Patent Application
20230119324
The Sequence Listing submitted Feb. 18, 2021 as a text file named “37759_0288P1_ST25.txt,” created on Feb. 01, 2021, and having a size of 1,995 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
Interferon regulatory factor 3 (IRF3) plays a central role in innate immunity. IRF3 is a transcription factor that is expressed constitutively and in response to viral infection, and induces the transcription of type I interferons. IRF3 is activated in response to cytosolic recognition of nucleic acids or tissue damage by a number of pattern recognition receptors (PRRs). In response to viral infection, IRF3 becomes phosphorylated leading to its dimerization and nuclear translocation, leading to induction of Type I interferons and orchestration of the antiviral response. IRF3 is activated by the TANK-binding kinase TBK1 and by IKKε. TBK1 is ubiquitously expressed and activated in response to activation of pattern recognition receptors (PRRs) and associated adaptor signaling proteins such as RIG-I/MAVS, cGAS-STING, and TLR3/4-TRIF.
Activation of IRF3 results in production of Type I interferons, cytokines essential for generating antiviral responses and activating innate immunity. Type I interferons include interferon-α and interferon-β and bind to the IFNAR, composed of IFNAR1 and IFNAR2 chains. Type I interferons play a tumor suppressive role. Indeed, IFNs have been used for treating certain types of cancer (kidney cancer, melanoma, chronic myeloid leukemia) and are thought to function through multiple mechanisms including promotion of anti-tumor immunity, anti-angiogenesis, promoting inflammation in the tumor microenvironment and a direct role in suppression of proliferation and apoptosis in tumor cells. Importantly, homozygous deletion of genes is common in 9p21.3, the locus for the type I interferon gene cluster. Homozygous deletion of the type I interferon genes is widespread in cancer, including about 10% of NSCLC. Importantly, Type IIFN loss confers a worse prognosis in multiple cancer types.
The EGFR is widely expressed in non-small cell lung cancer (NSCLC) and is an important target in NSCLC. EGFR inhibition using tyrosine kinase inhibitors (TKIs) is highly effective initially in the subset of patients with EGFR-activating mutations, who comprise about 10-15% of NSCLC patients in Western populations. However, the majority of NSCLC tumors express EGFR wild type (EGFRwt) and do not respond to EGFR inhibition. Nevertheless, EGFR ligands are commonly expressed in lung cancer. Furthermore, a constitutive overexpression-induced EGFRwt signaling has also been reported. Thus, it is possible that EGFRwt could play an oncogenic role in lung cancer. Moreover, secondary resistance inevitably develops in initially responsive EGFR mutant tumors. The major mechanisms of resistance to EGFR inhibition in NSCLC include EGFR mutations such as the T790M mutation and activation of other RTKs such as Met. In addition, EGFR inhibition triggers a rapid adaptive response in NSCLC that likely contributes to secondary resistance, but may also induce primary resistance to treatment. This adaptive response is broad and may involve bypass signaling pathways such as STAT3 or NF-κB. It was recently reported that a rapid TNF-driven adaptive response plays a key role in resistance to EGFR inhibition in NSCLC and in glioma. In this study we examine an adaptive response to EGFR inhibition in NSCLC mediated by upregulation of Type I IFNs.
Thus, there remains a need for compositions and methods for treating cancers that express EGFRwt and/or that are resistant to EGFR inhibition. These needs and others are met by the present invention.
In accordance with the purpose(s) of the invention, as embodied and broadly described herein, the invention, in one aspect, relates to compounds and compositions for use in the prevention and treatment of disorders of cancers such as, for example, sarcomas, carcinomas, hematological cancers, solid tumors, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, bladder cancer, thyroid cancer, testicular cancer, pancreatic cancer, endometrial cancer, melanomas, gliomas, leukemias, lymphomas, chronic myeloproliferative disorders, myelodysplastic syndromes, myeloproliferative neoplasms, and plasma cell neoplasms (myelomas).
Thus, disclosed are methods for treating cancer in a subject, the method comprising administering to the subject an effective amount of an agent that modulates epidermal growth factor receptor (EGFR) signaling, or a pharmaceutically acceptable salt thereof, and an agent that modulates interferon (IFN) signaling, or a pharmaceutically acceptable salt thereof.
Also disclosed are methods for treating cancer in a patient in need thereof, said method comprising administering to said patient an effective amount of erlotinib and anifrolumab.
Also disclosed are pharmaceutical compositions comprising: (a) an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof; (b) an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier, wherein at least one of the agent that modulates EGFR signaling and the agent that modulates IFN signaling is present in an effective amount.
Also disclosed are methods for making a pharmaceutical composition, the method comprising combining: (a) an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof; (b) an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier, wherein at least one of the agent that modulates EGFR signaling and the agent that modulates IFN signaling is present in an effective amount.
Also disclosed are kits comprising an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof, and an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof, and one or more of: (a) an agent associated with the treatment of cancer; (b) instructions for administering the agent that modulates EGFR signaling and/or the agent that modulates IFN signaling in connection with treating cancer; and (c) instructions for treating cancer.
While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects and together with the description serve to explain the principles of the invention.
Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or can be learned by practice of the invention. The advantages of the invention will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
The present invention can be understood more readily by reference to the following detailed description of the invention and the Examples included therein.
Before the present compounds, compositions, articles, systems, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.
While aspects of the present invention can be described and claimed in a particular statutory class, such as the system statutory class, this is for convenience only and one of skill in the art will understand that each aspect of the present invention can be described and claimed in any statutory class. Unless otherwise expressly stated, it is in no way intended that any method or aspect set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not specifically state in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, or the number or type of aspects described in the specification.
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein may be different from the actual publication dates, which can require independent confirmation.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a functional group,” “an alkyl,” or “a residue” includes mixtures of two or more such functional groups, alkyls, or residues, and the like.
As used in the specification and in the claims, the term “comprising” can include the aspects “consisting of” and “consisting essentially of.”
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
As used herein, the terms “about” and “at or about” mean that the amount or value in question can be the value designated some other value approximately or about the same. It is generally understood, as used herein, that it is the nominal value indicated ±10% variation unless otherwise indicated or inferred. The term is intended to convey that similar values promote equivalent results or effects recited in the claims. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but can be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about” or “approximate” whether or not expressly stated to be such. It is understood that where “about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.
References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.
A weight percent (wt. %) of a component, unless specifically stated to the contrary, is based on the total weight of the formulation or composition in which the component is included.
As used herein, “IC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, an IC50 can refer to the concentration of a substance that is required for 50% inhibition in vivo, as further defined elsewhere herein. In a further aspect, IC50 refers to the half-maximal (50%) inhibitory concentration (IC) of a substance.
As used herein, “EC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% agonism of a biological process, or component of a process, including a protein, subunit, organelle, ribonucleoprotein, etc. In one aspect, an EC50 can refer to the concentration of a substance that is required for 50% agonism in vivo, as further defined elsewhere herein. In a further aspect, EC50 refers to the concentration of agonist that provokes a response halfway between the baseline and maximum response.
As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
As used herein, the term “subject” can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Thus, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. In one aspect, the subject is a mammal. A patient refers to a subject afflicted with a disease or disorder. The term “patient” includes human and veterinary subjects.
As used herein, the term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the disease from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease, i.e., arresting its development; or (iii) relieving the disease, i.e., causing regression of the disease. In one aspect, the subject is a mammal such as a primate, and, in a further aspect, the subject is a human. The term “subject” also includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.).
As used herein, the term “prevent” or “preventing” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed.
As used herein, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein.
As used herein, the terms “administering” and “administration” refer to any method of providing a pharmaceutical preparation to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. In various aspects, a preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. In further various aspects, a preparation can be administered prophylactically; that is, administered for prevention of a disease or condition.
As used herein, the terms “effective amount” and “amount effective” refer to an amount that is sufficient to achieve the desired result or to have an effect on an undesired condition. For example, a “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease or condition.
