CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 62/826,626, filed March 29, 2019.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under grant number PC 190005 awarded by Department of Defense office of the Congressionally Directed Medical Research Programs (CDMRP). The United States government has certain rights in the invention.

Field of the Invention

This invention generally relates to a combination therapy for treating cancer. In particular, the combination therapy includes administration of at least one muscarinic receptor antagonist and at least one anti-cancer chemotherapeutic agent, and the cancers that are treated include chemotherapeutic resistant cancers.

BACKGROUND OF THE INVENTION

State of Technology

Prostate cancer (PCa) is a current epidemic that affects roughly 1 in 9 men. While treatment of early stages of this disease are very successful, a small percentage of patients have a recurrence of the disease that is much more aggressive. Current second line therapies include anti-androgens and chemotherapy yet eventually the disease becomes resistant to both therapies. Chemotherapy-resistant prostate cancer (ChRPC) remains a leading cause of cancer-related deaths in men as there are currently no therapeutics for PCa once it becomes resistant to chemotherapy.

One current line of thinking is that the tumor microenvironment plays a role in the progression of PCa. One molecule in the microenvironment that is released by peripheral neurons is the neurotransmitter acetylcholine (ACh), which activates nicotinic and muscarinic acetylcholine receptors. However, it is not known whether ACh has a role in protecting cells from chemotherapy-induced cell death and chemotherapy resistance.

SUMMARY OF THE EMBODIMENTS

Methods of treating cancer are disclosed herein. Generally, the methods involve administering a combination therapy to a subject in need thereof, the combination therapy including at least one muscarinic receptor antagonist and at least one chemotherapeutic agent. In some aspects, the cancer that is treated is recurrent and/or chemotherapy resistant cancer, and in further aspects, administration of the at least one muscarinic receptor antagonist resensitizes chemotherapy resistant cancer cells so that they are susceptible to killing by the at least one chemotherapeutic agent. In further aspects, the cancer is prostate cancer, e.g. recurrent and/or drug resistant prostate cancer that recurs after initial treatment, even when the initial treatment was or appears to have been successful. In other aspects, the cancer is lung cancer e.g. recurrent and/or drug resistant lung cancer that recurs after initial treatment e.g. recurrent and/or drug resistant lung cancer.

It is an object of the invention to provide a method for treating cancer in a subject in need thereof, comprising: administering to the subject a therapeutically effective dose of i) at least one agent that inhibits activity of at least one muscarinic ACh receptor; and ii) at least one anti-cancer chemotherapeutic agent. In some aspects, the at least one agent that inhibits activity of at least one muscarinic ACh receptor is an antagonist of the at least one muscarinic ACh receptor, or an agent that blocks expression of the at least one muscarinic ACh receptor. In additional aspects, the cancer is a chemotherapy resistant cancer and/or a recurrent cancer. In further aspects, the cancer is selected from the group consisting of: prostate cancer, lung cancer, pancreatic cancer, melanoma, breast cancer, bronchial cancer, colorectal cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or cancer of the pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, and osteosarcoma. In other aspects, the step of administering is performed orally, intravenously, intramuscularly, or by a combination of these. In yet further aspects, the at least one agent that inhibits activity of the at least one muscarinic ACh receptor and the at least one anti-cancer chemotherapeutic agent are administered as a single composition, or in separate compositions. In further aspects, the step of administering comprises: administering at least one first anti cancer chemotherapeutic agent, then administering at least one agent that inhibits activity of at least one muscarinic ACh receptor, and then administering at least one at least one second anti cancer chemotherapeutic agent, wherein the at least one first anti-cancer chemotherapeutic agent and the at least one at least one second anti-cancer chemotherapeutic agent are the same or different. In yet further aspects, the antagonist of the at least one muscarinic ACh receptor comprises at least one of: telenzepine, atropine, scopolamine, hydroxyzine, ipratropium, topicamide, pirenzepine, diphenhydramine, doxylamine, dimenhydrinate, dicyclomine, docet, flavoxate, oxybutynin, tiotropium, cycloopentolate, atropine methonitrate, trihexyphenidy, tolterodine, solifenacin, darifanacin, benztropine, mebeverine, procyclidine, and aclidinium bromide. In additional aspects, the at least one agent that blocks expression of the at least one muscarinic ACh receptor is siRNA.

The invention further provides a method of killing a cancer cell, comprising contacting the cancer cell with i) at least one agent that inhibits activity of at least one muscarinic ACh receptor; and ii) at least one anti-cancer chemotherapeutic agent. In certain aspects, the at least one anti-cancer chemotherapeutic agent is docetaxel. In additional aspects, the cancer cell is a prostate cancer cell or a lung cancer cell. In further aspects, the at least one agent that inhibits activity of the at least one muscarinic ACh receptor is i) an agent that blocks expression of the at least one muscarinic ACh receptor; or ii) an antagonist of the at least one muscarinic ACh receptor. In yet further aspects, the agent that blocks expression of the at least one muscarinic ACh receptor is shRNA. In other aspects, the antagonist of the at least one muscarinic ACh receptor is dicyclomine or darifenacin.

The invention also provides a composition comprising at least one muscarinic receptor antagonist and at least one anti-cancer chemotherapeutic agent. In certain aspects, the composition is formulated as a tablet, caplet, gel or capsule. In other aspects, the composition is formulated as a fluid that is iv administrable or orally administrable. In further aspects, the at least one muscarinic receptor antagonist and the at least one anti-cancer chemotherapeutic agent are dissolved in or suspended in a carrier fluid. In additional aspects, the composition is formulated as a topical formulation wherein the at least one muscarinic receptor antagonist and the at least one anti-cancer chemotherapeutic agent are suspended in or distributed in a carrier fluid or lotion.

