers, as well as latent tumors or micrometastases.
The term "anticancer therapy" refers to a therapy or therapeutic agent useful in the treatment of cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, for example, surgery, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents to treat cancer , such as anti-HER-2 antibodies (eg Herceptin™), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (eg a tyrosine kinase inhibitor), HER1/EGFR inhibitor (eg erlotinib (Tarceva™)), platelet-derived growth factor inhibitors (eg Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor (eg celecoxib), interferons, cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BLyS, APRIL, BCMA or VEGF receptor(s), TRAIL/Apo2 and other organic chemical agents and bioactives, etc. Combinations thereof are also included in the embodiments described herein.
[0051] The term "screening", as used herein, refers to the use of cells and tissues in the laboratory, and methods therewith, to identify agents with a specific function, for example, a modulating activity. In some embodiments, screening methods for identifying agents (e.g., compounds or drugs) that inhibit or otherwise modulate AhR activity are described herein.
[0052] As used herein, the term "comprising" or "comprises" is used in reference to compositions, methods and respective component(s) thereof that are essential to the invention, still open to the inclusion of elements unspecified, whether essential or not.
[0053] As used herein, the term "consisting essentially of" refers to those elements necessary for a particular embodiment. The term allows for the presence of additional elements that do not materially affect the basic and new or functional feature(s) of this embodiment of the invention.
[0054] The term "consisting of" refers to the compositions, methods and respective components thereof, as described herein, which are exclusive of any element not mentioned in said description of the embodiment.
[0055] As used in this specification and the appended claims, the singular forms "a", "an", "the" and "a" include plural references unless the context clearly indicates otherwise. Thus, for example, references to "the method" include one or more methods and/or steps of the type described herein and/or which will become apparent to those skilled in the art upon reading the present description and so forth.
[0056] Except as operative examples, or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified, in all cases, by the term "about". The term "about", when used in relation to percentages, can mean ± 1%.
[0057] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this description belongs. It should be understood that the present invention is not limited to the particular methodology, protocols and reagents, etc., described herein and, as such, may vary. The terminology used herein is for the purposes of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of terms common in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 18th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-63202182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Luttmann, published by Elsevier, 2006. Definitions of common terms in molecular biology are found in Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1986); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S.L. Berger and A.R. Kimmerl Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et al., ed., John Wiley and Sons, Inc.), Current Protocols in Protein Science (CPPS) (John E. Coligan et al., ed., John Wiley and Sons, Inc.) and Current Protocols in Immunology (CPI) (John E. Coligan et al., ed. John Wiley and Sons, Inc.), all of which are incorporated herein by reference in their entirety.
[0058] It is to be understood that the foregoing detailed description and the examples which follow are illustrative only and are not to be taken as limitations on the scope of the invention. Various changes and modifications in the described embodiments, which will be apparent to those skilled in the art, can be made without departing from the spirit and scope of the present invention. Furthermore, all patents, patent applications and publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention. These publications are provided solely for their dissemination prior to the filing date of this application. Nothing in this regard should be construed as an admission that inventors are not entitled to advance such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation of the content of these documents are based on the information available to Claimants and do not constitute an admission as to the accuracy of the dates or content of these documents. BRIEF DESCRIPTION OF THE FIGURES
[0059] Figure 1 shows that inhibition of AhR with AhRR upregulates p21 and decreases the growth of MCF-10F. Insertion: p21 protein levels in control or AhRR transduced cells.
[0060] Figure 2 demonstrates that inhibition of AhR with AhRR inhibits tumor invasion in matrigel.
[0061] Figure 3 demonstrates that down-regulation of AhR with AhR siRNA inhibits invasion into Boyden Chambers. The number of cells above and below the membrane containing Matrigel was determined after 48 hours and the percentage of cells in the lower chamber calculated. *p<0.04; **p < 0.03.
Figures 4A-4B show high throughput screening (HTS) for AhR antagonists and agonists. Figure 4A shows H1G1 cells expressing an AhR-targeted GFP reporter construct and treated with candidate AhR modifiers alone (dark line) or together with BNF, a known AhR agonist (light line). A dark circle identifies an AhR inhibitor, while a light circle indicates an agonist. Figure 4B shows data for the non-toxic compound identified under "A", which was titrated in the presence of BNF to calculate the IC50. More than 4,000 compounds were tested in this semi-automated assay.
Figures 5A-5B demonstrate that CH223191 and CMLD 2166 inhibit constitutive AhR activity and cell growth. Figure 5A shows data from immortalized malignant BP1 and MCF-10F cells that were transiently transfected with AhR-responsive pGudLuc and treated with vehicle (DMSO), CH2231 91 or 2166. Figure 5B shows data from BP1 and MCF-10F cells that were grown for 18 hours in the presence of DMSO, CH2231 91 or 2166 and the incorporation of 3 H-thymidine determined. *p < 0.05.
[0064] Figure 6 demonstrates that CB7993113 blocks AhR translocation to the nucleus after stimulation of cells with DMBA, an AhR agonist. Hepa-1 cells were treated with vehicle or CH223191 at 1-10 µM. One hour later, cells were treated with 0.1 µM DMBA (where indicated) and incubated for 30 minutes. Cells were collected and cytoplasmic and nuclear proteins were extracted. The extracted proteins were subjected to AhR-specific Western immunoblots or, as a loading control, β-actin. Data are representative of three independent experiments.
Figures 7A-7B demonstrate that CH223191, CMLD-2166 and CMLD-2186 inhibit tumor invasion. Hs578T cells were cultured in Boyden Chambers (Figure 7A) or in Matrigel (Figure 7B) in the presence of DMSO, 10-5 M CMLD-2166, CMLD-2186 (an enantiomer of 2166) or CH223191 and assayed for invasion into 2 (Boyden Chambers) or 7 (Matrigel) days later.
[0066] Figures 8A-8B demonstrate that CB7993113 is able to block the acute toxic effects of a potent prototypic AhR and PAH ligand, DMBA, in vivo and that three DMBA-affected bone marrow cell subpopulations are all rescued upon treatment with CB7993113. Figure 8A shows data from C57BL/6 mice (6/group) that were injected i.p. on day -1 and day 0 with vehicle (oil) or CB7993113 at 50 mg/kg. The mice were then injected with DMBA at 50 mg/kg. The mice were sacrificed 48 hours later. Bone marrow hematopoietic cells were purged from the bone and viable cells were counted. Data are presented as the mean number of viable white cells + SE. **p < 0.01, ***P < 0.001, Student's t-test. Figure 8B confirms the data shown in Figure 8A and further demonstrates that the three subpopulations of bone marrow cells affected by DMBA are all rescued upon treatment with CB7993113. C57BL/6 mice (6/group) were treated as in Figure 8A. The percentage and number of viable pro-B cells, pre-B cells and bone marrow neutrophils were determined 48 hours later by flow cytometry. Data are presented as the mean number of viable cells of each subtype + SE. (*p < 0.05, **P < 0.01, Student t-test).
[0067] Figure 9 describes computational strategies for searching for electrostatic similarity and format.
Figure 10 demonstrates that compounds CMLD-2166 and CB7993113 bind to human AhR. Compounds CMLD-2166 and CB7993113 were mixed with in vitro translated human AhR and radiolabeled TCDD (dioxin), a high affinity AhR ligand. The complexes were then separated over a sucrose gradient and the fractions were assayed for radioactivity. A decrease in signal (displacement of radiolabeled TCDD) can be seen in fractions 13-16, indicating binding of the test compound to AhR and displacement of TCDD. These results demonstrate that these compounds are competitive inhibitors of AhR.
[0069] Figure 11 demonstrates that AhR inhibitors do not significantly block PPARy-mediated transcription. Data were gathered from 4 experiments. Unlike their ability to block AhR-targeted reporter activity, AhR inhibitors had no effect on PPARy-targeted reporter activity.
[0070] Figure 12 demonstrates that AhR siRNA transfection blocks tumor invasion in matrigel. BP1 or Hs578T human breast tumor cells were transiently transfected with control siRNA or AhR siRNA to knock down AhR expression and cells were cultured in Matrigel for 6 days. Colonies of tumor cells that grow from individual cells during those 6 days are shown.
[0071] Figure 13 demonstrates that doxycycline-inducible AhR shRNA blocks tumor invasion in Matrigel. Hs578T cells were stably transduced with either doxycycline-inducible control shRNA (mixed/non-silent) (left) or AhR-specific shRNA (right). Cells were treated with doxycycline to activate both control and AhR shRNA vectors and cells were cultured in Matrigel for 5 days.
[0072] Figure 14 demonstrates that the AhR inhibitor CB993113 blocks tumor invasion in BP1 in matrigel. BP1 cells were cultured in the presence of vehicle (left) or CB7993113 at 5 µM in Matrigel for 5 days. CB7993113 is non-toxic at the highest doses tested, 10 μM.
[0073] Figure 15 is a schematic showing how AhR inhibitors and non-competitive agonists can inhibit tumor invasion.
Figures 16A-16B demonstrate that AhR agonists reduce human breast tumor (BP1) cell invasion into matrigel. BP1 cells were cultured on Matrigel in the presence of vehicle, DIM at 5 µM (16A) vehicle or CB7950998 at 5 µM (16B) for 5 days. DETAILED DESCRIPTION
[0075] New compositions and methods comprising agents that modulate AhR activity are described here. The inventors have discovered novel small molecule compounds of Formula (I) and (II), as described herein, which modulate constitutive AhR activity and can be used to treat and inhibit proliferative diseases, such as cancer, based on their ability to inhibit the growth of cancer cells and other parameters that characterize tumor metastasis, such as tumor cell invasiveness. Hydrocarbon Aryl Receiver and Hydrocarbon Modulators
The aryl hydrocarbon receptor ("AhR") is a ligand-dependent member of the basic helix-loop-helix family of transcription factors that have been found to be activated by several structurally diverse naturally occurring and synthetic compounds such as polycyclic aromatic hydrocarbons, indoles and flavonoids. In the absence of the bound ligand, the AhR is present in a latent conformation in the cytoplasmic compartment of the cell associated with two molecules of the heat shock protein molecular chaperone 90 ("hsp90") (Perdew, J. Biol Chem 263: 13802-13805 (1988) ) and Wilhelmsson et al., EMBO J. 9:69-76 (1990)), an immunophilin-like protein, XAP2 (Carver et al., J. Biol. Chem. 272:11452-11456 (1997); Ma and col., J. Biol. Chem. 272:8878-8884 (1997) and Meyer et al., Mol. Cell. Biol. 18:978-988 (1998)) and the hsp90 interacting protein, p23 (Kazlauskas et al. col., J. Biol. Chem. 274:13519-13524 (1999)). Ligand binding initiates a cascade of events that includes translocation to the nucleus, release of hsp90, and heterodimerization with ARNT (Schmidt et al., Annu. Rev. Cell. Dev. Biol. 12:55-89 (1996) and Rowlands et al., Crit. Rev. Toxicol. 27:109-134 (1997)). The AhR-ARNT linked ligand complex is able to recognize consensus sequences called dioxin response elements ("DRE") located in the promoter region of CYP1A1 and other responsive genes, thus activating transcription (Schmidt et al., Annu Rev. Cell. Dev. Biol. 12: 55-89 (1996) and Rowlands et al., Crit. Rev. Toxicol. 27: 109-134 (1997)). Known examples of AhR-associated proteins include, but are not limited to, hsp90, p23, XAP2, p60, hsp70 and p48.
The AhR protein contains several domains critical for function and is classified as a member of the basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) family of transcription factors. The bHLH motif is located at the N-terminus of the protein. Members of the bHLH superfamily have two functionally distinct and highly conserved domains. The first is the basic region (b) which is involved in the binding of transcription factor to DNA. The second is a helix-loop-helix (HLH) region that facilitates protein-protein interactions. Also contained in AhR are two PAS domains, PAS-A and PAS-B, which are stretches of 200-350 amino acids that exhibit high sequence homology to the protein domains that were originally found in the "period" (Per) and " single minded" (SIM) of Drosophila and in the dimerization partner of AhR, the aryl hydrocarbon receptor nuclear translocator (ARNT). PAS domains support specific secondary interactions with other PAS domain-containing proteins, as is the case with AhR and ARNT, so that heterozygous and homozygous protein complexes can form. The AhR ligand binding site is contained within the PAS-B domain and contains several conserved residues critical for ligand binding. Finally, a Q-rich domain is located in the C-terminal region of the protein and is involved in co-activator recruitment and transactivation.
[0078] Accordingly, the term "aryl hydrocarbon receptor" or "AhR", as used herein, refers to polypeptide of 848 amino acids having the amino acid sequence: MNSSSANITYASRKRRKPVQKTVKPIPAEGIKSNPSKRHRDRLNTELDRLASLL PFPQDVINKLDKLSVLRLSVSYLRAKSFFDVALQUSSPTERNGGQDNCRAANFREGLNL QEGEFLLQALNGFVLVVTTDALVFYASSTIQDYLGFQQSDVIHQSVYELIHTEDRAEFQ RQLHWALNPSQCTESGQGIEEATGLPQTVVCYNPDQIPPENSPLMERCFICRLRCLLDN SSGFLAMNFQGKLKYLHGQKKKGKDGSILPPQLALFAIATPLQPPSILEIRTKNFIFRT KHKLDFTPIGCDAKGRIVLGYTEAELCTRGSGYQFIHAADMLYCAESHIRMIKTGESGM IVFRLLTKNNRWTWVQSNARLLYKNGRPDYIIVTQRPLTDEEGTEHLRKRNTKLPFMFT TGEAVLYEATNPFPAIMDPLPLRTKNGTSGKDSATTSTLSKDSLNPSSLLAAMMQQDES IYLYPASSTSSTAPFENNFFNESMNECRNWQDNTAPMGNDTILKHEQIDQPQDVNSFAG GHPGLFQDSKNSDLYSIMKNLGIDFEDIRHMQNEKFFRNDFSGEVDFRDIDLTDEILTY VQDSLSKSPFIPSDYQQQQSLALNSSCMVQEHLHLEQQQQHHQKQVVVEPQQQLCQKMK HMQVNGMFENWNSNQFVPFNCPQQDPQQYNVFTDLHGISQEFPYKSEMDSMPYTQNFIS CNQPVLPQHSKCTELDYPMGSFEPSPYPTTSSLEDFVTCLQLPENQKHGLNPQSAIITP QTCYAGAVSMYQCQPEPQHTHVGQMQYNPVLPGQQAFLNKFQNGVL NETYPAELNNINN TQTTTHLQPLHHPSEARPFPDLTSSGFL (SEQ ID NO: 1), as described, for example, by NP_001612, along with any naturally occurring spliced allelic variants and processed forms thereof. Typically, AhR refers to human AhR. The term AhR is also used to refer to truncated forms or fragments of the AhR polypeptide that comprise, for example, specific domains of AhR. Reference to any such form of the AhR may be identified in the application, for example by "AhR (122224)".