As used herein, “dosage form” means a pharmacologically active material in a medium, carrier, vehicle, or device suitable for administration to a subject. A dosage forms can comprise inventive a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, in combination with a pharmaceutically acceptable excipient, such as a preservative, buffer, saline, or phosphate buffered saline. Dosage forms can be made using conventional pharmaceutical manufacturing and compounding techniques. Dosage forms can comprise inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonate, acetate, or citrate) and pH adjustment agents (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citrate or acetate, amino acids and their salts) antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene9-10 nonyl phenol, sodium desoxycholate), solution and/or cryo/lyo stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmotic adjustment agents (e.g., salts or sugars), antibacterial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsilozone), preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity-adjustment agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose) and co-solvents (e.g., glycerol, polyethylene glycol, ethanol). A dosage form formulated for injectable use can have a disclosed compound, a product of a disclosed method of making, or a salt, solvate, or polymorph thereof, suspended in sterile saline solution for injection together with a preservative.
As used herein, “kit” means a collection of at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose. Individual member components may be physically packaged together or separately. For example, a kit comprising an instruction for using the kit may or may not physically include the instruction with other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation.
As used herein, “instruction(s)” means documents describing relevant materials or methodologies pertaining to a kit. These materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the kit, trouble-shooting, references, technical support, and any other related documents. Instructions can be supplied with the kit or as a separate member component, either as a paper form or an electronic form which may be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. Instructions can comprise one or multiple documents, and are meant to include future updates.
As used herein, the terms “therapeutic agent” include any synthetic or naturally occurring biologically active compound or composition of matter which, when administered to an organism (human or nonhuman animal), induces a desired pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action. The term therefore encompasses those compounds or chemicals traditionally regarded as drugs, vaccines, and biopharmaceuticals including molecules such as proteins, peptides, hormones, nucleic acids, gene constructs and the like. Examples of therapeutic agents are described in well-known literature references such as the Merck Index (14th edition), the Physicians’ Desk Reference (64th edition), and The Pharmacological Basis of Therapeutics (12th edition), and they include, without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of a disease or illness; substances that affect the structure or function of the body, or pro-drugs, which become biologically active or more active after they have been placed in a physiological environment. For example, the term “therapeutic agent” includes compounds or compositions for use in all of the major therapeutic areas including, but not limited to, adjuvants; anti-infectives such as antibiotics and antiviral agents; anti-cancer and anti-neoplastic agents such as kinase inhibitors, poly ADP ribose polymerase (PARP) inhibitors and other DNA damage response modifiers, epigenetic agents such as bromodomain and extra-terminal (BET) inhibitors, histone deacetylase (HDAc) inhibitors, iron chelotors and other ribonucleotides reductase inhibitors, proteasome inhibitors and Nedd8-activating enzyme (NAE) inhibitors, mammalian target of rapamycin (mTOR) inhibitors, traditional cytotoxic agents such as paclitaxel, dox, irinotecan, and platinum compounds, immune checkpoint blockade agents such as cytotoxic T lymphocyte antigen-4 (CTLA-4) monoclonal antibody (mAB), programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) mAB, cluster of differentiation 47 (CD47) mAB, toll-like receptor (TLR) agonists and other immune modifiers, cell therapeutics such as chimeric antigen receptor T-cell (CAR-T)/chimeric antigen receptor natural killer (CAR-NK) cells, and proteins such as interferons (IFNs), interleukins (ILs), and mAbs; anti-ALS agents such as entry inhibitors, fusion inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs), nucleotide reverse transcriptase inhibitors, NCP7 inhibitors, protease inhibitors, and integrase inhibitors; analgesics and analgesic combinations, anorexics, anti-inflammatory agents, antiepileptics, local and general anesthetics, hypnotics, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics, antiarrhythmics, antihypertensive agents, hormones, and nutrients, antiarthritics, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants, antineoplastics, antipruritics, antipyretics; antispasmodics, cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics), antihypertensives, diuretics, vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or recombinantly produced); and nucleic acid molecules (polymeric forms of two or more nucleotides, either ribonucleotides (RNA) or deoxyribonucleotides (DNA) including both double- and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like), small molecules (e.g., doxorubicin) and other biologically active macromolecules such as, for example, proteins and enzymes. The agent may be a biologically active agent used in medical, including veterinary, applications and in agriculture, such as with plants, as well as other areas. The term “therapeutic agent” also includes without limitation, medicaments; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances which affect the structure or function of the body; or pro- drugs, which become biologically active or more active after they have been placed in a predetermined physiological environment.
The term “pharmaceutically acceptable” describes a material that is not biologically or otherwise undesirable, i.e., without causing an unacceptable level of undesirable biological effects or interacting in a deleterious manner.
As used herein, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.
As used herein, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.
Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acros Organics (Morris Plains, N.J.), Strem Chemicals (Newburyport, MA), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser’s Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd’s Chemistry of Carbon Compounds, Volumes 1-5 and supplemental volumes (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March’s Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).
Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including: matters of logic with respect to arrangement of steps or operational flow; plain meaning derived from grammatical organization or punctuation; and the number or type of embodiments described in the specification.
Disclosed are the components to be used to prepare the compositions of the invention as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the methods of the invention.
It is understood that the compounds and compositions disclosed herein have certain functions. Disclosed herein are certain structural requirements for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same function that are related to the disclosed structures, and that these structures will typically achieve the same result.
In one aspect, disclosed are pharmaceutical compositions comprising: (a) an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof; (b) an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier, wherein at least one of the agent that modulates EGFR signaling and the agent that modulates IFN signaling is present in an effective amount.
In various aspects, the compounds and compositions of the invention can be administered in pharmaceutical compositions, which are formulated according to the intended method of administration. The compounds and compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, a pharmaceutical composition can be formulated for local or systemic administration, intravenous, topical, or oral administration.
The nature of the pharmaceutical compositions for administration is dependent on the mode of administration and can readily be determined by one of ordinary skill in the art. In various aspects, the pharmaceutical composition is sterile or sterilizable. The therapeutic compositions featured in the invention can contain carriers or excipients, many of which are known to skilled artisans. Excipients that can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, polypeptides (for example, serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, water, and glycerol. The nucleic acids, polypeptides, small molecules, and other modulatory compounds featured in the invention can be administered by any standard route of administration. For example, administration can be parenteral, intravenous, subcutaneous, or oral. A modulatory compound can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for administration by drops into the ear, for injection, or for ingestion; gels or powders can be made for ingestion or topical application. Methods for making such formulations are well known and can be found in, for example, Remington’s Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, PA 1990.
In various aspects, the disclosed pharmaceutical compositions comprise the disclosed compounds (including pharmaceutically acceptable salt(s) thereof) as an active ingredient, a pharmaceutically acceptable carrier, and, optionally, other therapeutic ingredients or adjuvants. The instant compositions include those suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
In various aspects, the pharmaceutical compositions of this invention can include a pharmaceutically acceptable carrier and a compound or a pharmaceutically acceptable salt of the compounds of the invention. The compounds of the invention, or pharmaceutically acceptable salts thereof, can also be included in pharmaceutical compositions in combination with one or more other therapeutically active compounds.
The pharmaceutical carrier employed can be, for example, a solid, liquid, or gas. Examples of solid carriers include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. Examples of liquid carriers are sugar syrup, peanut oil, olive oil, and water. Examples of gaseous carriers include carbon dioxide and nitrogen.
In preparing the compositions for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques.
A tablet containing the composition of this invention can be prepared by compression or molding, optionally with one or more accessory ingredients or adjuvants. Compressed tablets can be prepared by compressing, in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.
The pharmaceutical compositions of the present invention comprise a compound of the invention (or pharmaceutically acceptable salts thereof) as an active ingredient, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents or adjuvants. The instant compositions include compositions suitable for oral, rectal, topical, and parenteral (including subcutaneous, intramuscular, and intravenous) administration, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The pharmaceutical compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.
Pharmaceutical compositions of the present invention suitable for parenteral administration can be prepared as solutions or suspensions of the active compounds in water. A suitable surfactant can be included such as, for example, hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof in oils. Further, a preservative can be included to prevent the detrimental growth of microorganisms.
Pharmaceutical compositions of the present invention suitable for injectable use include sterile aqueous solutions or dispersions. Furthermore, the compositions can be in the form of sterile powders for the extemporaneous preparation of such sterile injectable solutions or dispersions. In all cases, the final injectable form must be sterile and must be effectively fluid for easy syringability. The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, preferably should be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.
