METHOD OF TREATING GLIOMA CANCER USING TARP GAMMA 8-DEPENDENT AMPA RECEPTOR ANTAGONISTS

FIELD OF THE INVENTION

[0001] The embodiments of the present invention relate to methods for treating a malignant glioma comprising administering a therapeutically-effective amount of a TARP gd-dependent AMPA receptor antagonist.

BACKGROUND OF THE INVENTION

[0002] Glial-derived tumors ( i.e gliomas) are a common type of tumor originating in the brain rather than a metastatic brain cancer. About 33% of all brain tumors are gliomas, which originate in the glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes and ependymal cells. Gliomas are transformed cells that display increased metabolic activity as a result of the transformation process. Neoplastic transformation can occur in all glial cell types, thereby producing a large range of pathological and morphological variants. Most primary brain tumors derived from glial cells have lost growth control regulation, giving rise to astrocytomas, glioblastomas, or oligodendrocytomas. In the 2016 World Health classification of brain tumors, there were no less than 36 different subtypes of glioma identified, based on histology and various biomarkers. ¹

[0003] Glioblastoma multiforme (GBM), also known as glioblastoma, recurrent GMB, and/or grade IV astrocytoma, is an extremely aggressive tumor. Symptoms are similar to those of other brain tumors, and may include seizure, nausea and vomiting, headache, memory loss, hemiparesis, and progressive memory, personality, or neurological deficit due to temporal and frontal lobe involvement. Although any brain tumor can cause seizures, their development is most commonly associated with neuroepithelial tumors, including GBM. The incidence of epilepsy in patients with GBM varies between 30% and 62%, in about two thirds as presenting symptom and in one third developing during the course of the disease. ² [0004] It is very difficult to treat glioblastomas due to several complicating factors. GBM tumor cells are generally very resistant to conventional therapies. These tumors contain many different types of cells, wherein some cells may respond well to certain therapies, while others may not be affected at all. Another complicating factor is that normal brain cells are relatively susceptible to damage due to conventional therapy, and the brain has a very limited capacity to repair itself compared to other organs. The fact that many drugs cannot cross the blood-brain barrier to act on the tumor further limits the range of chemotherapies suitable for treating GBM patients.

[0005] GBM has the worst prognosis of any central nervous system (CNS) malignancy, despite multimodality treatment consisting of surgical resection of as much of the tumor as possible, with concomitant or sequential chemotherapy, radiotherapy, antiangiogenic therapy, immune therapy, gamma knife radiosurgery, and symptomatic management with corticosteroids. Prognosis is very poor, with a median survival time of approximately one year and the disease is almost invariably fatal, as only about 3% survive for more than 3 years. [0006] Accordingly, there is a need for new therapeutic targets and improved modalities for the treatment of malignant gliomas, including GBM.

BRIEF SUMMARY OF THE INVENTION

[0007] The embodiments of the present invention provide a method for treating a malignant glioma by administering an effective amount of a selective TARP g8 dependent AMPA receptor antagonist. In addition to its beneficial anti-seizure/anti-epileptic therapeutic effects, the administration of a selective TARP gd-dependent AMPA receptor antagonist to a subject afflicted with a malignant glioma results in one or more of: (a) an alleviation of one or more symptom of the glioma; (b) a delay or slowing of glioma tumor growth and/or metastasis; (c) a stabilized state of a glioma tumor or malignancy; and (d) an increased lifespan as compared to that expected in the absence of treatment. The malignant gliomas that can be treated by the methods of the present invention include, but are not limited to, astrocytoma, glioblastoma, oligodendrocytoma, pilocytic astrocytoma, diffuse intrinsic pontine glioma, ependymoma, oligo-astrocytoma, oligodendrogliocytoma, optic pathway glioma, and hypothalamic glioma.

[0008] In one embodiment, the method for treating a malignant glioma in a subject comprises administering to the patient a pharmaceutical composition comprising an effective amount of Formula I: or a pharmaceutically acceptable salt thereof.

[0009] In an alternate embodiment, the method for treating a malignant glioma in a subject comprises administering to the patient a pharmaceutical composition comprising an effective amount of Formula II: or a pharmaceutically acceptable salt thereof, wherein X is CH or N;

A is

R ¹ is selected from the group consisting of: hydrogen, deuterium, fluoro, methyl,

HO-(C1-C4)-alkyl, optionally substituted with one or two methyl or deuterium groups,

HO-(C1-C3)-alkoxy, optionally substituted with one or two methyl or deuterium groups, fluoro-(C1 -C3)-alkyl,

HO-(C1-C3)-alkoxy-methyl, optionally substituted with one or two methyl groups, cyano-(C1-C3)-alkoxy,

HO-(C1-C3)-alkylthio, optionally substituted with one or two methyl groups, HO-(C1-C3)-alkyl-NH-, HO-(C1-C3)-alkyl-N(CH3)-, methylsulfinyl, acyl, aminocarbonyl, methylcarbonylmethoxymethyl, aminomethylcarbonyloxyethoxy, triazolylmethyl,

1 -methyl-imidizol-2-ylthio, 5-hydroxymethyl-tetrahydrofuran-2-yl, 3-hyd roxy-3-methylazetidi n-1 -yl , 3-methoxy-azetidin-1 -yl

3-methoxy-3-methylazetidin-1 -yl

4-hydroxypiperidin-1-yl, 4-hydroxy-4-methyl-piperidin-1-yl, 4-hydroxy-4-vinyl-piperidin-1-yl, 4-hydroxymethyl-piperidin-1-yl, 4-(2-hydroxyethyl)-piperindin-1-yl, morpholin-4-yl,

2-hydroxymethyl-morpholin-4-yl, morpholin-4-yl-ethoxy, and tetrahydropyran-4-yl, provided that when A is then R1 is not unsubstituted HO-(C1-C3)-alkoxy, deuterium substituted HO-(C1- C3)-alkoxy ,or HO-(C1-C3)-alkythio.

[0010] In alternative embodiments, the pharmaceutical composition used in the treatment of a malignant glioma can further comprises one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0011] In alternative embodiments, the method of the present invention is administered concomitantly or sequentially with one or more of a surgical resection, chemotherapy, radiotherapy, antiangiogenic therapy, immune therapy, gamma knife radiosurgery, and symptomatic management with corticosteroids.

[0012] Other implementations are also described and recited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] For the purpose of illustration, certain embodiments of the present invention are shown in the drawings described below. It should be understood, however, that the invention is not limited to the precise arrangements, dimensions, and instruments shown. In the drawings: [0014] FIG. 1 shows provides a diagrammatic representation of the 96-well plates showing the wells containing Medium (labeled “M”), the wells containing PBS (labeled Έ”), and the final concentration map (mM) for the four test compounds: ES-481 and cisplatin concentrations are shown in FIG. 1A; temozolomide and perampanel concentrations are shown in FIG. 1B.

[0015] FIG. 2 shows the dose-response curves obtained in the LN-229 cell line with

Temozolomide ( ), Perampanel (- -M·- ----), ES-481 ), and cisplatin ( — ▼ — ).

[0016] FIG. 3 shows the dose-response curves obtained in the U-87 MG cell line with

Temozolomide ( — F — ), Perampanel ( — H — ), ES-481 and cisplatin ( — ▼ — ).