[0079] During canonical signaling, cytosolic AhR binds to a ligand, such as an appropriate small molecule, which facilitates AhR translocation to the nucleus and eventually results in further transcription of target genes. AhR target gene promoters contain the 5'-TNGCGTG-3' responsive element, termed "DRE" or "XRE" for "dioxin responsive elements" or "xenobiotic responsive elements". Genes for enzymes that metabolize xenobiotics (eg, cytochrome P450) are well known targets of AhR and are referred to herein as "battery genes of AhR". Hundreds of other genes also have DREs. The elucidation of the canonical signaling biochemistry of AhR revealed several parameters that can tune AhR activity. These include features of the ligand, adapter molecules, and transcription co-activators or co-repressors that regulate the extraordinary cell-specific activity of the AhR (C. Esser et al., Trends in Immunology 2009, Vol. 30: 9, pages 447 -454).
[0080] Alternative AhR signaling pathways have also been described. For example, AhR can bind to the retinoblastoma protein, the estrogen receptor (ER), the E2F1 transcription factor, and the RelA and RelB subunits of the NFKB pathway. Evidence of AhR interference with other signaling pathways, such as the kinase pathway (src, JNK, p38, MAPK) or competition for transcription cofactors has also been reported. AhR can act as a ubiquitin ligase, targeting the ER for proteosomal degradation. In these signaling pathways, the AhR and the other proteins sometimes mutually repress each other's function. Indeed, bioinformatics analysis points to the existence of complex signal interference between AhR and other transcription factors or transcription coactivators (C. Esser et al., Trends in Immunology 2009, Vol. 30: 9, Pages 447-454).
[0081] A number of low molecular weight chemicals qualify as endogenous or physiological "AhR binders", that is, they have binding dissociation constants (Kd) and effective concentrations at the level expected for a physiologically relevant AhR binder . Physical fluid shear stress (which causes the oxidation of low-density lipoproteins), second messengers cAMP and Ca2+, components of growth media and serum all activate AhR responses (C. Esser et al., Trends in Immunology 2009, Vol. 30: 9, pages 447-454). AhR has not yet been crystallized, so information on ligand-dependent structural changes does not currently exist. Ligand-protected protease digestion studies indicated that there is only one binding pocket for the linkers (S. Kronenberg et al., Nucleic Acids Res. 28 (2000), pages 2286-2291).
[0082] AhR binders need only meet minimum requirements for size and flat shape to fit in the AhR binding pocket. Consequently, a wide range of low molecular weight chemicals can activate AhR, albeit with different affinities ranging between 10-12 and 10-3 M. Many ligands have two carbon ring systems, such as tryptophan derivatives, flavonoids and biphenyls. The AhR system is genetically polymorphic and different alleles influence the responsiveness to AhR ligands (C. Esser et al., Trends in Immunology 2009, Vol. 30: 9, Pages 447-454). AhR ligands can generally be classified into two categories, synthetic or naturally occurring. The first binders to be discovered were synthetic and members of halogenated aromatic hydrocarbons (dibenzo-dioxins, dibenzofurans and biphenyls) and polycyclic aromatic hydrocarbons (3-methylcholanthrene, benzo(a)pyrene, benzanthracenes and benzoflavones).
[0083] Naturally occurring compounds that have been identified as AhR ligands include tryptophan derivatives such as indigo and indirubin, tetrapyrols such as bilirubin, arachidonic acid metabolites, lipoxin A4 and prostaglandin G, modified low-density lipoprotein, and various carotenoids dietetics.
[0084] Exemplary AhR ligands include, but are not limited to, endogenous ligands such as FICZ or 6-formylindole[3,2-b]carbazole and 6,12-diformylindole[3,2-b]carbazole or dFICZ ( tryptophan photoproducts), bilirubin (product of heme metabolism by the liver), lipoxin A4 (eicosanoid with anti-inflammatory properties), ITE [2-(1'H-indol-3'-carbonyl)-thiazole-acid methyl ester 4-carboxylic] (isolated from lung tissues); environmental pollutants (formed during combustion of organic material) such as 2,3,7,8-tetrachlorodibenzo-p-dioxine (TCDD) and Benz[a]pyrene; dietary binders such as quercetin (found in apples and onions), indole-3-carbinol (found in many Brassicaceae, for example cabbage), resveratrol (found in red wine) and curcumin (a spice); and (synthetic) drugs such as M50367 ({3-[2-(2-phenylethyl)-benzoimidazol-4-yl]-3-hydroxy-propanoic acid)} and VAF347 ({[4-(3-chloro-phenyl) )-pyrimidin-2-yl]}).
Two of the most potent and well characterized antagonists of AhR include the synthetic flavonoid 3'-methoxy-4'nitroflavone ("3M4NF") and the indole derivative 3,3'-diindolylmethane ("DIM"). These compounds have been shown to function through direct competition for binding to the binding site of the AhR ligand (Henry et al., Mol. Pharmacol 55: 716-725 (1999); Hestermann et al., Mol. Cell. Biol. 23 : 7920-7925 (2003)). The fate of AhR when binding these structurally distinct antagonists is very different. Binding of 3M4NF to AhR inhibits TCDD-mediated nuclear localization, ARNT dimerization and DNA binding (Henry et al., Mol. Pharmacol. 55: 716725 (1999)). 3M4NF is believed to inhibit a conformational change within the AhR complex necessary for exposure of the nuclear localization sequence, resulting in retention of the AhR in the cytoplasmic compartment of the cell. Conversely, binding of DIM to AhR allows for nuclear localization, dimerization of ARNT and subsequent binding to DNA. However, unlike the TCDD-linked AhR-ARNT dimer, this DIM-linked complex is incapable of recruiting the necessary cofactors responsible for initiating transcription ( Hestermann et al., Mol. Cell. Biol 23: 7920-7925 (2003) )). Halogenated and nitro-substituted flavones may exhibit structure-dependent aryl hydrocarbon receptor (AhR) agonist and antagonist activities comparable to those observed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (F. Lu et al. , Biochemical Pharmacology, 51: 1077 (1996)). AhR Modulators
[0086] The inventors have found that novel flavone and hydroflavone AhR modulating compounds described herein, such as small molecules of Formula (I) and Formula (II), eg, CB7993113, CB9950998 and CMLD-2166, modulate activity of constitutive AhR by functioning as AhR inhibitors or non-constitutive AhR agonists. Furthermore, they found that such AhR-modulating compounds can inhibit cancer cell growth as well as tumor invasion and are absorbed in vivo and can be used at pharmacologically effective doses to target peripheral organs such as bone marrow. Accordingly, novel small molecules modulating AhR and constitutive AhR signaling for use in therapeutic compositions and methods of treating and inhibiting cancer growth and tumor cell invasion, and other hyperproliferative disorders, are described herein.
AhR mediates a variety of functional responses including, but not limited to, new transcription of target genes or AhR battery genes that have a 5'-TNGCGTG-3' DRE or XRE responsive element. Alternative AhR signaling pathways have also been described, such as binding to the retinoblastoma protein, the estrogen receptor (ER), the E2F1 transcription factor and the RelA and RelB subunits of the NFKB pathway. AhR can also act as a ubiquitin ligase. Accordingly, the AhR signaling pathway comprises multiple pathways, including AhR constitutive and non-constitutive signaling pathways or signaling activity, as these terms are defined herein.
[0088] As used herein, "constitutive AhR signaling" refers to one or more signaling pathways mediated or regulated by the AhR that are activated or directed by one or more endogenous AhR ligands or one or more environmental ligands, such as toxins or pollutants, which cause constitutive or long-term translocation of the AhR to the nucleus, and activation or modulation of one or more AhR battery genes involved in dysregulated cell growth and proliferation, tumor cell invasiveness, or a combination thereof.
[0089] Consequently, a "constitutive AhR agonist" is an AhR agonist that selectively binds to AhR and can induce AhR activity or signaling or activate AhR function by itself, as measured or assayed using any method known to a person of skill in the art, such as an in vitro AhR reporter assay, and can cause dysregulated cell growth, tumor cell invasiveness, or a combination thereof. In some embodiments, a constitutive AhR agonist up-regulates the expression of CYP1A1, CYP1B1 or a combination thereof. Activation by an AhR agonist is achieved when the AhR activity value is at least 10% greater, at least 25% greater, at least 50% greater, at least 75% greater, at least 100% greater, at least 2 times larger, at least 5 times larger, at least 10 times larger, at least 25 times larger, at least 50 times larger, at least 100 times larger, at least 1000 times larger or more, compared to a control in the absence of the agonist of AhR.
[0090] As used herein, "non-constitutive AhR signaling" refers to one or more AhR-mediated or induced signaling pathways that do not cause constitutive or long-term translocation of the AhR to the nucleus, nor activation or modulation of one or plus AhR battery genes involved in dysregulated cell growth, tumor cell invasiveness, or a combination thereof. In some embodiments, non-constitutive AhR signaling does not cause upregulation of expression of CYP1A1, CYP1B1 or a combination thereof.
Accordingly, an "AhR modulator", as the term is used herein, refers to an agent, such as a small molecule of Formula (I), for example, small molecules of Formula (Ia) or of Formula (II), for example, small molecules of Formula (IIa), which modulate or cause or facilitate a qualitative or quantitative alteration, change or modification in one or more processes, mechanisms, effects, responses, functions, activities or pathways mediated by the receptor AhR. Such changes mediated by an AhR modulator, such as an inhibitor or a non-constitutive agonist of AhR described herein, may refer to a decrease or an increase in AhR expression, activity, or function, such as a decrease in, inhibition of or deviation of constitutive activity of AhR. The term "expression" refers to those cellular processes involved in the production of RNA and proteins and, as appropriate, secretion proteins including, where applicable, but not limited to, for example, transcription, translation, folding, modification and processing. "Expression products" include RNA transcribed from a gene and polypeptides obtained through translation of mRNA transcribed from a gene.
[0092] The term "modulate", in reference to an AhR modulator, is used consistently with its use in the art, for example, meaning to cause or facilitate a qualitative or quantitative alteration, change or modification in one or more processes, mechanisms, effects, responses, functions, activities, pathways or other biological phenomena of interest. Consequently, as used herein, modular refers to a qualitative or quantitative alteration, change or modification in one or more processes, mechanisms, effects, responses, functions, activities or pathways mediated by the AhR receptor.
The term "agent", as used herein in reference to an AhR modulator, means any compound or substance such as, but not limited to, a small molecule, nucleic acid, polypeptide, peptide, drug, ion, etc. An "agent" can be any chemical, entity or moiety including, but not limited to, naturally occurring, synthetic proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, a nucleic acid oligomer, an amino acid, or a carbohydrate, and includes, without limitation, proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNAs, lipoproteins, aptamers and modifications and combinations thereof, etc. In certain embodiments, as described herein, agents are small molecules having a chemical moiety. For example, chemical moieties include unsubstituted or substituted alkyl, aromatic or heterocyclyl moiety. Compounds may be known to have a desired activity and/or property, e.g., modulate AhR activity, or may be selected from a library of diverse compounds using, e.g., the screening methods described herein.
As used herein, the term "small molecule" refers to a chemical agent which may include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog , an aptamer, a nucleotide, a nucleotide analogue, an organic or inorganic compound (for example, including organometallic and hetero-organic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters and other pharmaceutically acceptable forms of such compounds.
[0095] In some embodiments, an AhR modulator selectively binds to AhR. As used herein, "selectively binds" or "specifically binds" refers to the ability of an AhR agonist or AhR inhibitor, such as an AhR antagonist described herein, to bind to a target, such as the RNA polypeptide. AhR, with a KD of 10-5 M (10000 nM) or less, eg 10-6 M or less, 10-7 M or less, 10-8 M or less, 10-9 M or less, 10- 10M or less, 1011M or less, or 10-12M or less. For example, if an agonist or inhibitor described herein binds to the AhR polypeptide with a KD of 10-5 M or less, but not to other molecules or a related homolog, then the agent is said to specifically bind to the AhR polypeptide. AhR. Specific binding can be influenced, for example, by the affinity and avidity of an agonist or inhibitor and the concentration of the agonist or inhibitor used. One of skill in the art can determine the proper conditions under which the agonists or inhibitors described herein selectively bind using any suitable methods, such as titration of an AhR agonist or AhR inhibitor in a suitable cell binding assay, such as those described here.
[0096] With respect to the AhR target, the term "ligand interaction site" in AhR means a site, epitope, antigenic determinant, part, domain or stretch of amino acid residues in AhR that is a site for binding a ligand , receptor or other binding partner, a catalytic site, a cleavage site, a site for allosteric interaction, a site involved in multimerization (such as homomerization or heterodimerization) of the AhR; or any other site, epitope, antigenic determinant, part, domain or stretch of amino acid residues in the AhR that is involved in a target's biological action or mechanism, ie, the AhR. More generally, a "ligand interaction site" can be any site, epitope, antigenic determinant, part, domain, or stretch of amino acid residues in the AhR polypeptide to which an inhibitor or agonist described herein can bind, so as that the activity and/or expression of AhR (and/or any pathway, interaction, signaling, biological mechanism or biological effect in which the AhR is involved) is modulated.
[0097] In some aspects of the compositions and methods described herein, AhR modulators are AhR inhibitors having the chemical structures of Formula (I), Formula (Ia), Formula (II) or Formula (IIa) described herein.
As used herein, the terms "AhR inhibitor" or "AhR inhibitor" refer to an agent or compound, such as the small molecules of Formula (I) or Formula (II) described herein, e.g., CB7993113 and CMLD-2166, respectively, which inhibit one or more downstream effector and AhR signaling pathways, including constitutive AhR signaling, as these terms are used herein. Thus, the term AhR inhibitor refers to an agent that inhibits the expression of the AhR polypeptide or polynucleotide encoding the AhR or one that binds to, partially or totally blocks stimulation, decreases, prevents, delays activation, inactive, desensitize or down-regulate the activity of the AhR polypeptide or polynucleotide encoding the AhR. Such AhR inhibitors can, for example, inhibit AhR expression, e.g. AhR translation, AhR post-translational processing, stability, degradation or nuclear or cytoplasmic localization of the AhR polypeptide, or transcription, post-transcriptional processing, stability or degradation of a polynucleotide that encodes the AhR or binds to, partially or completely blocks the stimulation, DNA binding or activity of the AhR transcription factor. An AhR inhibitor can act directly or indirectly. In some embodiments, an AhR inhibitor selectively binds to AhR.