Pharmaceutical compositions of the present invention can be in a form suitable for topical use such as, for example, an aerosol, cream, ointment, lotion, dusting powder, mouth washes, gargles, and the like. Further, the compositions can be in a form suitable for use in transdermal devices. These formulations can be prepared, utilizing a compound of the invention, or pharmaceutically acceptable salts thereof, via conventional processing methods. As an example, a cream or ointment is prepared by mixing hydrophilic material and water, together with about 5 wt% to about 10 wt% of the compound, to produce a cream or ointment having a desired consistency.
Pharmaceutical compositions of this invention can be in a form suitable for rectal administration wherein the carrier is a solid. It is preferable that the mixture forms unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art. The suppositories can be conveniently formed by first admixing the composition with the softened or melted carrier(s) followed by chilling and shaping in molds.
In addition to the aforementioned carrier ingredients, the pharmaceutical formulations described above can include, as appropriate, one or more additional carrier ingredients such as diluents, buffers, flavoring agents, binders, surface-active agents, thickeners, lubricants, preservatives (including anti-oxidants) and the like. Furthermore, other adjuvants can be included to render the formulation isotonic with the blood of the intended recipient. Compositions containing a compound of the invention, and/or pharmaceutically acceptable salts thereof, can also be prepared in powder or liquid concentrate form.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor. In a further aspect, the EGFR inhibitor is a tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib. In a still further aspect, the EGFR inhibitor is a monoclonal antibody.
In various aspects, the EGFR inhibitor is selected from erlotinib, afatinib, cetuximab, panitumumab, erlotinib HCl, gefitinib, lapatinib, neratinib, lifirafenib, HER2-inhibitor-1, nazartinib, naquotinib, canertinib, AG-490, CP-724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCl, pelitinib, AC480, AEE788, AP261 13-analog, OSI-420, WZ3146, WZ8040, AST-1306, rociletinib, genisten, varlitinib, icotinib, TAK-285, WHI-P154, daphnetin, PD168393, tyrphostin9, CNX-2006, AG-18, AZ5104, osimertinib, CL-387785, olmutinib, AZD3759, poziotinib, vandetanib, and necitumumab.
In various aspects, IFN signaling is Type IIFN signaling.
In various aspects, the agent that modulates IFN signaling is an interferon blocking antibody or an interferon neutralizing antibody. In a further aspect, the agent that modulates IFN signaling is an IFN inhibitor. Examples of IFN inhibitors include, but are not limited to, anifrolumab.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor and wherein the agent that modulates IFN signaling is an IFN inhibitor. In a further aspect, the agent that modulates EGFR signaling is erlotinib and wherein the agent that modulates IFN signaling is anifrolumab.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-formulated. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-packaged.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered concurrently. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are not administered concurrently.
In various aspects, the agent that modulates EGFR signaling is erlotinib and the agent that modulates IFN signaling is anifrolumab.
In a further aspect, an effective amount is a therapeutically effective amount. In a still further aspect, an effective amount is a prophylactically effective amount.
In various aspects, the effective amount is an individually effective amount of the agent that modulates EGFR signaling or the agent that modulates IFN signaling. In a further aspect, the effective amount is an individually effective amount of the agent that modulates EGFR signaling. In a still further aspect, the effective amount is an individually effective amount of the agent that modulates IFN signaling.
In various aspects, the effective amount is a combinatorically effective amount of the agent that modulates EGFR signaling and the agent that modulates IFN signaling.
In a further aspect, the pharmaceutical composition is administered to a mammal. In a still further aspect, the mammal is a human. In an even further aspect, the human is a patient.
In a further aspect, the pharmaceutical composition is used to treat cancers such as, for example, sarcomas, carcinomas, hematological cancers, solid tumors, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, bladder cancer, thyroid cancer, testicular cancer, pancreatic cancer, endometrial cancer, melanomas, gliomas, leukemias, lymphomas, chronic myeloproliferative disorders, myelodysplastic syndromes, myeloproliferative neoplasms, and plasma cell neoplasms (myelomas).
In a further aspect, the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).
In a further aspect, the cancer is NSCLC.
In a further aspect, the cancer expresses EGFR wild type. In a still further aspect, the cancer expresses EGFR mutant. In yet a further aspect, the cancer is resistant to EGFR inhibition.
In a further aspect, the cancer is a solid tumor.
In various aspects, the composition is a solid dosage form. In a further aspect, the composition is an oral solid dosage form. In a still further aspect, the solid dosage form is a tablet. In yet a further aspect, the solid dosage form is a capsule.
In various aspects, the composition is an injectable dosage form.
It is understood that the disclosed compositions can be prepared from the disclosed compounds. It is also understood that the disclosed compositions can be employed in the disclosed methods of using.
In one aspect, disclosed are methods for making a pharmaceutical composition, the method comprising combining: (a) an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof; (b) an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof; and (c) a pharmaceutically acceptable carrier, wherein at least one of the agent that modulates EGFR signaling and the agent that modulates IFN signaling is present in an effective amount.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor. In a further aspect, the EGFR inhibitor is a tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib. In a still further aspect, the EGFR inhibitor is a monoclonal antibody.
In various aspects, the EGFR inhibitor is selected from erlotinib, afatinib, cetuximab, panitumumab, erlotinib HCl, gefitinib, lapatinib, neratinib, lifirafenib, HER2-inhibitor-1, nazartinib, naquotinib, canertinib, AG-490, CP-724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCl, pelitinib, AC480, AEE788, AP261 13-analog, OSI-420, WZ3146, WZ8040, AST-1306, rociletinib, genisten, varlitinib, icotinib, TAK-285, WHI-P154, daphnetin, PD168393, tyrphostin9, CNX-2006, AG-18, AZ5104, osimertinib, CL-387785, olmutinib, AZD3759, poziotinib, vandetanib, and necitumumab.
In various aspects, IFN signaling is Type I IFN signaling.
In various aspects, the agent that modulates IFN signaling is an interferon blocking antibody or an interferon neutralizing antibody. In a further aspect, the agent that modulates IFN signaling is an IFN inhibitor. Examples of IFN inhibitors include, but are not limited to, anifrolumab.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor and wherein the agent that modulates IFN signaling is an IFN inhibitor. In a further aspect, the agent that modulates EGFR signaling is erlotinib and wherein the agent that modulates IFN signaling is anifrolumab.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-formulated. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-packaged.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered concurrently. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are not administered concurrently.
In various aspects, the agent that modulates EGFR signaling is erlotinib and the agent that modulates IFN signaling is anifrolumab.
In a further aspect, an effective amount is a therapeutically effective amount. In a still further aspect, an effective amount is a prophylactically effective amount.
In various aspects, the effective amount is an individually effective amount of the agent that modulates EGFR signaling or the agent that modulates IFN signaling. In a further aspect, the effective amount is an individually effective amount of the agent that modulates EGFR signaling. In a still further aspect, the effective amount is an individually effective amount of the agent that modulates IFN signaling.
In various aspects, the effective amount is a combinatorically effective amount of the agent that modulates EGFR signaling and the agent that modulates IFN signaling.
In various aspects, combining is co-formulating the agent that modulates EGFR signaling and the agent that modulates IFN signaling with the pharmaceutically acceptable carrier. In a further aspect, co-formulating provides an oral solid dosage form comprising the agent that modulates EGFR signaling, the agent that modulates IFN signaling, and the pharmaceutically acceptable carrier. In a still further aspect, the solid dosage form is a tablet. In yet a further aspect, the solid dosage form is a capsule.
In various aspects, co-formulating provides an injectable dosage form comprising the agent that modulates EGFR signaling, the agent that modulates IFN signaling, and the pharmaceutically acceptable carrier.
In one aspect, disclosed are methods for treating cancer in a subject, the method comprising administering to the subject an effective amount of an agent that modulates epidermal growth factor receptor (EGFR) signaling, or a pharmaceutically acceptable salt thereof, and an agent that modulates interferon (IFN) signaling, or a pharmaceutically acceptable salt thereof.
In one aspect, disclosed are methods for treating cancer in a patient in need thereof, said method comprising administering to said patient an effective amount of erlotinib and anifrolumab.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor. In a further aspect, the EGFR inhibitor is a tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib. In a still further aspect, the EGFR inhibitor is a monoclonal antibody.