[0017] FIG. 4 shows the dose-response curves obtained in the U-251 MG cell line with

Temozolomide ( ), Perampanel ES-481 ( and cisplatin ( — ▼ — ).

[0018] FIG. 5 shows the survival percentage curves obtained in the in vivo orthotopic

U-87MG human glioblastoma xenograft mouse model with the vehicle ( — ® — ) and 100 mg/kg Temozolomide given QD x 5/week for 2 weeks, p.o. ( ).

[0019] FIG. 6 shows the Kaplan-Meier life survival curves obtained in the in vivo orthotopic U-87MG human glioblastoma xenograft mouse model in mice treated with the vehicle

(Group 1 - - - ), 20 mg/kg ES-481 given QD x 5/week for 3 weeks, p.o. (Group 2 - ),

ES-481 and 30 mg/kg Temozolomide given QD x 5/week for 2 weeks, p.o. combined with 20 mg/kg ES-481 given QD x 5/week for 3 weeks, p.o. (Group 3 . ).

DETAILED DESCRIPTION OF THE INVENTION

[0020] The subject innovation is now described with reference to the drawings, wherein like reference numerals are used to refer to like elements throughout. In the following description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the present invention. It may be evident, however, that the present invention may be practiced without these specific details. In other instances, well- known structures and devices are shown in block diagram form in order to facilitate describing the present invention. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.

DEFINITIONS

[0021] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below.

The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0022] 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. For example, reference to "a cell" includes a combination of two or more cells, and the like.

[0023] As used herein, the term "approximately" or "about" in reference to a value or parameter are generally taken to include numbers that fall within a range of 5%, 10%, 15%, or 20% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value). As used herein, reference to "approximately" or "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to "about X" includes description of "X".

[0024] As used herein, the term “or” means “and/or.” The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;

A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0025] As used herein, the term "comprising" means that other elements can also be present in addition to the defined elements presented. The use of "comprising" indicates inclusion rather than limitation. [0026] The term "consisting of" refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0027] As used herein the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0028] The term "statistically significant" or "significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

[0029] As used herein, the term "subject" refers to a mammal, including but not limited to a dog, cat, horse, cow, pig, sheep, goat, chicken, rodent, or primate. Subjects can be house pets (e.g., dogs, cats), agricultural stock animals (e.g., cows, horses, pigs, chickens, etc.), laboratory animals (e.g., mice, rats, rabbits, etc.), but are not so limited. Subjects include human subjects. The human subject may be a pediatric, adult, or a geriatric subject. The human subject may be of either sex. The terms “subject” and “patient” are used interchangeably herein.

[0030] As used herein, the terms "effective amount" and “therapeutically-effective amount” include an amount sufficient to prevent or ameliorate a manifestation of disease or medical condition, such as brain cancer. It will be appreciated that there will be many ways known in the art to determine the effective amount for a given application. For example, the pharmacological methods for dosage determination may be used in the therapeutic context.

In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will depend on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds.

[0031] As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” when used in reference to a disease, disorder or medical condition, refer to therapeutic treatments for a condition, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a symptom or condition. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a condition is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation or at least slowing of progress or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of the deficit, stabilized ( i.e ., not worsening) state of a tumor or malignancy, delay or slowing of tumor growth and/or metastasis, and an increased lifespan as compared to that expected in the absence of treatment.

[0032] As used herein, the term "long-term" administration means that the therapeutic agent or drug is administered for a period of at least 12 weeks. This includes that the therapeutic agent or drug is administered such that it is effective over, or for, a period of at least 12 weeks and does not necessarily imply that the administration itself takes place for 12 weeks, e.g., if sustained release compositions or long acting therapeutic agent or drug is used. Thus, the subject is treated for a period of at least 12 weeks. In many cases, long-term administration is for at least 4, 5, 6, 7, 8, 9 months or more, or for at least 1 , 2, 3, 5, 7 or 10 years, or more. [0033] The administration of the compositions contemplated herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. In a preferred embodiment, compositions are administered parenterally. The phrases “parenteral administration” and “administered parenterally” as used herein refers to modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0034] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level {e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0035] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

[0036] As used herein, the term “cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues.

Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

[0037] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth ( i.e ., division beyond normal limits), invasion ( i.e ., intrusion on and destruction of adjacent tissues), and metastasis {i.e., spread to other locations in the body via lymph or blood). As used herein, the term “metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a “metastatic tumor” or a “metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. [0038] As used herein, the term "benign" or "non-malignant" refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.

[0039] A “cancer cell” or “tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0040] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Hemopoietic cancers, such as leukemia, are able to out-compete the normal hemopoietic compartments in a subject, thereby leading to hemopoietic failure (in the form of anemia, thrombocytopenia and neutropenia) ultimately causing death.

[0041] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non- Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non- Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome.

[0042] A “cancer cell” is a cancerous, pre-cancerous, or transformed cell, either in vivo, ex vivo, or in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is associated with, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, anchorage independence, malignancy, loss of contact inhibition and density limitation of growth, growth factor or serum independence, tumor specific markers, invasiveness or metastasis, and tumor growth in suitable animal hosts such as nude mice.

[0043] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g., a cancer) or one or more complications related to such a condition, and optionally, but need not have already undergone treatment for a condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition in need of treatment or one or more complications related to such a condition. For example, a subject can be one who exhibits one or more risk factors for a condition or one or more complications related to a condition or a subject who does not exhibit risk factors. A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

PHARMACEUTICAL COMPOSITIONS

[0044] The compositions and methods of the present invention may be utilized to treat an individual in need thereof. In certain embodiments, the individual is a mammal such as a human, or a non-human mammal. When administered to an animal, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In preferred embodiments, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration ( i.e ., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophile for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as a lotion, cream, or ointment.

[0045] A pharmaceutically acceptable carrier can contain physiologically acceptable agents that act, for example, to stabilize, increase solubility or to increase the absorption of a compound such as a compound of the invention. Such physiologically acceptable agents include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients. The choice of a pharmaceutically acceptable carrier, including a physiologically acceptable agent, depends, for example, on the route of administration of the composition. The preparation or pharmaceutical composition can be a self-emulsifying drug delivery system or a self-micro emulsifying drug delivery system. The pharmaceutical composition (preparation) also can be a liposome or other polymer matrix, which can have incorporated therein, for example, a compound of the invention. Liposomes, for example, which comprise phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

[0046] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0047] The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc;

(8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;

(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water;

(17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. [0048] A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water. Details of appropriate routes of administration and compositions suitable for same can be found in, for example, U.S. Patent Nos. 6,110,973, 5,763,493, 5,731,000, 5,541 ,231 , 5,427,798, 5,358,970 and 4,172,896, as well as in patents cited therein.

[0049] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.

[0050] Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0051] Formulations of the invention suitable for oral administration may be in the form of capsules (including sprinkle capsules and gelatin capsules), cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), lyophile, powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient. Compositions or compounds may also be administered as a bolus, electuary or paste.