[0099] The terms "inhibit", "decrease" and "reduce" are all used here generally to mean a decrease by a statistically significant amount. Consequently, AhR inhibition is obtained when the activity value of an AhR polypeptide or a polynucleotide encoding AhR is about at least 10% less, at least 20% less, at least 30% less, at least 40% less , at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, at least 95% less, at least 98% less, at least 99% less, including up to 100% or less, i.e. absent or undetectable, compared to a reference or control level in the absence of inhibitor. In some embodiments of the aspects described herein, AhR inhibitors inhibit the constitutive activity of AhR.
[0100] In some embodiments of these aspects, the AhR inhibitor is an "AhR antagonist". An AhR antagonist refers to an AhR inhibitor that does not elicit a biological response per se by specifically binding the AhR polypeptide or polynucleotide encoding AhR, but blocks or dampens ligand-mediated or agonist-mediated responses, i.e., an An AhR antagonist can bind, but does not activate the AhR polypeptide or polynucleotide encoding AhR, and the binding interrupts the interaction, displaces an AhR agonist, and/or inhibits the function of an AhR agonist. In some such embodiments, the AhR inhibitor is an AhR antagonist that prevents or inhibits the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the AhR. In some such embodiments, an AhR antagonist is identified as a small molecule compound that blocks AhR activation by a strong AhR activator, such as BNF or TCDD, but does not induce AhR activity or signaling by itself, as appropriate. measured or assayed using any method known to one of skill in the art, such as in vitro AhR reporter assays. Thus, as used here, an AhR antagonist does not function as an inducer of AhR activity when bound to AhR, that is, they function as pure AhR inhibitors. In some embodiments, an AhR antagonist selectively binds to AhR.
[0101] In some embodiments of these aspects, AhR inhibitors described herein, such as small molecules of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166, respectively, block AhR constitutive effector functions that mediate the growth and progression of established tumors. In other embodiments, small AhR inhibitory molecules of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166 described herein function as chemoprotective agents by blocking CYP1A1 induction and mutagenic agent production mediated by AhR when exposed to environmental binders.
[0102] In some embodiments of these aspects, small AhR inhibitory molecules of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166, described herein inhibit the initial contributions of constitutively active AhR in directing malignant transformation . In some embodiments, AhR inhibitory small molecules of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166, described herein inhibit cancer or tumor cell growth mediated by constitutive AhR signaling. In some embodiments, small molecules AhR inhibitors of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166, described herein inhibit tumor invasion mediated by constitutive AhR signaling in directing malignant transformation.
[0103] In other aspects, the AhR modulators described herein are non-constitutive agonists of AhR with the chemical structures of Formula (I) or Formula (II), such as CB7950998.
[0104] As used herein, the terms "non-constitutive AhR agonist", "non-constitutive AhR agonist", "non-constitutive AhR activator" or "non-constitutive AhR activator" refer to a small molecule of Formula ( I) or Formula (II) described herein, e.g., CB7950998, which binds to AhR and hyperactivates or shifts AhR constitutive signaling and downstream effector pathways, as these terms are used herein. Such non-constitutive AhR small agonist molecules of Formula (I) or Formula (II), e.g., CB7950998, can increase the expression of the AhR polypeptide or polynucleotide encoding the AhR or bind to the AhR and partially or completely bypass, the constitutive AhR signaling and downstream activity, such as cancer cell growth and tumor invasive properties. In some embodiments, a non-constitutive AhR agonist selectively binds to AhR.
[0105] In some embodiments of these aspects, the non-constitutive AhR agonist blocks or dampens endogenous ligand-mediated constitutive responses, that is, a non-constitutive AhR agonist can bind and signal through the AhR, but does not activate the constitutive activity of the AhR AhR polypeptide or polynucleotide encoding the AhR and binding disrupts the interaction and/or inhibits the function of an endogenous AhR ligand that activates the AhR constitutive pathway. Consequently, a non-constitutive AhR agonist of Formula (I) or Formula (II), e.g., CB7950998, can shift constitutive AhR signaling by at least 10% or more, at least 20% or more, at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, at least 90% or more, at least 95 % or more or at least 100% or completely compared to a reference or control level in the absence of non-constitutive AhR agonist.
In preferred embodiments of these aspects, non-constitutive AhR agonists of Formula (I) or Formula (II), e.g., CB7950998, described herein bind to AhR and mediate non-constitutive AhR signaling and inhibit or block functions constitutive effectors of AhR that mediate the growth and progression of established tumors. In other embodiments, non-constitutive AhR agonists of Formula (I) or Formula (II), e.g., CB7950998, described herein function as chemoprotective agents by blocking AhR-mediated induction of CYP1A1 and production of mutagenic agents when from exposure to AhR ligands that mediate constitutive AhR signaling. In some embodiments, non-constitutive AhR agonists of Formula (I) or Formula (II), e.g., CB7950998, described herein inhibit the initial contributions of constitutively active AhR in directing malignant transformation. In some embodiments, non-constitutive AhR agonists of Formula (I) or Formula (II), e.g., CB7950998, described herein inhibit or prevent cancer or tumor cell growth mediated, in part or entirely, by constitutive AhR signaling . In some embodiments, non-constitutive AhR agonists of Formula (I) or Formula (II), e.g., CB7950998, described herein inhibit or prevent tumor invasiveness mediated, in part or entirely, by constitutive AhR signaling.
[0107] In some embodiments, a non-constitutive AhR agonist is identified as a small molecule compound that: binds to AhR and can displace binding by an AhR activator, such as BNF or TCDD; can activate AhR as measured or assayed using any method known to a person skilled in the art; and does not cause, and preferably inhibit, the growth or proliferation of cancer cells or tumor invasiveness, as measured using any assay known to one of ordinary skill in the art. Activation of AhR by a non-constitutive agonist can be measured using, for example, any of the in vitro AhR reporter assays described herein. Assays to determine whether an AhR agonist functions as a non-constitutive AhR agonist that inhibits the growth and proliferation of cancer cells can be based on any method known to one of ordinary skill in the art, such as the Matrigel assays described in the identification of CB7950998 in Figures 14A-14B.
[0108] Accordingly, small molecules AhR inhibitory and non-constitutive AhR agonist compounds of Formula (I) are provided for use in the various aspects described herein:
FORMULA (I) wherein: Y is C or N; X is OR1, NHR1, SR1, CH2(n)R1, halo or H; n is 0-6; Z is O, S or NH; R1 and R2 are independently H, alkyl, alkenyl, alkynyl, amino, aminosulfonyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R 3 , R 4 , R 5 and R 6 are independently absent, H, halo, alkyl, alkenyl, alkynyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; pharmaceutically acceptable salts thereof.
[0109] Also provided for use in the various aspects described herein are compounds of Formula (II):
where: Y is C or N; X is OR1, NHR1, SR1, CH2(n)R1, halo or H; n is 0-6; Z is O, S or NH; R1 and R2 are independently H, alkyl, alkenyl, alkynyl, amino, aminosulfonyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R 3 , R 4 , R 5 and R 6 are independently absent, H, halo, alkyl, alkenyl, alkynyl, alkoxy, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; stereoisomers thereof; and pharmaceutically acceptable salts thereof.
[0110] As will be appreciated by those skilled in the art, various stereoisomers of Formula (II) can be prepared. For example, stereoisomers focused on the two chiral centers (marked 2 and 3 in Formula (II)), represent (2R, 3S), (2S, 3R), (2R, 3R) and (2S, 3S) stereoisomers of the compound. Whether a chiral center is oriented in an R configuration or an S configuration will obviously depend on the substituents assigned to the different variables. The following generic stereoisomers are examples of the various (2R, 3S), (2S, 3R), (2R, 3R) and (2S, 3S) configurations of Formula (II):

[0111] In some embodiments, Formula (I) may be represented as Formula (Ia):
wherein: X' is H, alkyl, aminosulfonyl, alkoxy, amino, acyl, aryl or heteroaryl, each of which may be optionally substituted; n is 0-6; R2 is H, alkyl, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R3, R4, R5 and R6 are independently H, alkyl, acyl, halo, aryl or heteroaryl, each of which may be optionally substituted; and pharmaceutically acceptable salts thereof.
[0112] In other embodiments of Formula (Ia), X' is alkyl, alkoxy, amino, aminosulfonyl (optionally substituted with aryl or aralkyl groups, such as toluene); n is 0 or 1; R2 is aryl, substituted aryl, heteroaryl or substituted aryl; and R3, R4, R5 and R6 are independently H, alkoxy, alkyl or halo.
[0113] Representative Compounds of Formula I
include the following compounds:
[0114] In other embodiments, Formula (II) may be represented as Formula (IIa):
X' is H, alkyl, aminosulfonyl, alkoxy, acyl, aryl or heteroaryl, each of which may be optionally substituted; n is 0-6; R2 is H, alkyl, acyl, aryl, heteroaryl, arylalkyl, cycloalkyl, heteroarylalkyl, heterocyclyl or haloalkyl, each of which may be optionally substituted; R3, R4, R5 and R6 are independently H, alkyl, acyl, halo, aryl or heteroaryl, each of which may be optionally substituted; stereoisomers thereof; and pharmaceutically acceptable salts thereof.
[0115] In other embodiments of Formula (IIa), X' is alkyl, alkoxy, aminosulfonyl (optionally substituted with aryl or aralkyl groups, such as toluene); n is 0 or 1; R2 is aryl, substituted aryl, heteroaryl or substituted aryl; and R3, R4, R5 and R6 are independently H, alkoxy, alkyl or halo.
[0116] Representative compounds of Formula II include the following compounds:

[0117] For simplicity, chemical moieties, as defined and mentioned throughout the document, may be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances evident to those skilled in the art. For example, in some embodiments, an "alkyl" moiety may refer to a monovalent radical (eg, CH3-CH2-) or, in other embodiments, a bivalent bond moiety may be "alkyl", in which case those versed. in the art it will be understood that alkyl is a divalent radical (eg, -CH 2 -CH 2 -), which is equivalent to the term "alkylene". Similarly, in circumstances where divalent moieties are required and are stated to be "alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic", "alkyl" "alkenyl", "alkynyl", "aliphatic" or "cycloalkyl", those of skill in the art will understand the terms "alkoxy", "alkylamino", "aryloxy", "alkylthio", "aryl", "heteroaryl", "heterocyclic", "alkyl ", "alkenyl", "alkynyl", "aliphatic" or "cycloalkyl" refer to the corresponding divalent moiety.
[0118] The term "halo" refers to any fluorine, chlorine, bromine or iodine radical.
[0119] The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl or heteroarylcarbonyl substituent, any of which may be further substituted by substituents. Exemplary acyl groups include, but are not limited to, (C1-C6)alkanoyl (e.g., formyl, acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.), (C3-C6)cycloalkylcarbonyl (e.g. , cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.), heterocyclic carbonyl (eg, pyrrolidinylcarbonyl, pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl, tetrahydrofuranylcarbonyl, etc.), aroyl (eg, benzoyl), and heteroaroyl (eg thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl, furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl, benzo[b]thiophenyl- 2-carbonyl, etc.). Furthermore, the alkyl, cycloalkyl, heterocycle, aryl and heteroaryl portions of the acyl group can be any of the groups described in the respective definitions.
[0120] The term "alkyl" refers to saturated non-aromatic hydrocarbon chains which may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include, without limitation, methyl, ethyl, propyl, butyl, pentyl, hexanyl), which can be optionally inserted with N, O, S, SS, SO2, C(O), C(O)O, OC(O), C(O)N or NC(O). For example, C1-C6 indicates that the group can have from 1 to 6 (inclusive) carbon atoms in it.
[0121] The term "alkenyl" refers to an alkyl radical that comprises at least one double bond. Exemplary alkenyl groups include, but are not limited to, e.g., ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
[0122] The term "alkynyl" refers to an alkyl radical that comprises at least one triple bond.
[0123] The term "alkoxy" refers to an -O-alkyl radical.
[0124] The term "aminoalkyl" refers to an alkyl substituted with an amino group.
[0125] The term "aryl" refers to a monocyclic, bicyclic or tricyclic aromatic ring system in which 0, 1, 2, 3 or 4 atoms of each ring may be replaced by a substituent. Exemplary aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like.
[0126] The term "arylalkyl" refers to an alkyl substituted with an aryl or aryl substituted with an alkyl.
[0127] The term "cycloalkyl" refers to saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons and, for example, 3 to 6 carbons, wherein the cycloalkyl group may additionally optionally be replaced. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, and the like.
[0128] The term "heteroaryl" refers to a 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic aromatic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic or 1-9 if heteroatoms if tricyclic, said heteroatoms selected from O, N or S (for example carbon atoms and 1-3, 1-6 or 1-9 heteroatoms of N, O or S if monocyclic, bicyclic or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be replaced by a substituent. Exemplary heteroaryl groups include, but are not limited to, pyridyl, furyl or furanyl, imidazolyl, benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, pyridazinyl, pyrazinyl, quinolinyl, indolyl, thiazolyl, naphthyridinyl, and the like.
[0129] The term "heteroarylalkyl" refers to an alkyl substituted with a heteroaryl.
[0130] The term "heterocyclyl" refers to a non-aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic, having 1-3 heteroatoms and monocyclic, 1-6 heteroatom ring system if bicyclic or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N or S (eg carbon atoms and 1-3, 1-6 or 1-9 heteroatoms of N, O, or S if monocyclic , bicyclic or tricyclic, respectively), in which 0, 1, 2 or 3 atoms of each ring may be replaced by a substituent. Exemplary heterocyclyl groups include, but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
[0131] The term "haloalkyl" refers to an alkyl group having one, two, three or more halogen atoms attached thereto. Exemplary haloalkyl groups include, but are not limited to, chloromethyl, bromoethyl, trifluoromethyl, and the like.
[0132] The term "optionally substituted" means that the specified group or moiety is not substituted or is substituted by one or more (typically 14) substituents independently selected from the group of substituents listed herein in the definition of "substituents" or otherwise , specified. Substituents can be "distinct" substituents, eg a halo group and an alkoxy group attached to different carbon atoms in a benzene ring, or the substituents can be "stacked" on top of each other, eg an acyl group (such as formyl), which is substituted with an aminosulfonyl group which is substituted with an arylalkyl (such as toluene).