In various aspects, the EGFR inhibitor is selected from erlotinib, afatinib, cetuximab, panitumumab, erlotinib HCl, gefitinib, lapatinib, neratinib, lifirafenib, HER2-inhibitor-1, nazartinib, naquotinib, canertinib, AG-490, CP-724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCl, pelitinib, AC480, AEE788, AP261 13-analog, OSI-420, WZ3146, WZ8040, AST-1306, rociletinib, genisten, varlitinib, icotinib, TAK-285, WHI-P154, daphnetin, PD168393, tyrphostin9, CNX-2006, AG-18, AZ5104, osimertinib, CL-387785, olmutinib, AZD3759, poziotinib, vandetanib, and necitumumab.
In various aspects, IFN signaling is Type IIFN signaling.
In various aspects, the agent that modulates IFN signaling is an interferon blocking antibody or an interferon neutralizing antibody. In a further aspect, the agent that modulates IFN signaling is an IFN inhibitor. Examples of IFN inhibitors include, but are not limited to, anifrolumab.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor and wherein the agent that modulates IFN signaling is an IFN inhibitor. In a further aspect, the agent that modulates EGFR signaling is erlotinib and wherein the agent that modulates IFN signaling is anifrolumab.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-formulated. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-packaged.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered concurrently. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are not administered concurrently.
In various aspects, the effective amount is a therapeutically effective amount. In a further aspect, the effective amount is a prophylactically effective amount.
In various aspects, the effective amount is an individually effective amount of the agent that modulates EGFR signaling or the agent that modulates IFN signaling. In a further aspect, the effective amount is an individually effective amount of the agent that modulates EGFR signaling. In a still further aspect, the effective amount is an individually effective amount of the agent that modulates IFN signaling.
In various aspects, the effective amount is a combinatorically effective amount of the agent that modulates EGFR signaling and the agent that modulates IFN signaling.
In a further aspect, the subject has been diagnosed with a need for treatment of cancer prior to the administering step. In a still further aspect, the subject is at risk for developing cancer prior to the administering step.
In a further aspect, the subject is a mammal. In a still further aspect, the mammal is a human.
In a further aspect, the method further comprises the step of identifying a subject in need of treatment of cancer.
In a further aspect, the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).
In a further aspect, the cancer is NSCLC.
In a further aspect, the cancer expresses EGFR wild type. In a still further aspect, the cancer expresses EGFR mutant. In yet a further aspect, the cancer is resistant to EGFR inhibition.
In a further aspect, the cancer is a solid tumor.
In a further aspect, the method further comprises the step of administering a therapeutically effective amount of at least one chemotherapeutic agent. In yet a further aspect, the chemotherapeutic agent is selected from an alkylating agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, and a mTor inhibitor agent.
In various aspects, the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.
In various aspects, the antimetabolite agent is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.
In various aspects, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.
In various aspects, the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharmaceutically acceptable salt thereof.
In various aspects, the mTor inhibitor agent is selected from everolimus, siroliumus, and temsirolimus, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.
The compounds and pharmaceutical compositions of the invention are useful in treating or controlling cancer. Examples of cancers include, but are not limited to, sarcomas, carcinomas, hematological cancers, solid tumors, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, bladder cancer, thyroid cancer, testicular cancer, pancreatic cancer, endometrial cancer, melanomas, gliomas, leukemias, lymphomas, chronic myeloproliferative disorders, myelodysplastic syndromes, myeloproliferative neoplasms, and plasma cell neoplasms (myelomas).
To treat or control cancer, the compounds and pharmaceutical compositions comprising the compounds are administered to a subject in need thereof, such as a vertebrate, e.g., a mammal, a fish, a bird, a reptile, or an amphibian. The subject can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig or rodent. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered. The subject is preferably a mammal, such as a human. Prior to administering the compounds or compositions, the subject can be diagnosed with a need for treatment of cancer.
The compounds or compositions can be administered to the subject according to any method. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, sublingual administration, buccal administration and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. A preparation can be administered therapeutically; that is, administered to treat an existing disease or condition. A preparation can also be administered prophylactically; that is, administered for prevention of cancer.
The therapeutically effective amount or dosage of the compound can vary within wide limits. Such a dosage is adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg or more, a daily dosage of about 10 mg to about 10,000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, as a continuous infusion. Single dose compositions can contain such amounts or submultiples thereof of the compound or composition to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
In one aspect, the invention relates to the use of a disclosed agent, a disclosed pharmaceutical composition, or a product of a disclosed method. In a further aspect, a use relates to the manufacture of a medicament for the treatment of cancer in a subject.
Also provided are the uses of the disclosed agents, compositions, and products. In one aspect, the invention relates to use of at least one disclosed agent, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof, or at least one disclosed composition. In a further aspect, the composition used is a product of a disclosed method of making.
In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed agent or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, for use as a medicament.
In a further aspect, the use relates to a process for preparing a pharmaceutical composition comprising a therapeutically effective amount of a disclosed agent or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, wherein a pharmaceutically acceptable carrier is intimately mixed with a therapeutically effective amount of the compound or the product of a disclosed method of making.
In various aspects, the use relates to a treatment of cancer in a subject. In one aspect, the use is characterized in that the subject is a human. In one aspect, the use is characterized in that the cancer is NSCLC.
In a further aspect, the use relates to the manufacture of a medicament for the treatment of cancer in a subject.
It is understood that the disclosed uses can be employed in connection with the disclosed agents, products of disclosed methods of making, methods, compositions, and kits. In a further aspect, the invention relates to the use of a disclosed agents or a disclosed product in the manufacture of a medicament for the treatment of cancer in a mammal. In a further aspect, the cancer is NSCLC.
In one aspect, the invention relates to a method for the manufacture of a medicament for treating cancer in a subject having cancer, the method comprising combining a therapeutically effective amount of a disclosed agent, composition, or product of a disclosed method with a pharmaceutically acceptable carrier or diluent.
As regards these applications, the present method includes the administration to an animal, particularly a mammal, and more particularly a human, of a therapeutically effective amount of the agents effective in the treatment of cancer. The dose administered to an animal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic response in the animal over a reasonable time frame. One skilled in the art will recognize that dosage will depend upon a variety of factors including the condition of the animal and the body weight of the animal.
The total amount of the agent of the present disclosure administered in a typical treatment is preferably between about 0.05 mg/kg and about 100 mg/kg of body weight for mice, and more preferably between 0.05 mg/kg and about 50 mg/kg of body weight for mice, and between about 100 mg/kg and about 500 mg/kg of body weight, and more preferably between 200 mg/kg and about 400 mg/kg of body weight for humans per daily dose. This total amount is typically, but not necessarily, administered as a series of smaller doses over a period of about one time per day to about three times per day for about 24 months, and preferably over a period of twice per day for about 12 months.
The size of the dose also will be determined by the route, timing and frequency of administration as well as the existence, nature and extent of any adverse side effects that might accompany the administration of the agent or composition and the desired physiological effect. It will be appreciated by one of skill in the art that various conditions or disease states, in particular chronic conditions or disease states, may require prolonged treatment involving multiple administrations.
Thus, in one aspect, the invention relates to the manufacture of a medicament comprising combining a disclosed agent, composition, or a product of a disclosed method of making, or a pharmaceutically acceptable salt, solvate, or polymorph thereof, with a pharmaceutically acceptable carrier or diluent.
In one aspect, disclosed are kits comprising an agent that modulates EGFR signaling, or a pharmaceutically acceptable salt thereof, and an agent that modulates IFN signaling, or a pharmaceutically acceptable salt thereof, and one or more of: (a) an agent associated with the treatment of cancer; (b) instructions for administering the agent that modulates EGFR signaling and/or the agent that modulates IFN signaling in connection with treating cancer; and (c) instructions for treating cancer.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor. In a further aspect, the EGFR inhibitor is a tyrosine kinase inhibitor. Examples of tyrosine kinase inhibitors include, but are not limited to, erlotinib. In a still further aspect, the EGFR inhibitor is a monoclonal antibody.
In various aspects, the EGFR inhibitor is selected from erlotinib, afatinib, cetuximab, panitumumab, erlotinib HCl, gefitinib, lapatinib, neratinib, lifirafenib, HER2-inhibitor-1, nazartinib, naquotinib, canertinib, AG-490, CP-724714, Dacomitinib, WZ4002, Sapitinib, CUDC-101, AG-1478, PD153035 HCl, pelitinib, AC480, AEE788, AP261 13-analog, OSI-420, WZ3146, WZ8040, AST-1306, rociletinib, genisten, varlitinib, icotinib, TAK-285, WHI-P154, daphnetin, PD168393, tyrphostin9, CNX-2006, AG-18, AZ5104, osimertinib, CL-387785, olmutinib, AZD3759, poziotinib, vandetanib, and necitumumab.