The foregoing and other objects, features, and advantages of the present disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Figure 1A-E. Docetaxel increases ACh secretion in PCa cells. A-B, Gene expression of ACh synthesis and secretion enzymes measured by RT-qPCR in PCa cells treated with DTX (1 nM, A) and in DTX -resistant cells (22RV1 ^r , DU145 ^r , B) normalized to GAPDH expression (n = 3). C-D, ACh secretion in PCa cells treated with DTX (1 nM, 48 hrs, C) and in DTX-resistant cells (D) measured by with the QuickDetect ACh ELISA Kit (n = 3). E, Representative Western Blot of 22Rvl cells pretreated with carbachol (CCh, 10 mM, 24 hrs) before addition of DTX (3 nM, 48 hrs) (n = 3). All data is represented as the average ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2A-M. Antimuscarinics decrease cell survival in chemosensitive and

chemoresistant PCa and NSCLC cells when combined with chemotherapy. A, CHRM1 and CHRM3 gene expression measured by RT-qPCR in 22Rvl cells treated with DTX (1 nM) and normalized to GAPDH expression (n = 3). B, Representative Western Blot of docetaxel- sensitive and -resistant cell lines (n = 3). C, CHRM1 and CHRM3 gene expression measured by RT-qPCR in A549 and A549 ^R cells and normalized to GAPDH expression (n = 3). D-E,

Inhibition of CHRM1 signaling in 22Rvl cells with Die (D) or siRNA targeting CHRM1 (E) in combination with DTX decreases cell survival compared to DTX alone (n = 4). F, Inhibition of CHRM1 signaling in 22Rvl cells with Die in combination with DTX decreases colony formation compared to DTX alone (n = 3). G-H, Inhibition of CHRM3 signaling in DU 145 cells with Dari (G) or siRNA targeting CHRM3 (H) in combination with DTX decreases cell survival compared to DTX alone (n = 4). I, Inhibition of CHRM3 signaling in DU145 cells with Dari in combination with DTX decreases colony formation compared to DTX alone (n = 3). J- L, Inhibition of CHRM1 or CHRM3 with Die or Dari in DU145 ^R (J), A549 (K), A549 ^R (L) cells in combination with DTX decreases cell survival compared to DTX alone (n = 4). M, Inhibition of CHRM1 or CHRM3 with Die or Dari in A549 ^R cells in combination with DTX decreases colony formation compared to DTX alone (n = 3).

Figure 3A-E. Antimuscarinics increase apoptosis in chemosensitive and chemoresistant PCa and NSCLC cells when combined with chemotherapy. A-C, Representative fluorescent images of a TUNEL assay of 22Rvl (A), DU145 ^R (B), and A549 ^R (C) cells pretreated with Die or Dari (24 hrs) before addition of DTX (48 hrs). Hoechst nuclear stain was used to mark each cell. Quantification of TUNEL-positive cells are to the right of the images (n = 3). D-E,

Representative Western Blot of PCa cells pretreated with Die (24 hrs, D) or Dari (24 hrs, E) before addition of DTX (48 hrs) (n = 3). All data is represented as the average ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. White bar = 200 pm

Figure 4A and B. Antimuscarinics decrease chemoresistant tumor growth when combined with chemotherapy. A, Tumor volume of 22RV1 ^R cell xenografts in SCID mice. Mice were treated with the following: Vehicle (0.9% saline, i.p., daily), Die (8mg/kg, i.p., daily), DTX (lOmg/kg, i.p, once a week), or a combination of Die and DTX. B, Tumors were removed and weighed at the end of treatment (Vehicle, n = 3; Die, n = 6; DTX, n = 6; DTX + Die, n = 8). All data is represented as the average ± SEM, * p < 0.05

Figure 5A and B. Antimuscarinics decrease cell survival in chemoresistant PCa and NSCLC cells when combined with the third generation taxane cabazitaxel. A-B, Inhibition of CHRM1 or CHRM3 with Die or Dari in combination with CTX in 22RV1 ^R (A), and A549 ^R (B) cells decreases cell survival compared to CTX alone (n = 4). All data is represented as the average ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001 Figure 6. Combined inhibition of CHRM1 and CHRM3 simultaneously has synergistic effects on cell survival when combined with chemotherapy. HSA synergy plot of various combinations of Die and Dari in combination with DTX. Positive numbers represent synergy while negative numbers represent antagonism between the 2 agents. All data is represented as the average ± SEM, * p < 0.05; ** p < 0.01; *** p < 0.001

DETAILED DESCRIPTION

Disclosed herein are methods for treating cancers, including recurrent and/or multi drug resistant cancers, in a subject in need thereof. The methods generally involve administering to the subject a combination of at least one anti-cancer chemotherapeutic agent and at least one antagonist of certain receptors and/or at least one inhibitor of the activity of certain receptors. One result is that the chemotherapy agent then advantageously and unexpectedly works against cells that it would not have worked against in the absence of the coordinated administration of the at least one receptor antagonist and/or at least one inhibitor of receptor activity. In some aspects, the receptors are muscarinic acetylcholine (ACh) receptors and co-administration with at least one muscarinic ACh receptor antagonist or with an inhibitor of the activity of one or more muscarinic ACh receptors (e.g. by inhibiting receptor expression) advantageously decreases the IC50 of at least one chemotherapeutic agent and/or resensitizes drug resistant cancer (tumor) cells to killing. When two or more muscarinic ACh receptor antagonists and/or inhibitors of the activity of the one or more muscarinic ACh receptors, such as two or more inhibitors of expression of the receptor, are administered, the result is a synergistic response by the cancer cell so that the ability of the at least one chemotherapeutic agent to kill the cancer cell is even further augmented. A combination of two muscarinic ACh receptor antagonists provides at least about a 2-fold increase in chemotherapy resensitization compared to the increase in resentitization that would be expected if the effects of the antagonists were merely additive. In other words, expressed as a percentage, the synergistic increase when two antagonists are administered together is at least about 50, 75, 100, 150 or 200%, including all multiples of 10 in between such as at least about 50%, 60%, 70%...up to about 180%, 190% or 200%.