[0052] To prepare solid dosage forms for oral administration (capsules (including sprinkle capsules and gelatin capsules), tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate;

(5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; (10) complexing agents, such as, modified and unmodified cyclodextrins; and (11) coloring agents. In the case of capsules (including sprinkle capsules and gelatin capsules), tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0053] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropyl methyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0054] The tablets, and other solid dosage forms of the pharmaceutical compositions, such as dragees, capsules (including sprinkle capsules and gelatin capsules), pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0055] Liquid dosage forms useful for oral administration include pharmaceutically acceptable emulsions, lyophiles for reconstitution, micro-emulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, cyclodextrins and derivatives thereof, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetra hydrofury I alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

[0056] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0057] Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. [0058] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

[0059] The ointments, pastes, creams and gels may contain, in addition to an active compound, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0060] Powders and sprays can contain, in addition to an active compound, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0061] Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body. Such dosage forms can be made by dissolving or dispersing the active compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel.

[0062] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraocular (such as intravitreal), intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. Pharmaceutical compositions suitable for parenteral administration comprise one or more active compounds in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0063] Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), 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. 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.

[0064] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

[0065] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0066] Injectable depot forms are made by forming microencapsulated matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.

[0067] For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably,

0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.

[0068] Methods of introduction may also be provided by rechargeable or biodegradable devices. Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals.

A variety of biocompatible polymers (including hydrogels), including both biodegradable and non-degradable polymers, can be used to form an implant for the sustained release of a compound at a particular target site.

[0069] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0070] The selected dosage level will depend upon a variety of factors including the activity of the particular compound or combination of compounds employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0071] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the patient's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art. See, e.g., Isselbacher et al. (1996). ³ [0072] In general, a suitable daily dose of an active compound used in the compositions and methods of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0073] If desired, the effective daily dose of the active compound may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In certain embodiments of the present invention, the active compound may be administered two or three times daily. In other embodiments, the active compound will be administered once daily.

[0074] The patient receiving this treatment is any animal in need, including primates, in particular humans; and other mammals such as equines bovine, porcine, sheep, feline, and canine; poultry; and pets in general.

[0075] In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent.

[0076] The present disclosure includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. In certain embodiments, contemplated salts of the invention include, but are not limited to, alkyl, dialkyl, trialkyl or tetra-alkyl ammonium salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, L-arginine, benenthamine, benzathine, betaine, calcium hydroxide, choline, deanol, diethanolamine, diethylamine, 2-(diethylamino)ethanol, ethanolamine, ethylenediamine, N-methylglucamine, hydrabamine, 1H-imidazole, lithium, L-lysine, magnesium, 4-(2-hydroxyethyl)morpholine, piperazine, potassium, 1-(2- hydroxyethyl)pyrrolidine, sodium, triethanolamine, tromethamine, and zinc salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In certain embodiments, contemplated salts of the invention include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, l-ascorbic acid, l-aspartic acid, benzenesulfonic acid, benzoic acid, (+)-camphoric acid, (+)-camphor-10-sulfonic acid, capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1 ,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, d-glucoheptonic acid, d-gluconic acid, d-glucuronic acid, glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, hydrobromic acid, hydrochloric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, l-malic acid, malonic acid, mandelic acid, methanesulfonic acid , naphthalene-1 ,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, nitric acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, proprionic acid, l-pyroglutamic acid, salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, l-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, triflu oroacetic acid, and undecylenic acid salts. [0077] The pharmaceutically acceptable acid addition salts can also exist as various solvates, such as with water, methanol, ethanol, dimethylformamide, and the like. Mixtures of such solvates can also be prepared. The source of such solvate can be from the solvent of crystallization, inherent in the solvent of preparation or crystallization, or adventitious to such solvent.

[0078] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0079] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0080] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy; ⁴ The Encyclopedia of Molecular Cell Biology and Molecular Medicine; ⁵ Molecular Biology and Biotechnology: a Comprehensive Desk Reference; ⁶ Immunology; ⁷ Janeway's Immunobiology; ⁸ Lewin's Genes XI; ⁹ Molecular Cloning: A Laboratory Manual; ¹⁰ Basic Methods in Molecular Biology; ¹¹ Laboratory Methods in Enzymology; ¹² Current Protocols in Molecular Biology (CPMB); ¹³ Current Protocols in Protein Science (CPPS); ¹⁴ and Current Protocols in Immunology (CPI). ¹⁵

[0081] Other terms are defined herein within the description of the various aspects of the invention. GLUTAMATE RECEPTORS

[0082] Glutamate is the primary excitatory neurotransmitter in mammalian brain.

Glutamatergic signaling participates in a wide range of neural functions including learning and memory, long-term potentiation and synaptic plasticity. Glutamate receptors can be divided into two families. The ionotropic glutamate receptors form ion channels that activate upon binding agonist, opening a pore through the plasma membrane through which cations can flow. The metabotropic glutamate receptors are G-protein-coupled receptors, activating intracellular signal transduction cascades. The ionotropic glutamate receptors can be further subdivided into four sub-families, based upon sequence homology and selectivity to exogenous agonists. These sub-families are the AMPA a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid), NMDA (N-methyl-D-aspartate), kainate, and delta receptors.

[0083] The AMPA subtype of glutamate receptors are glutamate sensitive ion channels on postsynaptic membranes of excitatory synapses in the central nervous system (CNS) and are largely responsible for mediating fast neurotransmission across synaptic gaps. AMPA receptors assemble as tetramers of subunits. Mammals express four AMPA-receptor subunits, called GluA1-GluA4. Each GluA subunit can be expressed in multiple splice variants; the two most prominent splice variants are called flop and flip. GluA subunits freely form functional homo- and hetero-tetramers. The majority of RNA encoding GluA2 subunits is edited post- transcriptionally, altering a genetically-encoded glutamine to arginine. This RNA editing causes AMPA receptors to preferentially form with two GluA2 units, and also prevents calcium entry through the activated receptor.

[0084] In their native environment, the pore-forming GluA tetramers directly or indirectly associate with numerous auxiliary proteins which modify the trafficking, localization, gating characteristics, and pharmacology of the AMPA receptor (AMPAR). These auxiliary subunits include cytoskeletal and anchoring proteins, other signaling proteins, and several intracellular and transmembrane proteins with unknown function. The wide variety of proteins which can participate in AMPA receptor complexes vastly increases the ability of a neuron to tune the response characteristics of its synapses.

[0085] Transmembrane AMPA Receptor Regulatory Proteins (TARPs) are a fairly recently discovered family of proteins that have been found to associate with and modulate the activity of AMPA receptors. Several TARPs are fairly regiospecific in the brain, leading to physiological differentiation of the AMPA receptor activity. For example, TARP g2 (stargazin) dependent AMPA receptors are primarily localized in the cerebellum and cerebral cortex and TARP g8 dependent AMPA receptors are localized primarily in the hippocampus, which region is particularly relevant to seizure origination and/or propagation. It has been theorized that targeting individual TARPs may enable selective modulation of specific brain circuits without globally affecting synaptic transmission. ¹⁶

AMPA RECEPTOR ANTAGONISTS

[0086] AMPA receptor antagonists are known anticonvulsant agents and their ability to down modulate excitatory neurotransmission is key to their anti-epileptic therapeutic potential. However, since AMPA receptor activity is so ubiquitous in the CNS, general antagonism affects most areas of the CNS resulting in undesired effects, such as ataxia, sedation, and/or dizziness, which are shared by all known general AMPA receptor antagonists. Typically, these general antagonists have very narrow therapeutic dosing windows, meaning that typically the doses needed to obtain anti-convulsant activity are close to or overlap with doses at which undesired effects are observed. ¹⁷

[0087] Levetiracetam ((S)-2-(2-oxopyrrolidin-1-yl)butanamide), gabapentin (2-[1-

(aminomethyl)cyclohexyl]acetic acid), topiramate (2,3:4,5-Bis-0-(1-methylethylidene)-beta-D- fructopyranose sulfamate), and carbamazepine (5H-dibenzo[b,f]azepine- 5-carboxamide) are current leading therapeutic drugs for epileptic seizures. None of the currently approved drugs appear to act entirely through modulation of AMPA receptors.