In various aspects, IFN signaling is Type I IFN signaling.
In various aspects, the agent that modulates IFN signaling is an interferon blocking antibody or an interferon neutralizing antibody. In a further aspect, the agent that modulates IFN signaling is an IFN inhibitor. Examples of IFN inhibitors include, but are not limited to, anifrolumab.
In various aspects, the agent that modulates EGFR signaling is an EGFR inhibitor and wherein the agent that modulates IFN signaling is an IFN inhibitor. In a further aspect, the agent that modulates EGFR signaling is erlotinib and wherein the agent that modulates IFN signaling is anifrolumab.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-formulated. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-packaged.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered concurrently. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are not administered concurrently.
In various aspects, the agent that modulates EGFR signaling is erlotinib and the agent that modulates IFN signaling is anifrolumab.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-formulated. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are co-packaged.
In a further aspect, the cancer is selected from a sarcoma, a carcinoma, a hematological cancer, a solid tumor, breast cancer, cervical cancer, gastrointestinal cancer, colorectal cancer, brain cancer, skin cancer, prostate cancer, ovarian cancer, thyroid cancer, testicular cancer, pancreatic cancer, liver cancer, endometrial cancer, melanoma, a glioma, leukemia, lymphoma, chronic myeloproliferative disorder, myelodysplastic syndrome, myeloproliferative neoplasm, non-small cell lung carcinoma, and plasma cell neoplasm (myeloma).
In a further aspect, the cancer is a solid tumor.
In a further aspect, the agent is a chemotherapeutic agent. In yet a further aspect, the chemotherapeutic agent is selected from an alkylating agent, an antimetabolite agent, an antineoplastic antibiotic agent, a mitotic inhibitor agent, and a mTor inhibitor agent.
In various aspects, the antineoplastic antibiotic agent is selected from doxorubicin, mitoxantrone, bleomycin, daunorubicin, dactinomycin, epirubicin, idarubicin, plicamycin, mitomycin, pentostatin, and valrubicin, or a pharmaceutically acceptable salt thereof.
In various aspects, the antimetabolite agent is selected from gemcitabine, 5-fluorouracil, capecitabine, hydroxyurea, mercaptopurine, pemetrexed, fludarabine, nelarabine, cladribine, clofarabine, cytarabine, decitabine, pralatrexate, floxuridine, methotrexate, and thioguanine, or a pharmaceutically acceptable salt thereof.
In various aspects, the alkylating agent is selected from carboplatin, cisplatin, cyclophosphamide, chlorambucil, melphalan, carmustine, busulfan, lomustine, dacarbazine, oxaliplatin, ifosfamide, mechlorethamine, temozolomide, thiotepa, bendamustine, and streptozocin, or a pharmaceutically acceptable salt thereof.
In various aspects, the mitotic inhibitor agent is selected from irinotecan, topotecan, rubitecan, cabazitaxel, docetaxel, paclitaxel, etopside, vincristine, ixabepilone, vinorelbine, vinblastine, and teniposide, or a pharmaceutically acceptable salt thereof.
In various aspects, the mTor inhibitor agent is selected from everolimus, siroliumus, and temsirolimus, or a pharmaceutically acceptable salt, hydrate, solvate, or polymorph thereof.
In various aspects, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered sequentially. In a further aspect, the agent that modulates EGFR signaling and the agent that modulates IFN signaling are administered simultaneously.
In various aspects, the agent that modulates EGFR signaling and the chemotherapeutic agent are administered sequentially. In a further aspect, the agent that modulates EGFR signaling and the chemotherapeutic agent are administered simultaneously.
In various aspects, the agent that modulates IFN signaling and the chemotherapeutic agent are administered sequentially. In a further aspect, the agent that modulates IFN signaling and the chemotherapeutic agent are administered simultaneously.
In various aspects, the agent that modulates EGFR signaling, the agent that modulates IFN signaling, and the chemotherapeutic agent are administered sequentially. In a further aspect, the agent that modulates EGFR signaling, the agent that modulates IFN signaling, and the chemotherapeutic agent are administered simultaneously.
The kits can also comprise compounds and/or products co-packaged, co-formulated, and/or co-delivered with other components. For example, a drug manufacturer, a drug reseller, a physician, a compounding shop, or a pharmacist can provide a kit comprising a disclosed compound and/or product and another component for delivery to a patient.
It is understood that the disclosed kits can be prepared from the disclosed compounds, products, and pharmaceutical compositions. It is also understood that the disclosed kits can be employed in connection with the disclosed methods of using.
The foregoing description illustrates and describes the disclosure. Additionally, the disclosure shows and describes only the preferred embodiments but, as mentioned above, it is to be understood that it is capable to use in various other combinations, modifications, and environments and is capable of changes or modifications within the scope of the invention concepts as expressed herein, commensurate with the above teachings and/or the skill or knowledge of the relevant art. The embodiments described herein above are further intended to explain best modes known by applicant and to enable others skilled in the art to utilize the disclosure in such, or other, embodiments and with the various modifications required by the particular applications or uses thereof. Accordingly, the description is not intended to limit the invention to the form disclosed herein. Also, it is intended to the appended claims be construed to include alternative embodiments.
All publications and patent applications cited in this specification are herein incorporated by reference, and for any and all purposes, as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. In the event of an inconsistency between the present disclosure and any publications or patent application incorporated herein by reference, the present disclosure controls.
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Here, it is reported that EGFR inhibition reprograms cellular signaling and results in a remarkable cooptation of antiviral signaling pathways in NSCLC. This antiviral response mediates resistance to EGFR inhibition in both EGFR wild type and EGFR mutant NSCLC. NSCLC cells respond to EGFR inhibition with a rapid increase in Type I interferon levels and the IFN upregulation was detected in all NSCLC cell lines examined, in animal tumor tissue, and in archival tissue from patients. In EGFRwt expressing NSCLCs, the increase in IFNs is sufficient to protect cells from loss of EGFR signaling. In NSCLCs with EGFR-activating mutations the IFN driven adaptive response is only partially protective and observed after treatment with low concentrations of EGFR inhibitors. This is also true for other adaptive bypass signaling mechanisms such as STAT3, or TNF-NF-κB that are triggered by EGFR inhibition in EGFR mutant NSCLC and do not inhibit the initial clinical response in patients but may play a role in the development of secondary resistance. Importantly, exogenous IFNα or IFNβ via activation of STAT1 protects NSCLC cells with mutant EGFR activating from cell death resulting from EGFR inhibition, further supporting an important role for Type I interferons in mediating resistance to EGFR inhibition in NSCLC.
The adaptive response to inhibition of RTK pathways is broad and leads to substantial reprogramming of signaling pathways that attempt to restore homeostasis. However, targeted inhibition of one or a small number of pathways may cripple the adaptive response and overcome therapeutic resistance in such cancers. Here, it is shown that combined inhibition of EGFR and type I IFN signaling is highly effective in suppressing the growth of NSCLC tumors in multiple animal models.
Although most patients with EGFR activating mutations initially respond to EGFR TKIs, they inevitably develop resistance, implying the persistence of subsets of cancer cells. Primary or intrinsic resistance to inhibition of EGFRwt could occur because an adaptive response prevents cell death in response to EGFR inhibition. Currently the EGFRwt does not appear to be a useful target for treatment, because EGFR inhibition is ineffective in EGFRwt expressing NSCLC. However EGFRwt is widely expressed and recent studies suggest that targeting the EGFR signaling network inhibition may also hold promise in EGFRwt/KRas NSCLC. Here, it is proposed that the primary resistance to EGFR inhibition in EGFRwt NSCLC does not necessarily indicate that EGFR signaling is irrelevant to the malignant phenotype. Rather, EGFR inhibition may not work in because an adaptive survival mechanism triggered by EGFR inhibition negates its effect. A combined inhibition of EGFR+adaptive response either unmasks a requirement for EGFR signaling for survival and/or sets up synthetic lethal conditions.