DEFINITIONS Antagonist: a substance that interferes with or inhibits the physiological action of another. A receptor antagonist is a type of receptor ligand or drug that blocks or dampens a biological response by binding to and decreasing the response of a receptor rather than activating it like an agonist. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex that is formed when the antagonist binds to the receptor and the nature of antagonist-receptor binding. Antagonists frequently achieve their potency by competing with endogenous ligands or substrates. Receptor antagonists are sometimes called“blockers”. Antagonists may be inverse agonists, partial agonists, silent antagonists, uncompetitive, non-competitive or competitive antagonists.

Muscarinic acetylcholine receptors, or mAChRs, are acetylcholine receptors that normally form G protein-coupled receptor complexes in the cell membranes of certain neurons and other cells and are expressed by cancer cells. They play several roles, including acting as the main end-receptor stimulated by acetylcholine released from postganglionic fibers in the parasympathetic nervous system. Five families of muscarinic ACh receptors are known: Ml (CHRM1), M2 (CHRM2), M3 (CHRM3), M4 (CHRM4) and M5 (CHRM5). These receptors are also known as cholinergic receptors.

As used herein, an inhibitor of the activity of a receptor is any substance that decreases or eliminates the biological effects of the normal functioning of the receptor. The inhibition may be direct (e.g. by the binding of an antagonist to the receptor) or indirect (e.g. by decreasing or eliminating expression of the receptor).

The term "cancer" refers to the growth or proliferation of a malignant neoplasm or cell. Cancer cells often form a tumor, but such cells may be a non-tumorigenic cancer cells in a subject, specimen, or sample.

“Synergy” refers to the interaction or cooperation of two or more substances or agents to produce a combined effect greater than the sum of their separate effects.

TYPES OF CANCER THAT ARE TREATED

Generally, the cancer that is treated is typically a caner that expresses or overexpresses one or more muscarinic receptors. In some aspects, cancer is or includes, but is not limited to, at least one of: various tumors, prostate cancer, pancreatic cancer, melanoma, breast cancer, lung cancer, bronchial cancer, colorectal cancer, stomach cancer, ovarian cancer, bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, oral or cancer of the pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small intestine or appendix cancer, salivary gland cancer, thyroid cancer, adrenal gland cancer, and osteosarcoma. In some aspects, the cancer that is treated is: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers, Kaposi Sarcoma, AIDS-Related Lymphoma, Primary CNS Lymphoma, Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Central Nervous System, Basal Cell Carcinoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Brain Tumor (e.g. Astrocytomas, Brain and Spinal Cord Tumors, Brain Stem Glioma, Central Nervous System Atypical Teratoid/Rhabdoid Tumor, Central Nervous System Embryonal Tumors, Central Nervous System Germ Cell Tumors, Craniopharyngioma, Ependymoma), Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Carcinoid Tumor, Gastrointestinal, Cardiac (Heart) Tumors, Central Nervous System (e.g. Atypical Teratoid/Rhabdoid Tumors, Embryonal Tumors, Germ Cell Tumors, Lymphomas), Cervical Cancer, Childhood Cancers, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic

Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma,

Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Embryonal Tumors,

Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing

Sarcoma, Extracranial Germ Cell Tumo, Extragonadal Germ Cell, Tumor, Eye Cancer,

Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Hairy Cell Leukemia, Head and Neck Cancer, Heart Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphom, Hypopharyngeal Cancer, Intraocular Melanoma, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney (Renal Cell, Wilms Tumor and Other Childhood Kidney Tumors), Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia (Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell), Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lung Cancer (Non-Small Cell, Small Cell), Lymphoma, Macroglobulinemia, Waldenstrom - see Non-Hodgkin Lymphoma, Malignant Fibrous Histiocytoma of Bone and Osteosarcoma, Melanoma, Intraocular (Eye), Merkel Cell Carcinoma, Mesothelioma, Malignant, Metastatic Squamous Neck Cancer with Occult PrimaryMouth Cancer, Multiple Endocrine Neoplasia Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (AML), Myeloma, Multiple, Myeloproliferative Neoplasms, Chronic, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Epithelial, Germ Cell Tumor, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (Ewing, Kaposi, Osteosarcoma,

Rhabdomyosarcoma, Soft Tissue, Uterine), Sezary Syndrome, Skin Cancer, Small Intestine Cancer, Squamous Cell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic Stomach (Gastric) Cancer, T-Cell Lymphoma, Cutaneous, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Vaginal Cancer, Vulvar Cancer, Wilms Tumor.

COMBINATION THERAPY

The term“combination therapy” refers to therapies which comprise administering to a subject at least one agent together with at least one additional (different) agent. The two may literally be administered together in a single composition, or separately as part of the same overall treatment regimen. As such, the posology of each of the two or more compositions or agents may differ, each potentially being administered at the same time or at different times. It will therefore be appreciated by those skilled in the art that the mixture may be administered sequentially or simultaneously, either in the same formulation, or in different formulations. The composition and the agent may be formulated for separate administration or may be formulated for administration together. The route of administration may also differ with those changes being appreciated by one skilled in the art.