[0133] The term "substituents" refers to a group that replaces a hydrogen on any atom of the substituted group or portion, as well as a "substituted" group on an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl, heteroaryl group, acyl, amino at any atom of this group. Suitable substituents include, without limitation, halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, aminosulfonyl, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxy, hydroxyalkylcarbonyl, , alkylthio, CF3, N-morphilino, phenylthio, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido. In some cases, the two substituents, along with the carbons to which they are attached, can form a ring.
[0134] In many cases, protecting groups are used during preparation of the compounds of the invention. As used herein, the term "protected" means that the indicated portion has a protecting group attached thereto. In some preferred embodiments of the invention, the compounds contain one or more protecting groups. A wide variety of protecting groups can be employed in the methods of the invention. In general, protecting groups make chemical functionalities inert to specific reaction conditions and can be added and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
[0135] Representative hydroxyl protecting groups, for example, are disclosed by Beaucage et al. (Tetrahedron 1992, 48, 2223-2311). Other hydroxyl protecting groups, as well as other representative protecting groups, are disclosed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 2, 2nd ed., John Wiley & Sons, New York, 1991 and Oligonucleotides and Analogues A Practical Approach , Ekstein, F. ed., IRL Press, NY, 1991. Examples of hydroxy protecting groups include, but are not limited to, t-butyl, t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy) ethyl, 2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl, p,p'-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl, t - butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, benzoyl formate, acetate, chloroacetate, trichloroacetate, trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate, 9-fluorenylmethyl carbonate, mesylate and tosylate.
[0136] Nitrogen or amino protecting groups stable to acid treatment are selectively removed through base treatment and are used to selectively make reactive amino groups available for replacement. Examples of amino protecting groups include, but are not limited to, carbamate protecting groups, such as 2-trimethyl-silyltoxiccarbonyl (Teoc), 1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl ( BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl (Fmoc) and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, trihaloacetyl, benzoyl and nitrophenylacetyl; sulfonamide protecting groups such as 2-nitrobenzenesulfonyl; imine and cyclic imide protecting groups such as phthalimido and dithiasuccinoyl. AhR Modulators: Therapeutic Uses and Pharmaceutical Compositions
[0137] Described herein are novel small AhR modulating molecules of Formula (I) or Formula (II) for use in methods of treating an individual having or at risk of developing a proliferative disease, such as cancer. Small AhR modulating molecules of Formula (I) or Formula (II) described herein, such as AhR inhibitors, e.g., CB7993113 and CMLD-2166, and non-constitutive AhR agonists, e.g., CB7950998, can be administered to an individual in need thereof by any appropriate route that results in effective treatment in the individual.
[0138] Consequently, in some aspects, methods of modulating AhR constitutive activity in an individual are provided here. Such methods comprise administering to a subject having AhR constitutive activity, a therapeutically effective amount of an AhR modulating small molecule of Formula (I) or Formula (II) described herein. In some embodiments of these aspects, the AhR modulator is an AhR inhibitor of Formula (I), e.g., CB7993113. In some embodiments of these aspects, the AhR modulator is an AhR inhibitor of Formula (II), e.g., CMLD-2166. In some embodiments of these aspects, the AhR modulator is a non-constitutive agonist of AhR of Formula (I) , for example, CB7950998, or Formula (II).
[0139] The terms "subject" and "individual" are used interchangeably herein and refer to an animal, eg, a human, that receives the small molecule AhR modulators of Formula (I) or Formula (II ) described herein, for example, AhR inhibitors such as CB7993113 and CMLD-2166, and non-constitutive AhR agonists such as CB7950998. For treatment of those disease states that are specific to a specific animal, such as a human being, the term "subject" refers to that specific animal. The terms "non-human animals" and "non-human mammals" are used interchangeably herein and include mammals such as rats, mice, rabbits, sheep, cats, dogs, cows, pigs and non-human primates. The term "subject" also includes any vertebrate animal including, but not limited to, mammals, reptiles, amphibians and fish.
[0140] In some aspects, provided herein are methods of treating an individual having a cancer or a cancerous condition or at risk of cancer or a cancerous condition, the methods comprising administering, to an individual having a cancerous condition or cancer or in risk of cancer or a cancerous condition, from a therapeutically effective amount of a small molecule AhR modulator of Formula (I) or Formula (II) described herein. In some embodiments of these aspects, the AhR modulator is an AhR inhibitor of Formula (I), e.g., CB7993113. In some embodiments of these aspects, the AhR modulator is an AhR inhibitor of Formula (II), e.g., CMLD-2166. In some embodiments of these aspects, the AhR modulator is a non-constitutive AhR agonist of Formula (I), e.g., CB7950998, or Formula (II).
[0141] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth or proliferation, which interferes with the normal functioning of the body's organs and systems. Consequently, the terms "cancer" or "tumor" as used herein refer to uncontrolled cell growth which interferes with the normal functioning of the body's organs and systems, including cancer stem cells and tumor vascular niches. An individual who has a cancer or a tumor is an individual who has objectively measurable cancer cells present in the individual's body. Cancers that migrate from their original location and spread to vital organs can eventually lead to the individual's death through functional deterioration of Organs affected organs. Hematopoietic cancers, such as leukemia, are able to compete with the normal hematopoietic compartments in an individual, thus leading to hematopoietic failure (in the form of thrombocytopenia, anemia, and neutropenia), ultimately causing death. This definition includes benign and malignant cancers, as well as latent tumors or micro-metastases.
[0142] A "metastasis" refers to the spread of the cancer from its primary site to other sites in the body. Cancer cells can spread from a primary tumor, enter lymph and blood vessels, circulate through the bloodstream, and grow into a distant focus (metastasis) to normal tissues elsewhere in the body. Metastasis can be local or distant. Metastasis is a sequential process, dependent on the spread of tumor cells from the primary tumor, which travel through the bloodstream and stop at a distant location. In the new location, the cells establish a blood supply and can grow to form a life-threatening mass. Both stimulatory and inhibitory molecular pathways within the tumor cell regulate this behavior and interactions between the tumor cell and host cells at the distant site are also significant.
[0143] Metastases are most often detected with the sole or combined use of magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, blood count and platelet counts, liver function studies, chest radiographs, and bone scintigraphy, in addition to monitoring specific symptoms.
[0144] Consequently, cancers that can be treated using the compositions and methods described in the various aspects of this document include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, but are not limited to, breast cancer, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer; brain and CNS cancer, peritoneum cancer, cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer, eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatoma; intraepithelial neoplasia, kidney or kidney cancer; laryngeal cancer; leukemia; liver cancer; lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and squamous cell carcinoma of the lung); lymphoma, including Hodgkin's and non-Hodgkin's lymphoma; melanoma; myeloma; neuroblastoma; glioblastoma, cancer of the oral cavity (eg, lip, tongue, mouth, and pharynx); Ovary cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; cancer of the uterus or endometrium; urinary system cancer; vulvar cancer, as well as other carcinomas and sarcomas, as well as B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade/follicular NHL; diffuse intermediate-grade NHL ; High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small cell uncleaved NHL; NHL by bulky disease; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), and Meig's syndrome.
[0145] In some embodiments, a cancer may be a solid tumor. As used herein, a "solid tumor" refers to an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells from which they are formed. Non-limiting examples of solid tumors are sarcomas, carcinomas and lymphomas. Leukemias and other blood cancers generally do not form solid tumors and therefore are not encompassed by the term "solid tumor" as used here.
[0146] In some embodiments of the aspects described here, an individual refers to a human who has a cancer or is at increased risk for a cancerous condition. An individual who has a cancer or a tumor is an individual who has objectively measurable cancer cells present in the individual's body. An individual who is at increased risk for a cancerous condition includes individuals with a known genetic risk factor for cancer or for whom there is a family history of cancer. In some embodiments of the aspects described here, the cancer is breast cancer. In some embodiments of the aspects described here, the cancer is a solid tumor.
[0147] In some embodiments of the aspects described herein, methods of treating cancer or a cancerous condition further comprise the step of selecting, diagnosing or identifying an individual with cancer or a cancerous condition. In such embodiments, an individual is identified as having cancer by the objective determination of the presence of cancer cells or a tumor in the individual's body by a person skilled in the art. Such objective determinations can be performed through the exclusive or combined use of tissue biopsies, blood count and platelet counts, urine analysis, magnetic resonance imaging (MRI) scans, computed tomography (CT) scans, liver function studies, radiographs of chest and bone scan, and monitoring for specific symptoms associated with cancer. Administration, Dosages and Durations
[0148] A small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, AhR inhibitors such as CB7993113 and CMLD-2166, and non-constitutive AhR agonists such as CB7950996, can be formulated, dosed and administered in a manner consistent with good medical practice for use in the treatment of the cancers and cancerous conditions described herein, such as breast cancer. Factors to consider in this context include the particular disorder or type of disorder, for example, the cancer being treated, the particular individual being treated, the individual's clinical condition, the cause of the disorder, the site of distribution of the agent, the method of administration, the schedule of administration and other factors known to physicians.
[0149] Consequently, the "therapeutically effective amount" of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, AhR inhibitors such as CB7993113 and CMLD-2166, and non-constitutive agonists of AhR, such as CB7950996, to be administered is governed by these considerations and, as used herein, refers to the minimum amount necessary to prevent, ameliorate, or treat, or stabilize a disorder or condition, such as one mediated by constitutive activity of AhR.
[0150] In these aspects and embodiments related to cancer or other proliferative disorders, the therapeutically effective amount of a small molecule AhR modulator of Formula (I) or Formula (II) described herein is the minimum amount necessary, for example, to increase the time to progression (progression-free survival duration), inhibiting or preventing tumor invasion, or treating or preventing the occurrence or recurrence of a tumor, a latent tumor, or a micro-metastasis. In some embodiments, a small molecule AhR modulator of Formula (I) or Formula (II) described herein is, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996 , is optionally formulated with one or more agents currently used to prevent or treat cancer or a risk of developing a cancer. The effective amount of these other agents depends on the amount of AhR inhibitor or non-constitutive AhR agonist present in the formulation, the type of disorder or treatment, and other factors discussed herein, and as understood by one of ordinary skill in the art. These are, in general, used at the same dosages and with routes of administration as used herein above, or about 1 to 99% of the dosages employed heretofore.
[0151] An effective amount, as used herein, also includes an amount sufficient to delay the development of a cancer symptom, alter the course of a cancer (for example, but not limited to, delay the progression of a cancer symptom, such as growth of a tumor) or reversing a symptom of the cancer or tumor. Thus, it is not possible to specify the exact "effective amount". However, for any given case, an adequate "effective amount" can be determined by a person skilled in the art using only routine experimentation.
[0152] Effective amounts, toxicity and therapeutic efficacy of small molecule AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-agonist. constitutive AhR, such as CB7950996, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the therapeutically effective dose in 50% of the population). The dosage may vary depending on the dosage form employed and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the LD50/ED50 ratio. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. In addition, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (ie, the concentration of AhR inhibitor or non-constitutive AhR agonist) that achieves half the maximal inhibition of symptoms). as determined in cell culture or in an appropriate animal model. Plasma levels can be measured, for example, by means of high performance liquid chromatography. The effects of any particular dosage can be monitored through a suitable bioassay. Dosage can be determined by a physician and adjusted as necessary according to the observed effects of treatment.
[0153] Depending on the type and severity of the disease, about 1 μg/kg to 100 mg/kg (eg 0.1-20 mg/kg) of a small molecule AhR modulator of Formula (I) or Formula ( II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or with a non-constitutive AhR agonist such as CB7950996, is an initial candidate dosage range for administration to the subject, for example, either by one or more separate administrations or by continuous infusion. A typical daily dosage might range from about 1 µg/kg to about 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the cancer is treated, as measured by methods described above or known in the art. However, other dosing regimens may be helpful. The progress of the therapeutic methods described herein is easily monitored by conventional techniques and assays, such as those described herein or known to one of skill in the art. In other embodiments, such a dosing regimen is used in combination with a chemotherapy regimen as first-line therapy for the treatment of locally recurrent or metastatic breast cancer.
[0154] The duration of the therapeutic methods described herein may continue for as long as is indicated by the physician or until a desired therapeutic effect (eg, those described here) is achieved. In certain embodiments, administration of an AhR modulator, i.e., "AhR inhibitor therapy" or "non-constitutive AhR agonist therapy" is continued for at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least one year, at least two years, at least three years, at least four years, at least five years, at least 10 years, at least 20 years or for at least a period of years until the individual's life expectancy.
[0155] The small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, can be administered to a subject, for example a human, in accordance with known methods, such as intravenous administration as a bolus or by continuous infusion over a period of time, by the intramuscular, intraperitoneal, intracerebrospinal, subcutaneous routes, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation. Local administration can be used if, for example, extensive side effects or toxicity are associated with the AhR inhibitor or non-constitutive AhR agonist. An ex vivo strategy can also be used for therapeutic applications.
[0156] Exemplary modes of administration of small molecule AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or with a non-constitutive AhR agonist , such as CB7950996 include, but are not limited to, administration by injection, infusion, inhalation (e.g., intranasal or intratracheal), ingestion, rectal and topical (including buccal and sublingual). The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, usually by injection. As used herein, "injection" includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular injection and infusion , subarachnoid, intraspinal, intracerebrospinal and intrasternal. The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered", as used herein, refer to the administration of a small molecule AhR modulator of Formula (I) or Formula (II) described herein , for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, but not directly to a target site, tissue or organ such as the lung, so that it it enters the individual's circulatory system and thus is subject to metabolism and other similar processes.
[0157] In some embodiments, small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, are administered by intravenous infusion or injection. In some embodiments where local treatment is desired, for example, at or near a tumor site, such as a breast tumor in an individual having breast cancer, the small AhR modulator molecules of Formula (I) or Formula ( II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996 can be administered via intralesional administration. Furthermore, in some embodiments, the AhR inhibitor or non-constitutive AhR agonist described herein may be administered via pulsed infusion, particularly with decreasing doses of the non-constitutive inhibitors or agonists. Preferably, dosage is provided by injections, more preferably intravenous or subcutaneous injections, depending, in part, on whether the administration is short or chronic.