EGFR inhibition results in an increase in Type I IFN levels via distinct mechanisms depending on whether EGFR is mutant or wild type. In EGFR mutant tumors, a RIGI-TRIM32-TBK1-IRF3 axis mediates induction of IFNs and resistance to EGFR inhibition. Here, it is shown that TRIM32, an E3-ubiquitin ligase, associates with TBK1 upon EGFR inhibition leading to K-63 linked ubiquitination of TBK1. TRIM32 is required for EGFR inhibition induced TBK1 and IRF3 phosphorylation and resistance to EGFR inhibition. In contrast, inhibition of EGFRwt tumors, upregulates Type I interferons via an NF-κB dependent pathway.
In a previous study it was demonstrated that inhibition of the EGFR in lung cancer cells resulted in a rapid increase in TNF secretion via an effect on TNF mRNA stability mediated by miR-21 and leading to a NF-κB driven survival pathway that protected cells from a loss of EGFR signaling. Here, it is shown that EGFR inhibition results in a distinct adaptive mechanism that activates an anti-viral signaling pathway mediated by upregulation of Type I IFNs. A combined inhibition of EGFR and type I interferons using the clinically available antibody anifrolumab enhances the effectiveness of EGFR inhibition in EGFR mutant cells and is able to overcome the primary resistance of EGFRwt NSCLC including the subset with KRas mutant. Intriguingly, it was also found that EGFR inhibition may lead to an upregulation of PD-L1 via an interferon dependent pathway, providing a possible explanation for the failure of immunotherapy in EGFR mutant NSCLC, and the possibility that anifrolumab may render such tumors responsive to immunotherapy.
The data herein indicate that Type IIFN signaling is a major targetable mechanism of resistance to EGFR TKI inhibition in EGFR mutant and EGFRwt NSCLC. The chromosomal locus for the Type I interferon genes is 9p23.1, one of the most common sites for homozygous deletions of tumor suppressive genes. Homozygous deletion of Type I IFN genes has been reported in multiple tumor types and in about 10% of NSCLC, and correlates with a worse prognosis. However, the data herein indicate that in the context of EGFR inhibition, type I interferons mediate therapeutic resistance and confer a worse prognosis. Whether the combined effect of Type I IFN and EGFR inhibition is synergistic in highly resistant EGFRwt/KRas mutant models and in EGFR mutant models when a low concentration of erlotinib is used was explored. Together these findings provide a therapeutic opportunity. Targeting a biologically significant upregulation of Type I interferons upon EGFR inhibition could greatly expand the reach and impact of EGFR targeted treatment in NSCLC. Thus, inhibiting the EGFR with a combination of TKI plus an IFN inhibitor such as the FDA approved anifrolumab may be effective in the treatment of NSCLCs that express EGFRwt. In tumors with EGFR activating mutations, a combined treatment with EGFR and IFN inhibition may result in a more effective elimination of tumor cells during the initial treatment and perhaps eliminate or delay secondary resistance to TKI treatment, and may also be useful in treating secondary resistance.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, articles, devices and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is at or near atmospheric.
The Examples are provided herein to illustrate the invention, and should not be construed as limiting the invention in any way. Examples are provided herein to illustrate the invention and should not be construed as limiting the invention in any way.
A549 and U87MG cells were purchased from American Type Culture Collection (ATCC). HCC827/ER3, HCC827/ER4(A), and HCC827/ER556 were obtained from Dr. Trever Bivona, University of California (San Francisco, CA). HCC827/ER4(B)55 and were obtained from Dr. Eric Haura, Moffitt Cancer Center (Tampa, FL). All other NSCLC cell lines were from the Hamon Center for Therapeutic Oncology Research at the University of Texas Southwestern Medical Center. NSCLC cells were cultured in RPMI-1640 containing 5% FBS, and U87MG in DMEM medium with 10% FBS. Cell lines were authenticated by DNA fingerprints for cell-line individualization using Promega StemElite ID system, a short tandem repeat (STR)-based assay, at UT Southwestern genomics core. Cells were tested for mycoplasma contamination using an e-Myco kit (Boca Scientific).
Western blot and immunoprecipitation were performed according to standard protocols. Western blot results are representative of at least 3 independent experiments. EGFR (06-847) antibody was from EMD Millipore (Billerica, MA); p-EGFR (Tyr1068) (2236), p-TBK1 (Ser172) (5483), TBK1 (3504), IKKε (2905), IRF3 (11904), K63-Ub (12930), RIG-I (3743), STAT1 (9172), p-STAT1 (Tyr701) (9167), AhR (83200), LAMIN A/C (4777), STING (3337), p-STING (Ser366) (19781), PD1 (86163), PD-L1 (13684), PD-L2 (82723) and IκBα (4814) antibodies were from Cell Signaling Technology (Danvers, MA); IFNAR1 (sc-7391), IFNGR1 (sc-12755), TNFR1 (sc-8346), and β-Actin (sc-47778) were from Santa Cruz Biotechnology (Dallas, TX); TRIM32 (Mab6515) antibody was from R&D (Minneapolis, MN); p-IRF3 (Ser386) (ab76493) antibody was from Abcam (Cambridge, MA).
Recombinant human IFNα1 (z02866) was purchased from Genscript (Piscataway, NJ); IFNβ1 (300-02BC) and TNFα (300-01A) was obtained from PeproTech (Rocky Hill, NJ). Mouse anti-mouse IFNAR1 antibody (BE0241) was purchased from Bioxcell (West Lebanon, NH). Anifrolumab, an anti-IFNARl antibody was obtained from Creative-Biolabs (Shirley, NY) (TAB-722). Entanercept (Enbrel), a fusion protein of TNF receptor and IgG, was purchased from Mckesson Medical Supply (San Francisco CA). NF-κB inhibitor BMS-345541 was obtained from MilliporeSigma (Burlington, MA). LPS (19661), TBK1 inhibitor BX795, and EGFR inhibitor erlotinib and afatinib for in vitro studies were obtained from Cayman Chemical (Ann Arbor, MI). Erlotinib for animal treatment was purchased from LC Laboratories (Woburn, MA). pCMV2-IRF3 plasmid was a kind gift from Dr. John Hiscott (McGill University, Montreal, Canada). NFκB luciferase reporter plasmid was provided by Dr. Ezra Burstein (UT Southwestern).
Cell viability assays were conducted with AlamarBlue Cell Viability Reagent from ThermoFisher (Waltham, MA), following the manufacturer’s protocol. Cells were cultured in Coming (Corning, NY) 96-well black plates with clear bottom and detected by the POLARstar Omega Microplate Reader (BMG LABTECH) (excitation at 544 nm and emission at 590 nm). At least 3 independent experiments were done. In oncogene addicted EGFR mutant cells, a lower dose of erlotinib 0.01 µM was used in cell viability assays to detect synergistic effects of combination therapy while a higher dose of 0.1 µM was used to detect the protection from erlotinib-induced cell death when IFNα or IFNβ were used.
Cells were plated in 48 well dishes followed by plasmid transfection with two reporters detecting activation of IRF3, ISRE-luciferase reporter or IFI27-ISRE luciferase reporter22, using lipofectamine 2000. A dual-luciferase reporter assay system was used according to the instructions of the manufacturer (Promega, Madison WI). Firefly luciferase activity was measured in the POLARstar Omega Microplate Reader (BMG LABTECH) and normalized based on Renilla luciferase activity. Three independent experiments were done in triplicate.
Total RNA was isolated by TRIzol Reagent (Fisher Scientific). cDNA Reverse Transcription was performed by using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). PCR primers were synthesized by IDT (Coralville, IA). Each PCR reaction was carried out in triplicate in a 20 µl volume using SYBR Green Master Mix (Applied Biosystems) for 15 minutes at 95° C. for initial denaturing, followed by 40 cycles of 95° C. for 15 s and 60° C. for 60 s in ViiA 7 Real-Time PCR System (Applied Biosystems). At least three independent experiments were done. Values for each gene were normalized to expression levels of ACTB (β-Actin) mRNA. Primer sequences were as follows.