Accordingly, combination therapy as used herein refers to administering agents belonging to at least two different classifications/categories (i.e. the agents are of at least two different types) to a subject in need thereof. In some aspects, the different types of agents are administered together in a single composition, i.e. a single composition contains both types of agents. Alternatively, in other aspects, the different types of agents are administered to the subject within overlapping time periods, e.g. one is administered first and then the other is administered afterwards. The period of time between administering the first type of agent and administrating the second type of agent can be very short (e.g. as soon as is practical, such as several seconds or a few minutes, e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes), or the time period may be longer (e.g. hours such as from about 1 to about 24 hours), or days (e.g. 1, 2, 3, 4, 5, 6, or 7 days apart), or even weeks or months apart. In some aspects, a first agent is administered to“load” the subject with that agent, and/or to achieve a desired result such as a desired level in the blood or plasma , or a desired effect (e.g. to give cancer cells time to be contacted by and/or absorb and react to the agent, such as to become sensitized or resensitized to chemotherapy).

In some aspects, one category of agents that is administered is muscarinic ACh receptor antagonists and/or inhibitors of muscarinic ACh receptor expression, and another category is anti-cancer chemotherapeutic agents.

COMPOSITIONS

The agents described herein are generally delivered (administered) as a pharmaceutical composition, either separately (i.e. in separate compositions) or together in the same

composition. Such pharmaceutical compositions generally comprise at least one of each type or class of agent, and may comprise more than one, i.e. a plurality of different agents from the same or different categories. For example, 1 or 2 or more such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more agents can be included in a single formulation. Accordingly, the present invention encompasses such formulations/compositions. The compositions generally include one or more substantially purified agents as described herein, and a pharmacologically suitable (physiologically compatible) carrier or carrier system. The term "carrier system" (including variations thereof such as the various specific injectable and infusible dosage forms) refers to compositions comprising one or more pharmaceutically suitable excipients, such as solvents like water and co-solvents, solubilizing compounds, wetting compounds, suspending compounds, thickening compounds, emulsifying compounds, chelating compounds, buffers, pH adjusters, antioxidants, reducing compounds, antimicrobial preservatives, bulking compounds, protectants, tonicity adjusters, and special additives, as described further below. The compositions may be timed release compositions and or compositions that are administered in vehicles that target the composition to an area of interest, such as a cancerous tumor. The administration of prodrug forms of the active agents is also encompassed.

In some aspects, such compositions are prepared as liquid solutions or suspensions, or as solid forms such as tablets, pills, powders and the like. Solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g. lyophilized forms of the compounds), as are emulsified preparations. In some aspects, the liquid formulations are aqueous or oil-based suspensions or solutions. In some aspects, the active ingredients are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g. pharmaceutically acceptable excipients and/or salts. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. If it is desired to administer an oral form of the composition, various thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders and the like are added. The composition of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration. The final amount of an agent in the formulations varies but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton). Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as twin 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene- polyoxypropylene-block polymers, methylcellulose, hydroxypropyl methylcellulose, wool fat, sugars such as lactose, glucose and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil and soybean oil;

glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

"Pharmaceutically acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts, and base addition salts, of compounds of the present invention.

These salts can be prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene- bis-.beta.-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfo nates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., "Pharmaceutical Salts,"

J. Pharm. ScL, 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include the sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. The sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia,

ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'- dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium

hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and

dicyclohexylamine, and the like.

The term“chemotherapeutic agent” refers to an agent or a composition comprising the agent that prevents or inhibits cell viability or causes cellular death. An anti-cancer or anti neoplastic agent prevents or inhibits cell viability and/or causes cellular death of cancer cells but may also prevent or inhibit cell viability and/or cause cellular death of normal, non-neoplastic cells, e.g. as a side effect of cancer treatment.

The anti-cancer chemotherapeutic agents that are present in a compositions disclosed herein include but are not limited to one or more of: busulfan, improsulfan, piposulfan, benzodepa, carboquone, meturedepa, uredepa, altretamine, triethylenemelamine,

triethylenephosphoramide, triethylenethiophosphoramide, trimethylolomelamine, chlorambucil, chlomaphazine, cyclophosphamide, estramustine, ifosfamide, mechlorethamine,

mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,

aclacinomycins, actinomycin F(l), anthramycin, azaserine, bleomycin, cactinomycin, carubicin, carzinophilin, chromomycin, dactinomycin, daunorubicin, docetaxel, cabazitaxel, paclitaxel, daunomycin, 6-diazo-5-oxo-l-norleucine, doxorubicin, epirubicin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, plicamycin, porfiromycin, puromycin,

streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, denopterin,

methotrexate, pteropterin, trimetrexate, fludarabine, 6-mercaptopurine, thiamiprine,

thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, fluororacil, tegafur, L-asparaginase, pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestrabucil, bisantrene, carboplatin, cisplatin, defofamide, demecolcine, diaziquone, elfomithine, elliptinium acetate, etoglucid, etoposide, flutamide, gallium nitrate, hydroxyurea, interferon-alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin, podophyllinic acid, 2- ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel, tamoxifen, teniposide, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, urethan, vinblastine, vincristine, and vindesine and other anti-neoplastic agents known to those skilled in the art.