[0088] Talampanel ((8R)-7-Acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1 ,3- dioxolo[4,5-h] [2,3]benzodiazepine), selurampanel (BGG492) (N-[7-isopropyl-6-(2-methyl-2H- pyrazol-3-yl)-2,4-dioxo-1 ,4-dihydro-2H-quinazolin-3-yl]methanesulfonamide), and perampanel (5'-(2-cyanophenyl)-1 '-phenyl-2, 3'-bipyridinyl-6'(1'H)-one) are general (non-TARP dependent/non-selective) AMPA receptor antagonists being tested as anti-epileptics.

[0089] Several selective, non-competitive TARP y8-dependent AMPA receptor antagonists were subsequently developed to be effective anti-seizure/anti-epileptic therapeutic drugs without the side effects ( e.g sedation, ataxia, and/or dizziness) of general (non-TARP dependent/non-selective) AMPA antagonists or TARP y2-dependent AMPA antagonists. Some selective, non-competitive TARP y8-dependent AMPA receptor antagonists are described in, for example, U.S. Patent Nos. 8,765,960 and 9,469,632.

[0090] AMPA receptor antagonists are known anticonvulsant agents, and their ability to down-modulate excitatory neurotransmission is critical to their antiepileptic therapeutic potential. However, because AMPA receptor activity is so ubiquitous in the central nervous system (CNS), a non-selective AMPA receptor antagonist approach affects many areas of the CNS, resulting in undesired effects such as ataxia, sedation, falls, and/or dizziness, which are shared by all known general or broad-spectrum AMPA receptor antagonists. Typically, the doses of these medications needed to obtain anticonvulsant activity are close to, or overlap with, doses at which undesired effects are observed.

[0091] TARPs are a fairly recently discovered family of proteins that have been found to associate with and modulate the activity of AMPA receptors. TARP-y8-dependent AMPA receptors are localized primarily in the hippocampus, a region of importance in complex partial seizures and particularly relevant to seizure origination and/or propagation. Research suggests that selectively targeting individual TARPs may enable selective modulation of specific brain circuits without globally affecting synaptic transmission, resulting in improved efficacy, safety, and tolerability.

[0092] ES-481 (previously known as CERC-611 , LY3130481 , LSN3130481 , 3130481) was discovered with the goal of retaining efficacy in epileptic patients but avoiding the motoric adverse events (AEs) that are a known liability of global AMPA receptor antagonists.

Importantly, ES-481 is the first molecule known to selectively target and functionally block region-specific AMPA receptors after oral dosing. This selectivity was engineered into ES-481 by chemical structure-activity relationship (SAR) studies to achieve selective blockade of AMPA receptors associated with the auxiliary protein TARP-y8 (high density in hippocampus, a region of importance in partial epilepsies) while sparing AMPA receptors associated with TARP-y2 (high density in cerebellum, which regulates the ataxia and falling associated with Fycompa ^® (perampanel)).

[0093] ES-481 is the first molecule to selectively target TARP-y8-containing AMPA receptors. Given the predominant hippocampal location of TARP-y8-dependent AMPA receptors, the efficacy and side effect profile ES-481 may represent an improvement compared to current antiepileptic drugs and may potentially treat high-grade gliomas..

GLIOMAS

[0094] Glial-derived tumors ( i.e gliomas) are a common type of tumor originating in the brain rather than a metastatic brain cancer. About 33% of all brain tumors are gliomas, which originate in the glial cells that surround and support neurons in the brain, including astrocytes, oligodendrocytes and ependymal cells. Gliomas are transformed cells that display increased metabolic activity as a result of the transformation process. They comprise a diverse group of neoplasms that differ in their morphology, their CNS location, their degree of invasiveness, their tendency for progression, and their growth characteristics. Neoplastic transformation can occur in all glial cell types, thereby producing a large range of pathological and morphological variants. Most primary brain tumors derived from glial cells have lost growth control regulation, giving rise to astrocytomas, glioblastomas, or oligodendrocytomas. In the 2016 World Health classification of brain tumors, there were no less than 36 different subtypes of glioma identified, based on histology and various biomarkers. ¹⁸

[0095] Astrocytomas are glial cell tumors developed from connective tissue cells called astrocytes and are the most common primary intra-axial brain tumor, accounting for nearly half of all primary brain tumors. They are most often found in the cerebrum (the large, outer part of the brain), but also in the cerebellum (located at the base of the brain).

[0096] Astrocytomas can develop in adults or in children. High-grade astrocytomas, called glioblastoma multiforme (GBM), are the most malignant of all brain tumors. Glioblastoma symptoms are often the same as those of other gliomas. Pilocytic astrocytomas are low-grade cerebellum gliomas commonly found in children. In adults, astrocytomas are more common in the cerebrum.

[0097] Brain stem gliomas, also called diffuse infiltrating brainstem gliomas or diffuse intrinsic pontine gliomas (DIPGs), are rare tumors found in the brain stem. They usually cannot be surgically removed because of their remote location, where they intertwine with normal brain tissue and affect the delicate and complex functions this area controls. These tumors occur most often in school-age children where they are responsible for the greatest number of childhood deaths from primary brain tumors.

[0098] Ependymomas develop from ependymal cells lining of the ventricles or in the spinal cord. Ependymomas are rare, accounting for just 2% to 3% of primary brain tumors. However, they account for about 8% to 10% of brain tumors in children and are more likely to affect those younger than 10 years old. The most location for ependymomas in children is near the cerebellum, where the tumor can block the flow of the cerebral spinal fluid and cause increased pressure inside the skull (obstructive hydrocephalus). These tumors can spread to other parts of the brain or spinal cord (drop-metastases) due to the flow of spinal fluid.

[0099] Mixed gliomas (also called oligo-astrocytomas) are made up of more than one type of glial cell. Their diagnosis as a distinct tumor type is controversial and may be resolved with genetic screening of tumor tissue. These tumors are often found in the cerebrum and are most common in adult men.

[0100] Oligodendrogliomas form from oliogodendrocytes, the supportive tissue cells of the brain and are usually found in the cerebrum. About 2% to 4% of primary brain tumors are oliogodendrogliomas. They are most common in young and middle-aged adults and more likely to occur in men. Seizures are a very common symptom of these gliomas (affecting 50% to 80% of patients), as well as headache, weakness, or problems with speech. Oligodendrogliomas typically have a better prognosis than most other gliomas. They are significantly more favorable in terms of their biological behavior and their prognosis for patients and can have survivorship measured of decades for folks with these tumors that have the most favorable biomarker profiles.

[0101] Optic pathway gliomas are a type of low-grade tumor found in the optic nerve or chiasm. They often infiltrate the optic nerves, which send messages from the eyes to the brain. People with neurofibromatosis are more likely to develop them. Optic nerve gliomas can cause vision loss and hormone problems, since these tumors are often located at the base of the brain where hormonal control is located. Gliomas affecting hormone function may be known as hypothalamic gliomas.