[0158] In some embodiments, small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, are administered locally, for example, by direct injection, when the disturbance or tumor location permits, and injections may be repeated periodically. The AhR inhibitors or non-constitutive AhR agonists described herein can also be delivered systemically to the individual or directly to tumor cells, for example, to a tumor or tumor bed, after surgical excision of the tumor, in order to prevent or reduce local recurrence. or metastasis, for example, from a latent tumor or micro-metastases. Pharmaceutical Formulations
[0159] Therapeutic formulations of AhR inhibitors can be prepared, in some aspects, by mixing a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers ( Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980) )), in the form of lyophilized formulations or aqueous solutions. Such therapeutic formulations of the AhR inhibitors or non-constitutive AhR agonists described herein include formulation into pharmaceutical compositions or pharmaceutical formulations for parenteral, e.g., intravenous, administration; through the mucosa, for example, intranasally; enteral, for example, orally; topical, for example transdermal; ocular, or other mode of administration.
[0160] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms that are, within the scope of good medical judgment, suitable for use in contact with human and animal tissues. no toxicity, irritation, excessive allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase "pharmaceutically acceptable carrier", as used herein, means a pharmaceutically acceptable material, composition or vehicle, such as a filler, excipient, diluent, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, talc, magnesium, calcium or zinc stearate or stearic acid) or solvent encapsulation material that is liquid or solid involved in maintaining activity or transport of the small AhR modulating molecules of Formula (I) or Formula (II) described herein , for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, from one organ or part of the body to another organ or part of the body.
[0161] Some non-limiting examples of acceptable vehicles, excipients or stabilizers that are not toxic to recipients at the dosages and concentrations employed include pH-buffered solutions such as phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, saffron oil, sesame oil, olive oil, corn oil and soybean oil; preservatives (such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3- pentanol and m-cresol); low molecular weight polypeptides (below about 10 residues); proteins such as serum albumin, gelatin, HDL, LDL or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including mannose, starches (corn starch or potato starch) or dextrins; cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; chelating agents such as EDTA; sugars such as glucose, sucrose, lactose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); glycols such as propylene glycol; polyols such as glycerin; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; polyesters, polycarbonates and/or polyanhydrides; C2-C12 alcohols such as ethanol; powdered tragacanth; malt; and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG); and/or other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives and antioxidants may also be present in the formulation.
[0162] In some embodiments, the therapeutic formulation comprising an AhR inhibitor comprises a pharmaceutically acceptable salt, typically, for example, sodium chloride, and preferably around physiological concentrations. Optionally, the formulations described herein can contain a pharmaceutically acceptable preservative. In some embodiments, the concentration of preservative ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methylparaben and propylparaben are examples of preservatives. Optionally, the formulations of the invention can include a pharmaceutically acceptable surfactant in a concentration of 0.005 to 0.02%.
[0163] In some embodiments of aspects described herein, a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or with an agonist non-constitutive AhR, such as CB7950996, may be specially formulated for administration of the compound to a subject in solid, liquid or gel form, including those adapted for the following: (1) oral administration, e.g., elixirs (solutions or suspensions aqueous or non-aqueous), lozenges, pills, capsules, pills, tablets (eg those intended for oral, sublingual and systemic absorption), cakes, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection such as, for example, a sterile solution or suspension, or an extended-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocular route; (7) transdermally; (8) transmucosal route; or (9) nasal route. Additionally, a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, can be implanted into a patient or injected using a drug delivery system. See, for example, Urquhart et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. "Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); United States Patent No. 3,773,919; and United States Patent No. 35 3,270,960. Examples of dosage forms include, but are not limited to: tablets; oblong tablets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; pills; pastilles; dragees; dispersions; suppositories; ointments; poultices (plasters); folders; powders; dressings; creams; plasters; solutions; stickers; aerosols (eg, nasal sprays or inhalers); gels; liquids, such as suspensions (for example, aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil emulsions); solutions and elixirs; and sterile solids (eg, crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms.
[0164] In some embodiments, parenteral dosage forms of the small molecule AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-agonist constitutive AhR, such as CB7950996, can be administered to an individual with a cancer or an increased risk of cancer through a variety of routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular and intra-arterial . Since administration of parenteral dosage forms typically overcomes a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, parenteral dosage forms with controlled release, and emulsions.
Suitable vehicles that can be used to deliver the parenteral dosage forms described herein are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as, but not limited to, sodium chloride for injection, Ringer's solution, dextrose for injection, dextrose and sodium chloride injection, and lactated Ringer's solution; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
[0166] In some embodiments, small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, are formulated to be suitable for oral administration, for example, as distinct dosage forms such as, but not limited to, tablets (including, without limitation, scored or coated tablets), pills, caplets, capsules, tablets chewables, powder sachets, lozenges, dragees, wafers, aerosol sprays or liquids such as, but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the described compounds and can be prepared by methods of pharmacy well known to those skilled in the art. See generally Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).
[0167] Due to their ease of administration, tablets and capsules represent the most advantageous solid oral dosage unit forms, in which case solid pharmaceutical excipients are used. If desired, tablets can be coated using standard aqueous or non-aqueous techniques. These dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by intimately and uniformly mixing the active ingredient(s) with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation. , if necessary.
Typical oral dosage forms of the compositions are prepared by combining the pharmaceutically acceptable salt of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD -2166, or a non-constitutive AhR agonist, such as CB7950996, in an intimate admixture with at least one excipient, according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms, depending on the form of composition desired for administration. For example, excipients suitable for use in aerosol dosage forms or oral liquids include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (eg powders, tablets, capsules and caplets) include, but are not limited to, starches, sugars, microcrystalline cellulose, kaolin, diluents, granulating agents, lubricants, binders and disintegration agents.
[0169] Suitable binders for use in the pharmaceutical formulations described herein include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, others alginates, powdered tragacanth, guar gum, cellulose and its derivatives (eg ethyl cellulose, cellulose acetate, calcium carboxy methyl cellulose and sodium carboxy methyl cellulose), polyvinylpyrrolidone, methyl cellulose, pregelatinized starch, hydroxypropylmethyl cellulose, (e.g. Nos. 2208, 2906, 2910), microcrystalline cellulose and mixtures thereof.
[0170] Examples of fillers suitable for use in the pharmaceutical formulations described herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol , silicic acid, sorbitol, starch, pregelatinized starch and mixtures thereof. The binder or filler material in the pharmaceutical compositions described herein are typically present in from about 50 to about 99 percent by weight of the pharmaceutical composition.
[0171] Disintegrants are used in the oral pharmaceutical formulations described herein to provide tablets that disintegrate when exposed to an aqueous environment. A sufficient amount of disintegrant that is neither too small nor too large to detrimentally alter the release of the active ingredient(s) should be used to form the solid oral dosage forms of the small-molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996. The amount of disintegrant used varies based on the type of formulation and is readily discernible to those skilled in the art. Disintegrators that can be used to form oral pharmaceutical formulations include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, potassium polacrylin, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, clays, other algins, other celluloses, gums and mixtures thereof.
[0172] Lubricants that can be used to form the oral pharmaceutical formulations of the AhR inhibitors described herein, include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (eg peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and oil soybean), zinc stearate, ethyl oleate, ethyl laurate, agar and mixtures thereof. Additional lubricants include, for example, a siloid silica gel (AEROSIL ® 200, manufactured by WR Grace Co. of Baltimore, Md.), a coagulated synthetic silica aerosol (marketed by Degussa Co. of Piano, Texas), CAB- O-SIL® (a fumed silicon dioxide product sold by Cabot Co. of Boston, Massachusetts) and mixtures thereof. If used generally, lubricants are typically used in an amount of less than about 1 percent by weight of the pharmaceutical compositions or dosage forms in which they are incorporated.
[0173] In other embodiments, lactose-free pharmaceutical formulations and dosage forms are provided, wherein such compositions preferably contain little or, if any, lactose or other mono- or disaccharides. As used herein, the term "lactose free" means that the amount of lactose present, if any, is insufficient to substantially increase the rate of degradation of an active ingredient. Lactose-free compositions of the disclosure can comprise excipients that are well known in the art and are listed in the USP (XXI)/NF (XVI), which is incorporated herein by reference.
[0174] Oral formulations of small molecules AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or with a non-constitutive AhR agonist, such as CB7950996, further encompass, in some embodiments, anhydrous pharmaceutical compositions and dosage forms comprising the AhR inhibitors or non-constitutive AhR agonists described herein as active ingredients, as water can facilitate the degradation of some compounds. For example, the addition of water (eg 5%) is widely accepted in pharmaceutical techniques as a means of simulating long-term storage in order to determine characteristics such as shelf life or stability of formulations over time. of time. See, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, NY: 1995). Anhydrous pharmaceutical compositions and dosage forms described herein can be prepared using ingredients containing low moisture or anhydrous and low moisture conditions. Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine are preferably anhydrous if substantial contact with moisture is expected during manufacturing, packaging and/or storage. Anhydrous compositions are preferably packaged using materials known to prevent exposure to water so that they can be included in suitable formulation kits. Examples of suitable packaging include, but are not limited to, hermetically sealed sheets, plastics, unit dose containers (e.g. vials), with or without desiccants, blister packs, and foil packs.
[0175] An AhR inhibitor described herein, such as a small molecule of Formula (I) or Formula (II), for example, CB7993113 and CMLD-2166, can be administered directly to the airways in the form of an aerosol or via of nebulization. Accordingly, for use as aerosols, in some embodiments, an AhR inhibitor described herein, such as a small molecule of Formula (I) or Formula (II), e.g., CB7993113 and CMLD-2166, can be packaged in an aerosol container. pressurized together with suitable propellants, for example hydrocarbon propellants such as propane, butane or isobutane with conventional adjuvants. In other embodiments, the AhR inhibitor can be administered in an unpressurized form, such as in a nebulizer or atomizer.
[0176] The term "nebulization" is well known in the art to include reducing liquid to a fine spray. Preferably, through such nebulization, small, uniformly sized liquid droplets are produced from a larger body of liquid in a controlled manner. Nebulization can be achieved by any suitable means, including using many known and currently marketed nebulizers. As is known, any suitable gas can be used to apply pressure during nebulization, with the preferred gases being those that are chemically inert to the small AhR modulating molecules of Formula (I) or Formula (II) described herein, for example, a an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996. Exemplary gases include, but are not limited to, nitrogen, argon, or helium.
[0177] In other embodiments, a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, can be administered directly to the airways as a dry powder. For use as a dry powder, an AhR inhibitor or non-constitutive AhR agonist can be administered using an inhaler. Exemplary inhalers include metered dose inhalers and dry powder inhalers.
[0178] Suitable powder compositions include, by way of illustration, powder preparations of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD -2166, or a non-constitutive AhR agonist, such as CB7950996, thoroughly mixed with lactose or other acceptable inert powders, for example, for intrabronchial administration. Powder compositions can be administered via an aerosol dispenser or encapsulated in a fragile capsule, which can be inserted by the individual into a device that pierces the capsule and spreads the powder out in a stable stream suitable for inhalation. The compositions can include propellants, surfactants and co-solvents and can be filled into conventional aerosol containers, which are closed off by a suitable metering valve.
[0179] Aerosols for delivery to the respiratory tract are known in the art. See, for example, Adjei, A. and Garren, J. Pharm. Res., 1: 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic as diagnostic agents to the respiratory tract" in Critical Reviews in Therapeutic Drug Carrier Systems, 6: 273-313 (1990); Anderson et al., Am. Rev. Respir. Dis., 140: 13171324 (1989)) and have potential for the systemic delivery of peptides as well as proteins (Patton and Platz, Advanced Drug Delivery Reviews, 8: 179-196 (1992)); Timsina et al., Int. J. Pharm., 101: 1-13 (1995); and Tansey, I.P., Spray Technol. Market, 4: 26-29 (1994); French, D.L., Edwards, D.A. and Niven, R.W., Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S. and R.H. Muller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, D.A., Drug Delivery, 2:10 1-20 1995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J.S. et al., Controlled Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R.W. et al., Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S. et al., Pharm. Res., 13(1): 80-83 (1996), the contents of all of which are hereby incorporated by reference in their entirety.
[0180] Topical dosage forms of the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or with a non-constitutive AhR agonist , such as CB7950996, are also provided in some embodiments and include, but are not limited to, creams, lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed, Mack Publishing, Easton, Pa. (1990) and Introduction to Pharmaceutical Dosage Forms, 4th Edition, Lea & Febiger, Philadelphia, Pa. (1985). For non-sprayable topical dosage forms, viscous to semi-solid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than that of water are typically employed. Suitable formulations include, without limitation, solutions, suspensions, emulsions, creams, ointments, powders, liniments, ointments and the like, which are, if desired, sterilized or mixed with auxiliary agents (for example, preservatives, stabilizers, wetting agents, buffers or salts) to influence various properties such as, for example, osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations in which the active ingredient, preferably in combination with an inert solid or liquid carrier, is packaged in a mixture with a pressurized volatile (eg, a gaseous propellant such as freon) or in a compression bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms, if desired. Examples of such additional ingredients are well known in the art. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990) and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985). Dosage forms suitable for treating mucosal tissues in the oral cavity may be formulated as mouthwashes, as oral gels, or as oral plasters. Additional transdermal dosage forms include "reservoir-type" or "matrix-type" patches, which can be applied to the skin and worn for a specified period of time to allow penetration of a desired amount of active ingredient.
[0181] Examples of transdermal dosage forms and administration methods that can be used to administer a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, include, but are not limited to, those disclosed in US Patent Nos. 4,624,665; 4,655,767; 4,687,481; 4,797,284; 4,810,499; 4,834,978; 4,877,618; 4,880,633; 4,917,895; 4,927,687; 4,956,171; 5,035,894; 5,091,186; 5,163,899; 5,232,702; 5,234.690; 5,273,755; 5,273,756; 5,308,625; 5,356.632; 5,358,715; 5,372,579; 5,421,816; 5,466,465; 5,494,680; 5,505,958; 5,554,381; 5,560,922; 5,585.111; 5,656,285; 5,667,798; 5,698,217; 5,741,511; 5,747,783; 5,770,219; 5,814,599; 5,817,332; 5,833,647; 5,879,322; and 5,906,830, each of which is incorporated herein by reference in its entirety.
[0182] Suitable excipients (e.g., vehicles and diluents) and other materials that can be used to provide transdermal and mucosal dosage forms of the inhibitors described herein are well known to those skilled in the pharmaceutical arts and depend on the particular tissue or organ to which a particular pharmaceutical composition or dosage form will be applied. Furthermore, depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with or subsequent to treatment with a small molecule AhR modulator of Formula (I) or Formula (II) described herein, e.g. an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996. For example, penetration enhancers can be used to aid in the delivery of active ingredients to or through tissue.