At least one muscarinic ACh receptor antagonist is administered as part of the combination therapy. In some aspects, the muscarinic ACh receptor antagonists that are administered include but are not limited to at least one of the following pharmacological agents: Telenzepine, Atropine, Scopolamine, Hydroxyzine, Ipratropium, Topicamide, Pirenzepine, Diphenhydramine, Doxylamine, Dimenhydrinate, Dicyclomine, Docet Flavoxate, Oxybutynin, Tiotropium, Cycloopentolate, Atropine Methonitrate, Trihexyphenidy, Tolterodine, Solifenacin, Darifanacin, Benztropine, Mebeverine, Procyclidine, and Aclidinium Bromide. In particular, antagonists of the Ml family of ACh receptors include but are not limited to: atropine, hyoscyamine, scopolamine, diphenhydramine, dimenhydrinate, dicycloverine, thorazine, tolterodine, oxybutynin, ipratropium, mamba toxin MT7, mamba toxin MT1, mamba toxin MT2, pirenzepine, telenzepine, chlorpromazine and haloperidol; antagonists of the M2 family of ACh receptors include but are not limited to: atropine, hyoscyamine, dicycloverine, thorazine, diphenhydramine, dimenhydrinate, tolterodine, oxybutynin, ipratropium, methoctramine, tripitramine, gallamine and chlorpromazine; antagonists of the M3 family of ACh receptors include but are not limited to: atropine, hyoscyamine, siphenhydramine, simenhydrinate, dicycloverine, tolterodine, oxybutynin, ipratropium, darifenacin and

tiotropium; antagonists of the M4 family of ACh receptors include but are not limited to:

atropine, diphenhydramine, dimenhydrinate, dicycloverine, tolterodine, oxybutynin,

ipratropium; and antagonists of the M5 family of ACh receptors include but are not limited to: atropine, diphenhydramine, dimenhydrinate, dicycloverine, tolterodine, oxybutynin and ipratropium mamba toxin MT1, mamba toxin MT2 and mamba toxin MT3.

In other aspects, the muscarinic ACh receptor antagonist is an antagonist antibody. Such antibodies may be polyclonal or monoclonal but are typically monoclonal. The terms "antibody" and "immunoglobulin" or "Ig" are used interchangeably herein, and are intended to include a polypeptide product of B cells within the immunoglobulin class of polypeptides that is able to bind to a specific molecular antigen and is composed of two identical pairs of polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa) and each amino-terminal portion of each chain includes a variable region of about 100 to about 130 or more amino acids and each carboxy-terminal portion of each chain includes a constant region (See, Borrebaeck (ed.) (1995) Antibody Engineering, Second Ed., Oxford University Press.; Kuby (1997) Immunology, Third Ed., W.H. Freeman and Company, New York). In specific embodiments, the specific molecular antigen can be bound by an antibody provided herein includes the target muscarinic ACh receptor, or a fragment or epitope thereof.

Antibodies also include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, multispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, intrabodies, anti-idiotypic (anti-id) antibodies, and functional fragments of any of the above, which refers a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment was derived. Non limiting examples of functional fragments include single-chain Fvs (scFv) (e.g., including monospecific, bispecific, etc.), Fab fragments, F(ab') fragments, F(ab)2 fragments, F(ab')2 fragments, disulfide-linked Fvs (sdFv), Fd fragments, Fv fragments, diabody, triabody, tetrabody and minibody. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, e.g., antigen binding regions/domains or molecules that contain an antigen-binding site that binds to a CD39 antigen (e.g., one or more complementarity determining regions (CDRs) of an anti-CD39 antibody). Such antibody fragments can be found described in, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989); Myers (ed.), Molec. Biology and Biotechnology: A Comprehensive Desk Reference, New York: VCH Publisher, Inc.; Huston et ah, Cell Biophysics, 22:189-224 (1993); Pluckthun and Skerra, Meth. Enzymok, 178:497-515 (1989) and in Day, E. D., Advanced Immunochemistry, Second Ed., Wiley-Liss, Inc., New York, N.Y. (1990). The antibodies provided herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. An anti-CD39 antibodies provided herein can be agonistic antibodies or antagonistic antibodies.

METHODS AND ADMINISTRATION

The disclosure provides methods of treating cancer in a subject in need thereof. The methods generally involve a step of identifying a suitable patient population for treatment. A member of the patient population is generally a subject with cancer. The cancer may be a primary cancer, a secondary (tertiary, etc.) metastatic cancer, or a recurrent cancer i.e. a cancer that has recurred, usually after initial treatment and after a period of time during which the cancer could not be detected. The cancer may come back to the same place as the original (primary) tumor or to another place in the body. In some aspects, the recurrent cancer is a drug resistant cancer.“Antineoplastic resistance”, often used interchangeably with“chemotherapy resistance”, is the resistance of neoplastic (cancerous) cells to damage or killing by a chemotherapeutic agent, and/or the ability of cancer cells to survive and grow despite anti cancer therapies. Such resistance may evolve in response to the administration of the chemotherapeutic agent. In some cases, cancers evolve resistance to multiple drugs, referred to as multiple (or multiply) drug resistance (MDR) cancers.

In addition, prior to beginning treatment, the cancer, e.g. a tumor, or tumor cells from the cancer, may be tested to determine that the cancer expresses or overexpresses at least one muscarinic Ach receptor that is susceptible to inhibition using the agents described herein; and/or to determine the level of expression of the one or more muscarinic Ach receptors as this information may guide the skilled practitioner, e.g. a physician, to determine how much of a particular agent or agents should be administered, i.e. the receptors can act as biomarkers and be used to determine which treatments to use. Generally, the subject is a mammal, such as a human. However, veterinary applications of this technology are also encompassed, e.g. the treatment of companion pets such as dogs and cats, etc. and or other animals, e.g. working animals, such as cattle, horses, etc.