[0102] The most important determinant of survival for gliomas is the "grade" of the glioma. Increasing grades represent increasing malignancy and decreasing differentiation, which is associated with increased mitotic activity and enhanced cell migration. ^19,20 Grade I tumors grow slowly and can sometimes be totally removed by surgery, while grade IV tumors are fast-growing, aggressive, and difficult to treat. High-grade gliomas account for 30% of primary brain tumors in adults and are the second most common cause of cancer death in children under 15 years of age. High-grade gliomas are divided by grade into two categories: anaplastic astrocytomas (WHO Grade III) and glioblastoma multiforme (GBM; WHO Grade IV). ²¹

[0103] GBM (Grade IV) tumors are characterized by the presence of areas of necrotizing tissue that are surrounded by anaplastic cells with numerous mitosis and endothelial proliferation. This characteristic, as well as the presence of hyperplastic abnormal blood vessels, differentiates the tumor from Grade III astrocytomas, which do not have these features. GBM tumors usually appear in the cerebral white matter, grow quickly, and can become very large before causing symptoms. Less than 10% form more slowly, following dedifferentiation of low-grade astrocytoma or anaplastic astrocytoma. These are called secondary GBM tumors and are more common in younger patients (mean age 45 versus 62 years). The tumor may extend into the meninges or ventricular wall, leading to high protein content in the cerebrospinal fluid (CSF) (>100 mg/dL), as well as an occasional pleocytosis of 10 to 100 cells, mostly lymphocytes. Malignant cells carried in the CSF may spread (rarely) to the spinal cord or cause meningeal gliomatosis. However, metastasis of GBM beyond the central nervous system is extremely unusual. About 50% of GBM tumors occupy more than one lobe of a hemisphere or are bilateral. Tumors of this type usually arise from the cerebrum and may rarely exhibit the classic infiltration across the corpus callosum, producing a butterfly (bilateral) glioma. [0104] GBM has the worst prognosis of any central nervous system (CNS) malignancy, despite multimodality treatment consisting of surgical resection of as much of the tumor as possible, with concomitant or sequential chemotherapy, radiotherapy, antiangiogenic therapy, immune therapy, gamma knife radiosurgery, and symptomatic management with corticosteroids. Prognosis is very poor, with a median survival time of approximately one year and the disease is almost invariably fatal, as only about 3% survive for more than 3 years.

GLIOMA-ASSOCIATED EPILEPSY

[0105] Glioma-associated epilepsy is a type of symptomatic focal epilepsy, which may manifest with focal seizures, generalized seizures, or both. Epilepsy occurs in >80% of patients with a low-grade glioma and 40-60% of patients with glioblastoma. ^22,23 It is often the first clear presentation of the glioma. Anti-neoplastic treatments often cause amelioration of epilepsy, possibly by reverting the pathophysiological pro-epileptogenic processes in the tumor.

[0106] Excessive glutamate release by glioma cells has been reported to induce pharmacologically-accessible neuronal hyperexcitation, including epilepsy. ²⁴ Two recent reports by Venkataramani et al. (2019) ²⁵ and Venkatesh et al. (2019) ²⁶ suggest that neuronal hyperexcitation stimulates bona fide glutamatergic synapses on glioma cells. Ionotropic glutamate receptors activate intercellular calcium signaling networks to orchestrate glioma cell growth and invasion, presumably by facilitating oncogenic signaling cascades and cytoskeletal remodeling.

[0107] Research has recently started to focus on these pathophysiological mechanisms and on the possibilities for new mechanism-based anti-epileptic treatments. Two recent investigations assessed the effects of perampanel (FYCOMPA ^® ), the first antiepileptic drug in the class of selective non-competitive antagonist of AMPA receptors, ²⁷ on glioma. Izumoto et al. (2019) ²⁸ assessed the effects of perampanel on the response to seizure and tumor- progression in 13 patients with gliomas and uncontrollable seizures. Perampanel (4 mg/day or 8 mg/day) was tested after surgery, radiation, and chemotherapy treatments. Epileptic seizures were reported to be eliminated in eight of the 13 patients and volume reduction was detected during the 6 month period. Unfortunately, the abstract does not specify if the radiation and chemotherapy overlapped with the perampanel treatment. The authors concluded that perampanel treatment was effective at controlling the epilepsy and inhibiting tumor progression. However, the abstract does not discuss the potential contributing effects of surgery, radiation, and chemotherapy treatments to the observed beneficial effects attributed to perampanel. In contrast to the alleged beneficial effects reported by Izumoto etal. (2019), a subsequent report by Mayer et al. (2020) ²⁹ indicated that perampanel inhibited epileptiform discharges in organotypic brain slices of glioma but failed to attenuate tumor growth or promote animal survival in rat C6 glioma model.

[0108] Even if perampanel was determined to be effective at attenuating tumor growth and/or promoting the survival of subjects afflicted with a glioma, it should be noted that this drug has significant adverse effects. The drug label has an FDA box warning that the drug cause may cause serious psychiatric and behavioral changes; it may cause homicidal or suicidal thoughts. ³⁰ Other side effects have included dizziness, somnolence, vertigo, aggression, anger, loss of coordination, blurred vision, irritability, and slurred speech. Perampanel also reduces the effectiveness of levonorgestrel oral contraceptives by about 40%. ³¹ Women who may get pregnant should not take it as studies in animals show it may harm a fetus. Perampanel is liable to be abused; very high doses produced euphoria responses similar to ketamine. As such, it is designated as a Schedule III controlled substance by the Drug Enforcement Administration. ³² Accordingly, these significant adverse effects would limit the therapeutic usefulness of perampanel for treating gliomas.

TARP Y8-DEPENDENT AMPA RECEPTOR ANTAGONIST FOR THE TREATMENT OF GLIOMAS [0109] Severe brain cancers have been shown that integrate into the brain’s wiring. ^33,34

High-grade gliomas, including glioblastoma, form synapses that hijack electrical signals from healthy nerve cells to drive their own growth. High-grade gliomas form synapses with healthy neurons that transmit electrical signals to the cancerous tissue. The tumors also contain cell-to- cell electrical connections known as gap junctions. Together, the two types of connections allow electrical signals from healthy nerve cells to be conducted into and amplified within the tumors.

[0110] High-grade gliomas include glioblastoma, a brain tumor seen in adults that has a five-year survival rate of 5%; diffuse intrinsic pontine glioma, a pediatric brain tumor with a five- year survival rate below 1%; and other diagnoses such as pediatric glioblastoma and diffuse midline gliomas occurring in the spinal cord and thalamus. Previous studies have indicated that high-grade gliomas use normal brain activity to drive their growth. ^35,36

[0111] Existing drugs that block electrical currents have been found to reduce growth of high-grade gliomas. Perampanel, a seizure medication that blocks activity of neurotransmitter receptors on the receiving end of a synapse, was reported to reduce proliferation of pediatric gliomas implanted into mice by 50%. Meclofenamate, a drug that blocks the action of gap junctions, resulted in a similar decrease in tumor proliferation. ³⁷

[0112] AMP-type glutamate receptors (AMPARs) are the principal transducers of fast synaptic transmission in the mammalian CNS. When high-grade gliomas form synapses with healthy neurons that transmit electrical signals to the cancerous tissues, these connections allow electrical signals from healthy nerves to be conducted and amplified within the tumor; allowing tumor growth. It was thus hypothesized that the use of selective TARP gd-dependent AMPA receptor antagonists has the potential for anti-tumorigenic effect by slowing down or inhibiting these electrical connections.