[0183] In some embodiments of the aspects described herein, pharmaceutical formulations comprising the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, may further comprise more than one active compound as needed for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, in some embodiments, it may be desirable to further provide antibodies that bind to EGFR, VEGF, VEGFR or ErbB2 (e.g., Herceptin™) in the formulation comprising the AhR inhibitor or non-constitutive AhR agonist of Formula (I) or Formula (II). In other embodiments, the formulation comprising the AhR inhibitor or non-constitutive AhR agonist of Formula (I) or Formula (II) may comprise a cytotoxic agent, cytokine, growth inhibitory agent and/or VEGFR antagonist. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.
[0184] In some embodiments, the active ingredients of formulations comprising AhR inhibitors and non-constitutive AhR agonists described herein may also be enclosed in microcapsules prepared, for example, by means of coacervation techniques or through interfacial polymerization, by example, hydroxymethyl cellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (eg liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980).
[0185] In some embodiments of these aspects, small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive agonist of AhR, such as CB7950996, can be administered to a subject through controlled or delayed release means. Ideally, the use of an optimally designed controlled release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum period of time. Advantages of controlled-release formulations include: 1) prolonged drug activity; 2) reduced dosing frequency; 3) increased patient acceptance; 4) use of less total drug; 5) reduction of local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in treatment effectiveness; 9) reduction of potentiation or loss of drug activity; and 10) improvement in the speed of control of diseases or conditions (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled-release formulations can be used to control the onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels of an AhR inhibitor. In particular, controlled or sustained release dosage forms or formulations can be used to ensure maximum effectiveness of the small AhR modulator molecules of Formula (I) or Formula (II) described herein, e.g., an AhR inhibitor, such such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, is obtained while minimizing potential adverse effects and safety concerns that can occur from either underdosing on a drug (i.e., is, below minimum therapeutic levels), as to exceed the drug's toxicity level.
[0186] A variety of known controlled or sustained release formulations, devices and dosage forms can be adapted for use with the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an inhibitor of AhR such as CB7993113 and CMLD-2166, or with a non-constitutive AhR agonist such as CB7950996. Examples include, but are not limited to, those described in U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591.767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each of which is incorporated herein by reference in its entirety. These dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymeric matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, California, USA)), coatings with multilayers, microparticles, liposomes or microspheres, or a combination thereof to provide the desired release profile in varying proportions. Furthermore, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the described compounds and thus effect controlled drug delivery. Examples of specific anion exchangers include, but are not limited to, Duolite® A568 and Duolite® AP143 (Rohm & Haas, Spring House, Pa. USA).
[0187] In some embodiments of aspects, small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive agonist of AhR, such as CB7950996, for use in the various therapeutic formulations and compositions and methods therewith described herein, are administered to a subject via sustained release or in pulses. Pulse therapy is not a form of discontinuous administration of the same amount of a composition over time, but comprises administering the same dose of the composition at a reduced frequency or administering reduced doses. Sustained-release or pulsed administrations are particularly preferred in chronic conditions, such as cancer, as each pulse dose can be reduced and the total amount of an AhR-inhibiting compound administered during the course of treatment to the patient is minimized.
[0188] The interval between pulses, when necessary, can be determined by a person skilled in the art. Often, the interval between pulses can be calculated by administering another dose of the composition when the composition or active component of the composition is no longer detectable in the individual prior to delivery of the next pulse. Intervals can also be calculated from the in vivo half-life of the composition. Ranges can be calculated as greater than the in vivo half-life or 2, 3, 4, 5 and even 10 times greater than the half-life of the composition. Various methods and apparatus for pulse administration of the compositions by infusion or other forms of administration to the patient are disclosed in the Patents. US 4,747,825; 4,723,958; 4,948,592; 4,965,251; and 5,403,590.
[0189] In some embodiments, sustained release preparations comprising the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or an agonist non-constitutive AhR such as CB7950996 can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the inhibitor, wherein the matrices are in the form of shaped pieces, for example, films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl-methacrylate) or (poly)vinyl alcohol), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and ethyl-L- glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid and leuprolide acetate copolymer) and poly-D-(- )-3-hydroxybutyric.
[0190] Formulations comprising the small AhR modulating molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, to be used for in vivo administration are preferably sterile. This is easily accomplished by filtration, for example, through sterile filtration membranes and other methods known to a person skilled in the art. Treatment Effectiveness
[0191] One of the main advantages of the methods, uses and compositions comprising the small AhR modulating molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, is the ability to produce marked anti-cancer effects in a human without causing significant toxicity or adverse effects. The effectiveness of the treatments described here can be measured by several parameters commonly used to evaluate cancer treatments including, but not limited to, tumor regression, tumor size or weight shrinkage, reduction in tumor growth rate, presence or opacity. size of a latent tumor, the presence or size of metastases or micro-metastases, degree of tumor or cancer invasion, size or number of blood vessels, time to progression, survival time, progression-free survival, overall response rate, response duration and quality of life. For example, tumor shrinkage of more than 50% on a two-dimensional analysis is the standard cutoff for declaring a response. However, in some embodiments, compositions comprising the small AhR modulating molecules of Formula (I) or Formula (II) described herein can be used to cause inhibition of metastasis spread without primary tumor retraction, or can simply exert a tumoristatic effect. . In the case of cancers, the therapeutically effective amount of compositions comprising the small AhR modulating molecules of Formula (I) or Formula (II) described herein can reduce the number of cancer cells; reduce tumor size; inhibit (i.e. reduce to some extent and preferably cease) the infiltration of cancer cells into peripheral organs; inhibit (that is, reduce to some extent and preferably cease) tumor metastasis; inhibit tumor growth to some extent; and/or alleviate to some extent one or more of the symptoms associated with the disorder. To the extent that compositions comprising the small AhR modulating molecules of Formula (I) or Formula (II) described herein can prevent the growth and/or kill existing cancer cells, they can be cytostatic and/or cytotoxic. For cancer therapy, in vivo efficacy can, for example, be measured by evaluating duration of survival, duration of progression-free survival (PFS), response rates (RR), duration of response and/or quality of life.
[0192] In some embodiments, methods of increasing the progression-free survival of a human susceptible to or diagnosed with a cancer are described here. "Time to disease progression", as used herein, is defined as the time from drug administration to disease progression or death. In preferred embodiments, methods of treatment described herein using small molecule AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor, such as CB7993113 and CMLD-2166, or a non-constitutive agonist of AhR, such as CB7950996 and, in some additional embodiments, one or more chemotherapeutic agents, significantly increase progression-free survival by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months when compared with no treatment or a chemotherapy-only treatment.
[0193] In other embodiments, the treatment methods described herein significantly increase the response rate in a group of humans susceptible to or diagnosed with a cancer who are treated with various therapeutic agents. "Response rate", as used here, is defined as the percentage of treated individuals who responded to treatment. In some such embodiments, the combination treatments described herein which comprise the use of a small molecule AhR modulator of Formula (I) or Formula (II), for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996 and, in some additional embodiments, one or more chemotherapeutic agents, significantly increase the response rate in the treated group of individuals compared to an untreated group or a group treated with chemotherapy alone.
[0194] In other embodiments of these methods, administering a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or an agonist non-constitutive AhR, such as CB7950996, is used to increase the duration of response in a human or a group of humans susceptible to or diagnosed with a cancer. As used herein, "response duration" is defined as the time from initial response to disease progression. In some embodiments, the AhR inhibitors and non-constitutive AhR agonists described herein can be used to increase the survival duration of a human subject susceptible to or diagnosed with a cancer.
[0195] As used herein, the terms "treat", "treatment", "treating" or "relief" refer to therapeutic treatments, where the objective is to reverse, alleviate, ameliorate, inhibit, delay or stop progression or severity of a condition associated with a disease or disorder. The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a chronic immune condition such as, but not limited to, a chronic infection or cancer. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is slowed or stopped. Thus, "treatment" includes not only improvement in symptoms or markers, but also an interruption or at least a delay in progression or worsening of symptoms, which would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, decrease in disease extent, stabilized disease state (i.e., no worsening), delay or decrease in disease progression, improvement or attenuation of disease. disease status and remission (whether partial or total), either detectable or undetectable. The term "treatment" of an illness also includes providing relief from the symptoms or side effects of the illness (including palliative treatment).
[0196] For example, in some embodiments, the methods described herein comprise administering an effective amount of the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, described herein to a subject, in order to alleviate a symptom of a cancer or other disorder. As used herein, "alleviating a symptom of a cancer" is ameliorating or reducing any condition or symptom associated with the cancer. When compared to an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100%, as measured using any standard technique. Ideally, the cancer is completely eliminated, as detected by any standard method known in the art, in which case the cancer is considered to have been treated. A patient being treated for cancer is one whose doctor has diagnosed as having the condition. Diagnosis can be done through any suitable means. Diagnosis and monitoring may involve, for example, detecting the level of cancer cells in a biological sample (eg a tissue or lymph node biopsy, blood test or urine test), detecting the level of a surrogate cancer marker in a biological sample, detection of symptoms associated with the specific cancer, or detection of immune cells involved in the immune response typical of such cancer infections. Combined Therapies
[0197] In some embodiments, compositions and methods comprising the novel small molecule AhR modulators of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, further comprises administration or treatment with one or more additional therapies for cancer. Examples of anti-cancer therapies include, without limitation, surgery, radiation therapy (radiotherapy), biotherapy, immunotherapy, chemotherapy or a combination of these therapies. In addition, cytotoxic agents, anti-angiogenic agents, and anti-proliferative agents can be used in combination with the AhR inhibitor(s).
[0198] For the treatment of cancer in such embodiments comprising combination therapies, the appropriate dosage of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996, will depend on the type of disease being treated, as defined above, on the severity and course of the disease, whether the AhR inhibitor or non-constitutive AhR agonist is administered for preventive or therapeutic purposes, prior therapy, the individual's medical history and response to an AhR inhibitor or non-constitutive AhR agonist, and at the discretion of the attending physician. The AhR inhibitor or non-constitutive AhR agonist is suitably administered to the individual at one time or over a series of treatments.
[0199] In those embodiments where a combination therapy regimen is applied, a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, and one or more anti-cancer therapeutic agents, as described herein, are administered in a therapeutically effective or synergistic amount. As used in such embodiments encompassing combination therapies, a therapeutically effective amount is such that the co-administration of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, and one or more other therapeutic agents, or administering a therapeutic composition or formulation comprising an AhR inhibitor or non-constitutive AhR agonist, as described herein, results in reduction or inhibition of cancer, as described here. A "therapeutically synergistic amount" is that amount of a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor, such as CB7993113 and CMLD-2166, or a non-constitutive agonist of AhR, such as CB7950996, and one or more other therapeutic agents necessary to synergistically or significantly reduce or eliminate conditions or symptoms associated with a particular cancer.
[0200] In some embodiments, a small molecule AhR modulator of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist, such as CB7950996, and one or more other therapeutic agents may be administered simultaneously or sequentially in an amount and for a time sufficient to reduce or eliminate the occurrence or recurrence of a tumor, a latent tumor or a micrometastasis. In some embodiments, the small AhR modulator molecules of Formula (I) or Formula (II) described herein, for example, an AhR inhibitor such as CB7993113 and CMLD-2166, or a non-constitutive AhR agonist such as CB7950996 , and one or more other therapeutic agents can be administered as maintenance therapy to prevent or reduce the likelihood of tumor recurrence.
[0201] As will be understood by those skilled in the art, appropriate doses of chemotherapeutic agents or other anticancer agents will generally be around those already employed in clinical therapies, for example, where chemotherapeutic agents are administered alone or in combination with other chemotherapeutic agents. Variation in dosage will likely occur depending on the condition being treated. The physician administering the treatment will be able to determine the appropriate dose for the individual.
[0202] In addition to the above therapeutic regimens, the individual may undergo radiotherapy.
[0203] The term "anticancer therapy" refers to a therapy useful for the treatment of cancer. Examples of anti-cancer therapeutic agents include, but are not limited to, for example, surgery, radiotherapy, chemotherapeutic agents, growth inhibitory agents, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treat cancer, such as anti-HER-2 antibodies (eg Herceptin™), anti-CD20 antibodies, an epidermal growth factor receptor (EGFR) antagonist (eg a tyrosine kinase inhibitor), an inhibitor of HER1/EGFR (eg erlotinib (Tarceva™)), platelet derived growth factor inhibitors (eg Gleevec™ (Imatinib mesylate)), a COX-2 inhibitor (eg celecoxib), interferons, cytokines, antagonists (eg, neutralizing antibodies) that bind to one or more of the following targets: ErbB2, ErbB3, ErbB4, PDGFR-beta, BLyS, APRIL, BCMA or VEGF, TRAIL/Apo2 receptors and other chemical agents organic and bioactives, etc. Combinations thereof are also included in the embodiments described herein.
[0204] The term "cytotoxic agent", as used herein, refers to a substance that inhibits or prevents cell function and/or causes cell destruction. The term is intended to include radioactive isotopes (eg, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial origin , fungal, plant or animal, including fragments and/or variants thereof.
[0205] As used herein, a "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN™ cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocone, meturedopa and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as the antibiotics enediin (for example, calicheamicin, especially calicheamicin gammaII and calicheamicin omegaI1 (see, for example, Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); A; bisphosphonates such as clodronate; an esperamycin; as well as neocarzinostatin chromophore and related chromoprotein antibiotic enediin chromophores); aclacinomisins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophylline, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™, including doxoorlinorubicin (doxorlinorubicin). doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, chelamincin, estreptomycin , zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; enyluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elformitin; elliptinium acetate; an epothilone; ethoglucid, gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pyrarubicin; losoxantrone; podophylinic acid; 2-ethyl hydrazide; procarbazine; PSK™; polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridine A and anguidine); urethane; vindesine; dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacitosin; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL™, paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™, nanoparticle-engineered paclitaxel formulation with albumin-free Cremophor (American Pharmaceutical Partners, Schaumberg, Illinois) and TAXOTERE™, doxetaxel (Rhone -Poulenc Rorer, Antony, France); chlorambucil; GEMZARTM gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin, oxaliplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including 5-FU and leucovorin treatment regimen of irinotecan); RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (for example, erlotinib (Tarceva™)) and VEGF-A, which reduce cell proliferation, and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0206] Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormonal action on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs) including, for example, tamoxifen (including tamoxifen NOLVADEX™ ), raloxifene, droloxifene, 4-hydroxytamoxifene, trioxifene, queoxifene, LY117018, onapristone and toremifene FARESTON; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate MEGASE™, exemestane AROMASIN™, formestane, fadrozole , RIVISOR™ vorozole, letrozole FEMARA™ and anastrozole ARIMIDEX™; and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analogue); antisense oligonucleotides, particularly those that inhibit gene expression in signaling pathways involved in abnormal cell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras; ribozymes, such as an inhibitor of VEGF expression (for example, ANGIOZYME™ ribozyme) and an inhibitor of HER2 expression; vaccines such as gene therapy vaccines, for example ALLOVECTIN™ vaccine, LEUVECTIN™ vaccine and VAXID™ vaccine; rIL-2 PROLEUKIN™; LURTOTECAN™ topoisomerase 1 inhibitor; rmRH ABARELIX™; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0207] The term "cytokine" is a generic term for proteins released by one population of cells that act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, monokines and traditional polypeptide hormones. Included among the cytokines are growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); epidermal growth factor; liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor alpha and beta; Mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon alpha, beta and gamma; colony stimulating factors (CSFs) such as macrophage CSF (M-CSF); granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12; a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit linker (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokines.