“Administering” or“administration” refers to any method of delivering a pharmaceutical composition or therapeutic agent to a subject, such as delivery to a specific region on the body, or systemically into the body of the subject or a system of the subject, such as the circulatory system. The compositions disclosed herein are administered in vivo by any suitable route including but not limited to: injection and/or infusion (e.g. intravenous, intraperitoneal, intramuscular, subcutaneous, intra-aural, intraarticular, intramammary, intratumoral, and the like), topical application (e.g. on areas such as eyes, skin, in ears, etc.) and by absorption through epithelial or mucocutaneous linings (e.g., nasal, oral, vaginal, rectal, gastrointestinal mucosa, and the like). Other suitable means include but are not limited to: inhalation (e.g. as a mist or spray), oral (e.g. as a pill, capsule, liquid, etc.), intravaginal, intranasal, rectal, as eye drops, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal, etc. without limitation. In some aspects, the mode of administration is oral or by injection, e.g. intravenously or intratumorally. In further aspects, administration of the therapy is systemic, enteral, peripheral, or a combination of two or more of these.

Generally, a therapeutically effective dose of an agent, combination of agents or a composition as described herein is administered. The term "therapeutically effective dose" (and variations thereof) refers to an amount, dose, or dosing regimen of a compound (i.e., active pharmaceutical ingredient, prodrug or precursor thereof), that upon interaction with a biological material is sufficient to treat at least one symptom of the disease or condition that is being treated. The terms "treating," "treatment," or "therapy" of a disease or disorder means slowing, stopping, or reversing progression of the disease or disorder, as evidenced by a reduction or elimination of one or more clinical or diagnostic symptoms, using the compositions and methods of the present invention as described herein.

The dose that is administered generally varies depending on a variety of factors e.g. on the form of the compound, the type of cancer and/or the severity thereof, the route of administration, and/or various patient-related factors such as age, weight, overall health and health history, gender, and the like.

Therapeutically effective amounts or doses of the anti-cancer therapeutic agents disclosed herein are generally known in the art. However, it should be noted that, as shown in the Examples section below, co-administration of an anti-cancer therapeutic agent and a muscarinic receptor antagonist advantageously results in a decrease in the IC50 of the anti cancer agent. Thus, the doses that are used can be lowered and the desired result can still be achieved. In some aspects, an individual single dose is lowered by e.g. about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or even 75% of the usual dose, i.e. about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35 or 30% of the usual dose is administered, compared to a prior art dose that is not given with one or more muscarinic receptor antagonists. Alternatively, a typical dose may be administered but administration may be less frequent. For example, doses that were administered 1-4 times per day may be administered daily, doses that were administered daily may be administered every 2, 3, 4, 5, 6, or 7 days, and/or regimens that were administered for e.g. 5 days of the month may be administered during only 2-3 days, etc., without decreasing the effectiveness of the treatment, e.g. the treatment of a primary tumor. This is highly advantageous since unwanted side effects of the anti-cancer agent are eliminated or at least lessened for the patient.

Similarly, therapeutically effective doses of the muscarinic receptor antagonists disclosed herein are also generally known in the art. Many of these agents are used medically for other purposes, e.g. diphenhydramine is an antihistamine, scopolamine is used to prevent nausea, tolterodine is used to treat overactive bladder, and dicyclomine and darifenacine are currently used in human patients for treatment of irritable bowel syndrome and overactive bladder symptoms, respectively. Thus, safe effective dosing amounts and regimens are known. The repurposing of these drugs for treatment of cancer is thus highly advantageous. In addition, the compositions may be administered in conjunction with other treatment modalities that are used to treat cancer, such as anti-nausea agents, appetite stimulants, substances that boost the immune system, various chemotherapeutic agents, antibodies that attack cancer cells, immune checkpoint inhibitors, surgery, radiation therapy, stem cell transplants, and the like.

PREVENTION OF DRUG RESISTANCE AND/OR RESENSITIZATION OF DRUG- RESISTANT CANCER CEUUS

As described above, in some aspects, a primary cancer is treated using the combination therapies described herein, the goal and result being to kill the primary cancer cells. In further aspects, a primary cancer is treated using the combination therapy and the goal and result is that recurrence of the cancer and/or the development of drug-resistant cancer is prevented, or at least slowed so that the period of time of remission is extended. The disclosure thus also provides methods of preventing the development of recurrent and/or drug resistant cancer (and thus extending remission and/or lengthening the survival time of the subject), by treating a primary cancer that is not recurrent cancer and is not yet drug resistant cancer with the disclosed combinations.

In additional aspects, a recurrent cancer is treated, and the recurrent cancer may not be or may be a drug-resistant cancer. Accordingly, provided herein are methods of resensitizing chemotherapy-resistant cancer cells to killing (e.g. by apoptosis) with an anti-cancer

chemotherapeutic agent. For example, the examples below show that pretreating chemotherapy resistant human PCa cells with antagonists of CHRM1 or CHRM3 receptors before treatment with DTX resensitized the cancer cells to DTX. Specifically, the CHRM1 antagonist dicyclomine and the CHRM3 antagonist darifenacin resensitized the drug-resistant cancer cells to killing by the anti-cancer agent DTX. In addition, pretreating chemotherapy-resistant human drug-resistant lung cancer cells with dicyclomine and/or darifenacin re-sensitized the cancer cells to DTX.

In some aspects of the present method, the scenario for treatment includes the following: at least one chemotherapy agent (e.g. a first chemotherapy agent) is administered to a subject, and it is that first agent that causes cancer cells in the subject to become chemotherapy resistant. Once it is determined that the cancer cells have become resistant to the first agent, one or more ACh receptor inhibitors is/are administered. Administration of the one or more ACh receptor inhibitors resensitizes the subject’s cancer cells to the first chemotherapy agent. Thereafter, the one or more ACh receptor inhibitors may be administered with the chemotherapeutic agent (e.g. to prevent further resistance) or a medical practitioner may choose to wait until signs of resistance are again present before administering the one or more ACh receptor inhibitors. This process may be repeated as need be whenever cancer cells become resistant to one or more chemotherapeutic agents, and/or when cancer recurs.