[0113] The present invention provides that selective TARP gd-dependent AMPA receptor antagonists can be effective therapeutics for the treatment of gliomas without the undesired effects ( e.g sedation, ataxia, and/or dizziness) of general (non-TARP dependent/non-selective) AMPA antagonists or TARP y2-dependent AMPA antagonists.

In addition to its beneficial anti-seizure/anti-epileptic therapeutic effects, the administration of a selective TARP gd-dependent AMPA receptor antagonist to a subject with a glioma will result in one or more of: (a) an alleviation of one or more symptom of the glioma; (b) a delay or slowing of glioma tumor growth and/or metastasis; (c) a stabilized state of a glioma tumor or malignancy; and (d) an increased lifespan as compared to that expected in the absence of treatment.

[0114] The present invention provides that TARP gd-dependent AMPA receptor antagonists useful for the treatment of gliomas include a compound of Formula I: i.e., 6-((S)-1-{1-[5-(2-hydroxy-ethoxy)-pyridin-2-yl]-1/-/-pyrazol -3-yl}-ethyl)-3/-/-1 ,3-benzothiazol- 2-one), or a pharmaceutically acceptable salt thereof. This antagonist, also known as ES-481 (previously known as CERC-611 , LY3130481 , LSN3130481 , 3130481 ), is a potent and selective antagonist of transmembrane alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptor regulatory protein (“TARP”)-Y8-dependent AMPA receptor that is being developed as an adjunctive therapy for the treatment of partial-onset seizures with or without secondarily generalized seizures in patients with epilepsy and for high-grade gliomas whose progression is robustly regulated by neuronal activity. The present invention also provides the use of a pharmaceutical composition comprising a compound of Formula I, or a pharmaceutically-acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0115] Additional TARP gd-dependent AMPA receptor antagonists useful for the method of the present invention for the treatment of gliomas include a compound of Formula II: or a pharmaceutically acceptable salt thereof, wherein X is CH or N;

A is

R ¹ is selected from the group consisting of: hydrogen, deuterium, fluoro, methyl,

HO-(C1-C4)-alkyl, optionally substituted with one or two methyl or deuterium groups,

HO-(C1-C3)-alkoxy, optionally substituted with one or two methyl or deuterium groups, fluoro-(C1 -C3)-alkyl,

HO-(C1-C3)-alkoxy-methyl, optionally substituted with one or two methyl groups, cyano-(C1-C3)-alkoxy,

HO-(C1-C3)-alkylthio, optionally substituted with one or two methyl groups, HO-(C1-C3)-alkyl-NH-, HO-(C1-C3)-alkyl-N(CH3)-, methylsulfinyl, acyl, aminocarbonyl, methylcarbonylmethoxymethyl, aminomethylcarbonyloxyethoxy, triazolylmethyl,

1 -methyl-imidizol-2-ylthio, 5-hydroxymethyl-tetrahydrofuran-2-yl, 3-hyd roxy-3-methylazetidi n-1 -yl , 3-methoxy-azetidin-1 -yl

3-methoxy-3-methylazetidin-1 -yl

4-hydroxypiperidin-1-yl, 4-hydroxy-4-methyl-piperidin-1-yl, 4-hydroxy-4-vinyl-piperidin-1-yl, 4-hydroxymethyl-piperidin-1-yl, 4-(2-hydroxyethyl)-piperindin-1-yl, morpholin-4-yl,

2-hydroxymethyl-morpholin-4-yl, morpholin-4-yl-ethoxy, and tetrahydropyran-4-yl, provided that when A is then R1 is not unsubstituted HO-(C1-C3)-alkoxy, deuterium substituted HO-(C1- C3)-alkoxy, or HO-(C1-C3)-alkythio.

[0116] The present invention also provides the use of a pharmaceutical composition comprising a compound of Formula II, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically-acceptable carriers, diluents, or excipients.

[0117] According to the present invention, selective TARP gd-dependent AMPA receptor antagonists can be used for the treatment of various gliomas including but not limited to, astrocytoma, glioblastoma, oligodendrocytoma, pilocytic astrocytoma, diffuse intrinsic pontine glioma, ependymoma, oligo-astrocytoma, oligodendrogliocytoma, optic pathway glioma, and hypothalamic glioma.

[0118] The main initial therapies for malignant gliomas and for glioblastomas typically start with a craniotomy and surgical resection for non-diffuse gliomas involving pre-op and intra operative imaging, awake craniotomies or real-time monitoring of neurological condition. A tissue sample then goes to a pathologist and a name and histology is given for the tumor and a grade. Importantly, biomarkers are identified that not only help classify tumors, but also are predictive of response to certain therapies. Treatment is tailored, as much as it can be, to the particular type of glioma but most patients will receive external beam radiation therapy. Patients with many of the tumor types will receive the radiation combined with the oral, alkylating agent temozolomide, an oral alkylating agent.

[0119] Accordingly, the method of the present invention can include a combination therapy. The administration of a selective TARP gd-dependent AMPA receptor antagonist for the treatment of a glioma can be done concomitantly or sequentially with one or more of a surgical resection, chemotherapy, radiotherapy, antiangiogenic therapy, immune therapy, gamma knife radiosurgery, and symptomatic management with corticosteroids.

[0120] Temozolomide (TMZ; brand names Temodar and Temodal and Temcad) is an oral chemotherapy drug. It is an alkylating agent widely used in the treatment of recurrent malignant gliomas. It is used as a second-line treatment for astrocytoma and as a first-line treatment for glioblastoma multiforme.

[0121] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0122] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0123] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

HUMAN EXPOSURE TO ES-481

[0124] ES-481 is currently being developed for the treatment of seizure by the Applicant of the present invention. In the first Phase 1 study performed, a single ascending dose (3 mg,

6 mg, 12 mg, 25 mg, 37 mg, 50 mg, 62 mg, 75 mg, 87 mg, and 100 mg) is being administered in healthy adult volunteers in order to assess the safety, tolerability, and pharmacokinetics of ES-481. To date, the single dose cohorts of 3 mg, 6 mg, 12 mg, 25 mg, 37 mg, and 50 mg of ES-481 have completed dosing in six active treatment (ES-481) and two placebo volunteers per cohort. All patients in the study are receiving continuous EEG monitoring prior to the administration of the dose and up to 24 post-dose. Only two mild adverse events were possibly related to the administration of ES-481 : mild emesis was reported by one volunteer after receiving the 12 mg dose and dysgeusia (altered taste) by one volunteer after receiving the 37 mg dose.

EXAMPLES

[0125] The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention and are not intended to limit the invention. EXAMPLE 1 CELL VIABILITY ASSESSMENT OF THREE CELL LINES FOLLOWING TREATMENT WITH ES-481 AND THREE OTHER TEST COMPOUNDS: TEMOZOLOMIDE, PERAMPANEL,

AND ClSPLATIN

[0126] The tumor growth attenuation potential of ES-481 and three other test compounds, temozolomide, perampanel, and cisplatin, were assessed using a CellTiter-Glo ^® Luminescent Cell Viability Assay (Promega) in three cell lines: LN-229, U-87 MG, and U-251 MG. The CellTiter-Glo ^® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells.