[0208] A "growth inhibitory agent", when used herein, refers to a compound or composition that inhibits the growth of a cell in vitro and/or in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells in S-phase. Examples of growth-inhibitory agents include agents that block cell cycle progression (other than S-phase), such as agents that induce both G1 arrest and M-phase arrest. Classic M-phase blockers include vincas (vincristine and vinblastine), TAXOL™, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that inhibit G1 also encompass S-phase inhibition, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. More information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell Cycle Regulation, Oncogenes and Antineoplastic Drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13.
[0209] The term "prodrug", as used herein, refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted in the most active original form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy", Biochemical Society Transactions, 14, pages 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery", Directed Drug Delivery, Borchardt et al., (ed.), pages 247-267, Humana Press (1985). Prodrugs of the present invention include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, prodrugs. glycosylated drugs, beta-lactam-containing prodrugs, optionally substituted phenoxy-acetamide-containing prodrugs or optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to the free cytotoxic drug more active. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use in the present invention include, but are not limited to, those chemotherapeutic agents described above.
[0210] By "radiotherapy" is meant the use of gamma rays or beta rays directed to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell completely. It will be appreciated that there are many ways known in the art to determine dosage and duration of treatment. Typical treatments are given as a single administration and typical dosages range from 10 to 200 Units (Grays) per day.
[0211] The present invention is further illustrated by the following examples which should not be considered as limiting. EXAMPLES
[0212] Aryl hydrocarbon receptor (AhR) binding and activation is the proximal signaling event through which abundant classes of environmental pollutants initiate breast tumorigenesis. The inventors demonstrated that AhR also plays an important role in late stages of tumorigenesis by regulating tumor growth and invasion. Inhibition of AhR constitutive activity through molecular or biochemical means has been found to significantly reduce immortalized tumor growth and invasion in multiple in vitro assays. Notably, the inventors have found that this AhR-mediated tumor progression occurs in the absence of environmental AhR ligands and is relevant to virtually all breast tumors, including "spontaneous" tumors not induced by environmental chemicals. The data further indicate that AhR appears to play a similar role in most solid cancers. Consequently, AhR inhibitors and non-constitutive AhR agonists for use as therapeutic agents for reducing tumor growth, invasion and metastasis are described herein. New therapeutic agents aimed at the treatment of breast cancer at any stage are described here. In some embodiments, these new targeted therapeutic agents are particularly useful in patients who have failed conventional untargeted chemotherapeutic agents or Her2/neu, ER, or PR dependent therapeutic agents.
[0213] High-throughput scanning of over 4000 compounds identified several AhR modifiers, including both AhR inhibitors and non-constitutive AhR agonists, at least three of which inhibit tumor invasion. "Hit-to-Lead" chemistry can be employed using the novel tumor aggressiveness biological assays described here to develop a suite of highly potent and specific AhR inhibitors for evaluation in preclinical animal studies. Using format-based and electrostatic screening, scaffold hopping, and computerized medicinal chemistry, AhR inhibitors and non-constitutive AhR agonists can be identified in commercial chemical libraries. Such AhR inhibitors and non-constitutive AhR agonists can be evaluated through HTS and, in an iterative process, chemically altered to produce ligands with improved affinity, potency, stability and binding solubility for AhR. Novel AhR inhibitors and synthetic non-constitutive AhR agonists can be evaluated using the secondary breast tumor growth and invasion assays described herein prior to evaluation of toxicity, specificity, stability, and in vitro absorption. The AhR inhibitors and synthetic non-constitutive AhR agonists described herein can be used in the synthesis of prototype compounds that significantly block tumor progression in vitro and exhibit a high level of efficacy and specificity in preclinical animal trials. AhR inhibitors and synthetic non-constitutive AhR agonists are useful in the treatment of advanced breast cancers, as a prevention for high-risk patients, and are applicable to the treatment of a variety of solid tumors. Fundamentals
[0214] Common AhR ligands initiate breast tumorigenesis by activating the transcription factor/aryl hydrocarbon receptor. In normal cells, cytosolic AhR can be "activated" by pollutants such as dioxins, non-ortho substituted polychlorinated biphenyls, and polycyclic aromatic hydrocarbons, resulting in AhR translocation to the nucleus and gene regulation. The inventors investigated the activation and function of this transcription factor/receptor in malignant cells and were the first to demonstrate a remarkable 50-fold increase in AhR mRNA in rat mammary tumors relative to normal tissue (2), in contrast to others studies describing acute toxicological outcomes of AhR activation in normal cells. The inventors have identified overexpression of AhR in mouse mammary tumors, human and mouse tumor lines, and primary human mammary tumors (3-15). The inventors demonstrated, in all of these systems, that AhR is constitutively active in the absence of environmental stimuli, indicating that AhR contributes to the initiation, growth and invasion of breast cancer. Other data correlate the transcriptional activity of constitutive AhR with tumor growth and invasiveness rates and with upregulated oncogenes associated with tumor growth and invasion (9, 12, 16) and by the direct demonstration that reduced AhR expression through techniques inhibits the growth of immortalized cells (through p21 upregulation) and tumor invasion in vitro (8, 12, 14, 17) (Figures 1-3). Given what is known from the biology of AhR and the wide therapeutic window afforded by overexpression of AhR in tumors, mechanism-based toxicity is a decreased risk and AhR inhibitors and non-constitutive AhR agonists may be protective for high individuals risk due to genetic predispositions or environmental exposures. Results
[0215] Using a high-throughput reporter-based AhR bioassay and cell viability assay, more than 4000 CMLD compounds, FDA drugs and extracts generated by the NCI were screened and 8 non-toxic AhR inhibitors (2 of the CMLD) and 19 AhR agonists (14 from CMLD) have been identified (as shown, for example, in Figures 4A-4B). IC50 values for inhibitors and EC50 values for agonists were determined (Table 1). One of these discovered inhibitors (CMLD-2166), its enantiomer (CMLD-2186), and a previously described lipid-soluble AhR inhibitor (CH223191) blocked tumor growth and invasion (Figures 5A-5B and 7A-7B).
[0216] To determine whether the inhibitory action of CB7993113 involves binding to the AhR and blocking its translocation to the nucleus, Hepa-1 cells were treated with CB7993113 at 1-10 μM. One hour later, they were stimulated with a strong prototypical AhR and PAH agonist, DMBA (1 µM). After an incubation period of 30 minutes, cells were collected and extracts of cell nuclei and cytoplasm were obtained. These extracts were then examined for AhR levels and, as a loading control, β-actin, by Western immunoblotting.
[0217] As can be seen in Figure 6, treatment of cells with the AhR inhibitor alone had no effect on AhR translocation to the nucleus (bands 3 and 6), confirming the lack of AhR agonist activity. In contrast, treatment with DMBA alone significantly induced nuclear translocation (band 5). Addition of CB7993113 at 1-10 µM completely blocked the DMBA-induced AhR translocation (bands 4 and 7). These results are consistent with the hypothesis that CB7993113 and similar competitive AhR inhibitors block AhR transcriptional activity by preventing AhR translocation to the nucleus.
[0218] To test the ability of one of the prototype compounds described here, CB7993113, to be absorbed and function effectively in vivo, C57BL/6 mice were treated with a markedly toxic dose (50 mg/kg) of DMBA, an agonist potent AhR, in the presence or absence of CB7993113 or another AhR inhibitor, CH223191. We had previously demonstrated that treatment of mice with DMBA at 50 mg/kg induces a rapid loss (within 48 hours) of hematopoietic cells from the bone marrow. These studies were confirmed here by demonstrating that treatment with DMBA resulted in a significant decrease in the total number of bone marrow cells 48 hours after i.p. of DMBA (Figure 8A). This reduction in the total number of bone marrow cells was prevented by i.p. of CB7993113 or CH223191 at 50 mg/kg.
[0219] Furthermore, previous experiments have been extended to demonstrate that acute treatment with DMBA resulted in specific loss of pro-B cells, pre-B cells, and neutrophils (Figure 8B). It is important to note that co-administration of either CH223191 or CB7993113 completely blocked this decrease in pro-B cells, pre-B cells and neutrophils. These findings are important as they demonstrate that the AhR inhibitors described herein are absorbed in vivo and that pharmacologically effective doses can be achieved in peripheral organs such as bone marrow.


[0220] Compounds shown in the high-throughput screen to inhibit or induce AhR without affecting the viability of the test cell line were titrated in the absence (agonists) or presence (antagonists) of 1 μM BNF, a known AhR agonist. IC50's or EC50's were calculated as shown in Figure 5.
[0221] Having established an initial dataset of AhR ligands (Table 1), these chemical structures can be used to predict and ultimately re-design higher activity AhR inhibitors with the goal of generating highly specific and soluble inhibitors effective in the 10-100 nanomolar range. For this purpose, commercial chemical libraries can be screened to identify AhR ligands and then, in an iterative process, chemical characteristics that enhance the potency of the AhR inhibitor while focusing on maintaining and improving specificity, stability and solubility.
[0222] Scaffold Hopping techniques based on computerized and electrostatic formatting are often employed in cases where ligand coupling models are not feasible due to a lack of X-ray crystallography data (18-22), as per is the case with AhR. To acquire critical chemical structure information needed to design more potent AhR inhibitors, shape-based screening and "Scaffold Hopping" can be applied to identify additional new AhR ligands in commercial libraries (see Figure 9 for exemplary details). Libraries containing over 700,000 compounds are available from ASDI and ChemBridge. These libraries represent a wide coverage of the pharmacophore space, while maintaining good drug and prototype-like properties. The Openeye OMEGA v2.2 program (23) can be used for this computational approach using our AhR ligands known as a training set. Identification of agonists as well as antagonists is important, as the structures of the agonists help define important portions for binding to the AhR, while a comparison of agonists and antagonists facilitates the identification of residues responsible for receptor inhibition.
[0223] Pharmacophore modeling can also be performed as a complementary computational approach. Using the Accelrys Discovery Studio computer package, structure and construct pharmacophores can be imported using our AhR ligand training kit. Three-dimensional maps of each structure with associated moiety functionality (eg hydrogen bond acceptors/donators, lipophilic regions) can be generated. Candidate compounds can then be re-selected from commercial chemical pools. While it is likely that non-overlapping “hit” sets can be identified for screening from pharmacophore and format-based modeling, those compounds predicted by both methods can be prioritized for purchase and screening in the high-throughput AhR bioassay. Confirmed hits will be titrated to establish IC50/EC50's. Antagonists can be characterized as competitive versus non-competitive for their ability to displace radiolabeled TCDD, as described (24).
[0224] The information collected from this first prediction/testing round can be used to design higher affinity AhR modifiers. Synthesis of second-generation modified compounds using either a commercial compound backbone or CMLD-BU can be performed. Synthesized compounds can be evaluated for potency (and toxicity) in the AhR reporter bioassay, modified again as guided by empirical results and pharmacophore modeling, and retested in the reporter assay. Secondary tests and ADMET in vitro. Prototype compounds shown to be active, but non-toxic, in the bioassays can further be examined for possible toxicity using vital dyes based on flow cytometry and a variety of primary human cells and cell lines available to the inventors, including breast epithelial cells primary human cells, lymphocytes, and hematopoietic stem cells. Specificity of up to 10 non-toxic prototype compounds can be assessed using targeted reporter assays for ERa, PPARy and NF-kB available to the inventors. The prototype compounds can then undergo exhaustive screening for off-target effects using Active Motif's commercial TransAm transcription factor kit. To confirm inhibition of AhR in human breast tumor cells, prototype compounds can be titrated in human breast tumor cell cultures BP1 and Hs578T and inhibition of AhR nuclear translocation can be assayed as described (24). The compound CMLD-2166, which has been shown to inhibit tumor invasion (Figures 7A-7B), can be used as a positive control. Compounds that inhibit constitutive AhR nuclear translocation can then be evaluated in secondary assays for tumor growth inhibition (3H-thymidine incorporation) and invasion (Matrigel morphology and Boyden Chamber migration) as described herein ( see Figures 5A-5B and 7A-7B).
[0225] Having established the potency, toxicity, specificity and in vitro efficacy, additional Absorption, Distribution, Metabolization, Excretion and Toxicity (ADMET) analyzes of the prototype compounds can be submitted to companies such as, for example, Apredica, Inc. Among other results, parameters that can be evaluated include, but are not limited to: 1) compound permeability using Caco-2 monolayers to predict human oral bioavailability; 2) metabolic disruption in the presence of liver microsomes or human/mouse plasma; and 3) stability in the presence of gastric and intestinal fluids. In addition, experimental measurement of compound solubility can be employed to supplement the computed values used during compound design and optimization cycles.
[0226] The studies described here are useful to promote product development from biological discovery to the prototype optimization phase. Furthermore, in vivo pharmacokinetics and efficacy studies can be performed using, for example, a human breast tumor xenograft model to quantify tumor growth and metastases in live mice in real-time in three dimensions (25,26).
[0227] In the United States, the age-adjusted incidence of breast cancer increased by about 1% per year between 1940 and 1990 (27,28) and by 0.4%/year between 1987 and 2002 (29) potentially, unintentionally be bound or bound by theory by virtue of exposure to environmental carcinogens, including AhR ligands (30-33). Breast cancer is now the second most common cancer (after skin cancer) in women, with 225,000 new cases in the US and 40,000 breast cancer-related deaths per year. One in eight women born this year will be diagnosed with breast cancer during her lifetime (34). The total number of women diagnosed with breast cancer tends to grow significantly over the next 20 years as population demography moves towards an older population (34). Thus, even subgroups of breast cancer patients treated with a specific therapeutic agent, such as those described here, represent a relatively large population.