RNA sequencing was performed at UT Southwestern Genomics and Microarray Core Facility. Total RNA was isolated by TRIzol Reagent (Fisher Scientific). RNA quality was determined by Agilent 2100 Bioanalyzer (RIN > 8), and quantity was measured by Qubit fluorometer. 1 µg RNA was then prepared with the TruSeq Stranded Total RNA LT Sample Prep Kit from Illumina. Poly-A RNA (mRNAseq) is purified and fragmented before strand specific cDNA synthesis. cDNA are then a-tailed and indexed adapters are ligated. After adapter ligation, samples are PCR amplified and purified with Ampure XP beads, then run on the Illumina NextSeq 500/550 system (Kits V2.5) with 75 bp single end reads to product about 25 Million reads per sample.
Sequencing data were further processed at UT Southwestern Bioinformatics Core Facility. Differential expression was analyzed by DESeq2. Pathway analysis was performed based on Gene Set Enrichment Analysis (GSEA, http://software.broadinstitute.org/gsea/index.jsp).
To detect IFNα and IFNβ levels in medium, cells were cultured in serum free medium and treated with indicated drugs for 48 hours. Supernatant was then collected and concentrated using a Pierce protein concentrator (Thermo-Fisher). To test IFNα and IFNβ in lysates, cell and tumor lysates were extracted using RIPA buffer. Total protein concentrations were determined by Pierce BCA Protein Assay Kit (Fisher Scientific). Then, the levels of IFNα and IFNβ protein were measured by ELISA using human IFNα ELISA kit (41100) and human IFNβ ELISA kit (41440), from PBL Assay Science (Piscataway, NJ) according to the manufacturer’s protocol.
Cells were cultured on coverslips in plates, after treatment cells were fixed with 4% Paraformaldehyde (PFA) followed by cell membrane permeabilization in 0.5 % Triton X-100/PBS and blocked in 1% BSA/PBS. The primary anti-AhR antibody (Santa Cruz Biotechnology, sc-101104, 1:200) was incubated at 4° C. overnight, followed with Alexa555-conjugated secondary antibodies (Cell Signaling, 4413, 1:500) at room temperature for 2 hours. Cell nuclei were counterstained with DAPI (Invitrogen) at 0.1 µg/ml. The cells were examined using fluorescence microscope.
HCC827 cells were treated with 0.1 µM erlotinib for 0, 2, 6, and 24 hours. Cell lysates were immunoprecipitated with TBK1 antibody. Antibody enriched protein samples were run on SDS-PAGE gels and submitted to UT Southwestern Proteomics Core Facility for Mass Spectrometry. Protein gel pieces were digested overnight with trypsin (Pierce) following reduction and alkylation with DTT and iodoacetamide (Sigma-Aldrich). The samples then underwent solid-phase extraction cleanup with Oasis HLB microelution plates (Waters) and the resulting samples were analyzed by LC/MS/MS, using an Orbitrap Fusion Lumos mass spectrometer (Thermo Electron) coupled to an Ultimate 3000 RSLC-Nano liquid chromatography systems (Dionex). Samples were injected onto a 75 µm i.d., 50-cm long EasySpray column (Thermo), and eluted with a gradient from 1-28% buffer B over 60 min. Buffer A contained 2% (v/v) ACN and 0.1% formic acid in water, and buffer B contained 80% (v/v) ACN, 10% (v/v) trifluoroethanol, and 0.1% formic acid in water. The mass spectrometer operated in positive ion mode with a source voltage of 2.55 kV and an ion transfer tube temperature of 275° C. MS scans were acquired at 120,000 resolution in the Orbitrap and up to 10 MS/MS spectra were obtained in the ion trap for each full spectrum acquired using higher-energy collisional dissociation (HCD) for ions with charges 2-7. Dynamic exclusion was set for 25 s after an ion was selected for fragmentation.
Raw MS data files were converted to a peak list format and analyzed using the central proteomics facilities pipeline (CPFP), version 2.0.3. Peptide identification was performed using the X!Tandem and open MS search algorithm (OMSSA) search engines against the human protein database from Uniprot, with common contaminants and reversed decoy sequences appended. Fragment and precursor tolerances of 20 ppm and 0.6 Da were specified, and three missed cleavages were allowed. Carbamidomethylation of Cys was set as a fixed modification and oxidation of Met was set as a variable modification. Label-free quantitation of proteins across samples was performed using SINQ normalized spectral index Software62.
siRNA knockdown was conducted with siRIG-I(sc-61480), siTRIM32(sc-61714), siTBK1(sc-39058), siIRF3(sc-35710), siIFNAR1(sc-35637), siSTAT1(sc-44123), siIFNGRl(sc-29357), siTNFR1(sc-29507), siSTING(sc-92042), and Control siRNA (sc-37007), purchased from Santa Cruz Biotechnology (Dallas, TX).
Lentiviruses for establishing stable cell lines used for xenograft experiments were obtained from Santa Cruz Biotechnology (Dallas, TX), including shTBK1(sc-39058-V), shIRF3(sc-35710-V), shIFNARl(sc-35637-V) Human Lentiviral Particles, and Control shRNA Lentiviral Particles-A(sc-108080). GFP adenovirus (1060) and IkBα (S32A/S36A)-DN (Dominant-negative) adenovirus (1028) were obtained from Vector Biolabs (Malvern, PA). A Multiplicity of infection (MOI) of 10 was used in the experiments. Cells were infected with shRNA lentiviral particles following the manufacturer’s protocol and 0.6 µg/mL puromycin was added for selecting stable clones.
Cell lines: Female nude mice (088) at four- to six-week-old were purchased from Charles River Laboratories (Wilmington, MA). One million A549 cells (1x106), or two million (2x106) HCC827 cells (including stable cell lines derived) were injected subcutaneously (s.c.) into the flanks of nude mice. About two weeks later, mice would develop subcutaneous tumors. The mice were randomly divided into indicated groups. Mice were treated with drugs using the doses described in the figure legends. For combination treatment, both drugs were given concurrently for indicated periods. Tumor dimensions were measured every 4 days and tumor volumes calculated by the formula: volume = 0.5 x length x width x width. Mice were sacrificed when tumors reached over 20 millimeter (mm) of length or after the indicated number of days.
Patient-derived xenograft (PDX): The NSCLC specimens (P0) for HCC4087 and HCC4190 PDXs were surgically resected from a patient diagnosed with NSCLC at UT Southwestern, after obtaining Institutional Review Board approval and informed consent. HCC4087 has KRAS G13C mutation but no EGFR activating mutations, HCC4190 harbors EGFR L858R mutation identified by Exome sequencing. 4 to 6 weeks old female NOD SCID mice (394) were purchased from Charles River Laboratories. The PDX tumor tissues were cut into small pieces (~20 mm3) and subcutaneously implanted in NOD SCID mice of serial generations (P1, P2, etc.). P4 tumor bearing SCID mice were used in this study.
LSL-Kras G12D mice (008179): were purchased from Jackson laboratories and the colony was expanded by breeding heterozygous LSL-Kras G12D mice with wildtype mice. Genotyping was performed per the protocol on Jackson website. Lung tumors were induced in mice carrying the LSL-Kras G12D allele with intranasal administration of 2.5 x 108 PFU Adeno-CMV-Cre (University of Iowa). Treatments were initiated once the tumors were confirmed by with Magnetic resonance imaging (MRI) at about 10-12 weeks after tumor induction.
MRI Imaging was conducted at UT Southwestern Mouse MRI Core, Advanced Imaging Research Center, using a 7T small animal MRI scanner (Bruker, Rheinstetten, Germany) equipped with a 40 mm quadrature Radiofrequency (RF) coil (ExtendMR LLC, Milpitas, CA). Under anesthesia by inhalation of 1.5 - 3% isoflurane mixed in with medical-grade oxygen via nose-cone, the animals were placed supine on a mouse holder, with a pneumatic respiratory sensor and electrocardiography (ECG) electrodes for cardiac sensing, head first with the lung centered with respect to the center of the RF coil. The mice’s chests were shaved and conducting hydrogels were applied to optimize ECG contact between electrodes and mouse. All MRI acquisitions were gated using both cardiac and respiratory triggering. The bore temperature was kept at 23 ± 2° C. to assure adequate and constant heart rate. Two-dimensional (2D) scout images on three orthogonal planes (transverse, coronal and sagittal) were acquired to determine the positioning. Then, lower resolution gradient echo (T1_FLASH) images were acquired on transverse plane to fine-adjust the slice position. Finally, higher resolution gradient echo images were recorded on the transverse plane, with the major parameters as follows: The repetition time (TR) = 200 ms (Note: the actual TR is changing according to ECG R-R interval, in range of 200 ms to 240 ms), the echo time (TE) = 1.966 ms, the flip angle (FA) = 45°, the number of average = 12, the field of view (FOV) = 32 x 32 mm2, the matrix size = 256 x 256, the slice number = 17-21 (changed upon the mouse lung size), and the slice thickness = 1 mm without any gap. The image analyses were performed using ImageJ.