DECREASING EXPRESSION OF THE MUSCARINIC ACH RECEPTOR

Other methods of inhibiting the activity of muscarinic ACh receptors so that anti-cancer chemotherapeutic agents may kill cancer cells, and/or so that the cancer cells can be resensitized to the anti-cancer agents, include decreasing or blocking, either partially or completely, the expression of the receptors in tumor cells. Examples of treatment that can be used to decrease expression of receptors include but are not limited to genetic manipulations e.g. by the use of inhibitory nucleic acids, as described in issued US patent 10,550,186, the complete contents of which is herein incorporated by referenced in entirety.

Inhibitory nucleic acids useful in the present methods and compositions include but are not limited to: antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, siRNA compounds, single- or double- stranded RNA interference (RNAi) compounds such as siRNA compounds, modified bases/locked nucleic acids (LNAs), antagomirs, peptide nucleic acids (PNAs), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of the target nucleic acid, i.e., a nucleic acid encoding the targeted receptor, and modulate its function. In some embodiments, the inhibitory nucleic acids include antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interference RNA (RNAi), short interfering RNA (siRNA); a micro, interfering RNA (miRNA); a small, temporal RNA

(stRNA); or a short, hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNAs (saRNAs), or combinations thereof, including chimeric oligonucleotides and modified forms thereof, e.g. forms that are resistant to nuclease digestion, forms with substituted sugar moieties, etc. For example, the examples below show that genetic manipulation of CHRM1 expression with siRNA knockdown enhanced DTX-induced cell death in drug-resistant prostate cancer cells.

KITS

The term“pharmaceutical kit” refers to an array of one or more-unit doses of a pharmaceutical composition or a plurality of pharmaceutical compositions together with dosing means and/or delivery means, all contained within common packaging. The pharmaceutical kit may optionally further comprise instructions for use. For example, kits of the present disclosure may comprise both one or more anti-neoplasitc agents and one or more muscarinic receptor antagonists, e.g. together in a single container such as a vial, ampule preloaded syringe, etc., or in separate containers.

It should be emphasized that the above-described embodiments and following specific examples of the present invention, particularly, any "preferred" embodiments, are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the invention. Many variations and modifications may be made to the above- described embodiment(s) of the invention without departing substantially from the spirit and principles of the invention. All such modifications and variations are intended to be included herein within the scope of this disclosure and the present invention and protected by the following claims. As used in this specification and the appended claims, the singular forms“a,” “an,” and“the” include plural referents unless the content clearly dictates otherwise.

The use of the word“a” or“an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of “one or more,”“at least one,” and“one or more than one.”

Throughout this application, the term“about” is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The use of the term“or” in the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and“and/or.”

As used in this specification and claim(s), the words“comprising” (and any form of comprising, such as“comprise” and“comprises”),“having” (and any form of having, such as “have” and“has”),“including” (and any form of including, such as“includes” and“include”), or“containing” (and any form of containing, such as“contains” and“contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

In addition, unless otherwise indicated, numbers expressing quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

In addition, unless otherwise indicated, numbers expressing quantities of ingredients, constituents, reaction conditions, and so forth used in the specification and claims are to be understood as being modified by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter presented herein. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the subject matter presented herein are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated. Additionally, it will be understood that any list of such candidates or alternatives is merely illustrative, and not limiting, unless implicitly or explicitly understood or stated otherwise.

EXAMPLES

EXAMPLE E Investigations with prostate cancer and lung cancer cells

Docetaxel increases ACh secretion in PCa cells

ACh is known to play a role in the PCa tumor microenvironment, but its involvement in the development and maintenance of chemotherapy resistance, if any, is not well understood.

To elucidate the role of ACh, we first measured the expression of key ACh synthesis and secretion enzymes with DTX treatment. Treating the chemotherapy- sensitive human prostate carcinoma cell lines 22Rvl and DU145 with a low dose of DTX (1 nM) over 48 hrs increases the expression of the ACh synthesizing enzyme choline acetyltransferase (CHAT), the enzyme responsible for packaging ACh into vesicles, vesicular acetylcholine transporter (VAChT), and the transporter responsible for reuptake of choline back into the cell, choline transporter 1 (CHT1) as seen in Figure 1A. This increase in ACh related enzyme gene expression is maintained in DTX-resistant cells as seen in Figure 2B . This increase correlates with an increase in ACh secretion as shown in Figure 1C-D. Stimulation with the ACh memetic carbachol (CCh) protects PCa cells from DTX-induced apoptosis as seen with decreased levels of the pro-apoptotic proteins cleaved Poly(ADP-Ribose) Polymerase 1 (PARP1), and cleaved caspase 3 as shown in Figure IE.

Antimuscarinics combined with docetaxel decrease cell survival

Next, to determine the ACh receptor involved in DTX-resistant we measured expression of the muscarinic ACh receptors (CHRMs). PCa cells treated with a low dose of DTX (1 nM) over 48 hrs increases the expression of CHRM1 and CHRM3 which is maintained in DTX- resistance and also occurs in DTX-resistant non-small cell lung cancer (NSCLC) cells (A549 ^R ) as seen in Figure 2A-C. To determine the role of CHRM1 in DTX sensitivity in PCa, we treated DTX-sensitive PCa cells with the CHRM1 antagonist dicyclomine (Die) or knockdown of the receptor with siRNA. Both treatments decreased cell survival and colony formation when challenged with DTX compared to DTX alone as seen in Figure 2D-F. To determine the role of CHRM3 in DTX sensitivity in PCa, we treated DTX-sensitive PCa with the CHRM3 antagonist darifenacin (Dari) or knockdown of the receptor with siRNA. Both treatments decreased cell survival and colony formation when challenged with DTX compared to DTX alone as seen in Figure 2G-I. We next show the combination treatment of Die + DTX and Dari + DTX resensitize DTX-resistant PCa cells to DTX-induced cell death where the combination of drugs acts in a synergistic manner as seen in Figure 2J.