[0228] While suitable as specific therapeutic agents for all breast cancer patients, the compounds described here can also be targeted to patients with aggressive "triple negative" breast cancers, eg, those patients who have failed conventional or therapeutic agents dependent on Her2/neu, ER or PR, since the constitutive activity of AhR increases with, and probably contributes to, increased levels of tumor aggressiveness (8). Since the small molecule compounds of Formula (I) and Formula (II) described herein are useful as specific therapeutic agents specifically targeting a protein (AhR) expressed at extremely high levels in cancer, and because modifiers are not expected to occur. AhR are associated with significant mechanism-based toxicities, in some embodiments, small molecule compounds of Formula (I) and Formula (II) can be used, for example, as an adjunctive therapeutic agent (e.g., postsurgical) for patients who desire a non-toxic alternative for improved quality of life or for patients who require targeted therapeutic agents because of accumulated organ toxicity.
[0229] In addition to contributing to stopping the stage of tumor progression, the studies described here and in rat and mouse models of mammary tumorigenesis indicate that AhR plays an early role in tumorigenesis before tumor formation manifests (24 , 8, 13). Therefore, in some embodiments, AhR therapy using the small molecule compounds of Formula (I) and Formula (II) described herein can be used as a long-term prevention for women at high risk for breast cancer. For example, in some embodiments, the compounds described herein are used as prophylactic therapeutic agents in women at high or increased genetic or environmental risk for breast cancer due to inheritance of mutated BRCA1, BRCA2, CHEK2, ATM, BRIP1 or PALB2 genes , family histories of reproductive organ cancers or chronic exposures to environmental carcinogens (36). In other embodiments, the small molecule compounds of Formula (I) and Formula (II) described herein can be used as long-term preventatives for the 42,000 women diagnosed as being at high risk each year with precancerous lesions (ductal carcinoma in situ/DCDIS), most of which will develop invasive adenocarcinomas (37). Importantly, the studies described here also indicate that AhR is overexpressed and constitutively active in virtually all types of cancer, including other epithelial tumors such as prostate, lung, and pancreatic ductal cell carcinomas. Accordingly, in some embodiments, the compounds described herein can be used in the treatment of cancer.
[0230] Figure 1 shows that AhR inhibition with AhRR upregulates p21 and decreases the growth of MCF-10F. MCF-10F cells were stably transduced with lentiviral vectors encoding GFP or GFP and AhR repressor (AhRR) to suppress AhR activity. AhRR-low and AhRR-GFP high cells were selected by flow cytometry. Cells were plated in equal numbers and cell numbers determined 1-6 days later. Insertion: p21 protein levels in control or AhRR transduced cells.
[0231] Figure 2 demonstrates that inhibition of AhR with AhRR inhibits tumor invasion in matrigel. BP1 and Hs578T malignant cells were transiently transfected with control (pSport) or AhR repressor plasmids to suppress AhR activity and plated on Matrigel. Photos were taken 5-7 days later.
[0232] Figure 3 shows that down-regulation of AhR with AhR siRNA inhibits invasion into Boyden Chambers. BP1 and Hs578T malignant cells were transiently transfected with control or specific siRNA for AhR and plated in the upper chamber in a serum-free medium. Medium containing serum was added to the lower chamber. The number of cells above and below the membrane containing Matrigel was determined 48 hours later and the percentage of cells in the lower chamber was calculated. *p<0.04; **p < 0.03.
[0233] Figures 4A-4B show high-throughput scanning (HTS) for AhR agonists and antagonists. Figure 4A shows H1G1 cells expressing an AhR-targeted GFP reporter construct that were plated in 384-well plates and treated with candidate AhR modifiers alone (dark line) or together with BNF, a known AhR agonist (light line) . 24 hours later, GFP fluorescence was measured and cells stained for viability. A dark circle identifies an AhR inhibitor, while a light circle indicates an agonist. Figure 4B shows data for the non-toxic compound identified under "A", which was titrated in the presence of BNF to calculate the IC50. More than 4,000 compounds were tested in this semi-automated assay.
[0234] Figures 5A-5B demonstrate that CH223191 and CMLD 2166 inhibit AhR constitutive activity and cell growth. Figure 5A shows data from immortalized MCF-10F and malignant BP1 cells that were transiently transfected with AhR-responsive pGudLuc and treated with vehicle (DMSO), CH223191 or 2166. pGudLuc activity was assayed 24 h later. Figure 5B shows data from BP1 and MCF-10F cells that were cultured for 18 hours in the presence of DMSO, CH223191 or 2166 and 3 H-thymidine incorporation was determined. *P < 0.05.
[0235] Figure 6 demonstrates that CB7993113 blocks AhR translocation to the nucleus after cell stimulation with DMBA, an AhR agonist. Hepa-1 cells were treated with vehicle or CH223191 at 1-10 µM. One hour later, cells were treated with 0.1 µM DMBA (where indicated) and incubated for 30 minutes. Cells were collected and proteins from the cytoplasm and nucleus were extracted. The extracted proteins were subjected to Western immunoblot specific for AhR or, as a loading control, β-actin. Data are representative of three independent experiments.
[0236] Figures 7A-7B demonstrate that CH223191, CMLD-2166 and CMLD-2186 inhibit tumor invasion. Hs578T cells were cultured in Boyden Chambers (Figure 7A) or in Matrigel (Figure 7B) in the presence of DMSO, 10-5 M CMLD-2166, CMLD-2186 (an enantiomer of 2166) or CH223191 and assayed for invasion 2 ( Boyden Chambers) or 7 (Matrigel) days later.
[0237] Figures 8A-8B demonstrate that CB7993113 is able to block the acute toxic effects of a potent prototypic AhR and PAH ligand, DMBA, in vivo, and that three bone marrow cell subpopulations affected by DMBA are all rescued by treatment with CB7993113. Figure 8A shows data from C57BL/6 mice (6/group) that were injected i.p. on day -1 and day 0 with vehicle (oil) or 50 mg/kg of CB7993113. The mice were then injected with 50 mg/kg of DMBA. The mice were sacrificed 48 hours later. Bone marrow hematopoietic cells were purged from the bone and viable cells were counted. Data are presented as the mean number of viable white cells + SE. **p < 0.01, ***P < 0.001, Student's t test. Figure 8B confirms the data shown in Figure 8A and further demonstrates that the three bone marrow cell subpopulations affected by DMBA are all rescued upon treatment with CB7993113. C57BL/6 mice (6/group) were treated as in Figure 8A. The percentage and number of pro-B cells, pre-B cells and viable bone marrow neutrophils were determined 48 hours later by flow cytometry. Data are presented as the mean number of viable cells of each subtype + SE. (*p < 0.05, **P < 0.01, Student's t test).
[0238] Figure 9 describes computational strategies for similarity scanning by shape and electrostatics. SMILES slices can be expanded to a 3D conformer database using the OpenEye OMEGA program (v.2.2.1), allowing an energy window of 8 kcal/mol above the basal state and a msd cutoff of 0.8 Â according to the method described in (1). The maximum rotational links for these experiments are 16. In order to speed up this calculation, a library of fragments can be pre-generated using the makefragmentlib program. The 3D conformer database can then be searched using ROCS (v.2.3.1), investigated against the 20 lowest energy conformations of the most powerful HTS hits, determined using OMEGA. The ROCS shape comparison program can be run using a built-in color force field and hits can be sorted based on the sum of their Tanimoto shape and color sorting (known as the “Combo Score”). The highest ranked ROCS overlays can be subjected to electrostatic overlay comparison using EON. For ranking the hit list, Tanimoto's Electrostatic Combo Score can be used for ranking. This is the sum of the Poisson-Boltzman Tanimoto and Electrostatic Tanimoto format. The top 200 compounds from each commercial pool can be searched and selected in high-throughput AhR functional assays.
[0239] Figure 10 demonstrates that compounds CMLD-2166 and CB7993113 bind to human AhR. Compounds CMLD-2166 and CB7993113 were mixed with in vitro translated human AhR and radiolabeled TCDD (dioxin), a high affinity AhR ligand. The complexes were then separated on a sucrose gradient and the fractions tested for radioactivity. A decrease in signal (displacement of radiolabeled TCDD) can be seen in fractions 13-16, indicating binding of the test compound to AhR and displacement of TCDD. These results demonstrate that these compounds are competitive inhibitors of AhR.
[0240] Figure 11 demonstrates that AhR inhibitors do not block PPAR-y mediated transcription. Cos7 cells were transfected with the peroxisome proliferator activator receptor-y (PPAR-y), its dimerization partner, RXR, a PPAR-y-targeted luciferase reporter construct and a control CMV-targeted GFP reporter for 24 hours. The cells were then treated with a vehicle, 10 µM AhR CH223191 inhibitor, 50 µM AhR CMLD-2166 inhibitor, or 10 µM AhR CB7993113 inhibitor. (These represent high doses of AhR inhibitors). Rosiglitazone (1 µM), a strong PPAR-y agonist was added 1 hour later and the cells were assayed 18-24 hours later for PPAR-y targeting reporter activity (luciferase intensity), normalized for the signal of GFP Data were gathered from 4 experiments. Unlike their ability to block AhR-targeted reporter activity, AhR inhibitors had no effect on PPAR-y-targeted reporter activity.
[0241] Figure 12 demonstrates that AhR siRNA transfection blocks tumor invasion in matrigel. Human breast tumor BP1 or Hs578T cells were transiently transfected with control siRNA or AhR siRNA to reduce AhR expression and cells were cultured in Matrigel for 6 days. Colonies of tumor cells that grow from individual cells during these 6 days are shown.
[0242] Figure 13 demonstrates that doxycycline-inducible AhR shRNA blocks tumor invasion in matrigel. Hs578T cells were stably transduced with either doxycycline-inducible control (mixed/non-silent) shRNA (left) or AhR-specific shRNA (right). Cells were treated with doxycycline to activate both control and AhR shRNA vectors and cells were cultured in Matrigel for 5 days.
[0243] Figure 14 demonstrates that the AhR inhibitor CB993113 blocks BP1 tumor invasion in matrigel. BP1 cells were cultured in the presence of vehicle (left) or CB7993113 at 5 µM in Matrigel for 5 days. CB7993113 is non-toxic at the highest doses tested, 10 μM.
[0244] Figure 15 is a schematic diagram describing how AhR inhibitors and agonists can inhibit tumor invasion.
[0245] Figures 16A-16B demonstrate that AhR agonists reduce human breast tumor cell (BP1) invasion in Matrigel. BP1 cells were cultured on Matrigel in the presence of vehicle, 5 µM DIM (16A), vehicle or 5 µM CB7950998 (16B) for 5 days.
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权利要求:
Claims (19)
[0001]
1. Pharmaceutical composition characterized in that it comprises an aryl hydrocarbon receptor (AhR) modulator of Formula (Ia) and a pharmaceutically acceptable excipient, vehicle or stabilizer
[0002]
2. Pharmaceutical composition according to claim 1, characterized in that the AhR inhibitor is CB7993113, which has a chemical structure:
[0003]
3. Pharmaceutical composition according to claim 1, characterized in that X' is an amino substituted with C1-C6-alkyl.
[0004]
4. Pharmaceutical composition according to claim 1, characterized in that R2 is an optionally substituted furanyl.
[0005]
5. Pharmaceutical composition according to claim 3, characterized in that R2 is a furanyl substituted with a halogen.
[0006]
6. Pharmaceutical composition according to claim 5, characterized in that the halogen is bromine.
[0007]
7. Pharmaceutical composition according to claim 1, characterized in that: X' is amino substituted with C1-C6-alkyl; n is 0 or 1; R2 is bromine-substituted furanyl; and R3, R4, R5 and R6 are independently H.
[0008]
8. Pharmaceutical composition according to claim 7, characterized in that n is 1.
[0009]
9. Pharmaceutical composition according to any one of claims 1 and 8, characterized in that the AhR modulator is an AhR inhibitor or a non-constitutive AhR agonist.
[0010]
10. Use of a pharmaceutical composition as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a drug for modulating the constitutive activity of AhR in an individual in need thereof.
[0011]
11. Use of a pharmaceutical composition as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a drug for treating a cancer or a cancerous condition.
[0012]
12. Use of a pharmaceutical composition as defined in any one of claims 1 to 9, characterized in that it is for the manufacture of a drug for inhibiting the invasiveness of tumor cells in an individual having a cancer or a cancerous disease.
[0013]
13. Use, according to any one of claims 11 to 12, characterized in that it further comprises the stage of selection of the individual having a cancer or a cancerous condition.
[0014]
14. Use according to any one of claims 11 to 13, characterized in that the cancer is breast cancer, squamous cell cancer, lung cancer, peritoneum cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, colon cancer, colorectal cancer, endometrial or uterine carcinoma, gland carcinoma salivary, kidney or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, head and neck cancer, B-cell lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL ), a hairy cell leukemia or a chronic myeloblastic leukemia.
[0015]
15. Use according to any one of claims 11 to 14, characterized in that the cancer is breast cancer.
[0016]
16. Use according to any one of claims 11 to 15, characterized in that it further comprises one or more additional anti-cancer therapies.
[0017]
17. Use according to claim 16, characterized in that the additional anticancer therapy comprises surgery, radiotherapy, biotherapy, immunotherapy or chemotherapy.
[0018]
18. Use according to any one of claims 11 to 17, characterized in that it further comprises the administration of one or more anticancer therapeutic agents.
[0019]
19. Use according to claim 18, characterized in that the anticancer therapeutic agent is a chemotherapeutic agent, a growth inhibitory agent, an anti-angiogenesis agent, a cytotoxic agent, an anti-hormonal agent, a pro- drug or a cytokine.

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法律状态:
2018-05-22| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2020-04-28| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-05-05| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-07-20| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-14| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/07/2011, OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF, QUE DETERMINA A ALTERACAO DO PRAZO DE CONCESSAO. |

优先权:

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申请号 | 申请日 | 专利标题

---

US36804210P| true| 2010-07-27|2010-07-27|

---

US61/368,042|2010-07-27|

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PCT/US2011/045526|WO2012015914A2|2010-07-27|2011-07-27|Aryl hydrocarbon receptormodifiers as novel cancer therapeutics|

---