MATERIALS Cell Lines

[0127] Three cell lines were used for the cell viability assessments: LN-229, U-87 MG, and U-251 MG. These three cell lines were derived from malignant gliomas and are commonly used for research on gliomas.

[0128] The LN-229 cell line (ATCC ^® CRL-2611 ™) ³⁸ is a human glioblastoma cell line that was established in 1979 from cells taken from a patient with right frontal parieto-occipital glioblastoma. U-87 MG (ATCC ^® HTB-14™) ³⁹ is one of a number of cell lines derived from malignant gliomas by J. Ponten and associates from 1966 to 1969. ⁴⁰ U-251 MG cell line (Sigma product no. 09063001 ) is a human cell line derived from a malignant glioblastoma tumor by explant technique. The U-251 cell line was established at the Wallenberg laboratory, Uppsala, Sweden, more than 40 years ago and was derived from a male patient with malignant astrocytoma. ⁴¹

[0129] The cell lines were cultured, and the cell viability was assessed in 96-wells plate

(Cat. No: 3610, Corning). The culture medium used, and the seeding numbers per well are provided in Table 1. All the cell lines are cultured at 37°C with 5% CO2.

Table 1

Reagents

[0130] The following reagents were used in the cell viability assays:

CellTiter-Glo (CTG) (Cat. No: G7573, Promega) DMEM (Cat. No: SH30243.01 , HyClone)

RPMI1640 (Cat. No: SH30809.01, Hyclone)

FBS (Cat. No: FND500, ExCell)

DMSO (Cat. No: 0231-500ml, Amresco)

NEAA (Cat. No: SH30238.01 , Hyclone)

MEM (Cat. No: SH30024.01)

0.25% Trypsin-EDTA (Gibco, Cat. No: 25200-072, Lot. No: 2063861).

Equipment

[0131] The following equipment was used in the cell viability assays:

Vi-Cell XR, Beckman Coulter (TACEL0030)

Envision 2104 Multilabel Reader, PerkinElmer (USA)

CO2 Incubator, SANYO

Reverse microscope, Chongguang XDS-1B, Chongqing Guangdian Corp..

Test Compounds

[0132] As mentioned above, ES-481 will be compared to three other test compounds, temozolomide, perampanel, and cisplatin in a cell viability assessment assay. ES-481 is the selective TARP gd-dependent AMPA receptor antagonist: 6-((S)-1-{1-[5-(2-hydroxy-ethoxy)- pyridin-2-yl]-1/-/-pyrazol-3-yl}-ethyl)-3/-/-1,3-benzothiazo l-2-one previously characterized for its anti-seizure/anti-epileptic effects. ⁴²

[0133] Temozolomide (TMZ; brand names Temodar and Temodal and Temcad) is an oral chemotherapy drug. It is an alkylating agent widely used in the treatment of recurrent malignant gliomas. It is used as a second-line treatment for astrocytoma and as a first-line treatment for glioblastoma multiforme.

[0134] Perampanel (sold under the brand name FYCOMPA ^® ) is an antiepileptic drug that is used in addition to other drugs to treat partial seizures and generalized tonic-clonic seizures for people older than 12 years. It was the first antiepileptic drug in the class of selective non-competitive antagonist of AMPA receptors. As described above, its therapeutic potential to attenuate tumor growth or promote animal survival has recently been investigated in human and rats with mixed success. However, perampanel’s well-documented significant adverse effects limit its potential usefulness in the treatment of glioma.

[0135] Cisplatin was employed as a positive control for this study. Cisplatin is one of the most effective chemotherapeutic agents to date used for the treatment of many malignancies, including glioma. ⁴³ Cisplatin causes tumor cell death by direct DNA damage and by generating reactive oxygen intermediates. Cisplatin is the most common platinum chemotherapy drug; it was first approved in the U.S. in 1978. But cisplatin is not without its problems. It has an extensive list of horrible side effects including severe nausea and vomiting. These two side effects are, in fact, so bad that the drug's development was almost stopped when it was first tested on people. In addition, tumors can become resistant to platinum-based chemotherapy drugs.

[0136] Relevant information on the four test compounds is provided in Table 2.

Table 2

METHODS

Day 1: Cell seeding

[0137] Each well of a 96-well plate contained 90 pl_ of cell suspension with standard medium. The number of cells to be seeded was determined on the basis of Cell Density Optimization Assays.

Day 2: Drug treatment

[0138] Test compounds were serially-diluted (1 :3) in 96-well plates at the concentrations indicated in FIG. 1. The test compounds (10 pL/well) were added to the corresponding plates (ES-481 and cisplatin are shown in FIG. 1A; temozolomide and perampanel are shown in FIG. 1B). The final volume was 100 pL/well for all the plates. The cell lines were cultured at 37°C with 5% C0 ₂ .

Day 5: Plate reading

[0139] After three days of incubation with the test compounds, cells were observed under a microscope to ensure that cells treated with the vehicle control were in good condition. Once confirmed, 50 mI_ of CellTiter-Glo ^® Reagent was added to each well. The contents were mix for 2 minutes on an orbital shaker to facilitate cell lysis. The plate were then allowed to incubate at room temperature for 10 minutes to stabilize luminescent signals and the luminescence was recorded using an EnVision Multi Label Reader. Data Analysis

[0140] Data analysis was performed using GraphPad Prism 7.0. In order to calculate the half maximal effective concentration (ECso) values, a dose-response curve was generated using a nonlinear regression model with a sigmoidal dose response. The formula of surviving rate is shown below, and the ECso values were automatically generated by GraphPad Prism 7.0.

RESULTS

[0141] The dose response curves for ES-481 , temozolomide, perampanel, and cisplatin in the cell lines LN-229, U-87 MG, and U-251 MG are shown in FIG. 2, FIG. 3, and FIG. 4, respectively, and the half maximal effective concentration (ECso) values are provided in Table 3.

Table 3

[0142] As expected, the most effective test compound was the positive control, cisplatin, followed by ES-481 , and then perampanel and temozolomide. Although cisplatin displayed the best tumor growth attenuation potential ( i.e the lowest ECso values), this compound has well- known extensive list of horrible side effects. ES-481 was the second most effective test compound in all three cell lines, ahead of perampanel and temozolomide. The least effective test compound was temozolomide; ECso values could not be calculated at the doses tested. [0143] These data suggest that, in addition to its beneficial anti-seizure/anti-epileptic therapeutic effects, the administration of a selective TARP gd-dependent AMPA receptor antagonist like ES-481 would attenuate tumor growth in subjects afflicted with a glioma thereby producing one or more of the following beneficial effects: (a) an alleviation of one or more symptom of the glioma; (b) a delay or slowing of glioma tumor growth and/or metastasis;

(c) a stabilized state of a glioma tumor or malignancy; and/or (d) an increased lifespan as compared to that expected in the absence of treatment. EXAMPLE 2 IN VIVO EFFICACY ASSESSMENT OF ES-481 AND A COMBINATION OF ES 481 AND TEMOZOLOMIDE IN THE ORTHOTOPIC U-87 MG HUMAN GLIOBLASTOMA CANCER XENOGRAFT MODEL IN FEMALE BALB/C NUDE MICE

[0144] The present study evaluated preclinically the in vivo therapeutic efficacy of

ES-481 and a combination of ES-481 and temozolomide in the orthotopic U-87 MG human glioblastoma cancer xenograft model in BALB/c nude mice.