Finally, we show that the combination of Die + DTX and Dari + DTX can decrease cell survival and colony formation in a synergistic manner compared to DTX alone in DTX- sensitive NSCLC cells and DTX-resistant NSCLC cells as seen in Figure 2K-M.

Antimuscarinics combined with docetaxel induce apoptosis

Next, to determine the ability of the antimuscarinic + chemotherapy treatment to induce programmed cell death, we measured apoptosis via TUNEL assay. We show that DTX-induced apoptosis is enhanced in DTX-sensitive and DTX-resistant PCa cells when first pre-treated with either Die or Dari as seen with increased DNA fragmentation shown in Figure 3A-B. The combination treatment also enhances apoptosis in DTX-resistant NSCLC cells compared to DTX alone as seen in Figure 3C. Furthermore, pretreatment with either Die or Dari enhances expression of the pro-apoptotic proteins cleaved PARP1 and cleaved caspase 3 compared to DTX alone as shown in Figure 3D-E.

Antimuscarinics combined with docetaxel decrease tumor growth

To determine if the combination therapy of Die + DTX works in an in vivo model, we injected DTX-resistant PCa cells into SCID mice. These mice were then treated with either vehicle, Die (8mg/kg/daily/i.p.), DTX (lOmg/kg/once per week/i.p.) or a combination of Die + DTX. The combination of Die + DTX decreased tumor volume and tumor mass compared to Die alone and DTX alone as seen in Figure 4A-B.

Antimuscarinics combined with cabazitaxel decrease cell survival

To determine if antimuscarinics can be combined with the third generation taxane chemotherapeutic, cabazitaxel (CTX), we treated DTX-resistant PCa and NSCLC with a combination of Die + CTX and Dari + CTX. The combination of antimuscarinic + CTX decreased cell survival compared to CTX alone as seen in Figure 5A-B.

Triple combination of two antimuscarinics and docetaxel act synergistically

To determine if multiple antimuscarinics can be used in a triple combination with chemotherapy, we measured synergy of Die + Dari + DTX using Combenefit and the HSA synergy model. We showed that a combination of a low concentration of Dari and variable concentration of Die when combined with DTX acts synergistically to decrease cell survival as seen in Figure 6.

MATERIALS AND METHODS

Cell Culture

The prostate cancer cell lines 22Rvl and DU145 and the NSCLC cells A549 were grown in RPMI media supplemented with 10% fetal bovine serum and 1%

penicillin/streptomycin. Docetaxel-resistant (DTXR) clones of these cell lines (22RV1 ^r , DU145 ^r , and A549 ^R ) were maintained in 3 nM DTX for PCa cells and 16 nM for NSCLC cells. For experiments measuring the effect of DTX on DTXR cells, docetaxel was omitted from the media until treatment with DTX was required.

RT-qPCR

Total RNA was isolated using Trizol and converted to cDNA with M-MLV reverse transcriptase. All genes were amplified using POWERUP™ SYBR green and normalized to GAPDH expression. Each condition was performed in triplicate. Acetylcholine Secretion

Cells were grown in serum starved media supplemented with neostigmine (100 mM) for 36 hrs before media was collected and frozen at -80°C. Media was lyophilized at -80°C and reconstituted with 1/5 volume water. Total ACh was measured with the QUICKDETECT™ ACh ELISA Kit. Each condition was performed in triplicate and run in duplicate.

Crystal Violet Assay

Cells were treated as described in the figure legends, and then stained with crystal violet for 20 min. Cells were washed, dried, and the remaining crystal violet was reconstituted in methanol. Absorbance was measured at 570 nm. Each condition was performed in

quadruplicate, and all experiments were repeated at least three times.

Western Blot Cells were treated as described in the figure legends, and total protein was collected using RIPA buffer supplemented with HALT™ Protease and Phosphatase Inhibitor Cocktail. Phosphorylated proteins were normalized to the total protein intensity, and non-phosphorylated proteins were normalized to actin. All experiments were repeated at least three times.

TUNEL Assay

Cells were treated as described in the figure legends and fixed using 4%

paraformaldehyde. Apoptotic cells were stained using the In Situ Cell Death Detection Kit according to the manufacturers protocol. All cells were marked with the Hoechst nuclear stain. Each condition was performed in quadruplicate, and images were taken in 5 random fields per condition.

Drug Synergy Assay

Cells were treated with varying concentrations of inhibitor, DTX, or a combination of inhibitor and DTX. Inhibitor was always added to the cells 24 hrs before addition of DTX. Cells were stained with crystal violet, and absorbances measured at 570 nm. Synergy between drugs was measured using the HSA synergy method using Combenefit. Each condition was performed in quadruplicate, and all experiments were repeated at least three times.

Colony Formation Assay

Cells were plated at a low concentration (1,000 cells/well) and treated with inhibitor for 24 hrs before addition of DTX. Cells were then challenged with DTX for 48 hrs and then media was replaced with fresh media without drug. Cells were then grown for an additional 3-7 days and then fixed and stained with crystal violet. Plates were imaged and colonies including >50 cells were counted with ImageJ. Each condition was performed in triplicate, and all experiments were repeated at least three times.