Before vs After treatment: Formalin fixed paraffin-embedded (FFPE) tissues from 23 NSCLC patients were obtained from The Jackson Laboratory or UT Southwestern according to IRB-approved protocols. Thirteen specimens were obtained from UT Southwestern and ten from The Jackson Laboratory. Thirteen patients had no EGFR TKI treatment, and ten patients had undergone EGFR TKI treatment.
To assess the effects of Interferons on Overall Survival we reviewed the medical records of NSCLC patients treated at UT Southwestern. 30 advanced (stages IIIB & IV) NSCLC patients harbored classical TKI-sensitive mutations, L858R or exon 19 deletion, but no T790M mutation at initial diagnosis, and all of them had TKI treatment history. Their FFPE tissues from initial diagnosis were collected for this study.
TCGA: Data were downloaded from https://portal.gdc.cancer.gov/. 42 TCGA-LUAD patients (any stages) with classical TKI-sensitive mutations, L858R or exon 19 deletion, but without T790M mutation, were achieved with their Copy Number Variation (CNV) and Survival data. 41/42 have RNAseq data.
All animal studies were done under IACUC-approved protocols at UT Southwestern and North Texas VA Medical Center (Dallas, Texas, USA). Patient tissues and medical records were obtained from UT Southwestern with IRB approval.
Error bars represent the means ± S.E.M. of 3 independent experiments unless indicated otherwise. The combination effects in vivo and in vitro were analyzed by two-way or three-way ANOVA with Bonferroni’s correction to adjust the significance level for multiple comparisons. One-way ANOVA with Dunnett’s test was used to determine adjusted p value for comparison between control and more than one treated sample. The familywise error rate (FWER) was set at 0.05. Kaplan-Meier survival curves were constructed and compared by log-rank test and Gehan’s test. The patient data comparison was shown as median ± IQR, analyzed by Kolmogorov-Smimov test. RNAseq data were analyzed by DESeq2 and GSEA. All other data were analyzed for significance between the indicated treated group and control group, with two-tailed two-sample Student’s t-test. All analyses above were performed using GraphPad Prism 8 software. A P value or an adjusted P value for multiple comparison less than 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; #: p>0.05, not statistically significant).
The Reproducibility information is provided in Reporting Summary, including sample size predetermination, randomization, blinding, and replication. There are no data exclusions.
RNA-seq data that support the findings of this study have been deposited in the Sequence Read Archive (SRA) under accession code PRJNA593064.
Mass spectrometry data have been deposited in ProteomeXchange with the primary accession code PXD016558.
The human lung adenocarcinoma data were derived from the TCGA Research Network: http://cancergenome.nih.gov/.
Uncropped western blot images and raw digital data have been proved in Source Data.
Information regarding codes used in this study have been proved in Reporting Summary. They are either commercially available or open-source.
To better understand the early adaptive response to EGFR inhibition, RNA sequencing was undertaken in the EGFRwt/KRas mutant A549 cells following exposure to erlotinib. The transcriptional response to EGFR inhibition is quite broad and affects a large number of genes (
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Next, it was found that siRNA knockdown of IFNAR1, the receptor for Type I interferons, resulted in enhanced sensitivity to EGFR inhibition in EGFR mutant NSCLC (
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Next, it was found that exogenous Type I IFNs protect from erlotinib-induced cell death in oncogene addicted cells in cell survival assays (
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Although the Type I interferons have a known role in cytotoxicity, they also have a pro-survival role and mediate resistance to radiation and chemotherapy, primarily through STAT1 activation. It was found that EGFR inhibition results in activation of STAT1 in multiple EGFR wild type and EGFR mutant NSCLC cell lines (
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The transcription factor IRF3 plays a central role in transcription of Type I interferons. IRF3 is activated by TBK1. EGFR inhibition led to a rapid and robust activation of TBK1 and IRF3 in NSCLC cell lines harboring EGFR-activating mutations (
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It was found that pharmacological inhibition of TBK1 with BX-795 or siRNA knockdown of TBK1 synergized with EGFR inhibition in multiple NSCLC cell lines with mutant EGFR in cell viability assays (
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To understand the mechanism of EGFR inhibition dependent TBK1 activation, proteins that associate with TBK1 in response to EGFR inhibition were analyzed using mass spectrometry (
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Retinoic acid inducible gene I (RIG-I) is a pattern sensing receptor that plays a key role in sensing RNA viruses. It was found that RIG-I is strongly induced by EGFR inhibition in multiple EGFR mutant NSCLC cell lines (
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cGAS/STING activates IRF3 leading to induction of Type I IFNs. STING is upregulated in EGFR TKI persister cells. However, increased phosphorylation of STING in response to erlotinib was unable to be detected in either EGFR mutant or EGFRwt cell lines (
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The aryl hydrocarbon receptor (Ahr) has been implicated as a mechanism of resistance to EGFR TKIs in EGFR mutant NSCLC. It was found that Ahr levels are not altered in response to erlotinib (
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Although EGFR inhibition leads to decreased levels of PD-L1 in EGFR mutant lung cancer cell lines, immunotherapy is not effective in EGFR mutant NSCLC. Since type I IFNs regulate PD-L1 expression, the PD-1 pathway was examine next, and it was found that erlotinib affects primarily PD-L1 levels (
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EGFR inhibition in EGFR mutant NSCLCs results in activation of a TBK1-IRF3 signaling axis. Also, pharmacological inhibition of TBK1 using BX-795 or siRNA knockdown of TBK1 or IRF3 completely suppressed erlotinib-induced upregulation of IFN in EGFR mutant cell lines (
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To investigate whether TNF-NF-κB signaling plays a role in the pro-survival effects of Type I IFNs in the context of EGFR inhibition, whether etanercept is able to inhibit the pro-survival effect of exogenous Type I IFNs was examined (
To examine the role of Type I IFN signaling in NSCLCs in secondary resistance to EGFR inhibition, NSCLC EGFR mutant cell lines rendered experimentally resistant to EGFR TKIs by two independent groups were examined. Four independent clones were analyzed. It was found that Type I IFN levels are high in all clones (
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Next, whether a combined inhibition of Type I IFNs and EGFR would influence sensitivity to erlotinib was examined in mouse xenograft models. The Type I interferon receptor IFNAR is composed of two subunits IFNAR1 and IFNAR2. HCC827 cells with stable silencing of IFNAR1 or control shRNA were generated and tested in cell viability assays followed by injection into the flanks of athymic mice to form subcutaneous tumors (
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Because the type I interferons play a key role in innate immunity, a well-established immunocompetent transgenic mouse model of KRas mutant lung cancer that is driven by Adeno-CMV-Cre-mediated induction of KRas G12D expression was used. These tumors express EGFRwt and previous studies have shown that ErbB receptors play an important role in driving the growth of KRas mutant tumors. Since anifrolumab is specifically directed against the human IFNAR, a Type IIFN neutralizing antibody directed against the mouse IFNAR was used. Once tumors were detected by magnetic resonance imaging (MRI) following adenovirus administration, treatment was started with control vehicle, erlotinib, IFN antibody, or erlotinib plus IFN antibody followed by periodic MRI imaging. While there is robust tumor growth in control, erlotinib alone, and IFN antibody treatment groups, a combination of erlotinib plus IFN antibody was highly effective in suppressing growth of these tumors resulting in significantly diminished tumor growth compared to other groups (
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Next, whether EGFR inhibition induces a Type I interferon upregulation in tumor tissue derived from patients was studied. 13 TKI naive and 9 TKI treated NSCLC patients were examined, and it was found that erlotinib treated patients had higher Type I IFN levels compared to TKI naive patients (
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It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This Application claims the benefit of U.S. Application No. 62/978,777, filed on Feb. 19, 2020, and U.S. Application No. 62/984,264, filed on Mar. 03, 2020, the contents of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/018718 | 2/19/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62978777 | Feb 2020 | US | |
| 62984624 | Mar 2020 | US |