EXPERIMENTAL METHODS Animals

[0145] Female Mus musculus BALB/c nude mice (n=30) were obtained from Beijing

Anikeeper Biotech Co., Ltd (Beijing, China). At the initiation of the study, mice were 7-8 weeks old (estimated age at inoculation) with a body weight of 17.0-21.9 g. Mice were housed in Polysulfone IVC cage (325 mm * 210 mm * 180 mm) up to 4 mice per cage with cardboard cylinder, tissue paper, and PC tube/club house for environment enrichment and crushed corncob bedding, autoclaved; changed weekly. The housing temperature was 20-26°C with a 40-70% humidity level and a light cycle of 12 hour light (7:00 am-7:00 pm) and 12 hours dark. Mice were provided 0.2 pm filtered, reverse osmosis, autoclaved water and fed standard rodent chow, irradiated, ad libitum.

Cell Culture

[0146] The U-87 MG tumor cells were maintained in vitro as a monolayer culture in

DMEM medium supplemented with 10% heat inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO2 in air. The cells, when in exponential growth phase, were harvested and quantitated by cell counter before tumor inoculation.

Selection of Dose

[0147] The dose of ES-481 selected was based on the anti-seizure doses previously documented in a mice seizure models. In addition, the dose of 20 mg/kg ES-481 was also chosen because the expected anti-seizure dose in humans is thought to range from approximately 80 mg to 150 mg per day of ES-481 and this dose should be sufficient to inhibit hyperactive electrical conduction in cancerous glioma cells. To assess the potential contribution of ES-481 in combination with temozolomide in this orthotopic xenograft model, a dose of 30 mg/kg x 5/week x 2 week of temozolomide was selected in combination with 20 mg/kg x 5/week x 3 weeks of ES-481. Tumor Inoculation

[0148] After anesthesia by intraperitoneal injection of Ketamine (0.1 mL/mouse), head skin of the mouse was sterilized with the 75% alcohol and then a 2 to 3 mm length incision was made just at the right of the midline and anterior to the interaural line. Mice were inoculated intracranially with the tumor cells (2 *10 ⁵ cells in 2 mI_ PBS) at the right frontal lobe 2 mm lateral from the bregma and 0.5 mm from the anterior at a depth of 3.5 mm. The incision was stitched using No.6 suture and then sterilized with povidone iodine solution. The mice were kept warm until they recovered from anesthesia. The treatments were started at day 5 after randomization. Each group consisted of 10 mice.

Randomization

[0149] Before grouping and treatment, all animals were weighed, body weight was used as numeric parameter to randomize selected animals into specified groups. Thus, the systematic error was minimized.

[0150] The randomization started at Day-5 after inoculation. Thirty (30) mice were enrolled in the study. All animals were randomly allocated to three treatment groups described in Table 4. Randomization is performed based on “Matched distribution” method/ “Stratified” method (StudyDirector™ software, version 3.1.399.19).

Table 4

* Vehicle: 20% (w/v) HPBCD in 25 mM phosphate buffer (pH 9)

ROA: Route of Administration is oral or per os (P.O.).

Observation and Data Collection

[0151] After tumor cells inoculation, the animals were checked daily for morbidity and mortality. During routine monitoring, the animals were checked for any effects of tumor growth and treatments on behavior such as mobility, food and water consumption, body weight gain/loss (body weights were measured every other day after randomization), eye/hair matting and any other abnormalities. Mortality and observed clinical signs were recorded for individual animals in detail. Body weights and tumor volumes were measured using StudyDirector™ software (version 3.1.399.19). EXPERIMENTAL TERMINATION Study Endpoints

[0152] The major endpoint was animal survival. The survival of all animals were monitored and the median survival time (MST) was calculated for each group. The increase in life-span (ILS) was calculated as follows:

II _ _{i nn} NVledian Survival Time of drug treated group

Median Survival Time of vehicle group ' °'

Study Termination

[0153] The observation time for the survival study was 40 days after inoculation. The brain tumor was removed after euthanasia and weighed. The differences in tumor weights among groups were analyzed for significance using the ANOVA test.

Body Weight Loss

[0154] The body weight of all animals were monitored throughout the study and animals were euthanized if they lost over 20% of their body weight relative to the weight at the first day of treatment.

General Animal Welfare Surveillance

[0155] All animals were monitored throughout the study and animals were euthanized if any of the following signs are observed: severe dehydration, hypothermia, abnormal/labored respiration, lethargy, obvious pain, diarrhea, skin lesions, neurological symptoms, impaired mobility (not able to eat or drink) due to significant ascites and enlarged abdomen, astasia, continuous prone or lateral position, signs of muscular atrophy, paralytic gait, clonic convulsions, tonic convulsions, persistent bleeding from body orifice.

Statistical Analysis

[0156] For survival analysis, Kaplan-Meier survival curves were generated, and a Log

Rank test was performed. The event of interest was the animal death. The survival time was defined as the time from the day of tumor cell inoculation until one day before animal death or ethical endpoint. For each group, the median survival time (MST), corresponding 95% confidence interval and the increased in life-span (ILS) were calculated. All data were analyzed using SPSS 18.0 and/or GraphPad Prism 5.0. P < 0.05 was considered statistically significant. RESULTS

Historical Data with this Model

[0157] Prior to initiating the present in vivo orthotopic U-87MG human glioblastoma xenograft mouse model study with ES-481 , it was well documented in this model that, with a temozolomide dose of 75 mg/kg (data not shown) and 100 mg/kg x 5days/week x 2 week (FIG. 5), no animal survived beyond Day 30.

ES 481 Alone or In Combination with Temozolomide

[0158] Although there was no statistically-significant difference in overall survival between vehicle (Group 1) and ES-481 (Group 2), there was a statistically-significant difference in the weight of the tumors between Group 1 and Group 2. More impressive was the fact that all of the animals in ES-481 and temozolomide (Group 3) survived and had no tumors presented when sacrificed at Day 41. Table 5 provides the tumor weights and FIG. 6 shows the overall survival curves for the three groups.

Table 5: Tumor Weights (mg)

Abbreviations: SD = Standard Deviation; Cl = Confidence Interval

[0159] These data suggest that, in addition to its beneficial anti-seizure/anti-epileptic therapeutic effects, the administration of a selective TARP gd-dependent AMPA receptor antagonist like ES-481 attenuates tumor growth in subjects afflicted with a glioma thereby producing one or more of the following beneficial effects: (a) an alleviation of one or more symptom of the glioma; (b) a delay or slowing of glioma tumor growth and/or metastasis;

(c) a stabilized state of a glioma tumor or malignancy; and/or (d) an increased lifespan as compared to that expected in the absence of treatment.

[0160] Surprisingly, the data further indicated that combining the treatment of ES-481 with temozolomide, an alkylating agent widely used in the treatment of recurrent malignant gliomas, significantly and drastically enhanced the tumor growth attenuation effects of ES-481 alone. Indeed, all of the animals treated with the combination of ES-481 with temozolomide survived the entire observation period of 40 days and none of these animals had any measurable tumor weight.

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[0161] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0162] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the present aspects and embodiments. The present aspects and embodiments are not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect and other functionally equivalent embodiments are within the scope of the disclosure. Various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects described herein are not necessarily encompassed by each embodiment. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.